【作者】 王强；

【导师】 权昌益；

【作者基本信息】 天津医科大学 ， 外科学 泌尿外（专业学位）， 2016， 硕士

【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。更多还原

【Abstract】 Objective: We package and prepare lentivirus by constructing a recombinant lentiviral vector interferring with YAP1. By virtue of high efficiency of lentiviral transfection, we establish a stable prostate cancer cell line with knock-down of YAP1.After that, we define roles of YAP1 in prostate cancer via detecting the growth of prostate cancer cell line and time needed for tumor formation in nude mice,providing tools for further research in genetic pathway related to YAP1.Methods: We detect several prostate cell lines for Yap1 protein expression by western blotting and determine one with highest expression level for research.Then we use plasmids ps PAX2, p MD2.G and the lentiviral vector which has been screened most effectively tranfecting 293 T cells to package lentivirus and collect the supernatant which contains lentivirus to infect C4-2 cell line. We screen further by puromycin and obtain a cell line with stable knock down of Yap1 expression. What’s more,we detect the knock-down efficiency by RT-PCR and western blotting.After that,MTT are used to detect cell viability and aptosis. Next we inoculate control and stable knock down cell line subcutaneously in nude mice,and then compare these two groups for their ability to form tumors.Results: 1.We detected RWPE-1,Lncap,PC3,C4-2 for Yap1 expression by western blotting, Among which C4-2 expressed the most.Then we transfected YAP1-sh RNA into C4-2 cells and found that the second sequence has higher gene silencing effect.2.After concentrating lentivirus with ultracentrifugation,we measure the titers of lentivirus by q PCR to ensure it meeting demand.72 hours after infecting C4-2 cells with concentrated lentivirus,we screened tranfected C4-2 cells by puromycin whch has been explored for the optimal concentration range for two weeks.And e GFP expression was close to 90 percent of all the tranfected C4-2 cells.By western blotting and RT-PCR, we detected nearly 85 percent konck-down efficiency.3.Next,we find deletion of Yap1 gene decreased cell viability and incresed the aptosis detected by MTT.4.Furthermore, after comparing two groups of nude mice,we found cells lacking Yap1 gene formed tumors much smaller than control group.Besides, fluorescence intensity was much weaker than the other one.Conclusions: In this experiment,we successfully construct a prostate cancer cell line with stable knock down of Yap1 expression by lentiviral vector technology,which slows down cell proliferation ability obviously.In vivo animal experiments shows that cells lack of Yap1 gene form tumors much slower than control group.It is of great significance to further study mechanisms of YAP1 in development,recurrence and metastasis of prostate cancer,and maybe it could be one of potential therapeutic targets.更多还原