Development of molecular and diagnostic of acute rejection after organ transplantation

器官移植的急性排斥反应是由细胞毒性T淋巴细胞(CTL)介导的，大量证据表明CTL颗粒中的丝氨酸蛋白酶(SE)或穿孔素等效应分子与细胞对靶细胞的杀伤过程有关。本研究的目的是探讨这些新发现是否有助于器官移植的诊断和免疫抑制治疗。通过裂解酯酶底物N-α-苄氧基甲酰基-L-赖氨酸硫代苯甲酸酯(BLT)来测定硒的活性。首先，我们研究了SE活性测定是否可以作为监测急性排斥反应的敏感工具。非治疗组外周血淋巴细胞(PBL)和移植物浸润性淋巴细胞(GIL)的SE活性在第6天及以后随排斥反应的进展而升高，而环孢菌素组无明显变化。其次，我们用连续柱层析步骤从大鼠血清中活体纯化了穿孔素抑制蛋白(P-1)。P-1呈剂量依赖性地抑制细胞毒性T淋巴细胞(CTL)的细胞毒活性。接受同种异体肾移植的LEW大鼠平均存活时间为7.9天(对照组：A组)。移植后给予P-1 2 mg/kg/d(B组)或4 mg/kg/d(C组)7天的受者存活时间明显延长(B组：14.5天；C组：22.9天)。结论：P-1可作为一种新型的免疫抑制剂，并可作为解决穿孔素在效应机制中实际作用的线索之一。

The acute rejection in organ transplantation lias been shown to be mediated by cytotoxic T lymphocytes (CTL) and lots of evidences revealed that the effectors molecules such as serine protease (SE) or perforin contained in graiitiles of CTL are related to the process of cell killing against the target cells. The aim of this study is to investigate the whether these new findings are useful for diagnosis and immunosuppressive therapy in organ transplantation. SE activity was measured by cleaving of the esterase substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). First we investigated whether SE activity assay could serve as a sensitive tool for monitering of acute rejection. The rapie increase in SE activily of peripheral blood lymphocytes (PBL) and graft infiltrating lymphocytes (GIL) according to progress of rejection was observed on and after the day 6 in non-treatment group in contrast with the changeless activity in cyclospolin group. Second, we live purified perforin inhibitor protein (P-1) from rat serum using serial column chromatographic steps. P-1 inhibited in a dose dependent manner the cytotoxic activity of cytotoxic T lympliocytes (CTL). LEW rat recipients receiving only a BN renal allografts survived for a mean survival time of 7.9 days (control : group A). Each group of recipients receiving P-1 of 2mg/kg/day (grotip B) or 4mg/kg/day (group C) for 7 days posttransplant showed extensive prolongation of survival in a dose dependent manner (group B : 14.5 days ; group C : 22.9 days). It is concluded that the P-1 could serve as a novel immunosuppressive agent and us one of the clue to resolve the actual roles of perforin in effector mechanisms.