PDX1过表达在胰腺癌细胞PANC-1对吉西他滨敏感性的探讨及IL-15对胰腺癌细胞PANC-1增殖和凋亡的影响
发布时间：2018-09-11 07:37
【摘要】：目的:探讨PDX1过表达是否能够影响PANC-1细胞对吉西他滨的敏感性。方法:(1)从Hela细胞中获取PDX1基因并将其构建到PCMV6-Entry空质粒上形成PCMV6-Entry-PDX1重组质粒。(2)培养人胰腺癌PANC-1细胞,待细胞生长状态良好时种入24孔板,分别将PCMV6-Entry空质粒和PCMV6-Entry-PDX1重组质粒利用脂质体Lipo3000转入PANC-1细胞中,48h后加入G418筛选2周形成稳定的过表达细胞。(3)将稳定的过表达细胞扩大培养,待细胞生长状态良好后种入24孔板中,培养72h提取总蛋白检测PDX1表达情况及对PI3K-AKT是否有影响(4)将转染细胞分别种入96孔板中,并用0,10,100,200,400u M浓度的吉西他滨分别作用48h和72h。MTS法在490nm处测定其吸光值。结果:(1)成功获取PDX1基因并将其构建到PCMV6-Entry空质粒上形成PCMV6-Entry-PDX1重组质粒,测序结果显示,重组质粒序列正确。(2)经Western blot分析显示成功构建了PDX1过表达PANC-1细胞,且其能够下调p-AKT的水平,说明其在一定程度上能够抑制PI3K-AKT信号通路。(3)MTS法分析吉西他滨对过表达细胞增殖的影响显示:与空质粒组相比较,48h和72h时PDX1过表达细胞增殖受到抑制,具有时间依赖性但是没有剂量依赖性。结论:PDX1能够提高胰腺癌细胞PANC-1对吉西他滨的敏感性,具有时间依赖性但是不具有剂量依赖性。目的:探讨IL-15对胰腺癌细胞PANC-1的增殖和凋亡的影响。方法:(1)利用PCR检测PANC-1细胞IL-15受体的表达情况。待细胞状态良好时,将其种入细胞板中。24h后分别用0,4,12,36,108,216 ng/ml浓度的IL-15作用细胞。(2)MTS法测不同浓度IL-15对PANC-1细胞增殖的影响。(3)流式细胞仪检测不同浓度IL-15作用PANC-1细胞72 h对细胞凋亡的影响。(4)Western blot分析不同浓度IL-15对PANC-1细胞凋亡蛋白Bax,Bcl-2,Caspase 3及周期蛋白Cyclin E等的变化。(5)利用单一浓度IL-15作用PANC-1细胞分别为0,1,6,12,24,48,72 h并检测凋亡蛋白Bax,Bcl-2,Caspase 3及周期蛋白Cyclin E等的变化。结果:(1)PANC-1细胞表达IL-15受体α亚单位而不表达β和γc亚单位。(2)MTS结果显示:不同浓度IL-15作用PANC-1细胞48h与空白对照组相比没有统计学差异,作用72 h与空白对照组比较低剂量没有抑制作用,而高剂量有抑制作用(3)流式分析结果显示:低剂量IL-15对PANC-1没有促进凋亡的作用而高剂量具有促进凋亡的作用,浓度越高凋亡越明显。(4)Western blot分析显示:IL-15能够上调Bax,Caspase 3的表达降低Bcl-2的表达,同时能够抑制p-AKT的表达。(5)结果显示:随着时间增加BAX和Caspase 3表达增加而Bcl-2降低。6,12,24,48h组Cyclin E表达增加而72 h时表达降低与空白对照组相比没有统计学差异。p-AKT在1,6,12,24,48,72 h表达降低与对照组相比有统计学差异,p-STAT3在24,48和72 h表达增加与对照组相比具有统计学差异。结论:IL-15能够抑制PANC-1细胞的增殖呈时间和剂量依赖性,也能够诱导PANC-1细胞的凋亡。
[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 伪 subunits but not 尾 and 纬 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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9
本文编号：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 伪 subunits but not 尾 and 纬 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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9
【参考文献】
相关期刊论文 前1条
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
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