發(fā)布時(shí)間：2018-02-15 06:59

  本文關(guān)鍵詞： 乙肝病毒X蛋白（HBx） 乙型肝炎病毒（HBV） 肝細(xì)胞癌（HCC） 細(xì)胞增殖 環(huán)氧合酶-2（COX-2）　出處：《福建醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】： 1.探討HBx蛋白的穩(wěn)定表達(dá)對(duì)HL7702肝細(xì)胞增殖以及細(xì)胞中COX-2表達(dá)的影響。 2.觀察COX-2選擇性抑制劑NS-398對(duì)HL7702/HBx細(xì)胞的影響，探討COX-2 在HBx蛋白促進(jìn)肝細(xì)胞增殖中的作用。 【方法】： HL7702/HBx細(xì)胞、HL7702/Mock細(xì)胞、HL7702細(xì)胞分別復(fù)蘇和培養(yǎng)，CCK-8法和克隆形成實(shí)驗(yàn)觀察和檢測(cè)上述三種細(xì)胞增殖能力；以β-actin為內(nèi)參，運(yùn)用RT-PCR檢測(cè)上述三種細(xì)胞中COX-2在mRNA水平的表達(dá)情況，Western blot檢測(cè)COX-2在蛋白水平的表達(dá)情況；相同濃度COX-2選擇性抑制劑NS398作用體外培養(yǎng)的各組細(xì)胞，并以DMSO組為空白對(duì)照，采用CCK-8法檢測(cè)經(jīng)處理24h、48h、72h后各組細(xì)胞的增殖活性；在50umol/L NS-398作用72h后，以DMSO作為對(duì)照組，Western blot檢測(cè)各組細(xì)胞中COX-2蛋白的表達(dá)水平。 【結(jié)果】： 細(xì)胞增殖實(shí)驗(yàn)及克隆形成實(shí)驗(yàn)顯示：與HL7702/Mock和HL7702兩組細(xì)胞相比較，HL7702/HBx增殖能力更強(qiáng)（P0.05），克隆形成率也更高（P0.05）。通過對(duì)各組細(xì)胞COX-2mRNA的半定量RT-PCR檢測(cè)發(fā)現(xiàn)，，相對(duì)于HL7702/Mock細(xì)胞、HL7702細(xì)胞，轉(zhuǎn)染HBV X基因的HL7702細(xì)胞內(nèi)COX-2的mRNA相對(duì)表達(dá)量明顯增高（P0.05）。Western blot檢測(cè)示：HL7702/HBx細(xì)胞中的COX-2蛋白相對(duì)表達(dá)水平較高（P0.05），而HL7702/Mock及HL7702之間則無明顯差異。NS-398能以時(shí)間依賴的方式抑制各組細(xì)胞的增殖能力，而在HL7702/HBx細(xì)胞中增殖抑制率在3個(gè)時(shí)間點(diǎn)均高于其他兩組細(xì)胞（P0.05）。經(jīng)50μmol/LNS-398處理后，各組細(xì)胞的COX-2蛋白水平顯著降低，而在HL7702/HBx細(xì)胞中更加明顯（P0.05）。 【結(jié)論】： 1. HBx蛋白可促進(jìn)HL7702細(xì)胞分裂增殖、促進(jìn)細(xì)胞生長；HBx蛋白可顯著升高肝細(xì)胞中COX-2的mRNA及蛋白表達(dá)水平；HBx蛋白能通過調(diào)控COX-2的表達(dá)而促進(jìn)肝細(xì)胞增殖，誘導(dǎo)肝細(xì)胞惡性轉(zhuǎn)化。 2. COX-2選擇性抑制劑NS-398能通過降低COX-2的表達(dá)，抑制高表達(dá)的COX-2的HL7702/HBx細(xì)胞的惡性增殖，阻止肝細(xì)胞在肝癌早期階段的癌變轉(zhuǎn)化。
[Abstract]:[purpose]:. 1. To investigate the effect of stable expression of HBx protein on the proliferation and COX-2 expression of HL7702 hepatocytes. 2. To observe the effect of NS-398, a selective inhibitor of COX-2, on HL7702/HBx cells and to explore COX-2. The role of HBx protein in promoting hepatocyte proliferation. [methods]:. Resuscitation and culture of HL-7702 / Mock cells from HL7702/HBx cells by CCK-8 method and clone formation assay were used to observe and test the proliferative ability of these three cells, 尾 -actin was used as the internal reference, and 尾 -actin was used as the internal reference, and 尾 -actin was used as the internal reference. RT-PCR was used to detect the expression of COX-2 at the mRNA level in the three kinds of cells, and Western blot was used to detect the expression of COX-2 at the protein level, and the same concentration of NS398, a selective inhibitor of COX-2, was used to treat the cultured cells in vitro, and DMSO group was used as the blank control. The proliferative activity of each group was detected by CCK-8 method after treatment for 24 h or 48 h or 72 h, and the expression level of COX-2 protein was detected with DMSO as control group after treatment with 50 mmol / L NS-398 for 72 h. [outcome]:. Cell proliferation assay and clone formation assay showed that HL7702 / HBx had stronger proliferative ability and higher clone formation rate than those of HL7702/Mock and HL7702 groups. By semi-quantitative RT-PCR detection of COX-2mRNA in each group, it was found that compared with HL7702/Mock cells, HL7702 cells were higher than HL7702 cells. The relative expression of COX-2 in HL7702 cells transfected with HBV X gene was significantly higher than that in HL7702 cells transfected with HBV X gene. Western blot detection showed that the relative expression level of COX-2 protein was higher in the cell line of 10 HL7702 / HBX, but there was no significant difference between HL7702/Mock and HL7702. NS-398 could inhibit the expression of COX-2 protein in a time-dependent manner. The proliferative ability of the group, The inhibitory rate of proliferation in HL7702/HBx cells at three time points was higher than that in the other two groups. After 50 渭 mol/LNS-398 treatment, the COX-2 protein level of the cells in each group was significantly decreased, and that in HL7702/HBx cells was more obvious. [conclusion]:. 1. HBx protein can promote the division and proliferation of HL7702 cells, and promote cell growth. HBX protein can significantly increase the level of mRNA and protein expression of COX-2 in hepatocytes. HBX protein can promote the proliferation of hepatocytes and induce malignant transformation of hepatocytes by regulating the expression of COX-2. 2. NS-398, a selective inhibitor of COX-2, can inhibit the malignant proliferation of HL7702/HBx cells with high expression of COX-2 by reducing the expression of COX-2, thus preventing the transformation of hepatocytes from cancerous transformation in the early stage of HCC.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.62

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本文編號(hào)：1512692