家蝇幼虫抗菌肽对肝癌HepG2细胞增殖及凋亡作用的研究

发布时间：2018-05-23 09:10

  本文选题：家蝇幼虫 + 抗菌肽　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:比较家蝇幼虫、家蝇幼虫抗菌肽纯化物I1、I2、I3、I4及其粗提物对肝癌HepG2细胞增殖的影响,选择对HepG2细胞抑制作用最强的组分进行体外抗肿瘤实验,并探索其可能的抗肿瘤机制。方法:1.采用MTT法检测家蝇幼虫及家蝇幼虫抗菌肽各组分对HepG2细胞增殖抑制的影响;2.采用Hoechst 33258荧光染色法观察家蝇幼虫抗菌肽粗提物作用后HepG2细胞核形态的改变;3.通过流式细胞术分别分析家蝇幼虫抗菌肽粗提物对HepG2细胞周期、细胞凋亡、线粒体膜电位及活性氧的影响。4.通过激光共聚焦显微镜及流式细胞术共同观察家蝇幼虫抗菌肽粗提物作用后HepG2细胞内Ga2+水平的变化。5.采用分光光度法检测家蝇幼虫抗菌肽粗提物作用后HepG2细胞中Caspase3、Caspase 9酶活性的改变。6.采用Western blot检测家蝇幼虫抗菌肽粗提物作用后凋亡相关蛋白:Cleaved Caspase 3、Cleaved Caspase 9、Bcl-2及Bax的表达情况;结果:1.MTT结果显示:家蝇幼虫抗菌肽粗提物及纯化物I1、I2、I4在各时段均能明显抑制HepG2细胞的增殖(P0.05),且随着时间的延长抑制效果愈加明显;当干预时间高于48h后粗提物组的抑制率明显高于其余各组(p0.05),抑制效果最为显著。不同浓度的家蝇幼虫抗菌肽粗提物对hepg2细胞均能产生明显抑制作用(p0.01),且随着浓度的增大抑制作用不断增强,呈剂量依赖特性。2.细胞周期检测结果显示:家蝇幼虫抗菌肽粗提物作用后处于g0/g1期的hepg2细胞比例明显升高(p0.01),而s期和g2/m期细胞比例下降明显。3.细胞凋亡检测结果显示:家蝇幼虫抗菌肽粗提物能够明显诱导hepg2细胞发生凋亡。从形态学上看,粗提物作用后部分细胞发生核凝集、染色质趋边及核碎裂等典型凋亡形态学特征的改变,颜色发白呈亮蓝色,而对照组细胞核形状规则,颜色分布均匀呈正常蓝色;从凋亡率看,60、240及480ug/ml的粗提物均能诱导hepg2细胞凋亡(p0.05),且随着浓度的增大,凋亡率也不断升高,呈剂量依赖性。4.线粒体膜电位检测结果显示:家蝇幼虫抗菌肽粗提物能够改变hepg2细胞线粒体膜电位的平衡状态,促进线粒体膜电位的下降。5.钙离子检测结果显示:家蝇幼虫抗菌肽粗提物能够明显提高hepg2细胞内的钙离子浓度。激光共聚焦显微镜下:粗提物干预后的各组细胞荧光强度明显高于对照组,且对比细胞形态可以看出,变圆、皱缩的细胞钙离子荧光强度明显高于正常形态的细胞;流式细胞术检测结果同样显示:粗提物各组细胞内的钙离子平均荧光强度明显高于对照组(p0.05),且随着浓度的增大,钙峰明显右移,荧光强度逐渐增强。6.ros检测结果显示:与对照组相比,240、480ug/ml的家蝇幼虫抗菌肽粗提物能够促进hepg2细胞内的ros的释放,差异具有统计学意义(p0.01)。7.caspase酶活性检测结果显示:与对照组相比,粗提物各组hepg2细胞内caspase3及caspase9的酶活性均明显升高,差异具有统计学意义(p0.05)。8.westernblot结果显示:家蝇幼虫抗菌肽粗提物能够上调cleavedcaspase3、cleavedcaspase9及促凋亡蛋白bax的表达,降低抑凋亡蛋白bcl-2的表达。结论:1.家蝇幼虫抗菌肽粗提物及纯化物i1、i2、i4能够明显抑制hepg2细胞的增殖,其中粗提物的抑制效果最为明显。2.家蝇幼虫抗菌肽粗提物对HepG2细胞的抑制作用呈剂量依赖效应,可将HepG2的细胞周期阻滞于G0/G1期,可能通过影响DNA的复制和有丝分裂,抑制细胞增殖。3.家蝇幼虫抗菌肽粗提物作用后HepG2细胞内ROS及钙离子浓度的增加可能作为启动细胞凋亡途径的上游信号分子共同推动细胞凋亡的发生。4.家蝇幼虫抗菌肽粗提物可能通过线粒体途径诱导HepG2细胞凋亡,其机制可能与其上调促凋亡蛋白Bax的表达、下调抑凋亡蛋白Bcl-2的表达有关。
[Abstract]:Objective: To compare the effects of I1, I2, I3, I4 and their crude extracts on the proliferation of hepatocellular carcinoma HepG2 cells by comparing the antimicrobial peptides of housefly larva and housefly larvae, select the anti tumor experiment in vitro and explore the possible anti-tumor mechanism of the most inhibitory components of HepG2 cells. Methods: 1. the MTT method was used to detect the antibacterial peptide of housefly larvae and housefly larvae. The effect of HepG2 cell proliferation inhibition was observed. 2. Hoechst 33258 fluorescence staining was used to observe the changes of HepG2 nucleus morphology after the action of the crude extracts of the antibacterial peptide of the housefly larvae. 3. through flow cytometry, the effects of the crude extracts of the antimicrobial peptide on the HepG2 cell cycle, the cell apoptosis, the mitochondrial membrane potential and the reactive oxygen species were analyzed by the flow cytometry, respectively,.4. passed through the flow cytometry. Laser confocal microscopy and flow cytometry were used to observe the changes of Ga2+ level in HepG2 cells after the action of the antibacterial peptide crude extracts from the larvae of housefly larvae..5. using spectrophotometric method to detect the activity of Caspase3, Caspase 9 enzyme in HepG2 cells after the action of the crude extracts from the larvae of housefly larvae, Caspase,.6. mining Western blot to detect the crude extract of antimicrobial peptides from the larvae of housefly Apoptosis related proteins after action: expression of Cleaved Caspase 3, Cleaved Caspase 9, Bcl-2 and Bax; results: 1.MTT results showed that the proliferation of HepG2 cells (P0.05) could be inhibited obviously in every time period of the crude extract of antibacterial peptide and the purified substance I1, I2 and I4 in the housefly larvae, and the inhibition effect was more obvious with the prolongation of time; when the intervention time was higher than 48 The inhibition rate of the crude extract group after h was significantly higher than that of the other groups (P0.05), and the inhibition effect was the most significant. The different concentrations of the crude extracts from the larva of the housefly larvae could have obvious inhibitory effects on HepG2 cells (P0.01), and the inhibition effect was increased with the increase of concentration. The results of the dose dependent.2. cell cycle detection showed that the larva of housefly The proportion of HepG2 cells in g0/g1 stage increased significantly (P0.01), while the proportion of S and g2/m cells decreased obviously in.3. cells apoptosis detection results showed that the crude extracts of housefly larvae can obviously induce apoptosis of HepG2 cells. The changes of typical morphological characteristics of typical apoptosis, such as edge and nuclear fragmentation, were bright blue in color, while the nucleus shape of the control group was regular and the color distribution was normal blue. From the rate of apoptosis, the 60240 and 480ug/ml crude extracts could induce apoptosis of HepG2 cells (P0.05), and the apoptosis rate was also increasing with the increase of concentration, showing a dose dependent manner. The results of the mitochondrial membrane potential of the.4. showed that the crude extracts of the housefly larvae can change the equilibrium state of the mitochondrial membrane potential of the HepG2 cells and promote the decrease of the mitochondrial membrane potential. The results of.5. calcium ion detection show that the crude extracts of the housefly larvae can obviously increase the concentration of calcium ions in the HepG2 cells. Laser confocal microscopy Under the microscope, the fluorescence intensity of the cells in each group was significantly higher than that of the control group, and the contrast cell morphology showed that the fluorescence intensity of the cell calcium ion was significantly higher than that of the normal cells, and the results of flow cytometry also showed that the average fluorescence intensity of calcium ions in the cells of the crude extracts was significantly higher than that of the control group. Group (P0.05), and with the increase of concentration, the calcium peak shifted to the right, and the fluorescence intensity was gradually enhanced by.6.ros detection. Compared with the control group, the crude extracts from 240480ug/ml's housefly larvae could promote the release of ROS in the HepG2 cells. The difference was statistically significant (P0.01) detection results of the activity of.7.caspase enzyme showed that it was with the control group. The enzyme activities of Caspase3 and caspase9 in the HepG2 cells of the crude extracts were significantly increased, and the difference was statistically significant (P0.05).8.westernblot results showed that the crude extracts of the housefly larvae can up regulate the cleavedcaspase3, cleavedcaspase9 and apoptotic protein Bax, and reduce the expression of apoptotic protein bcl-2. Conclusion: 1. Houseflies are young. I1, I2, and I4 can obviously inhibit the proliferation of HepG2 cells, and the inhibition effect of crude extracts is the most obvious effect on the inhibition of HepG2 cells by the crude extracts of.2. housefly larvae, which can block the cell cycle of HepG2 in G0/G1 phase, possibly by affecting the replication and mitosis of DNA. The increase of ROS and calcium ion concentration in HepG2 cells after cell proliferation of.3. housefly larvae may be the upstream signal molecules of the apoptotic pathway to promote cell apoptosis, which may induce apoptosis of HepG2 cells by mitochondrial pathway, and its mechanism may be related to its mechanism. The expression of apoptosis protein Bax was down regulated and the expression of Bcl-2 was down regulated.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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