【作者】 宋斌；

【导师】 王禾； 杨安刚； 秦卫军；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 【研究背景】肾癌( renal cell carcinoma, RCC)是泌尿生殖系统最常见的恶性肿瘤之一,虽然其发病率仅占全身恶性肿瘤的3%,但近来全世界的肾癌发病率以每年2%的速度在增长,每年死于肾癌者约10万多例。在我国,肾癌发病率仅次于膀胱癌居泌尿系肿瘤第二位,近年来有逐年上升的趋势,是威胁人民健康的最重要肿瘤之一。绝大多数肾癌为先天多药耐药,对化疗和放疗不敏感,除了早期肾癌手术治疗效果较好外,大多数中、晚期肾癌目前尚缺乏特异性的治疗方法。然而约50%的患者首次就诊时已属中、晚期,约40%的患者术后发生转移或复发,转移性肾癌预后差,平均存活时间小于1年,3年存活率低于5%。故寻找一种有效的、特异性的肾癌治疗方法,是泌尿外科临床治疗中迫切需要解决的问题。免疫基因治疗的出现给人们带来了新的希望,治疗基因和效应细胞的选择是免疫基因治疗的关键环节。转化生长因子β(TGF-β)是一种多功能的细胞因子,对细胞的增殖、分化、凋亡以及许多组织的发育形成有着十分重要的作用。TGF-β是很强的免疫抑制因子,很多肿瘤产生大量的TGF-β,通过TGF-β抑制宿主的免疫系统,主要包括抑制各种免疫细胞(T,B,巨噬细胞等)的增殖、分化,从而使这些免疫细胞无法识别、杀伤肿瘤细胞,导致所谓的免疫逃逸,从而加速了恶性肿瘤的发展。TGF-β受体分为三型,有高度同源性,通过其跨膜的受体复合物进行信号转导,这些受体分为Ⅰ型(55KD)、Ⅱ型(75KD)、Ⅲ型(280KD),Ⅰ型(TβR-Ⅰ)和Ⅱ型受体(TβR-Ⅱ)均属丝氨酸/苏氨酸活性的Ⅰ型跨膜糖蛋白,Ⅲ型受体(TβR-Ⅲ)不介导TGF-β信号转导,但调节TGF-β与受体结合,Ⅱ型受体能以较高的亲和性首先与TGF-β配体结合,继而介导其下游一系列的信号通路反应,使细胞的生物学行为受到调控。由此可见,TβR-Ⅱ是TGF-β信号通路中的关键分子,而CD8+T细胞又是肿瘤免疫中具有杀伤作用的效应细胞,在保证CD8+T细胞具有肿瘤反应性的前提下,通过RNA干扰敲除CD8+T细胞表面的TβR-Ⅱ,使这些肿瘤反应性的CD8+T细胞对TGF-β不敏感,降低TGF-β对CD8+T细胞分化、增殖的抑制作用,以提高CD8+T细胞对肿瘤细胞的杀伤作用,有望为临床中肾癌的治疗提供新的免疫基因治疗策略。【目的】构建含有针对小鼠TβR-Ⅱ基因shRNA的逆转录病毒载体,通过逆转录病毒介导的RNAi敲除肿瘤反应性CD8+T细胞表面的TβR-Ⅱ以制备肿瘤反应性的TGF-β不敏感的CD8+T细胞,观察其对小鼠肾癌Renca细胞系的体内外杀伤作用。【方法】1 siRNA的筛选以及MSCV-shRNAs逆转录病毒载体的构建1.1干扰靶点的选取根据小鼠TβR-Ⅱ基因(GenBank AF406755)的序列设计并合成三对siRNA,针对三个干扰位点,分别命名为siRNA.1、siRNA.2和siRNA.3。1.2 siRNA的筛选使用Oligofectamine?将三对siRNAs转染至小鼠成纤维细胞NIH3T3细胞系48小时后分别提取细胞总RNA和总蛋白,以RT-PCR及Western Blot鉴定干涉效果,选择干涉效果最好的一对siRNA用于后续实验。1.3 MSCV-shRNAs逆转录病毒载体的构建对应1.2中选取的siRNA合成相应的shRNA,并合成阴性对照shRNA,分别命名为shRNA-T和shRNA-N,将其分别插入使用ApaⅠ和EcoRⅠ线性化的pEGFP/U6载体,再以BamHⅠ和EcoRⅠ完整切取U6-shRNA-T和U6-shRNA-N ,插入经BamHⅠ和EcoRⅠ切除TβRIIDN后线性化的MSCV-TβRIIDN-IRES-GFP载体,所构建逆转录病毒载体分别命名为MSCV-shRNA-T和MSCV-shRNA-N,并经过测序鉴定所构建载体。2 TGF-β不敏感的肿瘤反应性CD8+T细胞的制备2.1 MSCV-shRNAs逆转录病毒的包装、滴定将MSCV-shRNAs质粒和pVSV-G质粒使用Lipofectamine?共转染泛嗜性GP293逆转录病毒包装细胞,37℃下共转染12小时,弃去上清后加入新鲜的DMEM(10%FBS)培养基37℃、5%CO2下继续培养48小时,收集上清,获得含有MSCV-shRNAs重组逆转录病毒的上清液,用于病毒滴度测定或者冻存于-80℃。2.2肿瘤反应性CD8+T细胞的分离、培养和鉴定制备BALB/c小鼠Renca肿瘤荷瘤模型,取其脾脏采用MagCellect小鼠CD8+ T细胞分离试剂盒(R&D公司)分离CD8+ T细胞,加入含Renca细胞裂解物、照射后的自体脾细胞、10% FBS、rmIL-2 (50 U/ml)、HEPE (25 mM)、L-glutamine (4 mM)、2-ME (25 mM)、以及抗CD3单克隆抗体(30 ng/ml, R&D)的RPMI-1640培养基于37℃、5%CO2下继续培养,并以流式细胞仪鉴定分离细胞的纯度。2.3 MSCV-shRNAs感染肿瘤反应性CD8+T细胞,获取肿瘤反应性TGF-β不敏感的CD8+T细胞MSCV-shRNAs重组逆转录病毒感染肿瘤反应性CD8+T细胞,5%CO2、37℃下孵化48小时后,获得敲除TβR-Ⅱ或未敲除TβR-Ⅱ的肿瘤反应性CD8+T细胞,同时将自未经任何处理的BALB/c小鼠脾脏中分离得到的CD8+T细胞设为对照组,三组细胞分别命名为M-T-CD8+T细胞、M-N-CD8+T细胞和N-CD8+T细胞。2.4 RT-PCR检测各组CD8+T细胞中TβR-ⅡmRNA表达情况2.5 Western Blot检测各组CD8+T细胞中TβR-Ⅱ蛋白表达情况3 TGF-β不敏感的肿瘤反应性CD8+T细胞对小鼠肾癌Renca细胞系的体内外杀伤作用3.1 Western Blot检测各组CD8+T细胞TGF-β信号通路中SMAD-2和磷酸化SMAD-2的表达3.2胸腺嘧啶脱氧核苷核素掺入法进行各组CD8+T细胞体外增殖抑制实验3.3 51Cr释放实验测定各组CD8+T细胞对Renca细胞的体外杀伤作用,黑色素瘤细胞B16-F1作为对照靶细胞3.4 TGF-β不敏感的肿瘤反应性CD8+T细胞体内抗肿瘤作用的评价建立小鼠肾癌Renca细胞皮下荷瘤BALB/c小鼠模型,皮下荷瘤小鼠30只,随机分为3组,每组10只,各组分别于腹腔内注射M-T-CD8+T细胞、M-N-CD8+T细胞和N-CD8+T细胞观察各组荷瘤小鼠的生存期和肿瘤的体积改变,于40天后处死全部存活小鼠,酶联免疫吸附(ELISA)测定细胞因子IL-2和IFN-γ变化,并分离各组小鼠脾脏中CD8+T细胞,流式细胞仪检测GFP阳性细胞百分率。【结果】1 siRNA的筛选以及MSCV-shRNAs逆转录病毒载体的构建三对siRNA转染小鼠成纤维细胞NIH3T3细胞系48小时后分别提取细胞总RNA和总蛋白,RT-PCR及Western Blot结果说明siRNA.1干涉组TβR-Ⅱ表达水平显著降低,是三者中干涉效果最好的,遂根据siRNA.1序列合成相应的shRNA,并合成阴性对照shRNA,分别命名为shRNA-T和shRNA-N,利用基因工程技术构建逆转录病毒载体,经DNA测序后证实成功构建了含有shRNAs的重组逆转录病毒载体,分别命名为MSCV-shRNA-T和MSCV-shRNA-N。2 TGF-β不敏感的肿瘤反应性CD8+T细胞的制备2.1 MSCV-shRNAs逆转录病毒的包装、滴定共转染MSCV-shRNAs质粒和pVSV-G质粒至泛嗜性GP293逆转录病毒包装细胞获得含有MSCV-shRNAs重组逆转录病毒的上清液后,以不同稀释浓度感染NIH3T3细胞,荧光显微镜下观察NIH3T3细胞绿色荧光,以流式细胞仪测定GFP阳性细胞数,计算病毒滴度为7.15×1010VP/L。2.2肿瘤反应性TGF-β不敏感的CD8+T细胞的获取自BALB/c小鼠Renca肿瘤荷瘤模型脾脏采用MagCellect小鼠CD8+ T细胞分离试剂盒(R&D公司)分离肿瘤反应性CD8+T细胞,流式细胞仪鉴定纯度为95.3% ;以逆转录病毒感染肿瘤反应性CD8+T细胞,MSCV-shRNA-T和MSCV-shRNA-N感染效率分别为93.1%和91.6%,同时将自未经任何处理的BALB/c小鼠脾脏中分离得到的CD8+T细胞设为对照组,三组细胞分别命名为M-T-CD8+T细胞、M-N-CD8+T细胞和N-CD8+T细胞。2.3各组CD8+T细胞中TβR-Ⅱ表达情况RT-PCR及Western Blot结果显示M-T-CD8+T细胞TβR-ⅡmRNA及蛋白水平明显降低,M-N-CD8+T细胞和N-CD8+T细胞TβR-ⅡmRNA及蛋白水平未见明显变化,表明成功获得敲除TβR-Ⅱ的肿瘤反应性CD8+T细胞。3 TGF-β不敏感的肿瘤反应性CD8+T细胞对小鼠肾癌Renca细胞系的体内外杀伤作用3.1各组CD8+T细胞TGF-β信号通路中SMAD-2和磷酸化SMAD-2的表达情况各组CD8+T细胞与10 ng/ml的TGF-β1孵育后,Western Blot在各组中均可以检测到Smad-2,M-T-CD8+T细胞中却没有检测到磷酸化的Smad-2。表明逆转录病毒介导的RNAi成功阻断了M-T-CD8+T细胞的TGF-β信号通路。3.2各组CD8+T细胞体外增殖抑制实验结果与TGF-β1共同培养72小时后,N-CD8+T细胞、M-N-CD8+T细胞和M-T-CD8+T细胞的脱氧胸腺嘧啶苷核素摄入抑制率分别为67.5%、63.5%和17%,M-T-CD8+T细胞的胸腺嘧啶脱氧核苷核素摄入抑制率与其他各组比较差异有显著统计学意义(P<0.05),表明M-T-CD8+T细胞对TGF-β1的抗增殖作用不敏感。3.3各组CD8+T细胞对Renca细胞的体外杀伤作用实验结果标准的51Cr释放实验测定各组CD8+T细胞对Renca细胞的体外杀伤作用,并以黑色素瘤细胞B16-F1作为对照靶细胞。实验结果显示:当靶细胞为Renca细胞时,M-T-CD8+T细胞具有最强的CTL杀伤活性(E/T比率为100:1时杀伤活性为41%),M-N-CD8+T细胞具有一定的CTL杀伤活性(E/T比率为100:1时杀伤活性为10%),N-CD8+T细胞杀伤活性最低;三组CD8+T细胞均对无关的黑色素瘤B16-F1细胞没有明显杀伤作用。结果表明阻断肿瘤反应性CD8+T细胞的TGF-β信号通路能增加肿瘤特异性杀伤活性。3.4各组CD8+T细胞体内抗肿瘤作用的评价成功地构建了小鼠肾癌Renca细胞皮下荷瘤BALB/c小鼠模型。与对照组相比,腹腔内注射M-T-CD8+T细胞、M-N-CD8+T细胞能够抑制荷瘤小鼠的肿瘤生长(P<0.01,P<0.05,vs.对照组),其中M-T-CD8+T细胞组抑制效果更明显,有2例肿瘤完全消失,这表明M-T-CD8+T细胞具有更强的抗肿瘤效果。40天后腹腔注射N-CD8+T细胞、M-N-CD8+T细胞和M-T-CD8+T细胞的荷瘤小鼠生存率分别为20%、30%和80%。Mantel-Haenszel log-rank统计分析表明:M-T-CD8+T细胞与其他2组相比差别有统计学意义(P<0.01)。这些结果说明TGF-β不敏感的肿瘤反应性CD8+T细胞能有效提高Renca皮下荷瘤小鼠的生存率。40天后处死全部存活小鼠,酶联免疫吸附(ELISA)测定细胞因子IL-2和IFN-γ变化,结果显示:相对于N-CD8+T细胞组,腹腔内注射M-T-CD8+T细胞和M-N-CD8+T细胞的荷瘤小鼠体内IL-2和IFN-γ水平明显升高,注射M-T-CD8+T细胞的荷瘤小鼠体内该两种细胞因子水平升高更明显。这些结果说明TGF-β不敏感的肿瘤反应性CD8+T细胞能够促进机体产生相应的细胞因子以协同对肿瘤细胞的杀伤作用。分离各组小鼠脾脏中CD8+T细胞,流式细胞仪检测GFP阳性细胞百分率,注射M-T-CD8+T细胞和M-N-CD8+T细胞的荷瘤小鼠脾脏分离的CD8+T细胞中GFP阳性细胞百分率分别为2.0%和0.2%,两组之间的差别有统计学意义(P<0.01)。结果说明TGF-β不敏感的肿瘤反应性CD8+T细胞能够在肿瘤的刺激下维持一定的时间。【结论】1成功构建了小鼠TβR-ⅡRNAi逆转录病毒表达载体。2采用自荷瘤小鼠分离CD8+T细胞的方法以及使用肾癌Renca细胞裂解产物作为抗原,诱导出了肾癌反应性的CD8+T细胞。3首次使用含有针对TβR-ⅡshRNA的逆转录病毒感染肾癌反应性的CD8+T细胞,敲除其表面的TβR-Ⅱ,阻断TGF-β信号通路。4 TGF-β不敏感的肿瘤反应性CD8+T细胞对TGF-β1的抗增殖作用不敏感,并具有较强的肿瘤特异性杀伤活性。5 TGF-β不敏感的肿瘤反应性CD8+T细胞能够在肿瘤的刺激下维持一定的时间,显著提高Renca皮下荷瘤小鼠的生存率,明显抑制肿瘤生长,而且有2例肿瘤完全消失。TGF-β不敏感的肿瘤反应性CD8+T细胞处理的荷瘤小鼠体内IL-2和IFN-γ水平明显升高。用逆转录病毒介导的RNAi敲除肿瘤反应性CD8+T细胞表面的TβR-Ⅱ,可以阻断这些CD8+T细胞中的TGF-β信号通路,降低TGF-β对CD8+T细胞分化、增殖的抑制作用,以提高CD8+T细胞对肿瘤细胞的杀伤作用,合理地利用RNAi技术以促进免疫效应细胞对肿瘤的杀伤作用,是肾癌免疫基因治疗的一个新方向。更多还原

【Abstract】 [Background]Renal cell carcinoma (RCC) is the most common malignancy in urogenital system. Although the incidence of RCC occupied only 3% of systemic malignant tumors, the incidence of RCC in the whole world increased 2% per year recently, and every year more than 100,000 people died of RCC. In our country, the incidence of RCC, which is inferior to the incidence of bladder cancinoma, occupys the second place in urogenital tumors. In recent years, there has been a ascending tendency of the RCC incidence and RCC is one of the most important tumors which threaten the people’s health. Most RCCs are congenitally multidrug resistant and insensitive to chemotherapy and radiotherapy. In addition to that surgical treatment of early stage RCCs can achieve a better therapeutic efficacy, there are no specific treatment methods to most advanced RCCs nowadays. Hower, about 50% patients with RCCs are diagnosed as intermediate or advanced stage of RCC for the first visit. In about 40% patients, metastasis and recurrence happened. The prognosis of metastatic RCCs is bad. The mean survival time of patients with metastatic RCCs was less than one year and the three-year survival rate of the patients was only less than 5%. Therefore, to find a effective and specific treatment method to RCCs is the exigent problem to resolve in the clinic therapy of urology.The development of immunogene therapy has brought the new hope to the people. Both the selection of target gene and the effector cell are the key points of immunogene therapy. Transforming growth factorβ(TGF-β) is a multifunctional cytokine. It is important to cell proliferation, differentiation, apoptosis and development of lots of tissues. TGF-βis a potent immunosuppressant. The overproduction of TGF-βby tumor cells and the resulting inhibition of immune effector cells (T cell, B cell, macrophage, etc) in proliferation and differentiation may lead to tumor evasion from the host immune surveillance and tumor progression. Three types of TGF-βreceptors with high homology have been identified: typeⅠ(TβR-Ⅰ, 55KD), typeⅡ(TβR-Ⅱ, 75KD), and typeⅢ(TβR-Ⅲ, 280KD). They perform the signal transduction via the transmembrane receptor compounds. TβR-Ⅰand TβR-Ⅱexhibit serine/threonine kinase activity in their intracellular domains. TβR-Ⅲfunctions by binding TGF-βand then transferring it to its signaling receptors, the typeⅠandⅡreceptors. The current understanding shows that TGF-βfirst binds to TβR-Ⅱwith high affinity, which initiates intracellular signaling by phosphorylating several transcription factors and regulates the cell biological behaviour.It can be seen that TβR-Ⅱis the key molecule in TGF-βsignaling and CD8+ T cell is one of the effctor cells with killing effect in tumor immunity. We hypothesized that based on the premise that CD8+ T cells were tumor-reactive, knock-out of TβR-Ⅱon these CD8+ T cells via RNAi to make these tumor-reactive CD8+ T cells TGF-β-insensitive would resist the inhibition effect of TGF-βon proliferation and differentiation of CD8+ T cells, so that the killing effect of CD8+ T cells on tumor cells would be improved. This new strategy of immunogene therapy is expected to be provided in clinic for the treatment of RCCs.[Objective]To construct a retrovirus vector which contains the shRNA to mouse TβR-Ⅱgene. To product tumor-reactive TGF-β-insensitive CD8+ T cells by knocking down TβR-Ⅱon tumor-reactive CD8+ T cell surface via MSCV-incuced RNAi. To observe the killing effect of these CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo.[Materials and methods]1 Selection of siRNAs and construction of MSCV-shRNAs retrovirus vectors1.1 Selection of the target sites of TβR-ⅡgeneBased on the mouse TβR-Ⅱgene (GenBank accession No. AF406755) sequence, three pairs of siRNAs to three different target sites were designed and synthesized, and named siRNA.1, siRNA.2 and siRNA.3.1.2 Selection of siRNAsThe three pairs of siRNAs were separately transfected to mouse fibroblast NIH3T3 cell line by Oligofectamine?. After 48h of transfection, cell total RNA and protein was extrcated for RT-PCR and Western Blot analysis to identify which pair of siRNA was the most effective one. Then this pair of siRNA was selected for the further experiments.1.3 Construction of MSCV-shRNAs retrovirus vectorCorrespondingly, shRNA to TβR-Ⅱgene, with the name of shRNA-T, was synthesized based on the selected siRNA sequence. Negative control shRNA, with the name of shRNA-N, was also synthesized. The two shRNAs were ligated into the linearized pEGFP/U6 vector by ApaⅠand EcoRⅠ. Then the whole U6-shRNA sequence was obtained by BamHⅠ/EcoRⅠdigestion and inserted into the MSCV-TβRⅡDN-IRES-GFP vector, which was also linearized by BamHⅠ/EcoRⅠdigestion. The reconstructed MSCV-shRNAs were named MSCV-shRNA-T and MSCV-shRNA-N respectively and were identified by DNA sequencing.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cells2.1 Package and titration of MSCV-shRNAs retrovirusMSCV-shRNA and pVSV-G were cotransfected to pantropic GP293 retroviral packaging cells. After cotransfection at 37℃for 12h, the supernatant was replaced by fresh DMEM medium containing 10% FBS for additioanl incubation at 37℃, 5% CO2 for 48h. The supernatant, which contained MSCV-shRNA reconstructed retrovirus, was then collected for determination of virus tite or for cryopreservation at -80℃.2.2 Isolation, cultivation and identification of tumor-reactive CD8+ T cellsRenca tumors were established in BALB/c mouse. Splenic CD8+ T cells were isolated by using MagCellect* mouse CD8+ T cell isolation kit (R&D Systems) and cultured at 37℃, 5% CO2 in the presence of Renca lysates and irradiated autologous splenocytes in a medium containing RPMI-1640 with 10% FBS, rmIL-2 (50 U/ml), anti-CD3+ moloclonal antibody (30 ng/ml, R&D), HEPE (25 mM), L-glutamine (4 mM), and 2-ME (25 mM). The cell purity was determined by flow cytometry.2.3 Production of tumor-reactive TGF-β-insensitive CD8+ T cells by infection of MSCV-shRNAs retrovirusTumor-reactive CD8+ T cells were infected by MSCV-shRNAs retrovirus and incubated at 37℃, 5% CO2 for 48h. Three types of CD8+ T cells were established. The first type was tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells (tumor-reactive CD8+ T cells infected with the MSCV-shRNA-T virus). The second type was tumor-reactive CD8+ T cells infected with the MSCV-shRNA-N virus. The third type was na?ve CD8+ T cells, which were freshly isolated from the spleen of na?ve donor animals without any treatment. The three types of CD8+ T cells were named M-T-CD8+T, M-N-CD8+T and N-CD8+T.2.4 The expression level of TβR-ⅡmRNA in each group of CD8+ T cells was determined by RT-PCR2.5 The expression level of TβR-Ⅱprotein in each group of CD8+ T cells was determined by Western Blot3 The killing effect of tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo3.1 Western Blot analysis for the expression of SMAD-2 and P-SMAD in each group of CD8+ T cells3.2 Thymidine incorporation assay for the analysis to each group of CD8+ T cells proliferation in vitro3.3 Detection of each group of CD8+ T cells’killing effect on Renca cell line or an irrelevant mouse melanoma B16-F1 cell line in vitro by a standard 51Cr release assay3.4 Assessment of anti-tumor effect presented by tumor-reactive TGF-β- insensitive CD8+ T cells in vivoSubcutaneous Renca tumors were established in BALB/c mouse. Total of 30 BALB/c tumor-bearing mice were divided into 3 groups randomly and inoculated i.p. with each group of CD8+ T cells respectively. Tumor growth and mouse survival were monitored post-inoculation. Forty days later, all of the survival mice were sacrificed and serum levels of IL-2 and IFN-γwere determined by enzyme-linked immunoabsorbant assay (ELISA). Splenic CD8+ T cells were isolated and the percentage of GFP-positive ones was determined by flow cytometry.[Results]1 Selection of siRNAs and construction of MSCV-shRNAs retrovirus vectors Three pairs of siRNAs transfected to mouse fibroblast NIH3T3 cell line. After 48h of transfection, cell total RNA and protein was extrcated. The results of RT-PCR and Western-Blot indicated that the expression level of TβR-Ⅱin siRNA.1 transfected cells decreased significantly and that siRNA.1 was the most effective one. Correspondingly, shRNA to TβR-Ⅱgene, with the name of shRNA-T, was synthesized based on the selected siRNA sequence. Negative control shRNA, with the name of shRNA-N, was also synthesized. By use of gene engineering technology and DNA sequencing, MSCV retrovirus vectors containing shRNAs were successfully reconstructed and named MSCV-shRNA-T and MSCV-shRNA-N, respectively.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cells2.1 Package and titration of MSCV-shRNAs retrovirusMSCV-shRNA and pVSV-G were cotransfected to pantropic GP293 retroviral packaging cells. Then the collected supernatant at different dilution, which contained MSCV-shRNA reconstructed retrovirus, infected NIH3T3 cells. The NIH3T3 cells with green fluorescence were observed by fluorescence microscope and the GFP-positive cell counts were determined by flow cytometry. The calculated virus tite was 7.15×1010VP/L.2.2 Production of tumor-reactive TGF-β-insensitive CD8+ T cellsSplenic tumor-reactive CD8+ T cells were isolated from BALB/c Renca tumor-bearing mouse by using MagCellect* mouse CD8+ T cell isolation kit (R&D Systems). The cell purity was 95.3% determined by flow cytometry. Tumor-reactive CD8+ T cells were infected with the MSCV-shRNAs retrovirus. The infection efficiency was determined by flow cytometry analysis. They were 93.1% and 91.6%, respectively, for the MSCV-shRNA-T and MSCV-shRNA-N retrovirus. MSCV-shRNA-T, MSCV-shRNA-N retrovirus infected tumor-reactive CD8+ T cells and na?ve CD8+ T cells freshly isolated from the spleen of na?ve donor animals without any treatment were established and named M-T-CD8+T, M-N-CD8+T and N-CD8+T respectively. 2.3 The expression level of TβR-Ⅱin each group of CD8+ T cellsRT-PCR and Western Blot indicated that TβR-Ⅱexpression in mRNA and protein level was decreased in the M-T-CD8+T. While in M-N-CD8+T and N-CD8+T, there was no significant decrease of TβR-Ⅱexpression. These results suggested that tumor-reactive TGF-β-insensitive CD8+ T cells were successfully established.3 The killing effect of tumor-reactive RNAi-induced TGF-β-insensitive CD8+ T cells to mouse renal cancer Renca cell line in vitro and in vivo3.1 Western Blot analysis for the expression of SMAD-2 and P-SMAD in each group of CD8+ T cellsSmad-2 and phosphorylated Smad-2 were detected by Western blot analysis after the three types of CD8+ T cells were treated with 10 ng/ml TGF-β1. The presence of Smad-2 was detected in all CD8+ T groups. But, phosphorylated Smad-2 was only detected in M-N-CD8+T and N-CD8+T in response to TGF-β1; absence of phosphorylated Smad-2 in M-T-CD8+T confirmed that TGF-βsignal transduction was successfully blocked by the MSCV-shRNA induced RNAi.3.2 Results of thymidine incorporation assayThe inhibitory rate of TGF-βon thymidine uptake was compared among the three types of CD8+ T cells after the addition of TGF-β1 for 72 h. TGF-β1 showed a dramatic antiproliferative effect on the established M-N-CD8+T and N-CD8+T, inhibiting uptake by a mean of 67.5% and 63.5% respectively. Whereas the mean inhibitory rate of thymidine uptake by M-T-CD8+T was 17%, the resistance to the antiproliferative effects of M-T-CD8+T was statistically significant when compared with the other groups (P<0.05). These results suggested that M-T-CD8+T were insensitive to the antiproliferative effect of TGF-β1.3.3 Detection of each group of CD8+ T cells’killing effect on Renca cell line in vitroThe ability of these CD8+ T cells to lyse Renca cells or irrelevant mouse melanoma B16-F1 cells was assayed in vitro using a standard 51Cr release assay. The results indicated that M-T-CD8+T showed the most potent Renca-specific CTL response (41% killing activity at an effector:target cell ratio of 100:1). M-N-CD8+T showed determinate Renca-specific CTL response (10% killing activity at an effector:target cell ratio of 100:1). N-CD8+T showed no significant CTL response. No apparent lysis was observed against irrelevant B16-F1 cells by these three types of CD8+ T cells. These results suggested that blocking TGF-βsignaling of tumor-reactive CD8+ T cells may improve the tumor-specific killing activity.3.4 Assessment of anti-tumor effect presented by tumor-reactive TGF-β- insensitive CD8+ T cells in vivoTo assess the antitumor effect of the M-T-CD8+T in vivo, Renca tumors were established in BALB/c mice. Compared with N-CD8+T group, adoptive i.p. transfer of M-T-CD8+T and M-N-CD8+T significantly suppressed the growth of the tumor (P<0.01,P<0.05,vs.control, respectively), with the M-T-CD8+T showing the more significant inhibitory effect. Complete tumor regression occurred in 20% of Renca-tumor-bearing mice that were treated with M-T-CD8+T. Forty days later, the survival rate of M-T-CD8+T, M-N-CD8+T and N-CD8+T treated mice was 80%, 30% and 20% respectively. Statistical analysis by using the Mantel-Haenszel log-rank test indicated a significant difference between the M-T-CD8+T and the other two control groups (P<0.01). These results demonstrated that the tumor-reactive TGF-β-insensitive CD8+ T cells were effective in improving the survival rate in mice bearing Renca tumors.Forty days after adoptive i.p. transfer of these three types of CD8+ T cells, all of the survival mice were sacrificed. Serum levels of IL-2 and IFN-γwere detected by enzyme-linked immunoabsorbant assay (ELISA). Compared with N-CD8+T treated group, the level of IL-2 and IFN-γin M-T-CD8+T and M-N-CD8+T treated mice increased significantly. A further increase in serum IL-2 and IFN-γwas observed in M-T-CD8+T group, suggesting the tumor-reactive TGF-β-insensitive CD8+ T cells promoted the host to product corresponding cytokines to kill the tumor cells robustly.Splenic CD8+ T cells were isolated and the percentage of GFP-positive ones was determined by flow cytometry. Adoptively transferred M-T-CD8+T cells were detected with a percentage of 2.0%, and 0.2% for M-N-CD8+T cells. The difference between the two groups was statistically significant, suggesting that these tumor-reactive TGF-β-insensitive CD8+ T cells were able to persist in recipient tumor-bearing hosts at least at the time of sacrifice, which occurred 40 days after the initial adoptive transfer.[Conclusions]1 We successfully constructed a retrovirus vector to deliver shRNA to mouse TβR-Ⅱgene.2 By use of the method to isolate CD8+ T cells from the tumor-bearing mice and make Renca cell lysate as antigens, we successfully induced the renal cancer reactive CD8+ T cells.3 MSCV retrovirus containing shRNA to TβR-Ⅱgene was used for the first time to infect renal cancer reactive CD8+ T cells. The TβR-Ⅱwas successfully knocked down so that the TGF-βsignaling was blocked.4 Tumor-reactive TGF-β-insensitive CD8+ T cells were insensitive to the antiproliferative effect of TGF-β1 and their tumor-specific killing activity was stronger.5 Tumor-reactive TGF-β-insensitive CD8+ T cells were able to persist for a period in recipient tumor-bearing hosts. These CD8+ T cells significantly suppressed tumor growth and increased survival rate of Renca tumor-bearing mice. Furthermore, complete tumor regression occurred in 2 treated mice. Serum levels of IL-2 and IFN-γin tumor-reactive TGF-β-insensitive CD8+ T cells treated mice were significantly improved. Taken together, by use of retrovirus induced RNAi to knock down TβR-Ⅱon the surface of tumor-reactive CD8+ T cells, the TGF-βsignaling was blocked, which decreased the inhibitory effect of TGF-βon the proliferation and differentiation of CD8+ T cells. Thus, the tumor-killing activity of CD8+ T cells were improved. It was demonstrated that reasonable use of RNAi technology to promote the tumor-killing activity of immune effctor cells could potentially be a new experimental approach for the immunogene therapy to renal cancer.更多还原