慢病毒法介导干扰YAP1表达的前列腺癌稳定细胞系的构建及作用研究

发布时间：2018-03-17 12:09

本文选题：YAP1　切入点：前列腺癌　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:通过构建干扰Yes相关蛋白(YAP1)表达的重组慢病毒载体,包装制备慢病毒,利用慢病毒高效转染的特性,建立稳定敲除YAP1表达的前列腺癌(PCa)C4-2细胞系。通过干扰YAP1表达后对前列腺癌细胞系生长以及裸鼠皮下成瘤时间的监测,确定YAP1在前列腺癌中的作用,从而为YAP1相关基因通路的进一步研究提供工具。方法:利用蛋白印迹(Western blot)的方法检测前列腺癌几种细胞系中YAP1表达含量最高的一株作为研究对象,之后采用将筛选出的干扰效果最佳的慢病毒载体采用三质粒系统共同转染于293T细胞,包装制备慢病毒。将收集的病毒液感染已选择的前列腺癌细胞系,待稳定表达后通过抗性标记嘌呤霉素进一步筛选,最终获得稳定干扰YAP1表达的C4-2细胞系,通过逆转录聚合酶链反应(RT-PCR)以及Western blot来检测敲除的效率。之后采用噻唑蓝比色法(MTT)检测稳定转染的C4-2细胞系的活力,并将正常以及稳定敲除YAP1表达的C4-2细胞系种植于裸鼠皮下,观察对比两组小鼠的成瘤情况。结果:1.通过Western blot方法检测RWPE-1,LNCaP,C4-2,PC3四个细胞系中的YAP1蛋白表达水平。结果显示C4-2细胞系中的YAP1含量表达最高。将YAP1短发夹RNA(YAP1-shRNA)表达载体成功转入C4-2细胞,Western blot结果显示第二条干扰序列具有更强的基因沉默效应。2.将包装好的慢病毒经过超速离心浓缩,采用绝对定量PCR法(qPCR)测定滴度,待达到要求后转染C4-2细胞,72h之后进一步通过梯度摸索确定的最佳浓度的嘌呤霉素筛选已转染的C4-2细胞系2周,荧光表达水平将近90%。Western blot以及RT-PCR检测敲除效率,可达85%。3.通过MTT法检测敲除YAP1表达后的C4-2稳定细胞系其细胞生长状态及细胞活力明显下降。4.两组裸鼠皮下成瘤体积对比发现,敲除YAP1表达的细胞成瘤体积明显小于对照组,荧光强度弱于对照组。结论:通过采用慢病毒技术,成功构建干扰YAP1表达的前列腺癌C4-2细胞系,明显降低了前列腺癌细胞的增殖能力,而体内试验证实干扰YAP1表达后的C4-2细胞系其裸鼠皮下移植瘤的生长速度远低于对照组。这对于进一步研究YAP1在前列腺癌疾病的发生发展甚至复发转移等过程中的机制具有重要意义,YAP1可能成为其中潜在的治疗靶点之一。
[Abstract]:Objective: to construct a recombinant lentivirus vector which interferes with the expression of Yes associated protein (YAP1), and to prepare lentivirus by packaging. To establish the prostate cancer cell line PCA C4-2, which stably knocks out the expression of YAP1, and to determine the role of YAP1 in prostate cancer by interfering with the expression of YAP1 and monitoring the growth of prostate cancer cell line and the time of subcutaneous tumorigenesis in nude mice. Methods: Western blotting was used to detect the highest YAP1 expression in several prostate cancer cell lines. After that, the lentivirus vector with the best interference effect was cotransfected into 293T cells with three plasmids. The collected liquid was infected with the selected prostate cancer cell line. After stable expression, C4-2 cell line which interfered with YAP1 expression was obtained by further screening with resistance marker purine mycin. The efficiency of knockout was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The activity of stable transfected C4-2 cell line was detected by thiazolyl blue colorimetry, and the normal and stable knockout YAP1 expressing C4-2 cell lines were planted subcutaneously in nude mice. Western blot method was used to detect the expression level of YAP1 protein in four cell lines of RWPE-1LNCaPnC4-2PC3. The results showed that the expression of YAP1 in C4-2 cell line was the highest. The expression vector of YAP1 short hairpin RNA-YAP1-shRNA was used as the expression vector of YAP1 short hairpin RNA-YAP1-shRNA. The results of Western blot showed that the second interference sequence had stronger gene silencing effect. 2. The packaged lentivirus was concentrated by ultracentrifugation. The titer of the transfected C4-2 cell line was determined by absolute quantitative PCR method. After 72 hours of transfection, the transfected C4-2 cell line was screened for 2 weeks by the optimal concentration of purinomycin determined by gradient search. The fluorescence expression level was nearly 90%. Western blot and RT-PCR detection of knockout efficiency could reach 85.3.The cell growth state and cell viability of C4-2 stable cell line after knockout YAP1 expression were detected by MTT method. The results of comparison of subcutaneous tumorigenic volume of two groups of nude mice showed that the cell growth state and cell viability of C4-2 stable cell line after knockout YAP1 expression were significantly decreased. The tumor forming volume of YAP1 knockout cells was significantly smaller than that of the control group, and the fluorescence intensity was weaker than that of the control group. Conclusion: by using lentivirus technique, the prostate cancer C4-2 cell line which interferes with the expression of YAP1 was successfully constructed. Significantly reduced the proliferation of prostate cancer cells, In vivo experiments confirmed that the growth rate of subcutaneous transplanted tumor in nude mice after interfering with YAP1 expression in C4-2 cell line was much lower than that in control group. This study was useful for further study on the role of YAP1 in the development of prostate cancer and even in the process of recurrence and metastasis of prostate cancer. YAP1 may be one of the potential therapeutic targets.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

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