【作者】 侍阳；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 外科学， 2008， 博士

【摘要】 目的⑴构建针对大鼠T淋巴细胞4-1BB基因编码区的小发夹RNA(shRNA)表达质粒和慢病毒载体系统,观察RNA(RNAi)对大鼠T淋巴细胞共刺激分子4-1BB基因表达和功能的影响。⑵探讨阻断T淋巴细胞4-1BB/4-1BBL共刺激通路在大鼠肝移植急性排斥反应中的作用。方法⑴根据RNAi作用原理,设计、合成4条T淋巴细胞4-1BB基因的shRNA(shRNA441、shRNA540、shRNA621、shRNA758),插入质粒pGPU6,构建4个编码目的shRNA的表达质粒(p441、p540、p621、p758),酶切及测序鉴定。设对照组,利用电穿孔法在体外将目的基因片段导入BN大鼠T淋巴细胞,48小时后,半定量逆转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM)检测其对T细胞4-1BB基因表达的抑制作用,并筛选、确定最佳效果的干扰质粒。⑵利用筛选出的质粒,PCR扩增其中的U6+shRNA序列,克隆至慢病毒转移质粒pcDNA-CMV-Lentivector并经酶切和测序鉴定,与包装质粒以脂质体介导共转染293T细胞,产生shRNA重组慢病毒。病毒颗粒感染BN大鼠T淋巴细胞6～8 h后,与经丝裂霉素处理的Lewis大鼠淋巴细胞作单项混合淋巴细胞反应,设对照组和干扰组。RT-PCR、FCM、Western bloting方法检测4-1BB表达,MTT、3H-TdR检测干扰后T细胞的增殖能力,FCM测定凋亡细胞数,ELISA法检测细胞因子IL-2、IL-10、IFN-γ水平。⑶“二袖套法”行Lewis鼠到Brown Norway(BN)鼠原位肝移植24例,大鼠随机分成实验对照组、阴性对照组和干涉实验组,每组8对。三组的受体鼠分别在移植前12 h经阴茎背静脉注射1 ml生理盐水、空病毒和重组病毒液。于术后第5天每组处死BN鼠3只,取脾脏,RT-PCR、FCM检测在体T细胞4-1BB表达;取下腔静脉血检测细胞因子和生化指标;取肝组织观察病理变化;剩余大鼠观察术后生存情况。结果⑴经酶切反应及测序证实构建成功重组干扰质粒p441、p540、p621和p758,转染T细胞后,4-1BB相对表达量分别为(33.4±2.3)%、(53.0±2.3)%、(62.1±3.1)%、(40.9±1.5)%,对照组4-1BB相对表达量为(67.2±1.5)%。其中p441沉默效果最佳(p<0.01)。⑵经酶切反应及测序证实,重组慢病毒转移质粒pLVs441序列正确,包装产生病毒液(LVs441)滴度在5×106 TU/ml,浓缩后约3×108 TU/ml。LVs441感染T细胞效率约90%,干涉组4-1BB表达量显著下降(p<0.01);T细胞凋亡较明显(p<0.05);T细胞增殖能力、IL-2,IFN-γ水平低于实验对照组。⑶干涉组BN鼠脾淋巴细胞4-1BB表达量低于对照组(p<0.05);两组IL-10、T-BIL水平无明显差别,但干涉组IL-2、IFN-γ和ALT、AST、LDH水平均低于对照组(p<0.05);对照组和干涉组大鼠肝组织急性排斥Banff评分分别为7.11±0.78、5.89±1.05(p=0.0384)、平均生存时间分别为10.4±1.95天和29±14.78天(p<0.05)。结论⑴siRNA可特异性抑制大鼠T淋巴细胞共刺激分子4-1BB基因转录和表达,诱发T细胞凋亡,抑制大鼠T细胞的增殖能力,降低IL-2,IFN-γ等细胞因子的分泌水平。⑵RNAi阻断T细胞4-1BB/4-1BBL共刺激通路,可抑制肝移植大鼠急性排斥反应,延长受体存活时间。⑶电穿孔技术是质粒载体转染原代T淋巴细胞的一种有效方法。⑷慢病毒载体系统是携带siRNA进入T细胞的理想载体。更多还原

【Abstract】 Objectives⑴To construct 4-1BB specific small hairpin RNA(shRNA) expression plasmids as well as lentiviral vector,and observe the expression of 4-1BB costimulatory molecule on rat lymphocytes inhibited by small interfering RNA(siRNA).⑵To investigate the role of blockade of 4-1BB/4-1BBL costimulatory pathway with RNA interference(RNAi) in acute rejection of rat orthotopic liver transplantation.Methods⑴According to the mechanism of RNAi, 4 expression sequences of shRNA target to rat lymphocyte 4-1BB were designed(shRNA441、shRNA540、shRNA621、shRNA758), and inserted into expression vector pGPU6. The recombined plasmid vectors(p441、p540、p621、p758) were identified by restriction enzyme analysis and sequencing,then, transfected into rat lymphocytes with electroporation. The changes of 4-1BB mRNA level and gene expression were determined by semi-quantitative Reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry(FCM)at 48 h post transfection, meanwhile, one of the recombined plasmids was judged optimization for following experiments.⑵The sequence of U6 + shRNA was obtained from the optimization recombined plasmid by PCR amplification, and cloned to lentivirus tansfer vector pcDNA-CMV- Lentivector. identified with restriction endonuclease digestion and sequencing, recombined tansfer vector pLVs/shRNA and other package plasmids were cotransfected to 293T cells with the help of lipofectamine 2000 to produce Lentivirus(LVs). Lymphocytes from BN rats as well as Lewis rats were respectively infected by LVs and pretreated by mitomycin C(MMC),then,the mixed lymphocyte reaction(MLR) were performed. RT-PCR, FCM and Western blot assay were employed to assess the expression of 4-1BB. Apoptosis was monitored by FCM also.The cell reproductive activity were detected by MTT and 3H-TdR.cytokine such as IL-2,IL-10 and IFN-γlevels were assayed by commercial ELISA kits.⑶24 cases of orthotopic liver transplantation were performed in Lewis to BN rats by using“the two-cuff technique”,and all rats were divided into experimental contrast group, negative control group and interference group randomly. The BN rats of 3 groups were injected saline, empty LVs and recombinant LVs via the dorsal penis vein respectively before operation. On the 5th day after transplantation, 3 rats in each group were killed. The lymphocytes were obtained from spleen to detect the 4-1BB expression by RT-PCR and FCM. blood samples were drawn from inferior vena cava for biochemistry tests and cytokine assay. liver lobes were excised to study the pathological changes. Postoperative survival time of the rats in each group was recorded also.Results⑴The interferential plasmid p441、p540、p621 and p758 were successfully constructed identified with restriction endonuclease digestion and sequencing. Relative expression of 4-1BB on activated T lymphocytes subjected to those plasmid were (33.4±2.3)%,(53.0±2.3)%,(62.1±3.1)% and (40.9±1.5)% respectively,while (67.2±1.5)% in control group. Silencing effect of p441 was the best.⑵Recombined lentivirus tansfervector LVs441 was also identified by restriction enzyme analysis and sequencing. The lentivirus was produced successfully by 293T cells with titer of 5×106 TU/ml,even 3×108 TU/ml if concentrating was carried out.90% T cells could be infected by LVs441, expression of 4-1BB was decreased(p<0.01), and the apoptosis T cell numbers increased in interference group, T cells reproductive activity and cytokine levels in interference group were lower than those in control group.⑶In vivo,the expression of 4-1BB in interference group was lower than those in control group,and levels of IL-2,IFN-γ,ALT,AST,LDH were decreased too.Banff scores calculated by the degree of acute cellular rejection in two groups were 7.11±0.78 and 5.89±1.05(p=0.0384),while the mean survival time were (11.00±4.28) and (12.75±5.57) days respectively(p<0.05).conclusions⑴siRNA could decrease the expression of 4-1BB mRNA and protein, induce T cells to apoptosis. furthermore, the proliferation of rat T cells and the levels of cytokine production were inhibited.⑵RNAi could inhibit T cell costimulatory pathway, prevent acute rejection,and prolong survival.⑶Electroporation technique was an effective method for transfecting plasmid to some difficult-transfection cells,such as T lymphocytes.⑷Lentivirus was an appropriate and efficient vector to deliver siRNA into T lymphocytes.更多还原