tonywujun wrote:
同志们，这段时间养小鼠骨髓来源的EPC出现奇怪现象，其中一些细胞4天后快速增殖，迅速形成片状集落，细胞可以排列形成线样结构，且可以融合，有些还有分支，形成的结构很像带有分支的血管，奇怪了，我从来没有见过这种细胞，请各位战友帮忙看看这可能是什么细胞。
一张100倍的图
应该是瓶子上的划痕留下的。细胞沿着划痕生长，看看我养的细胞，更漂亮。呵呵，当时觉得不可思议。其实，是个肝癌细胞而已。
（缩略图，点击图片链接看原图）
不过，我做实验，发现好多次，原代EPC培养的时候，确实会出现部分细胞连接成环状，几乎每次原代都可以发现。可以肯定，不是划痕所造成的，下图是比较典型的一张，偷懒，没有用摄像头拍，用数码相机拍的，所以拍摄效果不太好。‘
（缩略图，点击图片链接看原图）
个人同意yulinlong战友的观点。
yulinlong战友的图片又多又漂亮，与他相比真是逊色多了，
匿了。
首先我肯定的是这不是fibronectin划痕造成的结果，培养皿内形成的片状细胞团块很大，目前已经对此细胞进行了lectin和CD31的免疫荧光鉴定，不表达，基本上表明其不是内皮及其前体细胞，具体是什么细胞还不清楚，但从形态上讲，不像成纤维细胞、内皮细胞、平滑肌细胞、骨骼肌细胞、心肌细胞，不管怎样，以前从来没有见过，且比较了所有相关帖子上的细胞，没有见到相类似的细胞，真是郁闷呀，哪位高人提供一下线索呀。
用国产淋巴细胞分离液分离、提取白膜层后老是混有较多的红细胞，其影响单个核细胞的贴壁。本人所在实验室建议使用蒸馏水破除。
请问各位高手，你们是怎么处理这些红细胞的？
另外使用羟乙基淀粉法效果好吗？
小小鸟 wrote:
八戒兄：
你在实验中也能将外周血单核细胞浓度调整到1*10的9次方/L？真是羡慕啊。不过听我师姐说外周血单核细胞不多，要将10 ml的血分离出这么多单核细胞我还是有点诧异。请问八戒兄也是用10ml血分离的吗？
1*10的9次方/L只是个浓度问题，并不是个数量，10ml血分离1*10的9次方/L×
10ml,是不是？
1*10的9次方/L只是个浓度问题，并不是个数量，10ml血分离1*10的9次方/L×
10ml,是不是？
这个我明白，但我从文献上看到：成年循环EPCs数量非常有限（100～500个/ml），照1*10的9次方/L×10ml这样算，10ml外周血有10的7次方EPCs，还是跟文献不符啊
请教：
国内哪家公司代理有VEGF的EBM-2培养基。
是毕特博吗。
寒冰 wrote:
请教：
国内哪家公司代理有VEGF的EBM-2培养基。
是毕特博吗。
我查到的也就只有这家
wzmc225 wrote:一般是800G的离心力，既然你的机子是400G 那就把转速适当调高就是，比方现在是1500R/M,先调到2500R/M左右试试。,
在学习了playingstone 战友的2篇大作（一篇已经发表：生物医学工程与临床，2005，7，9（4）243～246.恭喜恭喜）之后，受益颇丰：
1.playingstone 战友培养方法比较有新意，综合了目前2种有争论的培养方法。我想问下48h内贴壁细胞和48h后贴壁细胞中内皮祖细胞的纯度比较问题，哪个更纯一些？也就是说哪个内皮祖细胞含量更多些？
2.playingstone 战友采用骨髓来源的培养方法，在用密度为1.071的淋巴细胞分离液（上海华精生物制品厂）进行梯度密度离心分离单核细胞，18℃,1500r/min离心23min后。想问下，实验室采用的是什么离心机，哪个厂家的。因为我们自己也在用这种淋巴细胞分离液，效果很不好，用的是Beck man高效离心机，400g，30min，效果很差，几乎看不到白膜层，连红细胞都没离心下来，试剂是刚买的，未过期。很是郁闷。
3.playingstone 战友在离心后，似乎未经过洗这一步，就直接制成细胞悬液，是不是在骨髓来源的培养方法中是这样的呢，有点疑问？呵呵
谢谢，欢迎讨论！
小小鸟 wrote:
八戒兄：
你在实验中也能将外周血单核细胞浓度调整到1*10的9次方/L？真是羡慕啊。不过听我师姐说外周血单核细胞不多，要将10 ml的血分离出这么多单核细胞我还是有点诧异。请问八戒兄也是用10ml血分离的吗？
我是10毫升分大约10的7 单个核细胞的
两个问题：
1.UEA-I和DiLDL双染色阳性细胞为正在分化的EPCs的原理是什么？而且我见一篇文献竟然用是否DiLDL染色阳性区别EPC和EC，不解
2.有用多聚赖氨酸代替Fn包板的吗？
请都各位，我准备培养大鼠的EPC，不知大家所用的VEGF和b-FGF是哪家公司的，有小包装吗？
下面是一篇blood上的如何培养EPC的文献，希望对大家有用。文件太大（475kb)，不能上传，所以给出全文摘要。
EPC生长特征.doc (35.0k)
我们买的VEGF是 sigma公司的，2微克包装的，500块钱，bFGF没买，我们用的是M199培养基＋VEGF＋PEX＋20％FBS养的兔的EPC，效果还行。
以前用的是EGM培养基，后来有一阵子EGM国内缺货，就改用了上述配方。
两种相比较，EGM培养基培养细胞的状态稳定一些，而且细胞生长较均匀，现在用的这这种培养基养的EPC原代的时候全部成簇状生长，也就是说一个培养瓶里长几大团细胞，没有细胞簇的地方几乎都是空的。长了6天以后有细胞的地方非常密，我们就消化，把细胞吹下来后还是放在原瓶里长，细胞就长得比较均匀了，生长速度也还可以。
以前用EGM时我们用的是5％血清浓度，现在用M199是20％血清浓度，但是感觉还是没有EGM时长得快。
请教大家：
加VEGF诱导骨髓细胞应在什么时候加？大概传几代啊？
寒冰 wrote:
请都各位，我准备培养大鼠的EPC，不知大家所用的VEGF和b-FGF是哪家公司的，有小包装吗？
下面是一篇blood上的如何培养EPC的文献，希望对大家有用。文件太大（475kb)，不能上传，所以给出全文摘要。
帮你把全文上传下
分成2部分，都下载后，放在同一个文件下，然后解压part1就可以了。
2577.part1.rar (244.14k)
22222222222222222222222222222
2577.part2.rar (211.6k)
请问cici_wl 师兄：
你是培养大鼠EPC吗，EGM是哪家公司的，培养板包被了吗，是自己包的吗？
谢谢。
playingstone前辈，请教您一下：
骨髓源的内皮祖细胞是本身就存在于骨髓的，而不是由MSCs分化而来的吗？如果是，它的前身是什么？
MSCs能不能分化为内皮祖细胞，还是只能分化为成熟内皮细胞？
我见到的文献好像不管是骨髓、外周血、脐血的内皮祖细胞，都是直接就用内皮细胞诱导培养基培养。就您所知，有没有先扩增MSCs，再将其诱导为EPCs的？
请问各位:你们消化EPC时候,觉得好消化吗?含EDTA的胰酶大概要作用多久啊?
我消化了15分钟(37度),吹打了几次才勉强下来!!
请问：
用DMEM低糖 和 高糖 的培养基对细胞的影响是什么呢？为什么MSC要用低糖的呢？
恳请指教。谢谢！
harry158:
你好，我也打不开下面这篇综述，能否发一份到邮箱(newbyte163@163.com)
综述 <Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells >
(blood ,106,1525-1531).
我已经分离了数次脐血里的EPC,大家说的这些我好象似曾见过,可鉴定时总是CD133(-)和CD34(-),但每次贴负在FIBRONECTIN上细胞也不少,我真不知道如果不是EPC会是什么,作了快3个月了,一直这样,都快崩溃了,大家帮我出出主意吧,多谢.
meini:
这片文献上有很漂亮的EPC集落 "Granulocyte Colony-Stimulating Factor Mobilizes Functional Endothelial Progenitor Cells in Patients With Coronary Artery Disease"(Arterioscler Thromb Vasc Biol. 2005;25:296-301.)
是pdf 文件,不知道怎么上传,如果有需要请告诉我邮箱.
我的邮箱是newbyte163@163.com,非常感谢.
求合购：
我现已问好小鼠CD 133 和FLK－1 上流式检测抗体，最小包装分别为25微克和50微克。（为Biosource公司产品）（都可以上100管以上）
由于本人只能用一半的量。价钱比较昂贵。想找位战友一起分享。我在武汉。有意者请与我联系。qq 358228927。 手机：13476210795
急！！急！！急！！
不好意思，才回贴。前一段时间出去开会。
现在EPC培养有些进展，也拍了图片，但我的图片都比较大，不能上传，如你感兴趣我可以发给你。从形态、FCM还是IHC均是EPC。
请问羟乙基淀粉沉淀红细胞怎么做？
不好意思，才回贴。前一段时间出去开会。
现在EPC培养有些进展，也拍了图片，但我的图片都比较大，不能上传，如你感兴趣我可以发给你。从形态、FCM还是IHC均是EPC。
请问羟乙基淀粉沉淀红细胞怎么做？
楼上的PDF文件均打不开，各位都能否MAIL给我。再次感激不禁。我有的东西，不会让各位失望的。多多交流。xiexie
my mail： dryudecai@hotmail.com
强烈建议我们开个网络会议！
方式： Hotmail MSN
时间待定：大家商量
我想一起聊天，可能效果更好
为表诚意，我先贴有关EPC的reriew。
多指教
Role of eNOS in neovascularization: NO for endothelial progenitor cells
Nitric oxide (NO) is a gaseous molecule with an astonishingly wide range of physiological and pathophysiological activities, including the regulation of vessel tone and angiogenesis in wound healing, inflammation, ischaemic cardiovascular diseases and malignant diseases. Recent data have revealed the predominant role of endothelial nitric oxide synthase (eNOS), an endothelial-cell-specific isoform of NO producing enzyme, in both angiogenesis (the development of new blood vessels derived from existing vessels) and vasculogenesis (blood vessel formation de novo from progenitor cells). In addition, successes in gene therapy, together with the recent development of an eNOS-specific inhibitor, suggest that the modulation of eNOS might be a potent new strategy for the control of pathological neovascularization.
Nitric oxide (NO) is produced by nitric oxide synthase (NOS), a haem-containing enzyme that is linked to NADPH-derived electron transport by flavin adenine dinucleotide and flavin mononucleotide. NOS catalyzes the oxidation of l-arginine to L-citrulline and NO, using tetrahydrobiopterin as an essential co-factor. There are three isoforms of NOS. Endothelial NOS (eNOS, also referred to as type III NOS) is constitutively expressed by vascular endothelial cells; it has a calcium-dependent activity and generates relatively low levels of NO. The NO produced by eNOS mediates a variety of physiological functions in vivo including neovascularization, regulation of blood vessel tone (vessel wall tension), platelet aggregation, vascular permeability and leukocyte–endothelial interaction. By contrast, inducible NOS (iNOS, type II NOS) is transcriptionally regulated by inflammatory cytokines and other stimuli, it is calcium-independent and it generates higher levels of NO (than does eNOS), which can induce cytostatic or toxic effects. Finally, neuronal NOS (nNOS, type I NOS) mediates the transmission of neuronal signals. In this article, we focus on the complex roles of eNOS during neovascularization, the process of new blood vessel formation.
Endothelial NO synthase in angiogenesis.
Pathological angiogenesis is a hallmark of cancer and of various ischaemic and inflammatory diseases. In addition to NO, more than 20 angiogenic factors have been discovered during the past two decades, including vascular endothelial growth factors (VEGF-A, B, C, D and E), placental-derived growth factor (PlGF), platelet-derived growth factors (PDGF-A, B, C and D), fibroblast growth factors (FGF-1 and 2), transforming growth factors (TGF- and ), hepatocyte growth factor (HGF), platelet-derived endothelial growth factor (PD-EGF), tumour necrosis factor- (TNF), interleukin-8 (IL-8), angiopoietins (Ang-1 and 2) and sphingosine 1-phosphate (S1P). The interactions among these molecules and their effect on vascular structure and function in different environments are currently under intensive investigation. As a central mediator, NO not only functions alone to induce endothelial migration and proliferation, but also modulates the effects of many of the aforementioned angiogenic factors.
VEGF, an endothelial-specific mitogen and survival factor, is one of the most potent angiogenic factors, and its signalling is crucial for both angiogenesis and vasculogenesis. In endothelial cells, VEGF activates eNOS by the induction of calcium flux, the recruitment of heat-shock protein 90 (Hsp90) and the phosphorylation of NOS via the phosphatidylinositol-3-OH-kinase [PtdIns(3)K]–Akt pathway. Predominantly mediated through eNOS, the intricate relationship between VEGF and NO has a major role in vascular permeability, vessel tone and angiogenesis during inflammation, wound healing and tumour growth. This makes the selective modulation of eNOS activity an attractive strategy for altering angiogenesis and vascular permeability. The first proof of this concept was recently published by Gratton, Sessa and colleagues, who showed that selective inhibition of eNOS by cavtratin blocks tumour vessel hyperpermeability and halts tumour progression.
Postnatal vasculogenesis: mechanisms of progenitor and stem-cell mobilization.
Over the last few years, several seminal studies have highlighted the key role of bone-marrow-derived cells in both normal and pathological states. Major advances have been made in the identification of haematopoietic stem cells (HSCs), the determination of their function and niche location, and the experimental expansion of HSC populations. Endothelial progenitor cells (EPCs), which are closely related to HSCs, carry out postnatal vasculogenesis and mediate post-ischaemia and tumour neovascularization in both xenografted models and (to a lesser extent) genetically induced tumours. The group of Rafii and collaborators showed that growth factors such as VEGF, PlGF, Ang-1 and stromal-derived factor-1 (SDF-1) induce the mobilization and expansion of EPC and HSC populations in the bone marrow. This was the consequence of both direct signalling through receptors on various subsets of precursor cells and of paracrine interactions with the bone marrow stromal cells. One crucial event for mobilization is the expression of matrix metalloproteinase 9 (MMP-9 or gelatinase in the bone marrow stromal-cell compartment. In turn, MMP-9 releases soluble c-Kit ligand (sKitL), which induces survival, mobilization and proliferation in the stem and progenitor cell compartments. In the bone marrow, VEGF itself conveys survival and mitogenic signals to both endothelial progenitor cells and hematopoietic stem cells through VEGF receptor 2 (VEGFR2 or flk-1/KDR). Furthermore, VEGF and PlGF contribute to cell mobilization for neovascularization and haematopoiesis reconstitution after 5 fluorouracil (5-FU) injury, via VEGF receptor 1 (VEGFR1 or flt-1), present on a variety of precursor cells and their progeny. In agreement with these reports, a recent study demonstrated that VEGFR1 signalling upregulated MMP-9 in lung endothelial cells. It is also of interest that MMP-9 is activated by NO.
Human Endothelial Progenitor Cells
Blood vessel development is a regulated process involving the
proliferation, migration, and remodeling of endothelial cells (ECs)
from adjacent pre-existing blood vessels (angiogenesis) or following
differentiation of endothelial progenitor cells (EPCs) or angioblasts
from mesodermal precursors (vasculogenesis). EPCs were originally
thought to be present only during embryonic development. However,
accumulating evidence in the past several years suggests that they can
persist into adult life. This has generated interest in the use of EPCs
for neovascularization of ischemic or injured tissue and for the clinical
assessment of risk factors for various diseases.
Phenotypic Characterization and Lineage of EPCs
Studies to purify and characterize EPCs from bone marrow (BM) or peripheral blood (P have been hampered by the absence of markers to phenotypically distinguish these cells from mature vascular wallderived ECs and from subsets of hematopoietic cells (Table 1). The identification of markers has in turn been hindered by the lack of functional assays to accurately evaluate EPCs. Subsequently, the term EPC has been used to describe different cell populations by different authors, with some recent papers calling the primary cells EPCs and others calling the progeny (cultured) cells EPCs. Although the precise differentiation pathway of an immature EPC to a mature EC is undefined, the loss of expression of CD133 is currently thought to represent a good marker to distinguish between an endothelial progenitor and a mature endothelial cell.1,5 At least some cells that express the antigens CD133, VEGFR2 and/or CD34 can differentiate into cells of the endothelial lineage in vitro, and contribute to neovascularization in animal models of ischemia.6-8 However, hematopoietic stem cells may also co-express these antigens. For this reason, some investigators believe that these cells represent the “hemangioblast”, a bipotential progenitor that can give rise to both endothelial and hematopoietic cells. Clonogenic studies by Pelosi et al.9 demonstrate that CD34+VEGFR2+ cells from BM and cord blood are able to form mixed hematopoietic-endothelial colonies in vitro as might be expected of the hemangioblast, although pure endothelial colonies were not shown. Lineage marker negative, CD117+ (c-kit+) BM cells, considered to contain hematopoietic stem cells, can also induce neovascularization of ischemic myocardium when delivered locally, thereby improving cardiac function in a mouse myocardial infarct model, suggesting that EPC are contained within that population.10 Reyes et al.11 have shown that multi-potent adult progenitor cells (MAPCs) were able to form cells capable of differentiating into cells expressing markers of mature endothelium (CD31, CD36, vWF). These cells formed vascular tube-like structures in Matrigel™ and, following injection into immunocompromised mice, contributed to tumor angiogenesis and participated in wound healing. The SP cell population (cells with the ability to efflux fluorescent dyes) contains hematopoietic stem cells and may also contain EPCs. SP cells may be derived from BM12 and other tissues.13 The time required for mature ECs to arise from cultured hematopoietic cells has been used as an alternative approach to the characterization of EPCs.14 In a study using sex-mismatched bone marrow transplant recipients, recipientderived cells expanded ~20 fold in endothelial culture conditions, whereas donor (bone marrow)-derived cells expanded ~1000 fold in the same conditions. The majority of EC outgrowth from recipient cells occurred in 5 - 9 days, while EC outgrowth from donor (bone marrow) cells occurred later (~1 month). These results suggest that EPC (with a greater proliferative potential) are bone marrow-derived and can be distinguished from mature EC by expansion potential and kinetics in culture. More recently, clonogenic studies have evaluated the proliferative potential of single cells derived from the adherent mononuclear cells of adult peripheral and umbilical cord blood.15 Adult PB was found to contain cells with the ability to expand for 20 – 30 population doublings, termed low proliferative potential endothelial colony-forming cells (LPP-ECFC). However cord blood contained cells that were able to form secondary and tertiary colonies (100 population doublings) containing cells expressing an array of EC surface proteins. These cells were termed high proliferative potential endothelial colony-forming cells (HPP-ECFC). The development of such clonogenic assays to evaluate the self-renewal and proliferative capacity of putative EPCs will be very helpful in determining the phenotype of the EPC and its differentiation pathway.
Isolation and Culture of EPCs
The majority of in vitro studies have focused on developing culture conditions to promote the differentiation of EPCs into mature, terminally differentiated ECs.7,8,14,16,17 However, there is currently no standardized procedure for the isolation and in vitro culture of EPCs. Most commonly, mononuclear cells are cultured on plastic (with either the adherent or the non-adherent fraction considered to contain the EPC), or CD34+ or CD133+ cells are positively selected and then cultured. The medium formulation, concentration of serum, growth factors utilized (VEGF, IGF-1, FGF-b, bovine brain extract), substrate upon which cells are grown (fibronectin, type I collagen, gelatin), and time in culture vary between researchers. This makes it very difficult to compare studies. Analysis of the EC phenotype of the outgrowth cells has been performed by flow cytometric analysis or immunostaining using the phenotypic markers described above, or by colony morphology at various time points (7 days to 4 weeks) following culture. This has led to contradictions in the estimation of EPC numbers in the circulation and may be problematic when using EPC quantitation as a surrogate marker in clinical studies (see below). For example, Kalka et al.18 suggested that EPCs (defined as CD34+VEGFR2+VE-cadherin+) are present at a frequency of ~ 3000 - 5000 cells per mL of blood based on flow cytometric analysis of peripheral blood mononuclear cells. In contrast, a study by Peichev et al.7 using CD34 magnetic beads for isolation estimated the frequency of EPCs (defined here as CD34+VEGFR2+AC133+) to be ~ 70 - 210 cells per mL of blood.
EPC in Disease States
There has been no systematic study of the number of EPCs in healthy individuals. Several studies have described the influence of pathological conditions, drugs, and growth factors on the number of “EPCs” (not exactly the same cells in each study) in vivo (Table 2). For example, the number of circulating EPCs and their migratory activity has been reported to be reduced in patients with risk factors for coronary artery disease19 or to negatively correlate with the Framingham cardiovascular risk score.20 EPCs from patients with diabetes mellitus type 2 are characterized by a decreased proliferative capacity, reduced adhesiveness, and reduced ability to form capillary tubes in vitro.21 The mechanism responsible for these findings are unknown but may be attributed to a decreased mobilization of EPCs from the BM, an increased consumption of EPCs at the injured site, and/or a reduced half-life of EPCs. In contrast, limb ischemia22 and acute myocardial infarction23 were associated with a rapid increase of EPCs in the circulation. Treatment with different hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) inhibitors (statins)24-25 and a number of growth factors including EPO,26 VEGF,18 G-CSF,7 and GM-CSF22 have also been reported to increase the number of EPCs in vivo. Preliminary evidence suggests that they may act by mobilizing EPCs from the BM and/or may improve the survival of EPCs by activation of the PI3 kinase/Akt pathway.6 Mancuso et al.4 found the numbers of resting and activated mature endothelial cells were increased in both breast cancer and lymphoma patients. More importantly, seven lymphoma patients who had chemotherapy and were in complete remission had similar numbers of mature ECs compared to healthy controls. These findings may indicate that quantitation of EPC or EC numbers could be used as a surrogate marker for evaluating response to treatment in specific diseases.
Summary
Although significant progress has been made in this exciting arena of research,
it is evident from the literature that a standardization of the procedures used for the
isolation, phenotypic characterization and culture of EPCs will be a prerequisite for
the use of EPCs in future therapies or for EPC quantitation as a diagnostic tool in
clinical studies.
References
1. Schmeisser A, Strasser RH: Phenotypic overlap between hematopoietic cells with suggested angioblastic
potential and vascular endothelial cells. Journal of Hematotherapy and Stem Cell Research 11: 69-79,
2002
2. Rafii R: Circulating endothelial precursors: mystery, reality, and promise. Journal of Clinical
Investigation 105: 17-19, 2000
3. Leucocyte Typing VI. White cell differentiation Antigens. Eds. Kishimoto, T. et al. Garland Publishing,
Inc, NY, 1998
4. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini F: Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 97: 3658-3661, 2001
5. Rafii S, Lyden D: Therapeutic stem and progenitor cell transplantation for organ vascularization and
regeneration. Nat Med 9: 702-712, 2003
6. Masuda H, Asahara T: Post-natal endothelial progenitor cells for neovascularization in tissue
regeneration. Cardiovasc Res 58: 390-398, 2003
7. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA,
Rafii S: Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of
functional endothelial precursors. Blood 95: 952-958, 2000
8. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner
JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964-967, 1997
9. Pelosi E, Valtieri M, Coppola S, Botta R, Gabbianelli M, Lilli V, Marzia G, Masella B, Muller R, Sgadari D,
Testa U, Bonanno G, Peschle C: Identification of the hemangioblast in postnatal life. Blood 100: 3203-
3208, 2002
10. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu
S: Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents
cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430-436, 2001
11. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM: Origin of endothelial progenitors in
human postnatal bone marrow. J Clin Invest 109: 337-346, 2002
12. Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA, Hirschi KK: Distinct progenitor
populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular
regeneration. J Clin Invest. 111: 71-79, 2003
13. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi
KK, Goodell MA: Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.
J Clin Invest. 107: 1395-1402, 2001
14. Lin Y, Weisdorf DJ, Solovey A, Hebbek RP: Origins of circulating endothelial cells and endothelial
outgrowth from blood. J Clin Invest 105: 71-77, 2000
15. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Yoder MC:
Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical
cord blood. Blood 104: 2752-2760, 2004
16. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA,
Storb RF, Hammond WP: Evidence for circulating bone marrow-derived endothelial cells. Blood 92: 362-
367, 1998
17. Quirici N, Soligo D, Caneva L, Servida F, Bossolasco P, Deliliers GL: Differentiation and expansion of
endothelial cells from human bone marrow CD133+ cells. Br J Haematol 115: 186-194, 2001
18. Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI,
Isner JM, Asahara T: Vascular endothelial growth factor165 gene transfer augments circulating
endothelial progenitor cells in human subjects. Circ Res 86: 1198-1202, 2000
19. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S: Number and
migratory activity of circulating endothelial progenitors cells inversely correlate with risk factors for
coronary heart disease. Circ Res 89: e1-e7, 2001
20. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T: Circulating endothelial
progenitor cells, vascular function and cardiovascular risk. N Engl J Med 348: 593-600, 2003
21. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC: Human
endothelial progenitor cells from type II diabetic exhibit impaired proliferation, adhesion and
incorporation into vascular structures. Circ 106: 2781-2786, 2002
22. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T:
Ischemia-and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for
neovascularization. Nat Med. 5: 434-438, 1999
23. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T:
Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circ 103: 2776-
2779, 2001
24. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher AM, Dimmeler S: Increase in circulating
endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circ. 103:
2885-2890, 2001
25. Llevadot J, Murasawa S, Kureishi Y, Uchida S, Masuda H, Kawamoto A, Walsh K, Isner JM, Asahara T.:
HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells. J Clin Invest
108: 399-405, 2001
26. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H,
Zeiher AM, Dimmeler S: Erythropoietin is a potent physiological stimulus for endothelial progenitor cell
mobilization. Blood 102:1340-1346, 2003
27. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, Imaizumi T:
Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization.
J Clin Invest 105: 1527-1536, 2000
28. Gill M, Dias S, Hattori K, Rivera ML, Hicklin D, Witte L, Girardi L, Yurt R, Himel H, Rafii S: Vascular
trauma induces rapid but transient mobilization of VEGFR2+AC133+ endothelial precursor cells.
Cir Res 88: 167-174, 2001
29. Kim HK, Song KS, Kim HO, Chung JH, Lee KR, Lee YJ, Lee DH, Lee ES, Kim HK, Ryu KW, Bae JM:
Circulating numbers of endothelial progenitor cells in patients with gastric and breast cancer.
Cancer Letters 198: 83-88, 2003
30. Carmeliet P: Angiogenesis in health and disease. Nat Med 9: 653-660, 2003
Morphological and phenotypical characterization of human endothelial progenitor cells in an early stage of differentiation
Lydia Bellik, Fabrizio Ledda, Astrid Parenti*
Summary
We demonstrate, for the first time, that PBMC cellular fraction in suspension, which is usually discarded or seeded again after a few days, displays comparable behaviour and may constitute a non-negligible target.
So it is more necessary and feasible to culture PMNCs in EGM-2 for the fourth day.
Abstract The exact phenotype and lineage of endothelial progenitor cells (EPCs) are still a matter of debate and different expansion protocols are used to obtain them. In this study, EPC expansion from peripheral blood mononuclear cells was analyzed within the first week of culture. Both the adherent and suspended cells, of which the latter usually discarded, were considered. We provide, for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture. Moreover, within the 2nd day, both cellular fractions displayed a significant increase in endothelial marker expression which correlated with EPC properties. _
2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
ex vivo EPC expansion protocols.
EPCs have been isolated from adult peripheral blood mononuclear cells (PBMCs) using magnetic bead selection against different stem cell antigens [1,3] or by primary adherence on fibronectin [4].
Unselected or selected EPCs have been cultured on fibronectin and non-adherent cells discarded or seeded again within 3–4 days; the immunophenotype of the adherent EPCs has been assessed after a culture period of several days to weeks [5,6].
infusion of ex vivo expanded EPCs derived from PBMCs in nude mice or rats has improved neovascularization in ischemia models [4] and initial pilot trials indicate that infusion of EPCs improve the blood supply to ischemic tissue and that this therapeutic approach is safe and feasible [7].
2. Materials and methods
2.1. Cell culture
PBMCs were isolated by Ficoll/Paque density gradient centrifugation from healthy human peripheral blood. 5*106 cells were stained immediately while remaining cells were cultured on fibronectin (Sigma)- coated dishes for 1, 2, 3 and 7 days in EBM-2 (Clonetics, San Diego) supplemented with EGM-2 MV Single-Quots and 10% FCS.
2.2. Flow cytometry analysis
An Epics XL flow cytometer (Beckman Coulter) was used for all analyses.
Light scatter profiles were collected on the cell using two electronic gates to discriminate among different PBMC populations. PBMCs were labelled and analyzed on day 0 and after 1, 2, 3 and 7 days of culture. At every time-point adherent and suspended PBMCs were separately recovered.
The following fluorescent antibodies were used: PECAM-1-FITC and aVb3 (BD-PharMingen; San Diego, CA), VE-cadherin-FITC and CD45-FITC (Chemicon; Temecula, CA), VEGF-R2-PE (R&D Systems, Minneapolis, MN), CD14-PE (Santa Cruz; Santa Cruz, CA). Results were expressed as percent positive cells. Isotype-identical antibodies served as controls and dead cells were excluded by 7-ADD. Each analysis included 100000 events. The mean fluorescence intensity (MFI) > 2 was considered positive.
2.3. Immunostaining
Cells were incubated with DiI–acLDL (Molecular Probes, Leiden, The Netherlands) and after fixation with FITC-UEA-1 (VECTOR Laboratories, Peterborough, England). Samples were viewed with an inverted fluorescent microscope [5].
2.4. RT-PCR analysis
Total RNA was extracted using Purescript RNA isolation kit (Gentra, Minneapolis, MN). RT-PCR was performed using 1ug of total RNA.
Primers for amplification:
VEGF-R2, sense-5’-AgACTTTgAGCATggAAg- 3’, antisense-5’-CCATTCCACCAAAAgATg-3’;
VECadherin sense-5’-AAgACATCAATgACAACTTCC-3’, antisense- 5’-CCTCCACAgTCAggTTATACC-3’ [9];
vWF sense-5’-gAggCTg- AgTTTgAAgTgC-3’, antisense-5’-CTgCTCCAgCTCATCCAC-3’ [9];
PECAM-1 sense-5’-CAACgAGAAAATgTCAgA-3’, antisense-5’- ggAgCCTTCCgTTCTAgAgT-3’ [10];
GAPDH sense-5’-CCATggAgAAggCTgggg- 3’, antisense-5’-CAAAgTTgTCATggATgACC-3’ [11].
The PCR programs: KDR (40 cycles); PECAM-1 (30 cycles); GAPDH (23 cycles), annealing at 56 _C; VE-Cadherin (40 cycles) and vWF (23 cycles) annealing at 60 _C. Quantitative evaluation was obtained as ratios between optical density of the target genes and GAPDH amplification products.
2.5. Migration assay
A modified Boyden chamber (48-well plates; Neuroprobe) was used as previously described [8]. Adherent and suspended cells (5 • 104), cultured for 2 and 3 days, were seeded in the upper wells of the chamber; SDF-1a (10 ng/ml) and VEGF (50 ng/ml) were added to lower wells. After 5 h at 37 _C, migrated cells were methanol-fixed, stained with Diff-Quick and counted by microscopic evaluation (400• in 10 random fields. Each experimental point was measured in triplicate.
2.6. Matrigel tubule assay
Twenty-four-well multidishes were coated with growth factor reduced Matrigel_ (50 ll/cm2, BD-PharMingen; San Diego, CA) according to the manufacturers instructions. Suspended and adherent (6.5 • 105) plated cells were suspended in EBM-2 and incubated at 37 _C. Network formation was observed using a phase contrast microscope. Human umbilical vein endothelial cells (HUVEC) were used as positive control.
2.7. Statistical evaluation
Data are reported as means ± S.E.M. Statistical analysis was performed using the ANOVA test (Bonferroni_s and Dunnett_s Multiple Comparison Test). P < 0.05 was considered significant.
3. Results
3.1. Morphologic analysis of the heterogeneous PBMC population
Flow cytometric morphological discrimination of PBMC populations was performed using their light scatter properties so as to identify them by their position in the light scatter plot (Fig. 1). The forward and right angle light scatter detectors were set so that lymphocytes and monocytes were observed in gate R1, while gate R2 was set using HUVEC with the light scatter characteristics of mature endothelial cells (Fig. 1A0). The starting population, confined in R1, showed lymphomonocytic morphology which was small and slightly granular. After 1 day of culture, 21 ± 4% of PBMCs were adherent and reached 33.5 ± 6.3% (P < 0.01 vs day 1, n = ;16) after 1 week, showing that the majority of cultured PBMCs were still in suspension. From day 2 to one week, a new population appeared in R2 with a different morphology which was large and granular (Fig. 1. This trend was highlighted by both the adherent and suspended cells. In gate R2, suspended cells (5%, day 1) doubled after 3 days (P < 0.01 vs day 1, n = 16). Likewise, the adherent cells (28.5 ± 3.45%, day 1) increased by 34% after 3 days (P < 0.05 vs day 1, n = 16).
3.2. Early endothelial marker expression in PBMCs
The immunophenotype of R1- and R2-gated cells, was selectively characterized during cell differentiation in the presence of angiogenic stimuli. The expression of monocytic marker CD14 of suspended fraction in R1 was 17% on day zero and decreased within the first 3 days (Fig. 2A). After a week there were no CD14+ suspended cells, while CD14+ expression significantly increased in R2-gated adherent PBMCs, representing 64% of the cellular population after 3 days. In gate R1, nearly 2% of both suspended and adherent PBMCs expressed VEGF-R2 (Fig. 2 during one week of culture, even if a significant difference was observed on day 2. Conversely, this receptor was highly expressed in R2-gated cells in which 3% of freshly isolated PBMCs expressed VEGF-R2, a value which increased up to 50% after 2 days. On the other hand, 40% of adherent cells expressed VEGFR2 on day 1 and reached the maximal expression on day 3, which was 68%. Nearly 5% of both suspended and adherent PBMCs were VE-cadherin+ in gate R1 (Fig. 2C), while in R2 there was a significant and time-dependent increase in VE-cadherin expression in both cell fractions. After 3 days 38% of suspended and 50% of adherent cells were VE-cadherin+. Multi-labeling analysis on R2-gated cells on day 2 demonstrated that coexpression of VEGF-R2 and VE-cadherin was displayed by 2.22 + 0.43% of suspended and 4.66 + 1.1% of adherent PBMCs. PECAM-1 was expressed in 34% of freshly isolated PBMCs in R1 while it was higher in R2-PBMCs (Table 1). R2-gated cells always expressed the highest percentage of CD31-positivity. The adherent and suspended PBMCs were also investigated for the expression of integrin aVb3. In our culture conditions this integrin was not expressed in suspended or in adherent PBMCs. The leukocyte marker CD45 was always highly expressed by all gated cells (70–80%) in both suspended or adherent PBMCs (data not shown).
Immunostaining performed on adherent cells after day 1 showed only UEA-1 positivity, while at day 2 also a weak capacity to take up acLDL appeared. After 3 days cells were strongly positive for both markers (Fig. 3). The same analysis was performed on suspended cells in which 1.9 ± 0.5% of R2- gated cells were UEA-1+/acLDL+ on day one, reaching 10.9 ± 1.3% of positivity after 3 days (P < 0.001 vs day 1, n = 7).
3.3. Gene expression of endothelial markers
Fig. 4 shows temporal expression of VEGF-R2, VE-cadherin, PECAM-1 and vWF mRNA in the first 3 days of culture. RT-PCR was performed in the whole suspended and adherent PBMCs without morphological discrimination of the two fractions. VEGF-R2 mRNA expression on adherent PBMCs significantly increased on days 2 and 3 compared with day 1. Likewise the expression of VE-cadherin mRNA signifi- cantly increased on days 2, only in the adherent PBMCs. PECAM-1 and vWF genes did not seem modulated in a timedependent manner, either in adherent or suspended PBMCs.
3.4. Migration of adherent and suspended PBMCs
After 2 days of culture, the ability of suspended and adherent PBMCs to respond to chemotactic factors was assessed by using SDF-1a and VEGF. Both the stimuli induced significant cell migration which was similar in suspended or adherent cells (Fig. 5). Moreover, VEGF induced a twofold increase in cell migration in suspended and adherent PBMCs. Similar findings were obtained using the cells after 3 days of culture in the presence of angiogenic stimuli.
3.5. Matrigel_ tubule assay
A Matrigel_ tubule assay was performed to assess the ability of adherent and suspended PBMCs to form cord and tubularlike structures. After 2 days of culture the two cellular fractions were separately seeded on basement matrix gel. After a few days of culture the formation of cord and tubular-like structures was observed in adherent cells (Fig. 6A and . The suspended PBMCs had similar behaviour with a slower kinetic (Fig. 6C and D).
4. Discussion
several groups have identified different sources which can gave rise to EPCs in peripheral blood [3,9]. Nevertheless a systematic description of phenotypic and functional changes occurring in the heterogeneous PBMC populations during the early stages of differentiation would be relevant and of interest for both basic research and clinical applications.
In this study, early differentiation of human PBMCs into EPCs under angiogenic conditions, was evaluated by flow cytometry analysis, which allows the simultaneous determination of both cell differentiation and cellular morphology. In this technique the shift of cellular light scatter properties represents a very sensitive indicator for changes in cell size and complexity. Cultured PBMCs displayed, between the 2nd and 3rd day, a shift in their light scatter properties moving from gate R1 to gate R2, thus suggesting that the shift displayed by PBMCs can be interpreted as the sequential acquisition of mature endothelial cell morphology. The selective immunophenotype analysis of the cells gated in R1 and R2 confirmed the initial hypothesis. After two days of culture, R2-gated cells showed 50% more expression of both VEGF-R2 and VE-cadherin, 50% of cells were still CD14+ and more than 70% were PECAM-1+ and CD45+. It is remarkable that only 2 days of culture were required to significantly change the immunophenotype on both suspended and adherent PBMCs. However, VEGF-R2 expression decreased in both non-adherent and adherent cells after 1 week of culture. This observation, which is in contrast with some previous findings [4] and in line with others [1], again demonstrates the tight correlation between EPC source, phenotype and culture conditions. It may be noted that 3-days cultured PBMCs were still CD14+ and CD45+ supporting the knowledge that EPCs share an endothelial and leukocyte phenotype [10]. Owing to the pivotal role of aVb3 integrin in angiogenesis its expression was investigated on PBMCs. In the early stages of differentiation PBMCs did not express aVb3. It seems that this molecule is expressed in EPCs after 14 days of culture (11). The EPC phenotype on day 3 was further confirmed by strong positivity for UEA-1 and acLDL displayed by 90% of adherent PBMCs and by a fourfold increase of UEA-1+/ acLDL+ suspended cells. Flow cytometry analysis and immunostaining suggested that endothelial commitment of PBMCs may occur earlier than generally expected, so gene expression of endothelial markers was also investigated by RT-PCR. Both VEGF-R2 and VEcadherin mRNA expression on 2 days-cultured adherent PBMCs increased significantly and was higher than in suspended cells. Probably slight differences in the expression of PECAM-1 and vWF were not detectable because suspended and adherent PBMCs fractions were considered without discriminating between R1 and R2 gates by RT-PCR analysis.
In summary, these results may provide a molecular explanation for the detection of VEGF-R2 and VE-cadherin cell surface expression on PBMCs and are in line with cytofluorimetric analysis which shows their expression already on day 2.
To determine a relationship between flow cytometry immunophenotype analysis of PBMCs and their functional characteristic, we assessed the migration of either suspended or adherent PBMCs, previously grown for 2 days, in response to SDF-1a and VEGF. The former is a chemokine involved in hematopoietic stem cell grafting in bone marrow [12], the latter is an angiogenic factor upregulated during ischemia and an EPC chemoattractant [11]. Both cellular fractions were able to significantly respond to the stimuli. Moreover the number of adherent cells involved in migration appeared to be greater than the suspended one, thus supporting a more advanced endothelial commitment as suggested by immunophenotype. This was further demonstrated by the ability of adherent PBMCs to form a network on Matrigel_ after a few days of culture while the suspended cells showed a slower response. These observations suggest that, although suspended PBMCs display a phenotype similar to those of the adherent cells, they still display different cellular characteristics.
Our findings are not an attempt to characterize the phenotype hierarchy of EPC during in vitro culture and expansion, but rather are a detailed and sequential description of endothelial differentiation of PBMCs in an angiogenic environment which strongly suggests that the endothelial commitment may occur earlier than previously expected. Certainly, different cellular types are present in PBMCs which might contribute to EPC expansion and support our results [9].
It is important to demonstrate the specific features of EPCs expanded in vitro from peripheral blood in order to improve their therapeutic use. Most recent pilot clinical trials have involved infusion of EPCs isolated from peripheral blood and cultured for 3 days. The present data provide both a quantifi- cation of PBMC populations committed to an endothelial phenotype in response to angiogenic stimuli, and a quantitative evaluation of endothelial marker expression in these different cellular fractions. We demonstrate, for the first time, that PBMC cellular fraction in suspension, which is usually discarded or seeded again after a few days, displays comparable behaviour and may constitute a non-negligible target.
Acknowledgement: Supported by the Italian Ministry of University and Scientific and Technological Research (FIR to Professor F. Ledda.
References
[1] Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman, G. and Isner, J.M. (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967.
[2] Takahashi, T., Kalka, C., Masuda, H., Chen, D., Silver, M., Kearney, M., Magner, M., Isner, J.M. and Asahara, T. (1999) Ischemia- and cytokine-induced mobilization of bone marrowderived endothelial progenitor cells for neovascularization. Nat. Med. 5, 434–438.
[3] Peichev, M., Naiyer, A.J., Pereira, D., Zhu, Z., Lane, W.J., Williams, M., Oz, M.C., Hicklin, D.J., Witte, L., Moore, M.A. and Rafii, S. (2000) Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood 95, 952–958.
[4] Kalka, C., Masuda, H., Takahashi, T., Kalka-Moll, W.M., Silver, M., Kearney, M., Li, T., Isner, J.M. and Asahara, T. (2000) Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA 97, 3422–3427.
[5] Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., Zeiher, A.M. and Dimmeler, S. (2001) Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ. Res. 89, e1–e7.
[6] Reyes, M., Dudek, A., Jahagirdar, B., Koodie, L., Marker, P.H. and Verfaillie, C.M. (2002) Origin of endothelial progenitors in human postnatal bone marrow. J. Clin. Invest. 109, 337–346.
[7] Scha¨chinger, V., Assmus, B., Britten, M.B., Honold, J., Lehmann, R., Teupe, C., Albolmaali, N.D., Vogl, T.J., Hofmann, W.-K., Martin, H., Dimmeler, S. and Zeiher, A. (2004) Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction. J. Am. Coll. Cardiol. 44, 1690–1699.
[8] Parenti, A., Donnini, S., Morbidelli, L., Granger, H.J. and Ziche, M. (1996) Br. J. Pharmacol. 119, 619–621.
[9] Ingram, D.A., Mead, L.E., Tanaka, H., Meade, V., Fenoglio, A., Mortell, K., Pollok, K., Ferkowicz, M.J., Gilley, D. and Yoder, M.C. (2004) Blood 104, 2752–2760.
[10] Schmeisser, A., Garlichs, C.D., Zhang, H., Eskafi, S., Graffy, C., Ludwig, J., Strasser, R.H. and Daniel, W.G. (2001) Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel_ under angiogenic conditions. Cardiovasc. Res. 49, 671–680.
[11] Kalka, C., Masuda, H., Takahashi, T., Gordon, R., Tepper, O., Gravereaux, E., Pieczek, A., Iwaguro, H., Hayashi, S.I., Isner, J.M. and Asahara, T. (2000) Circ. Res. 86, 1198–1202.
[12] Peled, A., Grabovsky, V., Habler, L., Sandbank, J., Arenzana- Seisdedos, F., Petit, I., Ben-Hur, H., Lapidot, T. and Alon, R. (1999) J. Clin. Invest. 104, 1199–1211.
斑竹能给我加几分？
谢谢，别忘了！
zhaoli463700 wrote:
两个问题：
1.UEA-I和DiLDL双染色阳性细胞为正在分化的EPCs的原理是什么？而且我见一篇文献竟然用是否DiLDL染色阳性区别EPC和EC，不解
2.有用多聚赖氨酸代替Fn包板的吗？
1.UEA-1 是EPCS的表面标记之一,但不具有特异性,CEC也可以有,
内皮来源正在分化生长(outgrowth)的细胞能够吞噬DiI-acLDL,
所以同时加上其它鉴定方法,可以这么说UEA-I和DiLDL双染色阳性细胞为正在分化的EPCs,
用是否DiLDL染色阳性区别EPC和EC,我想这里的EC应该是已经分化成熟的EC.
2.就我所看过的文献,在培养EPC的过程中没有专门用多聚赖氨酸代替Fn包板.有篇文献是直接分析FN对EPCS的作用的，具体题目,出处忘了,你可以查查.
感谢大家一直关注内皮祖细胞专业讨论区,欢迎大家踊跃发言讨论.
潜水高手要多支持啊