本文選題：家蠅幼蟲 + 抗菌肽��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:比較家蠅幼蟲、家蠅幼蟲抗菌肽純化物I1、I2、I3、I4及其粗提物對肝癌HepG2細(xì)胞增殖的影響,選擇對HepG2細(xì)胞抑制作用最強(qiáng)的組分進(jìn)行體外抗腫瘤實(shí)驗(yàn),并探索其可能的抗腫瘤機(jī)制。方法:1.采用MTT法檢測家蠅幼蟲及家蠅幼蟲抗菌肽各組分對HepG2細(xì)胞增殖抑制的影響;2.采用Hoechst 33258熒光染色法觀察家蠅幼蟲抗菌肽粗提物作用后HepG2細(xì)胞核形態(tài)的改變;3.通過流式細(xì)胞術(shù)分別分析家蠅幼蟲抗菌肽粗提物對HepG2細(xì)胞周期、細(xì)胞凋亡、線粒體膜電位及活性氧的影響。4.通過激光共聚焦顯微鏡及流式細(xì)胞術(shù)共同觀察家蠅幼蟲抗菌肽粗提物作用后HepG2細(xì)胞內(nèi)Ga2+水平的變化。5.采用分光光度法檢測家蠅幼蟲抗菌肽粗提物作用后HepG2細(xì)胞中Caspase3、Caspase 9酶活性的改變。6.采用Western blot檢測家蠅幼蟲抗菌肽粗提物作用后凋亡相關(guān)蛋白:Cleaved Caspase 3、Cleaved Caspase 9、Bcl-2及Bax的表達(dá)情況;結(jié)果:1.MTT結(jié)果顯示:家蠅幼蟲抗菌肽粗提物及純化物I1、I2、I4在各時段均能明顯抑制HepG2細(xì)胞的增殖(P0.05),且隨著時間的延長抑制效果愈加明顯;當(dāng)干預(yù)時間高于48h后粗提物組的抑制率明顯高于其余各組(p0.05),抑制效果最為顯著。不同濃度的家蠅幼蟲抗菌肽粗提物對hepg2細(xì)胞均能產(chǎn)生明顯抑制作用(p0.01),且隨著濃度的增大抑制作用不斷增強(qiáng),呈劑量依賴特性。2.細(xì)胞周期檢測結(jié)果顯示:家蠅幼蟲抗菌肽粗提物作用后處于g0/g1期的hepg2細(xì)胞比例明顯升高(p0.01),而s期和g2/m期細(xì)胞比例下降明顯。3.細(xì)胞凋亡檢測結(jié)果顯示:家蠅幼蟲抗菌肽粗提物能夠明顯誘導(dǎo)hepg2細(xì)胞發(fā)生凋亡。從形態(tài)學(xué)上看,粗提物作用后部分細(xì)胞發(fā)生核凝集、染色質(zhì)趨邊及核碎裂等典型凋亡形態(tài)學(xué)特征的改變,顏色發(fā)白呈亮藍(lán)色,而對照組細(xì)胞核形狀規(guī)則,顏色分布均勻呈正常藍(lán)色;從凋亡率看,60、240及480ug/ml的粗提物均能誘導(dǎo)hepg2細(xì)胞凋亡(p0.05),且隨著濃度的增大,凋亡率也不斷升高,呈劑量依賴性。4.線粒體膜電位檢測結(jié)果顯示:家蠅幼蟲抗菌肽粗提物能夠改變hepg2細(xì)胞線粒體膜電位的平衡狀態(tài),促進(jìn)線粒體膜電位的下降。5.鈣離子檢測結(jié)果顯示:家蠅幼蟲抗菌肽粗提物能夠明顯提高h(yuǎn)epg2細(xì)胞內(nèi)的鈣離子濃度。激光共聚焦顯微鏡下:粗提物干預(yù)后的各組細(xì)胞熒光強(qiáng)度明顯高于對照組,且對比細(xì)胞形態(tài)可以看出,變圓、皺縮的細(xì)胞鈣離子熒光強(qiáng)度明顯高于正常形態(tài)的細(xì)胞;流式細(xì)胞術(shù)檢測結(jié)果同樣顯示:粗提物各組細(xì)胞內(nèi)的鈣離子平均熒光強(qiáng)度明顯高于對照組(p0.05),且隨著濃度的增大,鈣峰明顯右移,熒光強(qiáng)度逐漸增強(qiáng)。6.ros檢測結(jié)果顯示:與對照組相比,240、480ug/ml的家蠅幼蟲抗菌肽粗提物能夠促進(jìn)hepg2細(xì)胞內(nèi)的ros的釋放,差異具有統(tǒng)計(jì)學(xué)意義(p0.01)。7.caspase酶活性檢測結(jié)果顯示:與對照組相比,粗提物各組hepg2細(xì)胞內(nèi)caspase3及caspase9的酶活性均明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。8.westernblot結(jié)果顯示:家蠅幼蟲抗菌肽粗提物能夠上調(diào)cleavedcaspase3、cleavedcaspase9及促凋亡蛋白bax的表達(dá),降低抑凋亡蛋白bcl-2的表達(dá)。結(jié)論:1.家蠅幼蟲抗菌肽粗提物及純化物i1、i2、i4能夠明顯抑制hepg2細(xì)胞的增殖,其中粗提物的抑制效果最為明顯。2.家蠅幼蟲抗菌肽粗提物對HepG2細(xì)胞的抑制作用呈劑量依賴效應(yīng),可將HepG2的細(xì)胞周期阻滯于G0/G1期,可能通過影響DNA的復(fù)制和有絲分裂,抑制細(xì)胞增殖。3.家蠅幼蟲抗菌肽粗提物作用后HepG2細(xì)胞內(nèi)ROS及鈣離子濃度的增加可能作為啟動細(xì)胞凋亡途徑的上游信號分子共同推動細(xì)胞凋亡的發(fā)生。4.家蠅幼蟲抗菌肽粗提物可能通過線粒體途徑誘導(dǎo)HepG2細(xì)胞凋亡,其機(jī)制可能與其上調(diào)促凋亡蛋白Bax的表達(dá)、下調(diào)抑凋亡蛋白Bcl-2的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

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