本文選題：YAP1　切入點：前列腺癌　出處：《天津醫(yī)科大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的:通過構建干擾Yes相關蛋白(YAP1)表達的重組慢病毒載體,包裝制備慢病毒,利用慢病毒高效轉染的特性,建立穩(wěn)定敲除YAP1表達的前列腺癌(PCa)C4-2細胞系。通過干擾YAP1表達后對前列腺癌細胞系生長以及裸鼠皮下成瘤時間的監(jiān)測,確定YAP1在前列腺癌中的作用,從而為YAP1相關基因通路的進一步研究提供工具。方法:利用蛋白印跡(Western blot)的方法檢測前列腺癌幾種細胞系中YAP1表達含量最高的一株作為研究對象,之后采用將篩選出的干擾效果最佳的慢病毒載體采用三質粒系統(tǒng)共同轉染于293T細胞,包裝制備慢病毒。將收集的病毒液感染已選擇的前列腺癌細胞系,待穩(wěn)定表達后通過抗性標記嘌呤霉素進一步篩選,最終獲得穩(wěn)定干擾YAP1表達的C4-2細胞系,通過逆轉錄聚合酶鏈反應(RT-PCR)以及Western blot來檢測敲除的效率。之后采用噻唑藍比色法(MTT)檢測穩(wěn)定轉染的C4-2細胞系的活力,并將正常以及穩(wěn)定敲除YAP1表達的C4-2細胞系種植于裸鼠皮下,觀察對比兩組小鼠的成瘤情況。結果:1.通過Western blot方法檢測RWPE-1,LNCaP,C4-2,PC3四個細胞系中的YAP1蛋白表達水平。結果顯示C4-2細胞系中的YAP1含量表達最高。將YAP1短發(fā)夾RNA(YAP1-shRNA)表達載體成功轉入C4-2細胞,Western blot結果顯示第二條干擾序列具有更強的基因沉默效應。2.將包裝好的慢病毒經過超速離心濃縮,采用絕對定量PCR法(qPCR)測定滴度,待達到要求后轉染C4-2細胞,72h之后進一步通過梯度摸索確定的最佳濃度的嘌呤霉素篩選已轉染的C4-2細胞系2周,熒光表達水平將近90%。Western blot以及RT-PCR檢測敲除效率,可達85%。3.通過MTT法檢測敲除YAP1表達后的C4-2穩(wěn)定細胞系其細胞生長狀態(tài)及細胞活力明顯下降。4.兩組裸鼠皮下成瘤體積對比發(fā)現(xiàn),敲除YAP1表達的細胞成瘤體積明顯小于對照組,熒光強度弱于對照組。結論:通過采用慢病毒技術,成功構建干擾YAP1表達的前列腺癌C4-2細胞系,明顯降低了前列腺癌細胞的增殖能力,而體內試驗證實干擾YAP1表達后的C4-2細胞系其裸鼠皮下移植瘤的生長速度遠低于對照組。這對于進一步研究YAP1在前列腺癌疾病的發(fā)生發(fā)展甚至復發(fā)轉移等過程中的機制具有重要意義,YAP1可能成為其中潛在的治療靶點之一。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.25

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