【作者】 杨波；

【导师】 焦顺昌；

【作者基本信息】 中国人民解放军军医进修学院 ， 肿瘤学， 2010， 博士

【摘要】 目的通过oligo化学合成及PCR扩增法合成人显性抑制TGF-βⅡ型受体(Dominant-negative transforming growth factor-βreceptorⅡ, DNTβRⅡ)目的基因,构建pIRES2-AcGFP-DNTβRⅡ真核表达质粒；利用Nucleofector技术将质粒转染NK-92细胞,观察阻断TGF-β信号通路的NK-92细胞的体外抗肿瘤效应；建立裸鼠荷Calu-6人肺癌细胞皮下移植瘤模型,观察阻断TGF-β信号通路的NK-92细胞过继性治疗的体内抗肿瘤效应。方法(1)根据Genebank数据库确定DNTβRⅡ目的基因,利用化学合成法进行单链oligo的合成,PCR法将合成的oligo拼接成完整的序列,装入PMD18-T载体并转染感受态细胞DH5α,经XhoI和EcoRI酶切后连接至目的载体pIRES2-AcGFP中,酶切电泳和DNA测序鉴定,转染COS-7细胞,倒置荧光显微镜观察GFP表达。(2)流式细胞术检测NK-92细胞TGF-βⅠ型和Ⅱ型受体表达情况,ELISA法检测Calu-6、A-549、MDA-MB231和HT-29四种肿瘤细胞系的TGF-β1分泌水平；采用Nucleofector技术将pIRES2-AcGFP-DNTβRⅡ质粒和pIRES2-AcGFP对照质粒转染NK-92细胞,流式细胞术、RT-PCR法及倒置荧光显微镜检测DNTβRⅡ在NK-92细胞的转染及表达,western blot险测转染后NK-92细胞Smad2蛋白的活化状态；将分泌水平最高的Calu-6细胞系与TGF-β1(终浓度10ng／m1)共孵育24小时,CCK-8法比较转染后的NK-92细胞对Calu-6人肺癌细胞系的体外杀伤活性。(3)采用细胞悬液接种法,将Calu-6细胞(1×106)接种于裸鼠背部皮下；成瘤实验同期经尾静脉分别输注转染pIRES2-AcGFP的NK-92细胞(B组)和转染pIRES2-AcGFP-DNTβRⅡ的NK-92细胞(C组),A组不予另外处理,接种后第10天比较各组成瘤率；抑瘤实验于接种后第10天、17天经尾静脉进行2次过继性输注,分为生理盐水组(a组)、NK92／pIRES2-AcGFP组(b组)和NK92／pIRES2-AcGFP-DNTβRⅡ组(c组),接种第56天处死动物；比较各组皮下移植瘤体积、生存期变化及外周血IFN-γ水平,肺转移情况。结果(1)合成DNA经测序验证与目的基因一致,真核表达质粒pIRES2-AcGFP-DNTβRⅡ转染后,倒置荧光显微镜证实DNTβRⅡ在COS-7细胞的表达。(2)Calu-6细胞系在四种肿瘤细胞系中TGF-β1表达水平最高；流式细胞术证实了NK-92细胞表面TβRⅠ、TβRⅡ的高表达；采用Nucleofector技术,pIRES2-AcGFP和pIRES2-AcGFP-DNTβRⅡ质粒对NK-92细胞的转染效率分别为28.53%和18.86%,RT-PCR和倒置荧光显微镜证实了DNTβRⅡ的表达,western blot显示虽有TGF-β的刺激,转染pIRES2-AcGFP-DNTβRⅡ质粒的NK-92细胞未见Smad2蛋白的活化。Calu-6细胞与TGF-β1共孵后,NK-92细胞对Calu-6细胞的杀伤活性较共孵前明显减弱(p<0.001)：转染DNTβRⅡ阳性质粒的NK-92细胞对Calu-6细胞杀伤活性明显高于GFP转染组(p<0.001)。(3)成瘤实验显示3组间成瘤率存在差异,C组成瘤率低于A组(p<0.05)。抑瘤实验表明转染DNTβRⅡ阳性质粒的NK-92细胞(c组)过继性治疗后裸鼠皮下移植瘤体积缩小,肺转移率下降,生存期明显延长,外周血IFN-γ水平明显增高。结论(1)成功构建pIRES2-AcGFP-DNTβRⅡ真核表达质粒,为阻断NK-92细胞的TGF-β信号通路提供了载体；(2)Nucelofector核转染技术实现了pIRES2-AcGFP-DNTβRⅡ质粒在NK-92细胞的有效转染和表达；(3)pIRES2-AcGFP-DNTβRⅡ质粒的转染在受体水平阻断了NK-92细胞的TGF-β信号通路；(4)阻断TGF-β信号通路的NK-92细胞能够在体外抵抗TGF-β的免疫抑制作用；(5)阻断TGF-β信号通路能够明显增强NK-92细胞过继性治疗的体内抗肿瘤效应；(6)改善肿瘤微环境中的免疫抑制作用,可能增强过继性免疫细胞治疗的临床疗效。更多还原

【Abstract】 Objective Transforming growth factor (TGF)-βis a potent suppressor in tumor microenvironment, which may lead to tumor evasion from the host immune surveillance and tumor progression. The present study is to synthesize the gene of human dominant-negative TGF beta receptorⅡ(DNTβRⅡ) and construct its eukaryotic expression vector pIRES2-AcGFP-DNTβRⅡ; block TGF-βsignaling pathway in NK-92 cells by transfection with pIRES2-AcGFP-DNTβRβⅡplasmid using Nucleofector technology; investigate the in vitro and in vivo antitumor effect of this TGF-βinsensive NK-92 cells against human Calu-6 lung cancer cells which secrete high levels of TGF-β1.Methods (1) According to the Genebank Database, the sequence of target gene DNTβRβⅡwas determined. The single chain oligo was synthesized chemically and then ligated into full length gene by PCR. The synthesized gene was cloned into plasmid PMD18-T and transferred into competent cells DH5α, then it was inserted into the corresponding restriction site on eukaryotic expression vector pIRES2-AcGFP, the DNTβRⅡgene was confirmed by double enzyme digesting and DNA sequencing analysis. After transfer into COS-7 cells, expression of DNTβRⅡwas identified by inverted fluorescent microscope. (2) The expression of TGF-βtypeⅠ(TβRⅠ) and typeⅡreceptors (TβRⅡ) in parental NK-92 cells were detected by fluorescence activated cell sorter (FACS). Mean levels of TGF-β1 secreted by 4 human tumor cell lines(Calu-6、A549、MDA-MB231 and HT-29) were determined by ELISA. NK-92 cells were transfected with recombinant plasmid pIRES2-AcGFP-DNTβRⅡand control plasmid pIRES2-AcGFP using Amaxa Nucleofector technology. RT-PCR and inverted fluorescent microscope were used to identify the expression of DNTβRⅡ, FACS was used to analyse the transfection efficiency, then western blot was used to detect the phosphorylated satus of Smad2. TGF-β1 was added at the final concentration of lOng/ml with Calu-6 cells for 24h. The cytotoxicity of two types transfected NK-92 cells against Calu-6 cells was detected and analyzed by CCK-8 kit. (3) According to the model of tumor formation,24 BALB/C-nu mice received a single subcutaneouly injection of 1x106 Calu-6 cells into the back; at the same time, 1x107 NK-92 cells transfected with DNTβRⅡpositive vector (group C) or GFP control plasmid (group B) were injected via tail vein while there was no intervention added in group A.10 days afer transplantion, tumor formation rate of mice bearing Calu-6 cells in 3 groups was compared. According to the model of tumor suppression, 1x106 Calu-6 cells were injected subcutaneouly into the back of BALB/C mice, then saline (group a)、1x107 NK-92 cells transfected with plasmid pIRES2-AcGFP (group b) or pIRES2-AcGFP-DNTβRⅡ(group c) were respectively injected via tail vein on the day 10,17 following transplantion. Serum IFN-y levels of tumor-bearing mice in different groups were detected by ELISA on the day next to the second adoptive transfer.56 days after transplantion, all animals were sacrificed and histologic examination was done.Results (1) The synthesized fragment was consistent with the target gene DNTβRⅡby DNA sequence analysis. After the recombinant expression plasmid pIRES2-AcGFP-DNTβRⅡtransferring into COS-7 cells, DNTβRⅡwas instantaneously transfected and expressed successfully. (2) The highest level of TGF-β1 was secreted by Calu-6 cell-line while the lowest level of TGF-β1 was secreted by HT-29 cell-line. High expression of TβRⅠand TβRⅡwas identified by FACS. Using nucleofector technology, the transfection efficiency was 18.80% for the plasmid pIRES2-AcGFP-DNTβRⅡand 28.53% for the control vector pIRES2-AcGFP to NK-92 cells. The expression of DNTβRⅡin NK-92 cells was confirmed by inverted fluorescent microscope and RT-PCR. By the irritation of TGF-β1, western blot detected inactive Smad2 status in NK-92 cells transfected with pIRES2-AcGFP-DNTβRⅡ. Parental NK-92 cells displayed lower cytotoxity against Calu-6 cells incubated with TGF-β1 than that without TGF-β1 (E:T ratio 10:1,32.93%±0.80% vs.17.90%±0.75%, p< 0.001; E:T ratio 20:1, 46.33%±1.40% vs.26.46%±1.10%, p<0.001). The cytotoxity of NK-92 cells transfeced with DNTβRⅡvector was higher than that with control GFP vector against Calu-6 cells cultured with TGF-β1(E:T ratio 29.73%±0.96% vs. 15.43%±0.97%, p<0.001; E:T ratio 20:1,45.0%±1.20% vs.24.83%±1.21%, p< 0.001). (3) The tumor formation rate of mice bearing Calu-6 cells in group C was lower than group A(p<0.05). After adoptive transfer, decreased tumor volume, increased survival, lower lung metastasis rate and higher levels of serum IFN-y were shown in group c which received adoptive transfer of NK-92 cells transfected with DNTβRⅡpositive vector.Conclusion (1) The eukaryotic expression vector pIRES2-AcGFP-DNTβRⅡwas constructed successfully, providing the basis for blocking TGF-βsignaling pathway in NK-92 cells. (2) The pIRES2-AcGFP-DNTβRⅡplasmid was successfully transferred and expressed in NK-92 cells by nucleofector technology. (3) pIRES2-AcGFP-DNTβRⅡplasmid transfection can block TGF-βsignaling pathway in NK-92 cells at the receptor level. (4) Blocking TGF-P signaling pathway in NK-92 cells can resist immunosupression effect of TGF-P in vitro. (5) Blocking TGF-βsignaling pathway can augment antitumor efficacy of adoptive NK-92 cell therapy in vivo. (6) Relieving immuosupression in tumor microenvironmet may enhance the efficacy of adoptive therapy in clinical practice.更多还原