PTD-mFoxp3抑制小鼠皮肤移植排斥反应的研究

PTD-mFoxp3 fusin protein inhibits skin allograft rejection in mice

目的:研究带有蛋白穿膜结构域的小鼠Foxp3融合蛋白(mouse Foxp3 fusin protein combining with transduc-tion domain,PTD-mFoxp3)对小鼠脾淋巴细胞活化增殖能力和皮肤移植排斥反应的影响。方法:取健康C57BL/6小鼠脾淋巴细胞,在体外经刀豆蛋白A(concanavaline A,ConA)活化,并分别给予环孢素A(cyclosporine,CsA)、不同浓度的PTD-mFoxp3,mFoxp3,PTD-GFP和培养基处理,检测脾淋巴细胞增殖指数。在此基础上进行从Balb/c小鼠到C57BL/6小鼠的皮肤移植试验,将40只C57BL/6受体小鼠随机分为4组,每组10只,分别以PTD-mFoxp3,CsA,0.9%氯化钠注射液(NS),PTD-GFP于手术当天开始连续腹腔注射8天为干预手段。术后第9天各组随机取2只移植小鼠,采集皮片标本,进行组织学检查。其余的8只用于观察移植皮瓣的存活时间。结果:PTD-mFoxp3和CsA相似,与其他处理方法相比明显抑制ConA活化的小鼠脾淋巴细胞增殖指数(P<0.05)。PTD-mFoxp3组,CsA组移植物的存活时间较各对照组显著延长(P<0.05);病理检查显示PTD-mFoxp3组和CsA组移植物淋巴细胞浸润明显轻于各对照组。结论:PTD-mFoxp3能抑制ConA活化的T淋巴细胞的增殖,且具有抑制小鼠皮肤移植排斥反应的作用。

Objective： To investigate the potential inhibiting ability to active lymphocytes proliferation and skin allograft rejection of the fusion protein of mouse forkhead box P3 combining with protein transduction domain （PTD-mFoxp3） in mice. Methods： The single-cell suspension of lymphocytes was prepared from mouse spleen. Polyclonal activator Con A was added to active the lymphocytes. Then the single-cell suspension of lymphocytes was incubated with different concentrations of PTD-mFoxp3, PTD-GFP, cyclosporine, and blank control for 48h, respectively. The bioactivity of inhibiting lymphocytes proliferation of each group was detected by incorporating of CCK-8 in vitro. Allogeneic full-thickness grafts from Balb/c mice were transplanted onto the back of C57BL/6 mice. Forty C57BL/6 mice were randomly divided into four groups, with each group having 10 mice and receiving a different treatment including PTD-mFoxp3, cyclos-porine, 0.9% physiological saline injection and PTD-GFP, respectively. On the same day of the skin trans-plantation, the treating regents were injected into C57BL/6 mice abdomen and did continuous injection for another 7 days. Two mice of each group were selected out randomly for histological observation till 9th day after skin transplantation. The survival time of other mice were observed. Results： The bioactivity of PTD- mFoxp3 was similar to eyclosporine, they could inhibit cells proliferation of mouse activated lymphocytes efficiently in vitro（ P 〈 0.05, comparing with blank control and PTD-GFP control） , they could prolong the mean survival time （MST） of skin allograft significantly（ P 〈 0.05, comparing with blank control and PTD- GFP control）. The infiltration of the lymphoeytes in the allografts of PTD-mFoxp3 treated groups and cyclosporine were reduced. Conclusion： The PTD-mFoxp3 can inhibit activated mouse lymphocytes proliferation in vitro and skin allograft rejection in vivo.