本文關(guān)鍵詞： USP18 肝癌細(xì)胞 siRNA 生物學(xué)活性　出處：《南昌大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:檢測USP18在肝癌細(xì)胞中的表達(dá)及對其生物學(xué)活性(細(xì)胞增殖、細(xì)胞克隆、細(xì)胞周期、細(xì)胞凋亡)的影響。方法:1、通過半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)、蛋白印跡(Western Blot)技術(shù)從肝癌細(xì)胞株HepG2、SMMC7721、Huh7,HepG2.2.15和Hep3B中篩選USP18基因高表達(dá)細(xì)胞株。2、設(shè)計并化學(xué)合成針對USP18的3對小干擾RNA(small interfering RNA,siRNA),采用陽離子脂質(zhì)體法瞬間轉(zhuǎn)染。篩選出高表達(dá)細(xì)胞株后,采用離體培養(yǎng)該高表達(dá)USP18肝癌細(xì)胞,并設(shè)Nomal Control組、Negative Control組及USP18-siRNA-1、USP18-siRNA-2、USP18-siRNA-3各轉(zhuǎn)染組。3、通過半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)和蛋白印跡(Western Blot)法檢測轉(zhuǎn)染后各組細(xì)胞中USP18在mRNA和蛋白水平表達(dá)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(zhuǎn)染后各組細(xì)胞增殖能力的變化。5、運(yùn)用Annexin V-FITC/PI染色流式細(xì)胞術(shù)檢測細(xì)胞凋亡和流式細(xì)胞術(shù)檢測細(xì)胞周期分布。結(jié)果:1、RT-PCR及Western blot結(jié)果顯示:五種肝癌細(xì)胞株中,USP18基因均有表達(dá),HBV陽性肝癌細(xì)胞中的表達(dá)高于HBV陰性肝癌細(xì)胞,其中Hep3B細(xì)胞表達(dá)最高,其次為HepG2.2.15、Huh7、SMMC7721,在HepG2細(xì)胞中表達(dá)最低。選擇USP18表達(dá)最高的Hep3B細(xì)胞進(jìn)行后續(xù)實驗。2、Hep3B細(xì)胞轉(zhuǎn)染USP18 siRNA 24 h后,在熒光顯微鏡下觀察綠色熒光蛋白的表達(dá)情況,隨機(jī)取10個低倍視野,計數(shù)帶有熒光蛋白的細(xì)胞占轉(zhuǎn)染細(xì)胞總數(shù)的百分比來評價轉(zhuǎn)染效率。結(jié)果顯示轉(zhuǎn)染效率達(dá)91±5.67%。3、3個特異性USP18-siRNA轉(zhuǎn)染組轉(zhuǎn)染48小時后,其USP18的mRNA和蛋白表達(dá)與另外2組比較受到明顯抑制,結(jié)果顯示轉(zhuǎn)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(zhuǎn)染干擾組與正常組及陰性對照組相比,細(xì)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(xì)胞術(shù)檢測細(xì)胞凋亡結(jié)果顯示:轉(zhuǎn)染干擾組與正常組及陰性對照組相比,細(xì)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(xì)胞術(shù)檢測細(xì)胞周期實驗顯示,轉(zhuǎn)染干擾組與正常組及陰性對照組相比,細(xì)胞周期分布差異有統(tǒng)計學(xué)差異(p0.05),出現(xiàn)G1期阻滯,S-G2期細(xì)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(jié)論:1、HepG2、SMMC7721、Huh-7,HepG2.2.15和Hep3B細(xì)胞中,Hep3B為USP18基因最高表達(dá)細(xì)胞株。2、siRNA干擾介導(dǎo)USP18基因沉默后,可抑制人肝癌Hep3B細(xì)胞的生長、增殖,促進(jìn)細(xì)胞凋亡,并阻滯細(xì)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.7

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相關(guān)碩士學(xué)位論文 前1條

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本文編號：1446410