中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2008年
11期
661-665
,共5页
鄢涛%王森%龚建平%彭勇%刘作金%石毓君%刘鸿翔
鄢濤%王森%龔建平%彭勇%劉作金%石毓君%劉鴻翔
언도%왕삼%공건평%팽용%류작금%석육군%류홍상
RNA干扰%白细胞介素2%转染%肝移植%移植物排斥
RNA榦擾%白細胞介素2%轉染%肝移植%移植物排斥
RNA간우%백세포개소2%전염%간이식%이식물배척
RNA interference,Interleukin-2%Transfection%Liver transplantation%Graft rejection
目的 探讨输注转染白细胞介素2(IL-2)基因同源短发夹型RNA(shRNA)的受者淋巴细胞对大鼠肝移植后急性排斥反应的影响.方法 构建针对大鼠活化T淋巴细胞IL-2基因编码区的shRNA表达质粒,体外转染受者(BN大鼠)淋巴细胞.以Lewis大鼠为供者、BN大鼠为受者,进行肝移植,干扰组的受者于移植术中经门静脉回输转染IL-2-shRNA的BN大鼠淋巴细胞;环孢素A组(CsA组)的受者移植术中经门静脉输入生理盐水,肝移植后应用CsA肌肉注射;生理盐水对照组的受者移植术中经门静脉输入生理盐水;空载体对照组的受者移植术中经门静脉输入转染空载体的BN大鼠淋巴细胞.术后第7天,处死BN大鼠,取移植肝组织,观察组织和细胞超微结构改变情况以及肝细胞凋亡情况,同时测定肝功能指标、肝组织中IL-2基因的表达以及血清IL-2、肿瘤坏死因子a(TNF-α)和γ干扰素(IFN-γ)水平.计算各组受者1周存活率.结果 移植后第7天,生理盐水对照组和空载体对照组的移植肝组织中可见中、重度急性排斥反应改变,而干扰组和CsA组病理切片中无或仅有轻度急性排斥反应征象.干扰组凋亡肝细胞数为(23±3.2)/mm2,CsA组为(26±4.0)/mm2,均明显低于生理盐水对照组和空载体对照组(P＜0.05).干扰组和CsA组的IL-2mRNA表达明显低于生理盐水对照组和空载体对照组(P＜0.05);干扰组和CsA组的血清IL-2、TNF-α和IFN-γ水平较空载体对照组和生理盐水对照组分别有不同程度降低(P＜0.05).干扰组和CsA组的丙氨酸转氨酶和总胆红素均明显低于空载体对照组和生理盐水对照组(P＜0.05).而白蛋白则高于空载体对照组和生理盐水对照组(P＜0.05).干扰组受者1周存活率为87.5%(7/8),明显高于生理盐水对照组的37.5%(3/8)和空载体对照组的37.5%(3/8,P＜0.05).结论 输注转染IL-2-shRNA的受者淋巴细胞可抑制大鼠肝移植后的急性排斥反应,但这种作用是短期的.
目的 探討輸註轉染白細胞介素2(IL-2)基因同源短髮夾型RNA(shRNA)的受者淋巴細胞對大鼠肝移植後急性排斥反應的影響.方法 構建針對大鼠活化T淋巴細胞IL-2基因編碼區的shRNA錶達質粒,體外轉染受者(BN大鼠)淋巴細胞.以Lewis大鼠為供者、BN大鼠為受者,進行肝移植,榦擾組的受者于移植術中經門靜脈迴輸轉染IL-2-shRNA的BN大鼠淋巴細胞;環孢素A組(CsA組)的受者移植術中經門靜脈輸入生理鹽水,肝移植後應用CsA肌肉註射;生理鹽水對照組的受者移植術中經門靜脈輸入生理鹽水;空載體對照組的受者移植術中經門靜脈輸入轉染空載體的BN大鼠淋巴細胞.術後第7天,處死BN大鼠,取移植肝組織,觀察組織和細胞超微結構改變情況以及肝細胞凋亡情況,同時測定肝功能指標、肝組織中IL-2基因的錶達以及血清IL-2、腫瘤壞死因子a(TNF-α)和γ榦擾素(IFN-γ)水平.計算各組受者1週存活率.結果 移植後第7天,生理鹽水對照組和空載體對照組的移植肝組織中可見中、重度急性排斥反應改變,而榦擾組和CsA組病理切片中無或僅有輕度急性排斥反應徵象.榦擾組凋亡肝細胞數為(23±3.2)/mm2,CsA組為(26±4.0)/mm2,均明顯低于生理鹽水對照組和空載體對照組(P＜0.05).榦擾組和CsA組的IL-2mRNA錶達明顯低于生理鹽水對照組和空載體對照組(P＜0.05);榦擾組和CsA組的血清IL-2、TNF-α和IFN-γ水平較空載體對照組和生理鹽水對照組分彆有不同程度降低(P＜0.05).榦擾組和CsA組的丙氨痠轉氨酶和總膽紅素均明顯低于空載體對照組和生理鹽水對照組(P＜0.05).而白蛋白則高于空載體對照組和生理鹽水對照組(P＜0.05).榦擾組受者1週存活率為87.5%(7/8),明顯高于生理鹽水對照組的37.5%(3/8)和空載體對照組的37.5%(3/8,P＜0.05).結論 輸註轉染IL-2-shRNA的受者淋巴細胞可抑製大鼠肝移植後的急性排斥反應,但這種作用是短期的.
목적 탐토수주전염백세포개소2(IL-2)기인동원단발협형RNA(shRNA)적수자림파세포대대서간이식후급성배척반응적영향.방법 구건침대대서활화T림파세포IL-2기인편마구적shRNA표체질립,체외전염수자(BN대서)림파세포.이Lewis대서위공자、BN대서위수자,진행간이식,간우조적수자우이식술중경문정맥회수전염IL-2-shRNA적BN대서림파세포;배포소A조(CsA조)적수자이식술중경문정맥수입생리염수,간이식후응용CsA기육주사;생리염수대조조적수자이식술중경문정맥수입생리염수;공재체대조조적수자이식술중경문정맥수입전염공재체적BN대서림파세포.술후제7천,처사BN대서,취이식간조직,관찰조직화세포초미결구개변정황이급간세포조망정황,동시측정간공능지표、간조직중IL-2기인적표체이급혈청IL-2、종류배사인자a(TNF-α)화γ간우소(IFN-γ)수평.계산각조수자1주존활솔.결과 이식후제7천,생리염수대조조화공재체대조조적이식간조직중가견중、중도급성배척반응개변,이간우조화CsA조병리절편중무혹부유경도급성배척반응정상.간우조조망간세포수위(23±3.2)/mm2,CsA조위(26±4.0)/mm2,균명현저우생리염수대조조화공재체대조조(P＜0.05).간우조화CsA조적IL-2mRNA표체명현저우생리염수대조조화공재체대조조(P＜0.05);간우조화CsA조적혈청IL-2、TNF-α화IFN-γ수평교공재체대조조화생리염수대조조분별유불동정도강저(P＜0.05).간우조화CsA조적병안산전안매화총담홍소균명현저우공재체대조조화생리염수대조조(P＜0.05).이백단백칙고우공재체대조조화생리염수대조조(P＜0.05).간우조수자1주존활솔위87.5%(7/8),명현고우생리염수대조조적37.5%(3/8)화공재체대조조적37.5%(3/8,P＜0.05).결론 수주전염IL-2-shRNA적수자림파세포가억제대서간이식후적급성배척반응,단저충작용시단기적.
Objective To investigate the inhibitory effects of RNA silencing via plasmidmediated interleukin-2 shRNA on acute rejection of rat liver transplantation.Methods pGenesil-2-IL2-shRNA vector plasmid expressing IL-2-shRNA was constructed,and transfected into freshly isolated rat lymphocytes with cationic liposome.At 24 h post-transfection,the production of IL-2 in the supematant was measured bv ELISA,and the change in the IL-2 mRNA levels was detected by using RT-PCR The 32 rats were randomly divided into 4 groups:contrast group in which recipients were injected by the branch of portal vein with the BN rat lymphocytes(1 ml,2×10/ml)carrying pGensil-2 in advance after anastomosis of infrahepatic veins,rejection group with isotonic Na chloride 1 ml.interference group with the lymphocytes carrying pGenesil-2-IL-2-shRNA,and CsA group with isotonic Na chloride during operation and Cyclosporine A(3.0 mg·kg-1·d-1)was intramuscularly injected daily for 7 days on the day O postoperatively.Recipients were sacrificed on 7th day postoperatively,and liver tissues and blood samples were collected.Recipient survival rate,histopathological and uhrastructural characteristics were observed.Apoptosis cells in the graft were measured by using TUNEL IL-2 mRNA expression in liver graft was detected by RT-PCR Serum levels of IL-2,TNF-α,IFN-γ,ALT,TBIL,and Alb were also determined.Results (1) pGenesil-2 IL-2-shRNA vector plasmid was successfully constructed and identified with digestion and sequencing.At 24th h post transfection.the results of ELISA and semi-quantitative RT-PCR assay indicated that the inhibition ratio was aboUt 82%,compared with the control(P＜0.05).(2)On the 7th day postoperatively.the results of IL-2 mRNA expression in liver graft,the serum levels of IL-2,TNF-α,IFN-γ,ALT,FBil,ALB,and the change of histopathological and ultrastructural characteristics,the detection of apoptosis cells in graft,all indicated that injection of lymphocytes carrying pGenesil-2-IL 2-shRNA during the operation of rat liver transplantation could effectively inhibit the acute rejection response.compared with the CsA group(P>0.05).Recipient survival rate on the 7th day postoperatively in interference group was 87.5%(survival/death,7/8),significantly higher than in rejection group(37.5%,3/8)and the control group(37.5%,3/8,P＜0.05).Conclusion This recombinant plasmids carrying IL-2-shRNA could sequence-specially reduce the expression of IL-2 mRNA level in rat activated T lymphocytes in vitro,and efficiently inhibit the acute rejection response in the liver locally after liver transplantation.
