本文關鍵詞： USP18 肝癌細胞 siRNA 生物學活性　出處：《南昌大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的:檢測USP18在肝癌細胞中的表達及對其生物學活性(細胞增殖、細胞克隆、細胞周期、細胞凋亡)的影響。方法:1、通過半定量逆轉錄聚合酶鏈反應(RT-PCR)、蛋白印跡(Western Blot)技術從肝癌細胞株HepG2、SMMC7721、Huh7,HepG2.2.15和Hep3B中篩選USP18基因高表達細胞株。2、設計并化學合成針對USP18的3對小干擾RNA(small interfering RNA,siRNA),采用陽離子脂質體法瞬間轉染。篩選出高表達細胞株后,采用離體培養(yǎng)該高表達USP18肝癌細胞,并設Nomal Control組、Negative Control組及USP18-siRNA-1、USP18-siRNA-2、USP18-siRNA-3各轉染組。3、通過半定量逆轉錄聚合酶鏈反應(RT-PCR)和蛋白印跡(Western Blot)法檢測轉染后各組細胞中USP18在mRNA和蛋白水平表達的變化,明確處理后USP18沉默的效果。4、通過MTT比色法描繪生長曲線和平板克隆形成實驗檢測轉染后各組細胞增殖能力的變化。5、運用Annexin V-FITC/PI染色流式細胞術檢測細胞凋亡和流式細胞術檢測細胞周期分布。結果:1、RT-PCR及Western blot結果顯示:五種肝癌細胞株中,USP18基因均有表達,HBV陽性肝癌細胞中的表達高于HBV陰性肝癌細胞,其中Hep3B細胞表達最高,其次為HepG2.2.15、Huh7、SMMC7721,在HepG2細胞中表達最低。選擇USP18表達最高的Hep3B細胞進行后續(xù)實驗。2、Hep3B細胞轉染USP18 siRNA 24 h后,在熒光顯微鏡下觀察綠色熒光蛋白的表達情況,隨機取10個低倍視野,計數帶有熒光蛋白的細胞占轉染細胞總數的百分比來評價轉染效率。結果顯示轉染效率達91±5.67%。3、3個特異性USP18-siRNA轉染組轉染48小時后,其USP18的mRNA和蛋白表達與另外2組比較受到明顯抑制,結果顯示轉染干擾組USP18-siRNA-1抑制效果最明顯(P0.05),而陰性對照組與正常組相比差異無顯著性(p0.05)。4、MTT比色法描繪生長曲線及平板克隆形成實驗顯示,轉染干擾組與正常組及陰性對照組相比,細胞增殖速度明顯減慢(p0.05),而陰性對照組與正常組相比差異無顯著性(p0.05)。5、Annexin V-FITC/PI染色流式細胞術檢測細胞凋亡結果顯示:轉染干擾組與正常組及陰性對照組相比,細胞凋亡率明顯增加(p0.05),而陰性對照組與正常組相比差異無顯著性(p0.05)。6、流式細胞術檢測細胞周期實驗顯示,轉染干擾組與正常組及陰性對照組相比,細胞周期分布差異有統計學差異(p0.05),出現G1期阻滯,S-G2期細胞顯著減少。而陰性對照組與正常組相比差異無顯著性(p0.05)。結論:1、HepG2、SMMC7721、Huh-7,HepG2.2.15和Hep3B細胞中,Hep3B為USP18基因最高表達細胞株。2、siRNA干擾介導USP18基因沉默后,可抑制人肝癌Hep3B細胞的生長、增殖,促進細胞凋亡,并阻滯細胞周期于G1期。
[Abstract]:Objective: to investigate the expression of USP18 in hepatocellular carcinoma (HCC) cells and its biological activity (cell proliferation, cell clone, cell cycle, apoptosis). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot technique were used to detect the expression of Huh7 from HepG2 cell line SMMC7721. The high expression cell line of USP18 gene was screened from HepG2.2.15 and Hep3B. Three pairs of small interfering RNA(small interfering siRNAs for USP18 were designed and chemically synthesized. The high expression USP18 hepatoma cell lines were screened by cationic liposome method. The cells were cultured in vitro, and Nomal Control group was set up. Negative Control and USP18-siRNA-1 were transfected with USP18-siRNA-3 and USP18-siRNA-3 respectively. By semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot). After transfection, the expression of USP18 in mRNA and protein was detected. The effect of USP18 silencing was determined. The growth curve was described by MTT colorimetry and the changes of proliferation ability of each group were detected by plate clone formation assay. Apoptosis was detected by Annexin V-FITC / Pi staining and cell cycle distribution was detected by flow cytometry. The results of RT-PCR and Western blot showed that the expression of USP18 gene was higher in HBV-positive hepatoma cells than in HBV negative HCC cells. The expression of Hep3B cells was the highest, followed by HepG2.2.15Huh7H7 SMMC7721. The Hep3B cells with the highest USP18 expression were selected to carry out the following experiment. 2Hep3B cells were transfected with USP18 siRNA for 24 h. The expression of green fluorescent protein (GFP) was observed under fluorescence microscope and 10 low-power visual fields were randomly selected. The transfection efficiency was evaluated by counting the percentage of the cells with fluorescent protein in the total number of transfected cells. The results showed that the transfection efficiency was 91 鹵5.67.3. The mRNA and protein expression of USP18 in the three specific USP18-siRNA transfection groups was significantly inhibited compared with the other two groups after 48 hours of transfection. The results showed that the inhibitory effect of USP18-siRNA-1 was the most obvious in the interference group, but there was no significant difference between the negative control group and the normal group. MTT colorimetric method was used to depict the growth curve and the plate clone formation experiment showed that compared with the normal group and the negative control group, the proliferation rate of the transfected interference group was significantly slower than that of the negative control group (P 0.05). However, there was no significant difference between the negative control group and the normal group (P 0.05). The results of flow cytometry with Annexin V-FITC / Pi staining showed that the rate of apoptosis in the transfected interference group was significantly higher than that in the normal group and the negative control group (p0.05). But there was no significant difference between the negative control group and the normal group. Flow cytometry analysis showed that the transfection interference group was compared with the normal group and the negative control group. The difference of cell cycle distribution was statistically significant (p0.05), and G1 phase arrest appeared. The S-G2 phase cells decreased significantly, but there was no significant difference between the negative control group and the normal control group (P 0.05). Conclusion: 1: 1 HepG2 SMMMC7721 Huh-7. In HepG2.2.15 and Hep3B cells, Hep3B is the highest expression cell line of USP18 gene. 2 siRNA interference mediates the silencing of USP18 gene. It can inhibit the growth and proliferation of human hepatoma Hep3B cells, promote cell apoptosis, and block the cell cycle in G1 phase.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.7

【參考文獻】

相關碩士學位論文 前1條

1 張玉波;ISG15/USP18在干擾素治療慢性乙型肝炎療效預測中的意義[D];南昌大學;2008年

，

本文編號：1446410