人骨髓间充质干细胞的鉴定:抗SH2和SH3单克隆抗体的制备与应用

Identification of Human Bone Marrow Mesenchymal Stem Cells: Preparation and Utilization of Two Monoclonal Antibodies against SH2, SH3

间充质干细胞(MSC)是能够自我更新和分化成一种或多种间叶组织的骨髓或其它组织中的一种干细胞,至今还没有类似于CD34识别造血干细胞一样的公认的抗原用于确认间充质干细胞。为了分离和鉴定MSC,本研究选取抗SH2和SH3这两种应用较广泛的抗体。从SH2和SH3杂交瘤细胞接种小鼠获得的腹水中提纯SH2和SH3两种抗体,并用流式细胞术和免疫组织化学的方法鉴定贴壁培养的骨髓间充质干细胞;用免疫磁珠分选方法从骨髓单个核细胞中分选SH2和SH3阳性的细胞;用流式细胞仪双标的方法检测SH2和CD105抗体在间充质干细胞上的阳性率。结果表明:SH2和SH3在间充质干细胞上的阳性率均在80%,分选阳性细胞中80%的细胞为间充质干细胞,阴性细胞中间充质干细胞的比例不足1%。标记了SH2后的间充质干细胞CD105的阳性率不足1%。结论:SH2和SH3是骨髓间充质干细胞的特异性的标记物,可用于间充质干细胞的分离和鉴定。抗CD105抗体和抗SH2抗体在分离和鉴定MSCs上具有同等效力。

Human mesenchymal stem cells （MSC） are one kind of adult stem cells that can self-renew and give rise to one or more mesenchymal tissues, existing in bone marrow and other tissues. Not similar to CD34 recognizing hematopoietic stem cells, no such marker can be used yet to identify MSC. To isolate and identify MSC from bone marrow, anti-SH2 and SH3 monoclonal antibodies as markers to identify MSC were used. Two monoclonal antibodies were purified from ascites of SH2 and SH3 hybridomas-inoculated mice, flow cytometry and immunohistochemistry were used to identify plastic-adherent cultured MSC. And SH2 and SH3 antigen positive cells were isolated from bone marrow mononuclear cells （BMMNC） by immunobeads covered with secondary antibodies. And anti-SH2 and CD105 McAbs were used to label MSC at the same time to clarify whether they recognize the same antigen. The results showed that about 80% of MSC were antigens SH3 and SH2 positive. The SH2 and SH3 positive-selected cells were MSC while MSC accounted for less than 1% of negative-selected cells. When cells were labeled by SH2 McAb, they could not be labeled by CDI05 simutaneously. In conclusion, antigens SH2 and SH3 are specific markers to identify and isolate MSC. Anti-SH2 McAb can replace anti-CDl05 McAb to identify the specific marker on MSC.