靶向CD40小RNA干扰抑制大鼠异体肢体移植急性排斥反应后细胞凋亡及P53，P21，Bcl-2，Bax的表达

Resistance to acute rejection and apoptosis in limbs allotransplantation of rats by RNA interference of CD40 costimulatory molecule and the expressions of P53, P21, Bcl-2 and Bax

背景：对于CD40通路的研究以往多集中在器官移植，涉及异体肢体方面的报道较少。目的：探讨靶向CD40的小分子RNA干扰对大鼠异体肢体移植急性排斥反应、细胞凋亡的影响，检测CD40通路阻滞后相关基因的表达变化。设计、时间及地点：细胞基因工程体内实验，于2007—09／2008—02在哈尔滨医科大学附属第一医院中心实验室完成。材料：清洁级10～12周龄雄性Wistar受鼠、SD供鼠各27只，行同种异体右后肢移植。真核表达质粒pSilencer4．1-CMV neo为Ambion公司产品。pSilencer4．1-CD40shRNA重组质粒由哈尔滨医科大学附属第一医院中心实验室保存。梭华-Sofastr^TM in vivo体内转染试剂为厦门太阳马生物工程有限公司产品。方法：27只Wistar受鼠异体肢体移植完成后随机分为3组，9只／组。干扰组经阴茎背静脉注入梭华-Sofast（15 μL）-siCD40—2／pSilencer（100μg）载体复合物600μL，空载体组等量注入梭华-Sofast（15μL）-pSilencer4．1-CMV neo（100μg）空载体复合物，对照组等量注入生理盐水，连续注射6d，1次／d。主要观察指标：观察移植物排斥反应征象及移植肢体存活时间，检测细胞凋亡情况，免疫组织化学染色检测p53，p21，Bcl-2，Bax的表达。结果：干扰组未见排斥反应征象，且移植物发牛排斥反应的时间及存活时间均显著长于空载体组、对照组（t=3．137～3．519，P〈0．01）；空载体组、对照组于移植后近期发生排斥反应。干扰组移植后第1，3，5，7天细胞凋亡指数均明显低于空载体组、对照组（t=3．636-4．786，P〈0．01）。干扰组移植后各时间点P53，P21，Bax的表达均明显低于空载体组、对照组（t=3．586-4．531，P〈0．01）；Bcl-2的表达均明显高于空载体组、对照组（t=3．664—4．644，P〈0．01）。结论：在不应用免疫抑制剂的情况下，靶向CD40的小分子RNA干扰可以减轻大鼠异体肢体移植后的急性排斥损伤，抑制细胞凋亡，可能与其对p53，p21，bcl-2，bax基因的调节密切相关。

BACKGROUND： Previous research of CD40 pathways mainly focuses on the organ transplantation, but few reports the limb allotransplantation. OBJECTIVE： To study the effect on the resistance to acute reaction and apoptosis of limb allotransplantation by using small RNA interference of CD40 costimulatory molecule in rats, and to detect the expressions of CD40 pathway-related genes. DESIGN, TIME AND SETTING： A genetic engineering trial in vivo was performed in the Central Laboratory of the First Affiliated Hospital of Harbin Medical University （Harbin, Heilongjiang, China） from September 2007 to February 2008. MATERIALS： Twenty-seven SD male rats were chosen as donors and 27 Wistar male rats were taken as recipients, of clean grade and aged 10-12 weeks. Eukaryotic expression plasmid pSilencer4.1-CMV neo was a product of Ambion Company. The pSilencer4.1-CD40shRNA recombinant plasmid was preserved in the Central Laboratory of the First Affiliated Hospital of Harbin Medical University. SofastTM transfection agent in vivo was a product of Xiamen Taiyangma Bioengineering Co., Ltd. METHODS： Totally 27 recipient rats were divided into 3 groups randomly after allogeneic transplantation, with 9 rats in each group. Rats in the interference group were injected with 600μ L composite of Sofast （15 μ L）-siCD40-2/pSilencer （100 μg） via vena dorsalis penis; Rats in the empty vector group received Sofast （15 μ L）-pSilencer 4.1-CMV neo （100 μ g） composite; In the control group, equal injection of sodium chloride was given. The injection was done every day for successive 6 days. MAIN OUTCOME MEASURES： The survival and rejection of the grafts were observed. Cell apoptosis was determined. The expressions of p53, p21, Bcl-2 and Bax were detected with immunohistochemistry. RESULTS： Rejection of grafts was never observed in the interference group, and emerged during a short term after transplantation in the empty vector and control groups. The interference group showed significantly longer time of the rejection and the survival than the empty vector group and the control group （t=3.137-3.519, P 〈 0.01）. The apoptotic index in the interference group was remarkably lower than that in other two groups （t=-3.636-4.786, P 〈 0.01）. The expressions of p53, p21, Bax in the interference group were obviously lower than other two groups （t=3.586-4.531, P 〈 0.01）, while the B cl-2 expression was obviously higher （t=3.664-4.644, P 〈 0.01）. CONCLUSION： Without any immunosuppressants after operation, the method of using small RNA interference of CD40 costimulatory molecule can relieve the acute rejection and inhibit the apoptosis following limb allotransplantation in rats. The mechanism may be related to the modulation of p53, p21, Bcl-2 and Bax.