免疫抑制剂FK506对大鼠移植肢体Bcl-2与Bax mRNA表达及细胞凋亡的影响

Effects of FK506 on Bcl-2 mRNA and Bax mRNA expressions as well as cell apoptosis in rat limb allografts

背景：肢体移植过程中，细胞凋亡是引起移植物丧失功能的主要机制之一，严重时可导致移植失败。假设免疫抑制剂的作用与细胞凋亡有关。目的：观察免疫抑制剂FK506对大鼠异体肢体移植模型Bcl-2 mRNA，Bax mRNA表达及细胞凋亡的影响。设计、时间及地点：随机对照动物实验，于2005—06／2006-11在哈尔滨医科大学附属第一医院实验动物中心实验室完成。材料：清洁级健康雄性SD受鼠及Wistar供鼠各56只。FK506为日本藤泽药品有限公司产品，批号100143G。方法：将供鼠右后肢自大腿上段离断，肝素盐水冲洗供肢内残血，对受鼠行右后肢原位异体移植建立损伤模型。造模后受体鼠随机分为两组：免疫抑制组注射FK5061mg／（kg·d），对照组不给予任何免疫抑制剂，28只/组。术后1，3，5，7d切取大鼠右后肢包含皮肤、皮下组织、肌肉、股动静脉的组织块待测。主要观察指标：RT-PCR法检测移植肢体Bcl-2 mRNA、Bax mRNA的表达，应用细胞凋亡原位末端标记技术检测细胞凋亡情况。结果：56只模型鼠均进入结果分析。FK506注射3，5，7d，免疫抑制组Bcl-2 mRNA的表达量显著高于对照组（t=7．18～21．20，P〈0．01）：Bax mRNA表达量显著低于对照组（t=3．35～14．01，P〈0．01或0．05）；细胞凋亡指数显著低于对照组（t=7．17-9．42，P〈0．01）。结论：FK506能使移植肢体中Bcl-2 mRNA表达增强，Bax mRNA表达减弱，抑制细胞凋亡的发生。

BACKGROUND： In the process of limb allograft, apoptosis of target cell is one of the main mechanisms of dysfunction of allografi, which might lead to the failure of allotransplantation. It is assumed that immunosuppressant may relate with cell apoptosis. OBJECTIVE： To investigate effects of FK506 on Bcl-2 mRNA and Bax mRNA expressions and cell apoptosis in rat limb allograft. DESIGN, TIME AND SETTING： The randomized controlled animal trial was performed at the Animal Experimental Center of First Hospital of Harbin Medical University from June 2005 to November 2006. MATERIALS： Fifty-six clean-grade healthy male SD recipient rats and 56 Wistar donor rats were selected. FK506 was product of Fujisawa, Japan （No. 100143G）. METHODS： Right hind limb was separated from the upper segment of thigh of SD rat （donor）, and washed using heparin saline. The recipient rates underwent limb allotransplantation from allogenetic Wistar to establish injury model. The recipients were randomly divided into two groups （n=28）： immunosuppressant group was injected with FK506 1 mg/kg per day, and the control group was not given any immunosuppressant. The right hind limb including skin, subcutaneous tissues, muscles and femoral arteriolar-venular tissue mass were harvested on postoperative days 1, 3, 5, and 7. MAIN OUTCOME MEASURES： Bcl-2 Mrna and Bax mRNA expression were detected using reverse transcription polymerase chain reaction （RT-PCR）, and cell apoptosis was detected using in situ terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling technique. RESULTS： Fifty-six model rats were included in final analysis. On the 3, 5, and 7 days after FK506 injection, Bcl-2 mRNA expression in immunosuppressant group was significantly higher than the control group （t=7.18-21.20, P 〈 0.01）, while Bax mRNA expression was significantly lower than the control group （t=3.35-14.01, P 〈 0.01 or 0.05）. In addition, the apoptosis index of allograft tissue was markedly lower than the control group （t=7.17-9.42, P 〈 0.01 ）. CONCLUSION： FK506 can increase Bcl-2 mRNA expression, decrease Bax mRNA expression and reduce apoptotic cells in allografts.