器官移植
器官移植
기관이식
OGRAN TRANSPLANTATION
2014年
4期
231-236
,共6页
尹东亮%孙罛%朱焕斌%李坤%李浩辉%张剑
尹東亮%孫罛%硃煥斌%李坤%李浩輝%張劍
윤동량%손고%주환빈%리곤%리호휘%장검
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
肝移植%排斥反應%腺病毒%骨髓間充質榦細胞%細胞毒性T淋巴細胞相關抗原4免疫毬蛋白
간이식%배척반응%선병독%골수간충질간세포%세포독성T림파세포상관항원4면역구단백
Liver transplantation%Rejection%Adenovirus%Bone marrow mesenchymal stem cell%Cytotoxicity T lymphocyte-associated 4-immunoglobulin
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。
目的：探討細胞毒性T淋巴細胞相關抗原4免疫毬蛋白（CTLA4-Ig）基因轉染骨髓間充質榦細胞（MSC）在抑製大鼠原位肝移植排斥反應的作用及機製。方法採用重組腺病毒（Ad）5-CTLA4-Ig轉染MSC。轉染72 h後，提取細胞總蛋白，採用蛋白質印跡法檢測轉染後MSC中CTLA4-Ig的蛋白錶達。採用細胞計數試劑盒（CCK）-8方法檢測未轉染和轉染後的MSC對外週血淋巴細胞增殖的抑製作用。以雄性Lewis大鼠為供體（40隻）；以雄性Brown Norway （BN）大鼠為受體（40隻）。採用改良的Kamada兩袖套法進行原位肝移植，建立大鼠原位肝移植急性排斥反應模型。40隻受體大鼠隨機分為4組，每組10隻。其中對照組（A組），于肝移植時門靜脈輸註生理鹽水；MSC治療組（B組），于肝移植時門靜脈輸註MSC；轉基因MSC治療組（C組），于肝移植時門靜脈輸註轉基因MSC；免疫抑製劑治療組（D組），于肝移植時門靜脈輸註生理鹽水，術後即給予環孢素（CsA）1.5 mg /（kg·d）肌內註射，連續8 d。每組大鼠取5隻觀察生存情況。每組其餘5隻于術後第9日處死，檢測外週血細胞因子白細胞介素（IL）-2、IL-4、榦擾素（IFN）-γ水平，光學顯微鏡下觀察肝組織病理學變化和排斥反應程度。結果重組Ad5-CTLA4-Ig轉染MSC 72 h後，蛋白質印跡法可檢測到轉染後的MSC中有CTLA4-Ig的蛋白錶達。噹未轉染的MSC∶外週血單覈細胞比例為1∶10、1∶20時，MSC抑製淋巴細胞增殖的作用分彆為85.60％、76.69％。重組Ad5-CTLA4-Ig轉染MSC 72 h後，在相同的數量比下，其抑製淋巴細胞增殖的作用分彆為90.50％、84.20％；與未轉染的MSC 比較，轉染後抑製淋巴細胞增殖的作用增彊（P＜0.05）。A、B、C、D組大鼠肝移植術後存活時間分彆為（13±3），（41±6），（90±15），（102±18）d。A、B、C組的大鼠術後存活時間比較差異有統計學意義（P＜0.05），C組和D組的大鼠術後存活時間比較差異無統計學意義（P＞0.05）。與A組比較， B組和C組的IL-4水平明顯升高；與B組比較，C組的IL-4水平明顯升高，差異均有統計學意義（均為P＜0.05）；C組和D組的IL-4水平比較，差異無統計學意義（P＞0.05）。與A組比較，B組和C組的IL-2、IFN-γ水平明顯降低，C組的IL-2、IFN-γ水平亦低于B組，差異均有統計學意義（均為P＜0.05），C組和D組的IL-2、IFN-γ水平比較，差異無統計學意義（P＞0.05）。大鼠肝組織病理檢查結果顯示，A組移植肝髮生重度排斥反應，B組移植肝亦髮生排斥反應，但與A組比較程度較輕。C組與D組移植肝有輕度排斥反應。結論重組Ad-CTLA4-Ig轉染MSC可抑製肝移植排斥反應，其效果優于MSC單獨應用。
목적：탐토세포독성T림파세포상관항원4면역구단백（CTLA4-Ig）기인전염골수간충질간세포（MSC）재억제대서원위간이식배척반응적작용급궤제。방법채용중조선병독（Ad）5-CTLA4-Ig전염MSC。전염72 h후，제취세포총단백，채용단백질인적법검측전염후MSC중CTLA4-Ig적단백표체。채용세포계수시제합（CCK）-8방법검측미전염화전염후적MSC대외주혈림파세포증식적억제작용。이웅성Lewis대서위공체（40지）；이웅성Brown Norway （BN）대서위수체（40지）。채용개량적Kamada량수투법진행원위간이식，건립대서원위간이식급성배척반응모형。40지수체대서수궤분위4조，매조10지。기중대조조（A조），우간이식시문정맥수주생리염수；MSC치료조（B조），우간이식시문정맥수주MSC；전기인MSC치료조（C조），우간이식시문정맥수주전기인MSC；면역억제제치료조（D조），우간이식시문정맥수주생리염수，술후즉급여배포소（CsA）1.5 mg /（kg·d）기내주사，련속8 d。매조대서취5지관찰생존정황。매조기여5지우술후제9일처사，검측외주혈세포인자백세포개소（IL）-2、IL-4、간우소（IFN）-γ수평，광학현미경하관찰간조직병이학변화화배척반응정도。결과중조Ad5-CTLA4-Ig전염MSC 72 h후，단백질인적법가검측도전염후적MSC중유CTLA4-Ig적단백표체。당미전염적MSC∶외주혈단핵세포비례위1∶10、1∶20시，MSC억제림파세포증식적작용분별위85.60％、76.69％。중조Ad5-CTLA4-Ig전염MSC 72 h후，재상동적수량비하，기억제림파세포증식적작용분별위90.50％、84.20％；여미전염적MSC 비교，전염후억제림파세포증식적작용증강（P＜0.05）。A、B、C、D조대서간이식술후존활시간분별위（13±3），（41±6），（90±15），（102±18）d。A、B、C조적대서술후존활시간비교차이유통계학의의（P＜0.05），C조화D조적대서술후존활시간비교차이무통계학의의（P＞0.05）。여A조비교， B조화C조적IL-4수평명현승고；여B조비교，C조적IL-4수평명현승고，차이균유통계학의의（균위P＜0.05）；C조화D조적IL-4수평비교，차이무통계학의의（P＞0.05）。여A조비교，B조화C조적IL-2、IFN-γ수평명현강저，C조적IL-2、IFN-γ수평역저우B조，차이균유통계학의의（균위P＜0.05），C조화D조적IL-2、IFN-γ수평비교，차이무통계학의의（P＞0.05）。대서간조직병리검사결과현시，A조이식간발생중도배척반응，B조이식간역발생배척반응，단여A조비교정도교경。C조여D조이식간유경도배척반응。결론중조Ad-CTLA4-Ig전염MSC가억제간이식배척반응，기효과우우MSC단독응용。
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 9 th 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-γin group B and C were lower significantly (P<0.05 ). The serum levels of IL-2 and IFN-γin 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-γbetween 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.
