Woodley S L, McMillan M, Shelby J, Lynch D H, Roberts L K, Ensley R D, Barry W H
Department of Medicine, University of Utah School of Medicine, Salt Lake City.
Circulation. 1991 Apr;83(4):1410-8. doi: 10.1161/01.cir.83.4.1410.
The mechanisms by which ventricular function is altered during cardiac transplant rejection are not well understood. Therefore, an in vitro model system has been developed to facilitate investigation of lymphocyte-mediated myocyte injury.
Splenic lymphoid cells were obtained from mice 8-10 days after placement of a vascularized abdominal cardiac allograft and were restimulated in vitro with irradiated donor-type splenocytes for 5 days. Cytotoxic effects of these allogenically stimulated lymphocytes on syngeneic and donor strain fetal cultured myocytes were determined by a 51Cr release assay at different lymphocyte to myocyte ratios. 51Cr release from donor strain myocytes was detectable within 1 hour of exposure, was maximal by 3-5 hours of coincubation with sensitized lymphocytes, and was allospecific. Cell injury manifest by 51Cr release was calcium dependent and was inhibited by pretreatment of lymphocytes with phorbol ester to deplete protein kinase C. Myocyte injury was also prevented by pretreatment of sensitized lymphocytes with anti-Thy 1.2 or anti-CD8 antibody plus complement but not by treatment with anti-CD4 antibody, indicating that CD8+ cytotoxic T cells are involved. Altered myocyte contractile motion preceded myocyte lysis (51Cr release), was characterized by an initial reversible decrease in amplitude of contraction, and was followed by rapid and irregular beating with eventual complete cessation of contraction. Contractile alterations induced by sensitized lymphocytes were inhibited by elimination of CD8+ cells.
Myocyte injury can be produced by sensitized cytotoxic T lymphocytes in vitro and is calcium and protein kinase C dependent. The contractile abnormalities produced appear to be similar to those observed in cardiac transplant patients undergoing rejection, and thus this model system promises to allow investigation of the mechanisms involved.
心脏移植排斥反应期间心室功能改变的机制尚未完全明确。因此，已开发出一种体外模型系统，以促进对淋巴细胞介导的心肌细胞损伤的研究。
在植入带血管蒂的腹部心脏同种异体移植物8 - 10天后从小鼠获取脾淋巴细胞，并在体外与经辐照的供体型脾细胞再刺激5天。通过51Cr释放试验在不同淋巴细胞与心肌细胞比例下测定这些同种异体刺激淋巴细胞对同基因和供体品系胎儿培养心肌细胞的细胞毒性作用。供体品系心肌细胞在暴露1小时内即可检测到51Cr释放，与致敏淋巴细胞共孵育3 - 5小时达到最大释放量，且具有同种特异性。51Cr释放所显示的细胞损伤依赖于钙，并且用佛波酯预处理淋巴细胞以耗尽蛋白激酶C可抑制这种损伤。用抗Thy 1.2或抗CD8抗体加补体预处理致敏淋巴细胞也可预防心肌细胞损伤，但用抗CD4抗体处理则无效，表明CD8 + 细胞毒性T细胞参与其中。心肌细胞收缩运动改变先于心肌细胞溶解（51Cr释放），其特征为收缩幅度最初可逆性降低，随后快速且不规则跳动，最终完全停止收缩。致敏淋巴细胞诱导的收缩改变可通过消除CD8 + 细胞而受到抑制。
致敏的细胞毒性T淋巴细胞可在体外导致心肌细胞损伤，且这种损伤依赖于钙和蛋白激酶C。所产生的收缩异常似乎与心脏移植排斥反应患者中观察到的异常相似，因此该模型系统有望用于研究其中涉及的机制。
