艾滋病毒Tat对HHV-8 K12基因诱导肿瘤形成的影响及其分子机制初探

发布时间：2018-05-21 21:03

本文选题：人类疱疹病毒8型 + Tat　；参考：《南京医科大学》2007年硕士论文

【摘要】： 本研究的目的是探索人类免疫缺陷病毒Ⅰ型(human immunodeficiency virus type 1，HIV-1，又称艾滋病毒)反式激活转录蛋白(transactivative transcription protein，Tat)对人类疱疹病毒8型(human herpesvims 8，HHV-8)K12基因诱导肿瘤形成的影响及其分子机制。实验首先构建了含UHV-8 K12基因的重组质粒K12-Flag-pcDNA3.1(+)(命名为pK12F)，将其与本室保存Tat-Flag-pcDNA3.1(+)(命名为pTatF)质粒单独或共转染NIH3T3细胞(Swiss小鼠成纤维细胞)，，同时以空载体pcDNA3.1(+)为对照，获得稳定转染目的基因的四株细胞，分别命名为N／K12、N／Tat、N／K12／Tat、N／pcDNA3.1。进而采用~3HTdR掺入实验及流式细胞检测法分别测定上述四株细胞的增殖能力和细胞周期。~3HTdR掺入实验结果显示，四株细胞的增殖能力从高到低依次为N／K12／Tat、N／K12、N／pcDNA3.1、N／Tat(P＜0.05)；流式细胞术结果显示，四株细胞的S期细胞比率(S-phase fraction，SPF)从高到低的排序与~3HTdR掺入实验结果一致。随后，软琼脂集落形成实验证实上述四株细胞均能体外诱导细胞转化。将四株细胞分别进行裸鼠体内成瘤实验的结果表明，自分泌和近距离旁分泌Tat蛋白能加速HHV-8 K12诱导肿瘤的形成(Log-rank检验，P＜0.05)，远距离旁分泌Tat蛋白不具备此功能(P＞0.05)。为进一步探索Tat和K12相互作用的机制，采用基因芯片技术对这四株细胞及其对应的肿瘤组织进行了与肿瘤形成相关15条信号通路96个标志物基因mRNA表达的检测，结果发现虽然细胞和组织结果存在差异，但均显示出Tat能促使K12基因激活更多的肿瘤信号基因。Western blot检测四株细胞中STAT3(signal transducer and activator of transcription 3，信号转导和转录激活因子3)蛋白的磷酸化水平，结果显示Tat可以促进K12激活磷酸化STAT3的表达。免疫组化检测裸鼠肿瘤组织中CyclinD1(周期蛋白D1)、VEGF(vascular endothelial growth factor，血管内皮细胞生长因子)和Survivin(存活素)的结果表明Tat能降低这些蛋白在K12诱导的肿瘤中的表达。本研究结果提示，在体外Tat能增强K12转化细胞的增殖能力；裸鼠体内成瘤实验中自分泌和近距离旁分泌Tat蛋白均能加速HHV-8 K12诱导肿瘤的形成；STAT3信号通路是参与Tat加速K12诱导肿瘤生成的肿瘤信号通路之一；同时Tat降低了VEGF、CyclinD1、Survivin在K12诱导肿瘤中的表达。
[Abstract]:The aim of this study was to investigate the effect of transactivative transcription protein (Tatatt) of human immunodeficiency virus type I (HIV-1) transactivated transcription protein on tumorigenesis induced by HHV-8K12 gene of human herpesvirus 8 (HHV-8) and its molecular mechanism. Firstly, a recombinant plasmid K12-Flag-pcDNA3.1 containing UHV-8 K12 gene was constructed. The four cells stably transfected with the target gene were named N / K12N / T / T / N / K 12 / T / pcDNA3.1, respectively. The proliferative ability and cell cycle of the four cell lines were measured by 3HTdR incorporation assay and flow cytometry, respectively. The results showed that the proliferative ability of the four cell lines was N / K 12 / TatK 12 / T K12N / pc3.1N / T T (P < 0.05), and the results of flow cytometry showed that the proliferation ability of the four cell lines was higher than that of the control group (P < 0.05), and the results of flow cytometry showed that the proliferative ability of the four cell lines was N / K 12 / Tatr / N% pc3.1N / T T (P < 0.05). The S phase ratio of S phase fractionated SPFs from high to low was consistent with the results of 3HTdR incorporation. Subsequently, the soft Agar colony formation assay confirmed that all the above four cell lines could induce cell transformation in vitro. The results of tumorigenesis in nude mice showed that autocrine and close paracrine Tat protein could accelerate the formation of tumor induced by HHV-8 K12 (P < 0.05), but the long-distance paracrine Tat protein did not have this function (P > 0.05). In order to further explore the mechanism of interaction between Tat and K12, the gene mRNA expression of 96 markers related to tumor formation in these four cell lines and their corresponding tumor tissues were detected by gene chip technique. It was found that although there were differences in cell and tissue outcomes, The results showed that Tat could induce K12 gene to activate more tumor signal genes. Western blot was used to detect the phosphorylation level of STAT3(signal transducer and activator of transcription 3, signal transduction and transcription activator 3 in four cell lines. The results showed that Tat could promote the expression of phosphorylated STAT3 activated by K12. The expression of cyclin D 1 (cyclin D 1) and survivin (survivin) in tumor tissues of nude mice was detected by immunohistochemistry. The results showed that Tat could decrease the expression of these proteins in K12 induced tumor. The results suggest that Tat can enhance the proliferation of K12 transformed cells in vitro. Autocrine and close paracrine Tat protein can accelerate tumor formation induced by HHV-8 K12 in nude mice. STAT3 signaling pathway is one of the tumor signaling pathways involved in Tat accelerating K12 induced tumor formation. At the same time, Tat decreased the expression of VEGF cyclin D 1 survivin in K 12 induced tumor.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373;R730.2

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