發(fā)布時(shí)間：2018-06-18 02:16

  本文選題：硒酸酯多糖(KSC) + 羥基喜樹堿(10-HCPT)��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：在腫瘤化療過程中,化療藥物的單獨(dú)應(yīng)用往往不能有效地殺死腫瘤,為了提高治療效果,人們?cè)谂R床上多使用聯(lián)合用藥方案,主要是通過選擇兩種或多種針對(duì)不同目標(biāo)靶點(diǎn)的藥物聯(lián)合來遏制腫瘤。硒是一種非金屬,它是機(jī)體每日所攝入的必要微量元素之一,它可以抗氧化,還會(huì)對(duì)惡性腫瘤細(xì)胞死亡和代謝有影響,從而發(fā)揮防癌、抗癌的重要作用。硒酸酯多糖(Kappa-selenocarrageenan,KSC))是一種人工制備的卡拉膠的硒化產(chǎn)物,具有非常顯著的抗腫瘤活性。羥基喜樹堿(10-hy droxycamptothecin,10-HCPT)具有較強(qiáng)的抗癌作用。10-HCPT的作用機(jī)制通常是通過影響惡性腫瘤細(xì)胞的拓?fù)洚悩?gòu)酶I(Topo-Ⅰ),所以也被稱為Topo-Ⅰ特異抑制劑。拓?fù)洚悩?gòu)酶I能使DNA的一條鏈發(fā)生斷裂和再連接。10-HCPT抑制拓?fù)洚悩?gòu)酶I在DNA合成中的作用,阻礙DNA的生成,最后使雙鏈DNA發(fā)生斷裂等情況。從而抑制腫瘤細(xì)胞的生存。本實(shí)驗(yàn)以人類肝癌Hep G-2細(xì)胞為研究對(duì)象,檢測(cè)KSC與羥基喜樹堿聯(lián)合對(duì)Hep G-2細(xì)胞的效應(yīng),為探討肝癌綜合治療方案提供新的理論依據(jù)。本研究采用MTT法考察硒酸酯多糖與羥基喜樹堿單獨(dú)使用及聯(lián)合使用對(duì)Hep G-2細(xì)胞的生長(zhǎng)抑制作用,建立回歸方程并求中效濃度IC50,同時(shí)通過計(jì)算q值判斷聯(lián)合用藥效果;使用倒置顯微鏡及熒光顯微鏡來觀察兩種藥物單獨(dú)使用及聯(lián)合使用對(duì)細(xì)胞形態(tài)的影響;使用流式細(xì)胞儀分析細(xì)胞周期及凋亡率;使用流式細(xì)胞儀及Western Blot法檢測(cè)細(xì)胞S期周期蛋白Chk2、Cyclin A、Cdc25A和Cdk2含量的變化。MTT結(jié)果顯示硒酸酯多糖與羥基喜樹堿均具有明顯的抑制Hep G-2細(xì)胞生長(zhǎng)作用,且具有時(shí)間—?jiǎng)┝康囊蕾囆?聯(lián)合用藥組較2種藥物單獨(dú)組作用更加顯著。硒酸酯多糖和羥基喜樹堿單獨(dú)作用72小時(shí)時(shí)IC50分別為46.79mg/L和1.70nmol/L,1nmol/L 10-HCPT和30mg/L KSC聯(lián)合作用72小時(shí)后IC50降至27.89mg/L和0.15nmol/L。硒酸酯多糖和10-HCPT聯(lián)合使用后得出的q值大于0.85小于1.15,這說明聯(lián)合用藥具有協(xié)同效應(yīng)。倒置顯微鏡觀察結(jié)果顯示對(duì)照組細(xì)胞基本都貼壁生長(zhǎng),胞集落生長(zhǎng)特性明顯。給藥組使用藥物48h之后,惡性腫瘤細(xì)胞體積變小、形狀邊緣、細(xì)胞間隙變大、甚至出現(xiàn)細(xì)胞脫落等情況。還有一部分的細(xì)胞體積驟然增大,細(xì)胞膜破裂呈現(xiàn)壞死狀態(tài)。其中聯(lián)合給藥組活細(xì)胞數(shù)少于單獨(dú)給藥組。熒光顯微鏡觀察細(xì)胞結(jié)果顯示,藥物作用于細(xì)胞48小時(shí)后,可以發(fā)現(xiàn)一些細(xì)胞出現(xiàn)的典型的形態(tài)學(xué)改變:細(xì)胞核凝固縮小,染色質(zhì)出現(xiàn)凝集甚至片段化,致密強(qiáng)熒光及凋亡小體形成等;但聯(lián)合給藥組與單獨(dú)給藥組凋亡細(xì)胞數(shù)并無明顯差異。流式細(xì)胞儀得出的結(jié)論是,硒酸酯多糖和10-HCPT主要影響Hep G-2細(xì)胞細(xì)胞周期的G1期下降、S期上升,而G2/M期無明顯的變化。聯(lián)合用藥之后,G1期會(huì)下降,而S期明顯上升,這不難看出硒酸酯多糖及10-HCPT均能影響細(xì)胞S期。硒酸酯多糖組24小時(shí)時(shí)凋亡率為零,但48小時(shí)時(shí)出現(xiàn)凋亡峰。羥基喜樹堿組細(xì)胞死亡率會(huì)受藥物濃度和作用時(shí)間的影響,即時(shí)間—?jiǎng)┝恳蕾囆�。�?lián)合用藥組的細(xì)胞死亡個(gè)數(shù)要低于單獨(dú)給藥組,但是聯(lián)合用藥組S期細(xì)胞比例數(shù)值比單獨(dú)給藥組要高,說明硒酸酯多糖與羥基喜樹堿聯(lián)合用藥主要通過誘導(dǎo)細(xì)胞周期阻滯從而抑制腫瘤細(xì)胞生長(zhǎng)。在硒酸酯多糖與羥基喜樹堿聯(lián)合誘導(dǎo)Hep G-2細(xì)胞S期阻滯機(jī)制研究中,流式細(xì)胞儀檢測(cè)結(jié)果顯示用藥48小時(shí)后,空白組Cyclin A蛋白表達(dá)量為52.2%,1nmol/L10-HCPT、120mg/L KSC和1nmol/L 10-HCPT+120mg/L KSC各組Cyclin A蛋白表達(dá)分別為61.5%、89.5%和91.1%;空白組Chk2蛋白表達(dá)為49.6%,其余各組Chk2蛋白表達(dá)分別為58.1%、91.3%和92.2%,和對(duì)照組進(jìn)行比較,可以看出存在一定的差異,具有統(tǒng)計(jì)學(xué)意義(P0.01),聯(lián)合用藥組和羥基喜樹堿單獨(dú)用藥組具有明顯差異,和硒酸酯多糖組沒有顯著改變。Western Blot結(jié)果顯示給藥后細(xì)胞內(nèi)Cdc25A和Cdk2蛋白表達(dá)量均下降,聯(lián)合給藥組表達(dá)量最低。綜上所述:硒酸酯多糖與羥基喜樹堿聯(lián)合對(duì)Hep G-2細(xì)胞具有相加的生長(zhǎng)抑制作用,并且可誘導(dǎo)Hep G-2細(xì)胞S期阻滯。硒酸酯多糖與羥基喜樹堿誘導(dǎo)Hep G-2細(xì)胞S期阻滯的原因?yàn)槁?lián)合用藥激活了細(xì)胞周期S期檢控點(diǎn),Chk2被激活后催化Cdc25A,并使其磷酸化,甚至出現(xiàn)降解。這樣Cdc25A就無法去掉Cdk2上的位點(diǎn),Cdk2激活受到阻礙,不能很好的結(jié)合Cyclin A,由此引發(fā)S期被抑制的情況,這就影響了整個(gè)細(xì)胞周期。
[Abstract]:In the course of tumor chemotherapy, the individual use of chemotherapeutic drugs can not effectively kill the tumor. In order to improve the therapeutic effect, people use a combination of drugs in clinical practice, mainly by choosing two or more drugs combined with different target targets to prevent cancer. Selenium is a non metal, it is the daily intake of the body. One of the essential trace elements, it can be antioxidant, and also affects the death and metabolism of malignant tumor cells, which plays an important role in cancer prevention and cancer. Selenate polysaccharide (Kappa-selenocarrageenan, KSC)) is a prepared carrageenan selenide product with very significant antitumor activity. Hydroxycamptothecin (10-hy droxyc). Amptothecin, 10-HCPT), which has a strong anti-cancer effect of.10-HCPT, is usually known as a specific inhibitor of I (Topo- I) affecting the malignant tumor cells, so it is also known as a specific inhibitor of Topo- I. Topoisomerase I can break a chain of DNA and re connect the.10-HCPT inhibition of topoisomerase I in DNA synthesis. Hinder the formation of DNA and finally make the double strand DNA break and so on. Thus inhibiting the survival of tumor cells. In this experiment, the effects of KSC and hydroxycamptothecin on Hep G-2 cells were detected by the combination of KSC and Hydroxycamptothecin in human hepatoma Hep G-2 cells, and a new theoretical basis was provided to explore the comprehensive treatment scheme for liver cancer. This study was used to investigate selenate ester by MTT method. The inhibitory effect of polysaccharide and hydroxycamptothecin on the growth of Hep G-2 cells, the regression equation was established and the medium effect concentration IC50 was established, and the effect of combined use of Q was calculated by calculating the value of Q; the effects of the two drugs alone and combined use on the cell morphology were observed by the inverted microscope and the fluorescence microscope. Cell cycle and apoptosis rate were analyzed by cytometer. The changes of S periodic protein Chk2, Cyclin A, Cdc25A and Cdk2 content were detected by flow cytometer and Western Blot method. The results showed that both selenate polysaccharide and hydroxycamptothecin had obvious inhibition of Hep G-2 cell growth and had time and dose dependence and combined use. The effect of the drug group was more significant than that of the 2 drugs alone. IC50 was 46.79mg/L and 1.70nmol/L respectively when selenate polysaccharide and hydroxycamptothecin acted alone for 72 hours. After the combination of 1nmol/L 10-HCPT and 30mg/L KSC for 72 hours, IC50 was reduced to 27.89mg/L and 0.15nmol/L. selenate polysaccharide was combined with 10-HCPT, and the Q value was less than 0.85 less than 1.15, This shows that the combined use of drugs has synergistic effects. The results of the inverted microscope show that the cells in the control group are basically adhered to wall growth and the colony growth characteristics are obvious. After the use of drug 48h in the drug group, the volume of the malignant tumor cells becomes smaller, the shape edge, the cell gap becomes larger, even the cell fall off. However, the number of living cells in the combined drug group was less than that of the single drug group. The results of the fluorescence microscope observation showed that after 48 hours the drug acted on the cell, the typical morphological changes of some cells could be found: the nuclear coagulation was reduced, the chromatin was agglutinated and even fragmented, and the density was strong. Fluorescent and apoptotic bodies were formed, but there was no significant difference in the number of apoptotic cells in the combined administration group and the individual group. The flow cytometer concluded that selenate polysaccharide and 10-HCPT mainly affected the G1 phase of the cell cycle of Hep G-2 cells, the S phase increased and the G2/M phase had no obvious changes. After the combination, the G1 phase decreased and the S phase was obvious. Up, it is not difficult to see that both selenate polysaccharide and 10-HCPT can affect the cell S phase. The apoptosis rate of the selenate polysaccharide group is zero at 24 hours, but the apoptotic peak is found at 48 hours. The cell mortality of HCPT group is affected by the drug concentration and time, that is, time dose dependence. Only drug group, but the proportion of S phase cells in the combination group is higher than that in the single drug group. It shows that the combination of selenate polysaccharide and hydroxycamptothecin can inhibit the growth of tumor cells mainly by inducing cell cycle arrest. In the study of the S phase blocking mechanism of Hep G-2 cells induced by selenate polysaccharide and hydroxycamptothecin, flow cytometry After 48 hours, the expression of Cyclin A protein in blank group was 52.2%, 1nmol/L10-HCPT, 120mg/L KSC and 1nmol/L 10-HCPT+120mg/L KSC were expressed as 61.5%, 89.5% and 91.1%, and Chk2 protein in the blank group was 49.6%. The expression of Chk2 protein in the other groups was 58.1%, 91.3% and 92.2%, respectively, and the control group entered the control group. Comparison, we can see that there is a certain difference, with statistical significance (P0.01), the combination group and hydroxycamptothecin alone group has obvious differences, and selenate polysaccharide group has no significant changes in.Western Blot results showed that the expression of Cdc25A and Cdk2 protein in the cells decreased, the combination of drug group expression was the lowest. The combination of selenate polysaccharide and hydroxycamptothecin can inhibit the growth of Hep G-2 cells, and induce the S phase block of Hep G-2 cells. The reason for the S phase arrest of Hep G-2 cells induced by selenate polysaccharide and hydroxycamptothecin is to activate the S phase control point of the cell cycle by combined use of drugs, and Chk2 is activated and catalyzes Cdc25A, and its phosphorus is activated. Acidification, even degradation. So Cdc25A can not remove the site on Cdk2, Cdk2 activation is hindered, not a good combination of Cyclin A, resulting in the inhibition of the S phase, which affects the whole cell cycle.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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