广西眼镜蛇毒蛋白natrin在体外诱导人肿瘤细胞SMMC-7721和SKOV3凋亡的研究

发布时间：2018-10-10 07:58

【摘要】：目的用凝胶层析，离子交换层析，，FPLC分离的方法，从广西眼镜蛇的毒液中分离纯化得到高纯度的natrin；观察natrin在体外对人肝癌SMMC-7721细胞和人卵巢癌SKOV3细胞的增殖抑制作用和凋亡诱导作用，并研究natrin诱导SMMC-7721细胞凋亡的机制。方法（1）广西眼镜蛇的毒液经Sephadex G50凝胶过滤及CM SepharoseCL-6B阳离子交换柱，再通过FPLC mono-S柱层析分离纯化得到natrin；再用SDS-PAGE和质谱分析测定natrin的纯度和相对分子质量。（2）以不同浓度的natrin作用于SMMC-7721细胞和SKOV3细胞24h后，用MTT法检测细胞的生长抑制率。（3）应用透射电子显微镜法、AO/EB双染色法观察细胞凋亡形态。（4）流式细胞仪检测细胞凋亡率及细胞周期。（5）FQ-PCR法分析被natrin作用24h后，人肝癌SMMC-7721细胞中凋亡相关基因bax、bcl-2、p53、caspase-3mRNA的表达量变化。结果（1）从广西眼镜蛇毒中分离纯化出高纯度蛋白组分natrin，经质谱测定分子量为24.937ku。（2）Natrin对人肝癌细胞SMMC-7721和人卵巢癌细胞SKOV3有体外抑制作用，随着药物浓度的增大抑制率也随之增大，24h半数抑制浓度IC50分别为138.69mg.L-1和26.13mg.L-1。（3）给药后，荧光显微镜下与透射电子显微镜下两种细胞呈现典型的凋亡形态改变。（4）流式细胞仪检测到随着药物浓度的增大凋亡细胞百分数也随之增大。（5）Natrin能诱导SMMC-7721细胞发生S期和G2/M期阻滞。（6）Natrin能降低bax、bcl-2、p53、caspase-3mRNA在人肝癌SMMC-7721细胞中的表达量，随着剂量的增大，各基因的mRNA表达量随之降低。结论从广西眼镜蛇毒中分离纯化得到的蛋白组分natrin在体外能抑制人肿瘤细胞SMMC-7721和SKOV3增殖并诱导其凋亡；其中诱导SMMC-7721细胞凋亡的机制可能与natrin引起S期、G2/M期阻滞和降低细胞中bcl-2mRNA表达量有关。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965;R735.7

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