PDX1過表達在胰腺癌細胞PANC-1對吉西他濱敏感性的探討及IL-15對胰腺癌細胞PANC-1增殖和凋亡的影響
發(fā)布時間：2018-09-11 07:37
【摘要】：目的:探討PDX1過表達是否能夠影響PANC-1細胞對吉西他濱的敏感性。方法:(1)從Hela細胞中獲取PDX1基因并將其構建到PCMV6-Entry空質(zhì)粒上形成PCMV6-Entry-PDX1重組質(zhì)粒。(2)培養(yǎng)人胰腺癌PANC-1細胞,待細胞生長狀態(tài)良好時種入24孔板,分別將PCMV6-Entry空質(zhì)粒和PCMV6-Entry-PDX1重組質(zhì)粒利用脂質(zhì)體Lipo3000轉入PANC-1細胞中,48h后加入G418篩選2周形成穩(wěn)定的過表達細胞。(3)將穩(wěn)定的過表達細胞擴大培養(yǎng),待細胞生長狀態(tài)良好后種入24孔板中,培養(yǎng)72h提取總蛋白檢測PDX1表達情況及對PI3K-AKT是否有影響(4)將轉染細胞分別種入96孔板中,并用0,10,100,200,400u M濃度的吉西他濱分別作用48h和72h。MTS法在490nm處測定其吸光值。結果:(1)成功獲取PDX1基因并將其構建到PCMV6-Entry空質(zhì)粒上形成PCMV6-Entry-PDX1重組質(zhì)粒,測序結果顯示,重組質(zhì)粒序列正確。(2)經(jīng)Western blot分析顯示成功構建了PDX1過表達PANC-1細胞,且其能夠下調(diào)p-AKT的水平,說明其在一定程度上能夠抑制PI3K-AKT信號通路。(3)MTS法分析吉西他濱對過表達細胞增殖的影響顯示:與空質(zhì)粒組相比較,48h和72h時PDX1過表達細胞增殖受到抑制,具有時間依賴性但是沒有劑量依賴性。結論:PDX1能夠提高胰腺癌細胞PANC-1對吉西他濱的敏感性,具有時間依賴性但是不具有劑量依賴性。目的:探討IL-15對胰腺癌細胞PANC-1的增殖和凋亡的影響。方法:(1)利用PCR檢測PANC-1細胞IL-15受體的表達情況。待細胞狀態(tài)良好時,將其種入細胞板中。24h后分別用0,4,12,36,108,216 ng/ml濃度的IL-15作用細胞。(2)MTS法測不同濃度IL-15對PANC-1細胞增殖的影響。(3)流式細胞儀檢測不同濃度IL-15作用PANC-1細胞72 h對細胞凋亡的影響。(4)Western blot分析不同濃度IL-15對PANC-1細胞凋亡蛋白Bax,Bcl-2,Caspase 3及周期蛋白Cyclin E等的變化。(5)利用單一濃度IL-15作用PANC-1細胞分別為0,1,6,12,24,48,72 h并檢測凋亡蛋白Bax,Bcl-2,Caspase 3及周期蛋白Cyclin E等的變化。結果:(1)PANC-1細胞表達IL-15受體α亞單位而不表達β和γc亞單位。(2)MTS結果顯示:不同濃度IL-15作用PANC-1細胞48h與空白對照組相比沒有統(tǒng)計學差異,作用72 h與空白對照組比較低劑量沒有抑制作用,而高劑量有抑制作用(3)流式分析結果顯示:低劑量IL-15對PANC-1沒有促進凋亡的作用而高劑量具有促進凋亡的作用,濃度越高凋亡越明顯。(4)Western blot分析顯示:IL-15能夠上調(diào)Bax,Caspase 3的表達降低Bcl-2的表達,同時能夠抑制p-AKT的表達。(5)結果顯示:隨著時間增加BAX和Caspase 3表達增加而Bcl-2降低。6,12,24,48h組Cyclin E表達增加而72 h時表達降低與空白對照組相比沒有統(tǒng)計學差異。p-AKT在1,6,12,24,48,72 h表達降低與對照組相比有統(tǒng)計學差異,p-STAT3在24,48和72 h表達增加與對照組相比具有統(tǒng)計學差異。結論:IL-15能夠抑制PANC-1細胞的增殖呈時間和劑量依賴性,也能夠誘導PANC-1細胞的凋亡。
[Abstract]:Aim: to investigate whether overexpression of PDX1 can affect the sensitivity of PANC-1 cells to gemcitabine. Methods: (1) PDX1 gene was obtained from Hela cells and constructed into PCMV6-Entry empty plasmid to form PCMV6-Entry-PDX1 recombinant plasmid. (2) Human pancreatic cancer PANC-1 cells were cultured. PCMV6-Entry empty plasmids and PCMV6-Entry-PDX1 recombinant plasmids were transfected into PANC-1 cells by liposome Lipo3000 for 48 h and then G418 was added to screen them for 2 weeks to form stable overexpression cells. (3) stable overexpression cells were cultured and planted into 24-well plates when the cells grew well. After 72 h culture, the total protein was extracted to detect the expression of PDX1 and its effect on PI3K-AKT. (4) the transfected cells were seeded into 96 well plates respectively. The absorptivity of the transfected cells was measured by using gemcitabine (010100200400um) for 48h and 72h.MTS at 490nm for 48h. Results: (1) PDX1 gene was successfully obtained and constructed into empty PCMV6-Entry plasmid to form PCMV6-Entry-PDX1 recombinant plasmid. Sequencing results showed that the recombinant plasmid sequence was correct. (2) PDX1 over-expressed PANC-1 cells were successfully constructed by Western blot analysis, and they could down-regulate the level of p-AKT. (3) the effect of gemcitabine on the proliferation of overexpression cells was analyzed by MTS assay. The results showed that the proliferation of overexpression cells of PDX1 was inhibited at 48h and 72h compared with the blank plasmid group. Time dependent but no dose dependent. Conclusion the sensitivity of PANC-1 to gemcitabine is time-dependent but not dose-dependent. Objective: to investigate the effect of IL-15 on the proliferation and apoptosis of pancreatic cancer cell line PANC-1. Methods: (1) PCR was used to detect the expression of IL-15 receptor in PANC-1 cells. When the cells are in good condition, (2) MTS assay was used to detect the effect of different concentrations of IL-15 on the proliferation of PANC-1 cells. (3) flow cytometry was used to detect the effect of different concentrations of IL-15 on the apoptosis of PANC-1 cells for 72 h. The changes of apoptosis protein (Bax,Bcl-2,Caspase 3) and cyclin Cyclin E (Cyclin E) in PANC-1 cells with different concentrations of IL-15 were analyzed. (5) the apoptosis protein Bax,Bcl-2,Caspase 3 and the cycle protein Cyclin E were detected by single concentration IL-15 treatment on PANC-1 cells for 0 ~ 1 ~ (12) ~ 12 ~ (24) ~ (24) ~ 872 h, respectively. Results: (1) PANC-1 cells expressed IL-15 receptor 偽 subunits but not 尾 and 緯 c subunits. (2) MTS results showed that there was no significant difference between PANC-1 cells treated with different concentrations of IL-15 for 48 h and control group. At 72 h, compared with the control group, the low dose had no inhibitory effect, while the high dose had the inhibitory effect. (3) Flowflow analysis showed that low dose IL-15 did not promote apoptosis of PANC-1, but high dose had the effect of promoting apoptosis. (4) Western blot analysis showed that IL-15 could up-regulate the expression of Bax,Caspase 3 and decrease the expression of Bcl-2. At the same time, the expression of p-AKT was inhibited. (5) the results showed that with the increase of time, the expression of BAX and Caspase 3 increased, but the expression of Cyclin E increased in the group of Bcl-2 decreased, but the expression decreased at 72 h. There was no significant difference in the expression of p-AKT between the control group and the control group. The expression of p-STAT3 increased at 24 h and 72 h compared with the control group, and the expression of p-STAT3 was significantly higher than that of the control group. Conclusion: IL-15 can inhibit the proliferation of PANC-1 cells in a time-and dose-dependent manner, and can also induce apoptosis of PANC-1 cells.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.9
本文編號：2236036
[Abstract]:Aim: to investigate whether overexpression of PDX1 can affect the sensitivity of PANC-1 cells to gemcitabine. Methods: (1) PDX1 gene was obtained from Hela cells and constructed into PCMV6-Entry empty plasmid to form PCMV6-Entry-PDX1 recombinant plasmid. (2) Human pancreatic cancer PANC-1 cells were cultured. PCMV6-Entry empty plasmids and PCMV6-Entry-PDX1 recombinant plasmids were transfected into PANC-1 cells by liposome Lipo3000 for 48 h and then G418 was added to screen them for 2 weeks to form stable overexpression cells. (3) stable overexpression cells were cultured and planted into 24-well plates when the cells grew well. After 72 h culture, the total protein was extracted to detect the expression of PDX1 and its effect on PI3K-AKT. (4) the transfected cells were seeded into 96 well plates respectively. The absorptivity of the transfected cells was measured by using gemcitabine (010100200400um) for 48h and 72h.MTS at 490nm for 48h. Results: (1) PDX1 gene was successfully obtained and constructed into empty PCMV6-Entry plasmid to form PCMV6-Entry-PDX1 recombinant plasmid. Sequencing results showed that the recombinant plasmid sequence was correct. (2) PDX1 over-expressed PANC-1 cells were successfully constructed by Western blot analysis, and they could down-regulate the level of p-AKT. (3) the effect of gemcitabine on the proliferation of overexpression cells was analyzed by MTS assay. The results showed that the proliferation of overexpression cells of PDX1 was inhibited at 48h and 72h compared with the blank plasmid group. Time dependent but no dose dependent. Conclusion the sensitivity of PANC-1 to gemcitabine is time-dependent but not dose-dependent. Objective: to investigate the effect of IL-15 on the proliferation and apoptosis of pancreatic cancer cell line PANC-1. Methods: (1) PCR was used to detect the expression of IL-15 receptor in PANC-1 cells. When the cells are in good condition, (2) MTS assay was used to detect the effect of different concentrations of IL-15 on the proliferation of PANC-1 cells. (3) flow cytometry was used to detect the effect of different concentrations of IL-15 on the apoptosis of PANC-1 cells for 72 h. The changes of apoptosis protein (Bax,Bcl-2,Caspase 3) and cyclin Cyclin E (Cyclin E) in PANC-1 cells with different concentrations of IL-15 were analyzed. (5) the apoptosis protein Bax,Bcl-2,Caspase 3 and the cycle protein Cyclin E were detected by single concentration IL-15 treatment on PANC-1 cells for 0 ~ 1 ~ (12) ~ 12 ~ (24) ~ (24) ~ 872 h, respectively. Results: (1) PANC-1 cells expressed IL-15 receptor 偽 subunits but not 尾 and 緯 c subunits. (2) MTS results showed that there was no significant difference between PANC-1 cells treated with different concentrations of IL-15 for 48 h and control group. At 72 h, compared with the control group, the low dose had no inhibitory effect, while the high dose had the inhibitory effect. (3) Flowflow analysis showed that low dose IL-15 did not promote apoptosis of PANC-1, but high dose had the effect of promoting apoptosis. (4) Western blot analysis showed that IL-15 could up-regulate the expression of Bax,Caspase 3 and decrease the expression of Bcl-2. At the same time, the expression of p-AKT was inhibited. (5) the results showed that with the increase of time, the expression of BAX and Caspase 3 increased, but the expression of Cyclin E increased in the group of Bcl-2 decreased, but the expression decreased at 72 h. There was no significant difference in the expression of p-AKT between the control group and the control group. The expression of p-STAT3 increased at 24 h and 72 h compared with the control group, and the expression of p-STAT3 was significantly higher than that of the control group. Conclusion: IL-15 can inhibit the proliferation of PANC-1 cells in a time-and dose-dependent manner, and can also induce apoptosis of PANC-1 cells.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.9
【參考文獻】
相關期刊論文 前1條
1 Quan-Jun Lin;Feng Yang;Chen Jin;De-Liang Fu;;Current status and progress of pancreatic cancer in China[J];World Journal of Gastroenterology;2015年26期
，本文編號：2236036
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