發(fā)布時間：2018-05-04 00:24

  本文選題：食管鱗癌 + 調控�。� 參考：《新疆醫(yī)科大學》2017年碩士論文

【摘要】：目的:初步闡述Bad在食管鱗癌細胞中對Dad1表達的調控作用,了解上調和下調Bad對食管鱗癌細胞周期和凋亡的影響。方法:(1)培養(yǎng)人食管鱗癌ECA109和KYSE450細胞,構建Bad過表達載體重組質粒GV142-Bad,轉染食管鱗癌細胞組作為Bad高表達組,轉染空載體的細胞組作為陰性對照組,完全培養(yǎng)基處理的細胞組作為空白對照組。轉染24h后,熒光倒置顯微鏡觀察細胞中綠色熒光蛋白的表達情況,提取總RNA和蛋白并使用qPCR及Western blot檢測Bad在基因轉錄和蛋白水平的表達來驗證轉染效率。同時檢測轉染細胞中Dad1在基因轉錄和蛋白水平的表達。GV142-Bad載體轉染48 h后,使用流式細胞儀檢測上調Bad的表達對細胞周期和凋亡的影響。(2)人工合成針對人Bad基因的si RNA(si RNA-Bad),轉染食管鱗癌ECA109和KYSE450細胞,作為Bad的低表達組,轉染陰性對照si RNA(negative-si RNA)的細胞組為陰性對照組,完全培養(yǎng)基處理的細胞組作為空白對照組。轉染24h后,提取總RNA和蛋白,使用qPCR以及Western blot檢測Bad在基因轉錄水平和蛋白水平的表達,驗證轉染效率。同時檢測Dad1在基因轉錄和蛋白水平的表達,驗證下調Bad表達是否調控了Dad1的表達。si RNA-Bad轉染48 h后,使用流式細胞儀檢測下調Bad表達對細胞凋亡的影響。結果:(1)成功構建Bad過表達載體GV142-Bad,GV142-Bad過表達重組質粒轉染食管鱗癌細胞ECA109和KYSE450后可以上調Bad在基因轉錄和蛋白水平的表達。同時檢測到Dad1在基因轉錄和蛋白水平表達被下調。(2)成功合成針對人Bad基因的si RNA,轉染食管鱗癌ECA109和KYSE450細胞后能夠下調Bad在基因轉錄和蛋白水平的表達。同時檢測到Dad1在基因轉錄和蛋白水平的表達被上調。(3)上調Bad基因的表達后觀察到ECA109細胞周期G2/M期被延長,同時S期有縮短趨勢,但統(tǒng)計結果差異不顯著(P0.05)。而KYSE450細胞周期阻滯于G2/M期并伴有S期縮短,差異有統(tǒng)計學意義(P0.05)。上調Bad基因的表達后發(fā)現(xiàn)兩種細胞的凋亡比率顯著增加,差異有統(tǒng)計學意義(P0.05)。下調Bad基因表達后兩種細胞的凋亡率有下降趨勢但不顯著,三組之間差異無統(tǒng)計學意義(P0.05)。結論:促凋亡基因Bad可能調控抗凋亡基因Dad1的表達,兩者之間可能存在負調控關系,Bad基因可能通過將癌細胞周期阻滯于G2/M并且促進其凋亡來,抑制其增殖。
[Abstract]:Aim: to investigate the role of Bad in the regulation of Dad1 expression in esophageal squamous cell carcinoma cells and to understand the effects of up-regulation and down-regulation of Bad on cell cycle and apoptosis in esophageal squamous cell carcinoma cells. Methods Human esophageal squamous cell carcinoma (ECA109) and KYSE450 cells were cultured. Bad overexpression vector GV142-Badwas constructed. The transfected esophageal squamous carcinoma cells group was used as the high expression group of Bad and the empty vector group was used as the negative control group. The cells treated with complete culture medium were used as blank control group. After 24 hours of transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence inverted microscope, the total RNA and protein were extracted, and the expression of Bad at gene transcription and protein level was detected by qPCR and Western blot to verify the transfection efficiency. At the same time, the expression of Dad1 at gene transcription and protein level in transfected cells was detected 48 h after transfection with GV142-Bad vector. The effect of upregulation of Bad expression on cell cycle and apoptosis was detected by flow cytometry (FCM). Si RNA(si RNA-Badsite targeting human Bad gene was synthesized and transfected into ECA109 and KYSE450 cells of esophageal squamous cell carcinoma as the low expression group of Bad. The cells transfected with si RNA(negative-si of negative control group were negative control group, and the cell group treated with complete culture medium served as blank control group. After 24 hours of transfection, the total RNA and protein were extracted, and the expression of Bad at the level of gene transcription and protein was detected by qPCR and Western blot to verify the transfection efficiency. At the same time, the expression of Dad1 at gene transcription and protein level was detected to verify whether the down-regulation of Bad expression regulated the expression of Dad1. Si RNA-Bad was transfected for 48 h. The effect of down-regulation of Bad expression on apoptosis was detected by flow cytometry. Results Bad overexpression vector GV142-BadGV142-Bad was successfully constructed and transfected into esophageal squamous cell ECA109 and KYSE450, which could up-regulate the expression of Bad at gene transcription and protein levels. The siRNAs targeting human Bad gene were successfully synthesized and transfected into ECA109 and KYSE450 cells of esophageal squamous cell carcinoma, which could down-regulate the expression of Bad at gene transcription and protein level. At the same time, it was found that Dad1 up-regulated the expression of Bad gene at the level of gene transcription and protein. After upregulating the expression of Bad gene, it was observed that the G2 / M phase of ECA109 cell cycle was prolonged and the S phase was shortened, but there was no significant difference in the statistical results (P 0.05). The cell cycle of KYSE450 was arrested at G _ 2 / M phase with S phase shortening, and the difference was statistically significant (P < 0.05). After upregulating the expression of Bad gene, it was found that the apoptotic ratio of the two kinds of cells was significantly increased, and the difference was statistically significant (P 0.05). After down-regulation of Bad gene expression, the apoptosis rate of the two kinds of cells decreased, but not significantly. There was no significant difference between the three groups (P 0.05). Conclusion: the pro-apoptotic gene Bad may regulate the expression of anti-apoptotic gene Dad1, and there may be a negative regulatory relationship between the two genes, which may inhibit the proliferation of cancer cells by blocking the cell cycle at G _ 2 / M and promoting its apoptosis.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

【參考文獻】

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