本文選題：CerS2基因 + BCPAP　； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:探討CerS2基因過(guò)表達(dá)對(duì)甲狀腺乳頭狀癌BCPAP細(xì)胞生長(zhǎng)、增殖的影響。方法:1)采用pAdxsi重組腺病毒骨架載體構(gòu)建人源重組CerS2腺病毒表達(dá)載體,并確認(rèn)pAdxsi-CerS2-GFP腺病毒表達(dá)載體對(duì)BCPAP細(xì)胞株感染的最佳感染復(fù)數(shù)(MOI)、過(guò)表達(dá)高峰時(shí)間;2)將pAdxsi-GFP、pAdxsi-CerS2-GFP腺病毒表達(dá)載體分別感染BCPAP細(xì)胞,培養(yǎng)特定時(shí)間后,分別提取空白組(blank group)、空載組(pAdxsi-GFP)、實(shí)驗(yàn)組(pAdxsi-CerS2-GFP)細(xì)胞總RNA與總蛋白,實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Q-PCR)、蛋白質(zhì)印跡法(WB)檢測(cè)各組細(xì)胞中CerS2 mRNA、蛋白表達(dá)水平;3)噻唑藍(lán)比色法(MTT)檢測(cè)CerS2基因不同作用時(shí)間對(duì)各組細(xì)胞體外增殖的影響;4)流式細(xì)胞術(shù)PI法分析各組細(xì)胞周期變化;5)Annexin V-APC/7-AAD雙染法檢測(cè)各組細(xì)胞凋亡。結(jié)果:1)經(jīng)酶切、測(cè)序鑒定,克隆片段大小、序列與NM_022075一致,其感染BCPAP細(xì)胞最佳MOI值為240,最佳作用時(shí)間48h;2)Q-PCR、WB結(jié)果提示實(shí)驗(yàn)組細(xì)胞CerS2 mRNA在24h、48h、72h相對(duì)表達(dá)量依次為空白組/空載組的134.57倍、774.26倍、347.29倍(均P0.001),其蛋白48h表達(dá)水平也較空白組、空載組明顯增加(P0.01),空白組與空載組CerS2 mRNA相對(duì)表達(dá)量、蛋白表達(dá)水平均無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05);3)MTT法檢測(cè)生長(zhǎng)曲線發(fā)現(xiàn):24h、48h實(shí)驗(yàn)組相對(duì)空載組、空白組,細(xì)胞OD值明顯降低(均P0.001),空白組與空載組比較細(xì)胞OD值無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05);4)流式細(xì)胞術(shù)檢測(cè)三組細(xì)胞周期,結(jié)果提示:空白組與空載組細(xì)胞周期時(shí)相分布無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);G0/G1期、S期實(shí)驗(yàn)組細(xì)胞數(shù)分別為(40.38±3.43)%、(18.83±1.18)%,空載組細(xì)胞數(shù)分別為(56.63±1.95)%、(27.28±0.52)%,實(shí)驗(yàn)組較空載組G0/G1期、S期細(xì)胞數(shù)下降(均P0.01),實(shí)驗(yàn)組G2/M期細(xì)胞數(shù)(40.79±4.50)%較空載組(16.09±1.44)%明顯增高(P0.001);5)流式細(xì)胞術(shù)檢測(cè)空載組細(xì)胞凋亡率相對(duì)空白組無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05),實(shí)驗(yàn)組細(xì)胞凋亡率(29.64±0.20)%較空白組(5.70±0.19)%、空載組(6.07±0.17)%明顯增高(均P0.001)。結(jié)論:本研究采用pAdxsi重組腺病毒骨架載體構(gòu)建pAdxsi-CerS2-GFP人源重組腺病毒表達(dá)載體,成功感染甲狀腺癌BCPAP細(xì)胞,并分別從mRNA、蛋白層次驗(yàn)證BCPAP細(xì)胞CerS2基因過(guò)表達(dá)成功。研究結(jié)果顯示CerS2基因過(guò)表達(dá)對(duì)BCPAP細(xì)胞增殖有抑制作用,其主要機(jī)制可能與導(dǎo)致BCPAP細(xì)胞G2/M期阻滯,進(jìn)而誘導(dǎo)細(xì)胞凋亡發(fā)生有關(guān),繼續(xù)深入研究其作用機(jī)制,可望為甲狀腺癌的臨床診治及基礎(chǔ)研究提供依據(jù)。
[Abstract]:Aim: to investigate the effect of CerS2 gene overexpression on the growth and proliferation of BCPAP cells in papillary thyroid carcinoma. Methods pAdxsi recombinant adenovirus skeleton vector was used to construct human recombinant CerS2 adenovirus expression vector. It was also confirmed that pAdxsi-CerS2-GFP adenovirus expression vector could infect BCPAP cell line with the best number of infection plural moi (over peak expression time: 2) pAdxsi-GFPFP-pAdxsi-CerS2-GFP adenovirus expression vector was respectively infected with BCPAP cell line, and cultured for a specific time, and the expression vector of pAdxsi-GFPFP-pAdxsi-CerS2-GFP adenovirus expression vector was infected with BCPAP cell line respectively. Total RNA and total protein were extracted from blank group, pAdxsi-GFPN, pAdxsi-CerS2-GFPFP, respectively. Detection of CerS2 mRNAs and protein expression levels of CerS2 mRNAs by Real-time fluorescence quantitative Polymerase chain reaction (PCR) and Western blot (WB) Detection of the effect of CerS2 Gene on Cell Proliferation in Vitro Cell cycle changes in each group were analyzed by Pi method. Apoptosis was detected by Annexin V-APC-7-AAD double staining. Results the cloned fragment size was identical to that of NM022075 by restriction endonuclease digestion and sequencing. The optimal moi of BCPAP cells infected with BCPAP was 240. The results of the optimal time of 48h treatment showed that the relative expression of CerS2 mRNA in the experimental group was 134.57 times, 774.26 times and 347.29 times as much as that in the blank group / no-load group, and the protein expression level at 48 hours was also higher than that in the blank group, and the relative expression of CerS2 mRNA in the control group was 134.57 times, 774.26 times and 347.29 times as much as that in the blank group. The relative expression of CerS2 mRNA and protein expression in the blank group and the no-load group were not significantly different. The growth curve was detected by MTT assay and the control group was compared with the no-load group at 48 h after 24 hours of growth. The results showed that there was no significant difference in the expression of CerS2 mRNA and protein between the blank group and the blank group, and there was no significant difference between the control group and the blank group. The OD value of cells decreased significantly (all P 0.001g, compared with the blank group and the no-load group, there was no significant difference in the OD value between the blank group and the no-load group). Flow cytometry was used to detect the cell cycle in the three groups. The results showed that there was no significant difference in cell phase distribution between the blank group and the no-load group. The number of cells in the experimental group was 40.38 鹵3.43 and 18.83 鹵1.18 respectively in G _ 0 / G _ 0 / G _ 1 phase, while the cell number in the no-load group was 27.28 鹵0.522. The number of cells in the G _ 0 / G _ 1 phase in the experimental group was significantly lower than that in the no-load group (all P 0.01), and the number of the cells in the no-load group was 27.28 鹵0.52. The number of cells in G _ 2 / M phase in the test group was 40.79 鹵4.50%, which was significantly higher than that in the no-load group (16.09 鹵1.44%). There was no significant difference in the cell apoptosis rate between the no-load group and the blank group by flow cytometry. The apoptotic rate of the experimental group was 29.64 鹵0.20% higher than that of the blank group (5.70 鹵0.19%), and that of the no-load group was 6.07 鹵0.17% (all P 0.001). Conclusion: in this study, the recombinant adenovirus vector pAdxsi-CerS2-GFP was constructed by using pAdxsi recombinant adenovirus skeleton vector to successfully infect thyroid carcinoma BCPAP cells, and the overexpression of CerS2 gene in BCPAP cells was verified by mRNA-protein level. The results show that the overexpression of CerS2 gene can inhibit the proliferation of BCPAP cells, and its main mechanism may be related to the G _ 2 / M phase arrest of BCPAP cells, and then induce apoptosis. It is expected to provide basis for clinical diagnosis, treatment and basic research of thyroid carcinoma.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R736.1

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