抗人间充质干细胞单克隆抗体的制备及生物学特性研究

Preparation of monoclonal antibodies against human mesenchymal stem cells

目的:制备抗人间充质干细胞(hM SC s)的单克隆抗体,为鉴定、分选hM SC s奠定基础。方法:以多供体hM SC s免疫BALB/C小鼠,采用杂交瘤技术制备单克隆抗体,并以间接免疫荧光法、免疫组织化学法和流式细胞术等检测单克隆抗体在细胞及组织中的表达。结果:建立了5个能分泌抗人间充质干细胞的单克隆抗体杂交瘤细胞株,该组单抗能与人间充质干细胞特异性结合,而对所检测的人间充质和非间充质来源的组织和细胞均未见交叉反应,5个单抗的相对亲和力分别为:ZUB1 1×106、ZUB4 1×105、ZUC 3 1×106、ZUE 12 1×105、ZUF10 1×105;表位分析:ZUB1、ZUE 12、ZUF10针对相同的表位,ZUC 3和ZUB4则分别针对不同的表位。流式细胞分析显示分离培养的hM SC s阳性表达率为ZUB1 87.39%、ZUB4 88.07%、ZUC 3 88.12%、ZUE 12 69.89%和ZUF10 83.67%。结论:5株杂交瘤分泌的单克隆抗体均能与人间充质干细胞发生特异性结合,为深入研究hM SC s的生物学功能奠定了基础。

Objective： To prepare monoclonal antibodies （McAbs） against human mesenchymal stem cells （hMSCs） and to study their biological characteristics. Methods： BALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry. Results ： Five hybridoma cell lines were successfully established,which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells,the relative affinities of 5 McAbs were 1 × 10^6（ZUB1）,1× 10^5（ZUB4）,1× 10^6（ZUC3）,ZUE12 （1× 10^5） and 1× 10^5（ZUF10）,respectively. Isotype analysis showed that ZUB1,ZUE12,ZUF10 against the same isotype,while ZUC3,ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87. 39%（ZUB1）,88. 07%（ZUB4）,88. 12% （ZUC3）, 69.89% （ZUE12） and 83.67% （ZUF10）. Conclusion： The prepared five McAbs can specifically react against hMSCs,which can be used for selection and study of hMCSs.