Bone marrow mesenchymal stem cell modified by CTLA4-Ig gene can inhibit the rejection of liver transplantation in rats
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摘要:
目的 探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4-Ig)基因转染骨髓间充质干细胞(MSC)在抑制大鼠原位肝移植排斥反应的作用及机制。 方法 采用重组腺病毒(Ad)5-CTLA4-Ig转染MSC。转染72 h后,提取细胞总蛋白,采用蛋白质印迹法检测转染后MSC中CTLA4-Ig的蛋白表达。采用细胞计数试剂盒(CCK)-8方法检测未转染和转染后的MSC对外周血淋巴细胞增殖的抑制作用。以雄性Lewis大鼠为供体(40只);以雄性Brown Norway(BN)大鼠为受体(40只)。采用改良的Kamada两袖套法进行原位肝移植,建立大鼠原位肝移植急性排斥反应模型。40只受体大鼠随机分为4组,每组10只。其中对照组(A组),于肝移植时门静脉输注生理盐水;MSC治疗组(B组),于肝移植时门静脉输注MSC;转基因MSC治疗组(C组),于肝移植时门静脉输注转基因MSC;免疫抑制剂治疗组(D组),于肝移植时门静脉输注生理盐水,术后即给予环孢素(CsA)1.5 mg/(kg·d)肌内注射,连续8 d。每组大鼠取5只观察生存情况。每组其余5只于术后第9日处死,检测外周血细胞因子白细胞介素(IL)-2、IL-4、干扰素(IFN)-γ水平,光学显微镜下观察肝组织病理学变化和排斥反应程度。 结果 重组Ad5-CTLA4-Ig转染MSC 72 h后, 蛋白质印迹法可检测到转染后的MSC中有CTLA4-Ig的蛋白表达。当未转染的MSC:外周血单核细胞比例为1:10、1:20时,MSC抑制淋巴细胞增殖的作用分别为85.60%、76.69%。重组Ad5-CTLA4-Ig转染MSC 72 h后,在相同的数量比下,其抑制淋巴细胞增殖的作用分别为90.50%、84.20%;与未转染的MSC比较,转染后抑制淋巴细胞增殖的作用增强(P<0.05)。A、B、C、D组大鼠肝移植术后存活时间分别为(13±3),(41±6),(90±15),(102±18)d。A、B、C组的大鼠术后存活时间比较差异有统计学意义(P<0.05),C组和D组的大鼠术后存活时间比较差异无统计学意义(P>0.05)。与A组比较,B组和C组的IL-4水平明显升高;与B组比较,C组的IL-4水平明显升高,差异均有统计学意义(均为P<0.05);C组和D组的IL-4水平比较,差异无统计学意义(P>0.05)。与A组比较,B组和C组的IL-2、IFN-γ水平明显降低,C组的IL-2、IFN-γ水平亦低于B组,差异均有统计学意义(均为P<0.05),C组和D组的IL-2、IFN-γ水平比较,差异无统计学意义(P>0.05)。大鼠肝组织病理检查结果显示,A组移植肝发生重度排斥反应,B组移植肝亦发生排斥反应,但与A组比较程度较轻。C组与D组移植肝有轻度排斥反应。 结论 重组Ad-CTLA4-Ig转染MSC可抑制肝移植排斥反应,其效果优于MSC单独应用。 -
关键词:
- 肝移植 /
- 排斥反应 /
- 腺病毒 /
- 骨髓间充质干细胞 /
- 细胞毒性T淋巴细胞相关抗原4免疫球蛋白
Abstract:Objective To investigate the effects and mechanism of bone marrow mesenchymal stem cell(MSC) modified by cytotoxicity T lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) gene on the rejection of orthotopic liver transplantation(OLT) in rats. Methods MSC was infected with recombinant adenoviruses(Ad)5 containing CTLA4-Ig gene. After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h, the total proteins were extracted. The protein expression of CTLA4-Ig was assessed by Western-blot. The suppression to lymphocyte proliferation by MSC and transgenic MSC were tested by cell counting kit(CCK)-8 analysis. Forty models of acute rejection after OLT in rats were established by modified Kamada's two-cuff technique, with male Lewis and BN rats serving as liver donors and recipients respectively. Forty recipient rats were randomly divided into 4 groups with 10 rats in each group including control group (group A, only saline solution was injected into portal venous during transplantation), MSC group(group B, MSC was injected into portal venous during transplantation), transgenic MSC group(group C, transgenic MSC was injected into portal venous during transplantation), immunosuppressant group[group D, saline solution was injected into portal venous during transplantation, and ciclosporin(CsA) was administered intramuscularly at a dose of 1.5 mg/(kg·d)for 8 days]. On the 9th day after operation, 5 rats were killed randomly in every group, then the levels of interleukin(IL)-2, interferon(IFN)-γ,IL-4 in peripheral blood were measured and the pathological changes and rejection expression of liver tissues were observed by light microscope. The survival condition of other 5 rats in 4 groups was observed. Results After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h, the protein expression of CTLA4-Ig gene in MSC infected with Ad5-CTLA4-Ig could be detected by Western-blot.When the ratios of MSC:peripheral blood monouclear cell(PBMC) were 1:10 and 1:20, the rates of suppression to lymphocyte proliferation were 85.60% and 76.69% respectively. When the ratios of transgenic MSC:PBMC were 1:10 and 1:20, the rates of suppression to lymphocyte proliferation were 90.50% and 84.20% respectively. Compared with MSC, MSC infected with Ad5-CTLA4-Ig had stronger effect on suppression to lymphocyte proliferation (P<0.05). The survival time after liver transplantation of rats in group A, B, C, D was (13±3), (41±6), (90±15), (102±18) d respectively. There were significant differences among group A, B, C (P<0.05) and there was no significant difference between group C and D (P>0.05). Compared with group A, the serum levels of IL-4 in group B and C were significantly higher(P<0.05). The serum levels of IL-4 in group C were significantly higher than that in group B (P<0.05). There was no significant difference in the serum levels of IL-4 between group C and D (P>0.05). Compared with group A, the serum levels of IL-2 and IFN-γ in group B and C were lower significantly (P<0.05). The serum levels of IL-2 and IFN-γ in group C were significantly lower than those in group B(both in P<0.05). There were no significant differences in the serum levels of IL-2 and IFN-γ between group C and D(P>0.05). The pathological result of liver tissues of rats showed that the grafts of group A developed severe rejection, and the grafts of group B developed moderate rejection. And the grafts of group C and D developed slight rejection. Conclusions MSC infected with recombinant Ad5-CTLA4-Ig can inhabit the rejection in liver transplantation, and the effect is superior to MSC alone. -
图 1 重组Ad5-CTLA4-Ig转染MSC后CTLA4-Ig蛋白的表达
注：条带1样本为MSC裂解液；条带2样本为表达增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的重组Ad(Ad-eGFP)转染MSC 72 h后的裂解液；条带3为Ad5-CTLA4-Ig转染MSC 72 h后的裂解液
Figure 1. The protein expression of CTLA4-Ig in transgenic MSC infected with recombinant Ad5-CTLA4-Ig
图 3 4组大鼠术后第9日的肝组织病理学变化(HE，×100)
注：A图为SD大鼠正常肝组织；B图为A组大鼠肝组织，可见汇管区及其周围大量炎症细胞浸润，伴中重度静脉炎和肝细胞坏死； C图为B组大鼠肝组织，可见汇管区内胆管和血管被较多炎症细胞浸润，但程度明显减轻；D图和E图分别为C组和D组大鼠肝组织，汇管区内胆管和血管仅见少量炎症细胞浸润
Figure 3. Pathological changes of the liver tissues of rats in 4 groups on the 9th day after transplantation
表 1 4组大鼠肝移植术后细胞因子水平的比较
Table 1. Comparison of the levels of cytokines of the rats in 4 groups after liver transplantation ( (x±s)，ng/L)
组 别 n IL-2 IFN-γ IL- 4 A组 5 86±10 239±16 31±6 B组 5 42±6a 140±15a 52±7a C组 5 29±4a,b 52±10a,b 89±15a,b D组 5 27±4 56±8 86±14 注：与A组比较，aP<0.05;与B组比较，bP<0.05 
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