内皮祖细胞和骨髓基质干细胞的培养实践精华

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请问内皮祖细胞和骨髓基质干细胞的细胞特异性标记分别是什么？如何在体外分离？谢谢大家！！！

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内皮祖细胞可用cd34或cd133标记，骨髓基质干细胞的细胞特异性标记还没有哦，体外分离可用流式细胞仪，或免疫磁珠法，分步贴壁法，胰酶不同时间消化法等哦。

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cd133还是ad133?如果没有特异性标记，那如何在体外把两者分离哪？能不能讲详细点？

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cd133又称为ad133。二者一个标记两种称呼。在体外把两者分离的方法可有这样啊：如你有经费，1，用荧光耦联的抗cd133或cd34抗体与细胞温育，然后过流式细胞仪，流式细胞仪能将有荧光的细胞和无荧光的细胞分开。2 g; T/ y) {. ^: j; o
2 用磁珠耦联的抗cd133或cd34抗体与细胞温育，然后过磁珠分离器，能将有磁珠的细胞和无磁珠的细胞分开。
7 Y2 B, i, A2 V: l如经费不足：3 可利用二者不同的贴壁时间分离，骨髓基质干细胞的贴壁时间短，一般4h－48h贴壁，内皮祖细胞一般48h以后，先将细胞混合物置于皿中，48h后，将上悬液移入另一皿，前皿细胞大多为骨髓基质干细胞。后皿大多为内皮祖细胞。+ m. E* l' ]# u3 _
4 骨髓基质干细胞对胰酶反应较内皮祖细胞敏感。骨髓基质干细胞对胰酶反应时间通常为4分钟左右，内皮祖细胞通常为10分钟左右。可在贴壁混合细胞中加胰酶，5－7分钟后，去液体，留下的大部分为内皮祖细胞。* n3 f8 X) A- B+ P  ^" ?, f
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1，2法要钱，但细胞较纯，0 l! d( H- F$ D
3，4法简单，便宜，但细胞不纯哦。4 `- l) N2 U8 c% O# B

" f# y. E% ~4 S以上是我的一些个人经验，可能还有更好的方法哦。欢迎各位战友指教咯.

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主要参考文献：7 \. l3 f  u6 @% G+ i! y% J
1..Endothelial Progenitor Cells Isolation and Characterization Mihail Hristov,* Wolfgang Erl, and Peter C. Weber.Trends Cardiovasc Med 2003;13:201–206* ~4 S; o. w9 W# I8 c/ R% ^
http://www.ncbi.nlm.nih.gov/entr ... i/S105017380300077X
' o: N3 \* O" G, t1 G2..Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo % B: A! k' a6 x9 [, T) n% M2 f- q
S Kaushal, G E Amiel, K J Guleserian, O M Shapira, T Perry, F W Sutherland, E Rabkin, A M Moran, F J Schoen, A Atala, S Soker, J Bischoff & J E Mayer Jr Nat Med 2001,7(9), 1035 – 1040., K$ A2 E4 t9 u1 w' s5 w
http://www.nature.com/cgi-taf/dy ... al/v7/n9/index.html
2 e+ P8 S5 `" L' u' c' R1 W3..Isolation, characterization, and biologic features of bone marrow endothelial cells.7 \6 Z5 F7 V( t. H3 _' u, j% {
Almeida-Porada G, Ascensao JL.J Lab Clin Med. 1996 Oct;128(4):399-407. / ]3 Z, r! p# L$ {2 E& B4 A* |
http://gateway1.ovid.com/ovidweb ... n1_TZWnFRAV0sqKvq3v
4 X- F4 r8 x# c9 N( R4..Isolation and characterization of endothelial progenitor cells from mouse embryos." |  S' ?& c+ g/ N- y% V/ t
Hatzopoulos AK, Folkman J, Vasile E, Eiselen GK, Rosenberg RD.Development. 1998 Apr;125:1457-68. ' {5 W/ W: c' h% V; W
http://dev.biologists.org/cgi/reprint/125/8/1457  Z" J- E( ]/ o9 P$ i' |' X: i
5..Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Rafii S, Lyden D.Nat Med. 2003 Jun;9:702-12. 1 n' D7 _, S6 A
http://www.ncbi.nlm.nih.gov/entr ... 9&dopt=Abstract

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Phenotypic and functional identification of human and mouse endothelial progenitors.5 F0 J! {: |3 v

3 |( c1 ~; ~& C1 A1 d& ^CEPs originate from bone marrow–derived EPCs or resident progenitors within organs, such as skeletal muscle or vascular parenchyma. Numerous markers, including VEGFR2 (KDR) and CD133, are expressed on human EPCs and CEPs. Differentiation of EPCs to mature adherent endothelial monolayers results in the loss of CD133 expression. Subsets of the Sca1+c-Kit+LinNeg population of mouse pluripotent cells are composed of EPCs that also express VEGFR2+ (Flk1+). Whether mouse CEPs and EPCs also express CD133 is not known. The potential for formation of late-outgrowth endothelial colonies CFU-EC (colony forming units-endothelial cells) identifies both mouse and human EPCs and CEPs. The proliferative potential of endothelial cells derived from EPCs and CEPs are several fold higher than the vascular wall–derived CECs. EPCs, CEPs and CECs also express the sialomucin CD146 (S-endo-1, P1H12, MCAM/MUC18, Mel-CAM) that is present on endothelial cells during embryonic development and on adult microvasculature57. Certain markers, including CD31 (PECAM), von Willebrand Factor (vWF) and the capacity to take up Ac-LDL or bind to lectins are shared by hematopoietic cells, EPCs, CEPs and CECs.

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Cross-talk between EPCs, CEPs, HSCs and HPCs.
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Throughout vascular development, cellular collaboration between hematopoietic cells and their endothelial counterparts is essential for angiogenesis. HSCs, HPCs and their progeny release angiogenic factors that support proliferation, survival, motility and remodeling of endothelial cells. Release of factors including VEGF-A, VEGF-C, FGF and angiopoietins supports neovessel formation and stabilization, whereas release of thrombospondin-1 and platelet factor-4 (PF4) convey signals that inhibit angiogenesis.& P% J! f8 k: {8 I) P4 [/ |

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我现在作的是用家兔模型，因此要求从兔骨髓中分离出内皮前体细胞，之前按照老板的想法是仿效人的CD34+标志筛选得到骨髓源性EPC的方法，寻找出类似标记做兔骨髓细胞的筛选。
1 d! u, p. P7 I# e5 s. ]0 T6 t3 Y可是我寻找了很长时间，也没见到有类似的标记分子，直至现在仍有些不甘，因此请问版主可有这方面的资料。

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我最进在着手内皮祖细胞诱导的实验，以后各位多多指教交流。
, Y, j! C$ m4 L. r: |, U" t7 B内皮细胞：vWf，UEA-1，CD105，KDR，CD31，CD44+，vWf，并摄入乙酰化LDL，CD45-。
- E2 n) ?5 e# h& P; c最近多家研究团体证明非粘附性CD133+细胞可定向诱导分化为内皮细胞，并形成血管样结构，认为CD133+可能是内皮祖细胞。其表型为CD34，CD45，CD14，抗-GFAD，抗-O4，抗-A2B5。并表达PECAM及VEGR受体) h7 D5 L* ]3 a+ A/ L7 Z5 O
HSC表型为CD34+DR-Lin-，并有与内皮细胞共同的Marker，如Tie，Tek，Flk-1。但CD34+细胞不表达vVf。但究竟HSC是否本身或其某一亚群既具有内皮祖细胞的活性或二者有着共同的前体，仍不确定。
7 Q' \6 p: h& m+ k, D( m5 y! k最近更有研究者认为骨髓基质中的MAPC可分化为成血管样细胞并可分化为内皮细胞，但未最后确定，MSC的定向诱导为内皮细胞可能会是今后研究重点。