二甲双胍对胰腺癌细胞以及胰腺癌干细胞ALDH1表达的影响

发布时间：2018-08-29 17:42

【摘要】：背景据美国癌症统计调查显示,2016年癌症新发病例数为1,685,210人,相当于每天约4,600人被确诊为恶性肿瘤。在众多类型的恶性肿瘤中,胰腺癌的死亡率是最高的,其总生存率为8%,而5年生存率只为2%,这主要是由于大多数胰腺癌患者在确诊时往往已经有远处转移。目前治疗胰腺癌仍认为手术是最有效的手段,但即使经过积极的治疗后,其5年生存率也只有25%~35%。近年来发现乙醛脱氢酶(aldehyde dehydrogenases,ALDHs)在胰腺癌中高表达,并且还和胰腺癌干细胞的特性紧密相关。另外,它不仅能够作为鉴定和分离胰腺癌干细胞(cancer stem cells,CSCs)的表面标志物,还与胰腺癌治疗过程中产生的耐药、转移、复发以及患者的预后等均有一定关系。二甲双胍是目前世界上使用最广泛的抗糖尿病的口服药物之一。它能够通过抑制肝脏糖异生,同时增加外周组织中胰岛素的敏感性来降低血糖和胰岛素浓度。一项回顾性研究发现,二甲双胍可以降低糖尿病患者的肿瘤患病率和死亡率。另外,还有研究发现二甲双胍不仅可以通过哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin,mTOR)通路抑制胰腺癌细胞的增殖,还能够抑制胰腺癌动物模型中ALDH1的表达,但其作用机制尚不清楚。在本实验中,我们通过使用mTOR抑制剂--雷帕霉素进而阻断mTOR信号通路的激活,观察二甲双胍以及雷帕霉素对胰腺癌细胞和胰腺癌干细胞中ALDH1的表达水平的影响,并初步探究胰腺癌中ALDH1的表达与mTOR的关系。目的观察二甲双胍对胰腺癌PANC-1细胞以及胰腺癌干细胞中ALDH1表达的影响,探讨其相关的作用机制。方法采用CCK-8法和Western Blot方法分别检测不同浓度二甲双胍对胰腺癌PANC-1细胞的增殖及对ALDH1和p-mTOR蛋白表达的影响。使用mTOR抑制剂雷帕霉素,通过Western Blot方法,免疫荧光法(Immunofluorescence)以及荧光实时定量PCR(Quantitative real-time PCR,q RT-PCR)来检测二甲双胍组,雷帕霉素组以及联合用药组中PANC-1细胞ALDH1、4EBP1、p-mTOR蛋白及m RNA的表达。采用超低黏附培养板悬浮培养胰腺癌细胞,观察不同药物干预对胰腺癌细胞成球能力的影响,进一步采用Western Blot方法检测胰腺癌干细胞ALDH1的表达情况;通过建立胰腺癌裸鼠移植瘤模型来比较PANC-1细胞和胰腺癌成球细胞之间成瘤能力的不同。结果1.CCK-8结果:使用浓度分别为1、2、5、10、15和20 mmol/L的二甲双胍干预胰腺癌PANC-1细胞,24 h后的细胞增殖抑制率分别为:(5.23±2.03)%、(10.23±4.19)%、(16.89±4.19)%、(20.60±5.24)%、(29.40±2.05)%、(35.38±1.70)%;48 h后的细胞增殖抑制率分别为:(8.30±3.48)%、(14.20±3.76)%、(22.10±6.10)%、(31.20±2.03)%、(42.33±2.17)%、(49.58±1.84)%;72 h后的细胞增殖抑制率分别为:(19.02±2.67)%、(22.13±6.11)%、(25.83±2.69)%、(36.65±6.30)%、(49.67±3.80)%、(53.70±3.13)%。这些结果表明,相比于对照组,二甲双胍能够明显抑制PANC-1细胞的增殖,并且其抑制作用随着二甲双胍剂量的增加和干预时间的延长而逐渐增强。计算后得到二甲双胍的最适干预时间是48 h,IC50值约为20 mmol/L。经统计学分析数据后发现,每个实验组的细胞增殖抑制率与对照组之间均有差异,且具有统计学意义(P均0.05)。2.胰腺癌细胞Western Blot结果显示:使用浓度分别为2,5,10,15,20mmol/L的二甲双胍干预胰腺癌PANC-1细胞后,细胞中ALDH1和p-mTOR蛋白表达随着二甲双胍浓度的增加均呈下降趋势,并且呈现出显著的剂量依赖性。在作用机制的研究结果中显示:二甲双胍以及雷帕霉素组均能够明显降低ALDH1,4EBP1以及p-mTOR的蛋白水平;联合用药组的抑制作用会更显著。二甲双胍对ALDH1的蛋白表达的抑制作用要明显强于雷帕霉素,其两者之间的差异具有统计学意义(P0.05);然而二甲双胍对PANC-1细胞中4EBP1以及p-mTOR的表达与雷帕霉素相比较未见显著变化。3.q RT-PCR结果:相比于对照组,二甲双胍以及雷帕霉素明显抑制胰腺癌PANC-1细胞中ALDH1,4EBP1以及p-mTOR m RNA的转录水平;然而,二甲双胍对PANC-1中ALDH1的表达的抑制作用明显强于雷帕霉素,并且差异具有统计学意义(P0.05)。4.免疫荧光法结果示:相比于对照组,二甲双胍组能够明显降低ALDH1的表达,雷帕霉素也有类似的作用。5.成球实验的结果显示:对照组胰腺癌PANC-1的细胞球数为85.00±5.12,二甲双胍干预后的细胞球数为43.00±3.66,雷帕霉素干预后细胞球数为55.00±3.21。我们使用Western Blot的方法检测二甲双胍针对胰腺癌细胞球中ALDH1蛋白表达的作用时发现:相对于胰腺癌细胞系中ALDH1的表达水平,胰腺癌细胞球ALDH1的表达明显增高;在使用二甲双胍和雷帕霉素干预胰腺癌细胞球后发现:相比于对照组,二甲双胍以及雷帕霉素组均能够抑制ALDH1蛋白的表达,差异有统计学意义(P0.05)。6.成瘤能力的比较结果显示:胰腺癌PANC-1细胞的成瘤能力(7/10)明显低于胰腺癌细胞球的成瘤能力(10/10)。不仅如此,胰腺癌细胞球接种后形成的肿瘤体积要显著大于胰腺癌PANC-1细胞,且两者的差异有统计学意义(P0.05)。结论1.二甲双胍显著抑制胰腺癌PANC-1细胞的增殖能力以及ALDH1的表达,并且随着二甲双胍剂量的增加和干预时间的延长,抑制作用越明显。2.二甲双胍以及雷帕霉素均能够抑制胰腺癌PANC-1细胞中ALDH1的表达,其机制可能是通过mTOR通路。3.二甲双胍以及雷帕霉素均能够抑制胰腺癌PANC-1细胞球的形成,还能够抑制胰腺癌成球细胞中ALDH1的表达,其机制可能是通过mTOR通路。
[Abstract]:Background According to the U.S. Cancer Survey, the number of new cancer cases in 2016 was 1,685,210, equivalent to about 4,600 people diagnosed as malignant tumors per day. Surgery is still considered the most effective treatment for pancreatic cancer, but even after aggressive treatment, the 5-year survival rate is only 25%-35%. Aldehyde dehydrogenases (ALDHs) have been found to be highly expressed in pancreatic cancer in recent years, and are closely related to the characteristics of pancreatic cancer stem cells. Metformin is one of the most widely used oral antidiabetic drugs in the world. It can be used to identify and isolate pancreatic cancer stem cells (CSCs). A retrospective study found that metformin could reduce cancer morbidity and mortality in diabetic patients. In addition, other studies have found that metformin can pass not only mammalian rapamycin target protein (ma). The mmalian Target of Rapamycin (mTOR) pathway inhibits the proliferation of pancreatic cancer cells and the expression of ALDH1 in pancreatic cancer animal models, but its mechanism is not clear. Objective To investigate the effect of metformin on the expression of ALDH1 in pancreatic cancer PANC-1 cells and pancreatic cancer stem cells and the relationship between the expression of ALDH1 and mTOR. The effects of metformin on the proliferation of PANC-1 cells and the expression of ALDH 1 and p-mTOR proteins were measured by Western Blot, Immunofluorescence and Quantitative real-time PCR (q RT-PCR). The expression of ALDH1, 4EBP1, p-mTOR protein and m RNA in PANC-1 cells was detected by Western Blot assay. Pancreatic cancer cells were cultured in suspension with ultra-low adhesion plate to observe the effect of different drugs on the ability of pancreatic cancer cells to globe. Results 1. CCK-8 Results: The inhibitory rates of proliferation of PANC-1 cells treated with metformin at concentrations of 1,2,5,10,15 and 20 mmol/L after 24 hours were (5.23+2.03)%, (10.23+4.19)%, (16.89+4.19)%, (20.60+5.24)%, (20.60+5.24)% respectively. 29.40 [(29.40 [(2.05)%, (35.38 [(1.70)%, (35.38 [1.70)%, (35.38 [1.70)%; (8.30 [3.48)%, (14.20 [3.76)%, (14.20 [(14.20 [3.766.76)%, (22.10 [22.10 [6.10 [(22.10 [6.10)%, (31.20 [(31.20 [2.20 [2.03)%, (42.33 [2.22.33 [2.17 [2.17 [(49.58 [1.58 [1.84)%, (49.58 80%, (53.70+3.13)%. These knots The results showed that metformin significantly inhibited the proliferation of PANC-1 cells compared with the control group, and the inhibitory effect increased with the increase of metformin dose and the prolongation of intervention time. The results of Western Blot showed that the expression of ALDH 1 and p-mTOR protein in pancreatic cancer PANC-1 cells increased with the concentration of metformin after the intervention of metformin at 2,5,10,15,20 mmol/L, respectively. Metformin and rapamycin could significantly reduce the levels of ALDH1, 4EBP1 and p-mTOR, and the inhibitory effect of metformin on the expression of ALDH1 was stronger than that of Ray. However, the expression of 4EBP1 and p-mTOR in PANC-1 cells was not significantly changed by metformin compared with rapamycin. 3. Q RT-PCR results: Compared with the control group, metformin and rapamycin significantly inhibited the expression of ALDH1, 4EBP1 and p-mTOR m RNA in PANC-1 cells. However, the inhibitory effect of metformin on the expression of ALDH1 in PANC-1 was significantly stronger than that of rapamycin, and the difference was statistically significant (P 0.05). 4. Immunofluorescence assay showed that metformin significantly decreased the expression of ALDH1 compared with the control group, and rapamycin had a similar effect. 5 In the control group, the number of PANC-1 globules was 85.00 (+ 5.12), 43.00 (+ 3.66) after metformin treatment, and 55.00 (+ 3.21) after rapamycin treatment. Compared with the control group, metformin and rapamycin could inhibit the expression of ALDH1 protein, and the difference was statistically significant (P 0.05). The tumor-forming ability of PANC-1 cells (7/10) was significantly lower than that of pancreatic cancer cells (10/10). Moreover, the tumor volume of PANC-1 cells inoculated with pancreatic cancer cells was significantly larger than that of PANC-1 cells, and the difference was statistically significant (P 0.05). Conclusion 1. Metformin significantly inhibited the proliferation of pancreatic cancer PANC-1 cells. Metformin and rapamycin can inhibit the expression of ALDH 1 in pancreatic cancer PANC-1 cells, which may be through the mTOR pathway. 3. Metformin and rapamycin can inhibit the globules of pancreatic cancer PANC-1 cells. It can also inhibit the expression of ALDH1 in pancreatic cancer spheroid cells. The mechanism may be through the mTOR pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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