【摘要】：目的:構(gòu)建RNAi干擾肝癌細(xì)胞株HePG-2,通過實(shí)驗(yàn)觀察各種肝素酶不同表達(dá)水平的肝癌細(xì)胞對血管內(nèi)皮細(xì)胞粘附分子表達(dá)的影響,完善并豐富肝素酶在肝癌侵襲轉(zhuǎn)移中的分子學(xué)研究。方法:(1)選用肝癌細(xì)胞株HePG-2作為研究的實(shí)驗(yàn)細(xì)胞,運(yùn)用抑制肝素酶的基因?qū)ePG-2進(jìn)行細(xì)胞轉(zhuǎn)染,通過qRT-PCR及Western-blot等實(shí)驗(yàn)方法檢測各組轉(zhuǎn)染細(xì)胞中HPSE基因表達(dá)的水平;(2)通過上述方法選擇出相應(yīng)的HePG-2細(xì)胞分為轉(zhuǎn)染組(RNAi干擾的HePG-2細(xì)胞)、陰性對照組(轉(zhuǎn)染陰性的HePG-2細(xì)胞)、空白對照組(未轉(zhuǎn)染的HePG-2細(xì)胞)三組實(shí)驗(yàn)細(xì)胞進(jìn)行細(xì)胞粘附實(shí)驗(yàn),通過qRT-PCR及Western-blot等實(shí)驗(yàn)方法分析各實(shí)驗(yàn)組細(xì)胞粘附分子表達(dá)的影響。結(jié)果:(1)運(yùn)用qRT-PCR及Western-blot等實(shí)驗(yàn)方法分析刷選,成功得到最佳干擾的HePG-2細(xì)胞;(2)qRT-PCR和Western-blot結(jié)果:未轉(zhuǎn)染的HePG-2細(xì)胞對血管內(nèi)皮細(xì)胞粘附分子表達(dá)有明顯增強(qiáng);RNAi干擾的HePG-2細(xì)胞對血管內(nèi)皮細(xì)胞粘附分子表達(dá)有明顯降低;轉(zhuǎn)染陰性的HePG-2細(xì)胞對血管內(nèi)皮細(xì)胞粘附分子表達(dá)相對空白對照組有所降低,但高于RNAi干擾的HePG-2細(xì)胞組。結(jié)論:通過qRT-PCR及Western-blot等實(shí)驗(yàn)方法證實(shí)肝素酶對肝癌細(xì)胞在與血管內(nèi)皮細(xì)胞粘附的作用過程中發(fā)揮了相關(guān)重要的的作用,參與介導(dǎo)了肝癌細(xì)胞與血管內(nèi)皮細(xì)胞的粘附。
[Abstract]:Objective: to construct a RNAi interfering hepatoma cell line HePG-2, and observe the effect of different levels of heparinase expression on the expression of adhesion molecules in vascular endothelial cells (VECs). To improve and enrich the molecular studies of heparanase in the invasion and metastasis of liver cancer. Methods: (1) Hepatocellular carcinoma cell line HePG-2 was selected as the experimental cell, and HePG-2 was transfected with the gene that inhibited heparinase. QRT-PCR and Western-blot were used to detect the expression of HPSE gene in the transfected cells. (2) the corresponding HePG-2 cells were selected to be divided into transfection group (RNAi interfering HePG-2 cells) and negative control group (transfected negative HePG-2 cells). Three groups of untransfected HePG-2 cells in blank control group were tested for cell adhesion. The effects of cell adhesion molecules on the expression of adhesion molecules were analyzed by means of qRT-PCR and Western-blot. Results: (1) the best interfering HePG-2 cells were successfully obtained by qRT-PCR and Western-blot. (2) the results of qRT-PCR and Western-blot: the expression of vascular endothelial cell adhesion molecules was significantly increased in untransfected HePG-2 cells, and decreased by RNAi interfering HePG-2 cells. Compared with the blank control group, the expression of adhesion molecules in the transfected HePG-2 cells was lower than that in the control group, but higher than that in the HePG-2 cells with RNAi interference. Conclusion: heparanase plays an important role in the adhesion of HCC cells to vascular endothelial cells by means of qRT-PCR and Western-blot. It plays an important role in mediating the adhesion of hepatoma cells to vascular endothelial cells.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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