【摘要】：目的用凝膠層析，離子交換層析，，F(xiàn)PLC分離的方法，從廣西眼鏡蛇的毒液中分離純化得到高純度的natrin；觀察natrin在體外對(duì)人肝癌SMMC-7721細(xì)胞和人卵巢癌SKOV3細(xì)胞的增殖抑制作用和凋亡誘導(dǎo)作用，并研究natrin誘導(dǎo)SMMC-7721細(xì)胞凋亡的機(jī)制。 方法（1）廣西眼鏡蛇的毒液經(jīng)Sephadex G50凝膠過濾及CM SepharoseCL-6B陽離子交換柱，再通過FPLC mono-S柱層析分離純化得到natrin；再用SDS-PAGE和質(zhì)譜分析測定natrin的純度和相對(duì)分子質(zhì)量。（2）以不同濃度的natrin作用于SMMC-7721細(xì)胞和SKOV3細(xì)胞24h后，用MTT法檢測細(xì)胞的生長抑制率。（3）應(yīng)用透射電子顯微鏡法、AO/EB雙染色法觀察細(xì)胞凋亡形態(tài)。（4）流式細(xì)胞儀檢測細(xì)胞凋亡率及細(xì)胞周期。（5）FQ-PCR法分析被natrin作用24h后，人肝癌SMMC-7721細(xì)胞中凋亡相關(guān)基因bax、bcl-2、p53、caspase-3mRNA的表達(dá)量變化。 結(jié)果（1）從廣西眼鏡蛇毒中分離純化出高純度蛋白組分natrin，經(jīng)質(zhì)譜測定分子量為24.937ku。（2）Natrin對(duì)人肝癌細(xì)胞SMMC-7721和人卵巢癌細(xì)胞SKOV3有體外抑制作用，隨著藥物濃度的增大抑制率也隨之增大，24h半數(shù)抑制濃度IC50分別為138.69mg.L-1和26.13mg.L-1。（3）給藥后，熒光顯微鏡下與透射電子顯微鏡下兩種細(xì)胞呈現(xiàn)典型的凋亡形態(tài)改變。（4）流式細(xì)胞儀檢測到隨著藥物濃度的增大凋亡細(xì)胞百分?jǐn)?shù)也隨之增大。（5）Natrin能誘導(dǎo)SMMC-7721細(xì)胞發(fā)生S期和G2/M期阻滯。（6）Natrin能降低bax、bcl-2、p53、caspase-3mRNA在人肝癌SMMC-7721細(xì)胞中的表達(dá)量，隨著劑量的增大，各基因的mRNA表達(dá)量隨之降低。 結(jié)論從廣西眼鏡蛇毒中分離純化得到的蛋白組分natrin在體外能抑制人腫瘤細(xì)胞SMMC-7721和SKOV3增殖并誘導(dǎo)其凋亡；其中誘導(dǎo)SMMC-7721細(xì)胞凋亡的機(jī)制可能與natrin引起S期、G2/M期阻滯和降低細(xì)胞中bcl-2mRNA表達(dá)量有關(guān)。
[Abstract]:Objective to isolate and purify high purity natrin; from the venom of cobra in Guangxi by gel chromatography and ion exchange chromatography. To observe the inhibitory effect of natrin on the proliferation and apoptosis of human hepatoma SMMC-7721 cells and human ovarian cancer SKOV3 cells in vitro, and to study the mechanism of natrin induced apoptosis of SMMC-7721 cells. Methods (1) the venom of cobra in Guangxi was purified by Sephadex G50 gel filtration and CM SepharoseCL-6B cation exchange column, and then purified by FPLC mono-S column chromatography. The purity and relative molecular weight of natrin were determined by SDS-PAGE and mass spectrometry. (2) SMMC-7721 and SKOV3 cells were treated with different concentrations of natrin for 24 h. The cell growth inhibition rate was detected by MTT method. (3) the morphology of cell apoptosis was observed by transmission electron microscope (TEM) and AOP / EB double staining. (4) flow cytometry was used to detect the cell apoptosis rate and cell cycle. (5) FQ-PCR assay was used to analyze 24 h after natrin treatment. Expression of apoptosis-related gene bax,bcl-2,p53,caspase-3mRNA in human hepatocellular carcinoma SMMC-7721 cells. Results (1) natrin, a highly purified protein component, was isolated from the venom of Naja naja atra, Guangxi, and its molecular weight was 24.937ku. (2) Natrin could inhibit the expression of SMMC-7721 and SKOV3 in human hepatoma cells and ovarian cancer cells in vitro. With the increase of drug concentration, the inhibition rate also increased. The half inhibitory concentration (IC50) at 24h was 138.69mg.L-1 and 26.13 mg 路L ~ (-1), respectively. (3) after administration, (4) flow cytometry showed that the percentage of apoptotic cells increased with the increase of drug concentration. (5) Natrin could induce the occurrence of S in SMMC-7721 cells. (6) Natrin could reduce the expression of bax,bcl-2,p53,caspase-3mRNA in human hepatoma SMMC-7721 cells. With the increase of dose, the mRNA expression of each gene decreased. Conclusion natrin, a protein component isolated from Naja naja atra venom in Guangxi, can inhibit the proliferation and induce apoptosis of human tumor cells SMMC-7721 and SKOV3 in vitro. The mechanism of inducing apoptosis of SMMC-7721 cells may be related to the arrest of G _ 2 / M phase and the decrease of bcl-2mRNA expression in S phase G _ 2 / M phase induced by natrin.
【學(xué)位授予單位】：廣西醫(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)】：R965;R735.7

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