猪睾丸Sertoli细胞抵御人血清中天然抗体介导的补体杀伤作用

目的 探讨新生猪睾丸Sertoli细胞抵御人血清中灭然抗体介导的补体杀伤作用及其主要机制.方法 分离培养10～15口龄的湖北白猪睾丸Sertoli细胞;以猪血管内皮细胞(PED)为对照,采用流式细胞仪(FACS)及免疫荧光检测并比较两种细胞天然抗原α-Gal的表达;FACS枪测并比较两种细胞分别与正常人血清(NHS)中天然抗体IgM、IgG的结合情况;乳酸脱氢酶(LDH)检测20%NHS对Serto-li细胞及PED细胞的杀伤;MTT法检测两种细胞在20%NHS中培养的活性;细胞免疫荧光检测补体C3c、C4d在Sertoli细胞、PED细胞的沉积;细胞免疫组化及免疫荧光检测补体C5b-9在两种细胞上的沉积.结果 猪睾丸Sertoli细胞表达ot-Gal,但明显弱于PED细胞,猪睾丸Sertoli细胞能与天然抗体(主要是IgM)结合;20%NHS对Sertoli细胞、PED细胞的杀伤率分别为24.38%±0.50%、53.13%±14.53%,P<0.01;两种细胞在20%NttS中的活性分别为98.73%±18.84%、52.43%±8.08%,P<0.01;免疫荧光及组化可见PED细胞上有补体C3c、C4d、C5b-9的沉积;但Sertoli细胞仅有C3c、C4d沉积,而未见C5b-9沉积.结论 猪睾丸Sertoli细胞可以显著抵御人血清巾天然抗体介导的杀伤作用,其机制可能与Sertoli细胞低表达α-Gal并抑制补体攻膜复合物形成有关.

Resistance of porcine Sertoli cells to xenoreactive antibodies mediated complement lysis

Objective To investigate whether porcine Sertoli cells eould resist xenoreactive antibodies mediated complement lysis. Methods Sertoli cells were isolated from testes of 10 to 15 day-old landrace pigs. Α-Gal expression on Sertoli cells was measured by FACS and cytoimmunofluorescence. The binding of human se-rum IgG and IgM with Sertoli cells was assayed by FACS. After the incubation of the cultured Sertoli cells with 20% human B serum in vitro, the cellular lysis and cytotoxicity assay were detected with CytoTox-ONETM homogeneous membrane integrity assay and MTT method, and then activation of the complement cascade was examined by immunohistochemistry and immunofluorescence. The SV40-porcine endothelium cells line (SV40-PED) was served as control cells. Results α-Gal expression was found on Sertoli cells by FACS and cytoimmunofluorescence. After incubation with 20% human serum, cellular lysis ratio of the SV40-PED was 53. 13% ± 14.53%, while lysis ratio of the Sertoli cells was significantly lower (24.38% ±0.50%, P<0.01), the viability of Sertoli cells and SV40-PED were 98.73% ± 18.84% and 52.43% ± 8.08%, respectively. With immunohistochemistry and immunofluorescence, C3c and C4d were found binding on both the Sertoli cells and SV40-PED cells, however, C5b-9 was only detected on SV40-PED cells. Conclusion In vitro, compared with the SV40-PED, Sertoli cells could resist xenoreactive antibodies of human serum mediated complement lysis by preventing the C5b-9 formation.