重组人p53腺病毒联合氟维司群对乳腺癌细胞MCF-7凋亡的影响

发布时间：2018-07-09 13:23

本文选题：乳腺癌 + p53基因　；参考：《安徽医科大学》2015年硕士论文

【摘要】：研究目的自1979年p53基因被发现以来,其结构与功能已被广泛认知,其转录后编码的p53蛋白已成为肿瘤细胞发生发展过程中重要的参与者。在100多种人类肿瘤中,有60%以上的肿瘤发生与p53基因突变有关,目前已知人乳腺癌p53基因突变率为20%,而其余80%的乳腺癌均含有野生型的p53,与胃癌、大肠癌、肺癌等肿瘤类型不同,乳腺癌发生和发展的原因除了抑癌基因p53的基因突变外,大部分是由于基因序列正常的p53(野生型p53)没有发挥应有的作用所致。通过腺病毒载体将外源性肿瘤抑制基因(p53基因)导入肿瘤内,通过细胞凋亡、旁观者效应、抑制多药耐药基因表达等多种抗肿瘤机制,抑制肿瘤组织的生长,促进肿瘤细胞的凋亡,已成为一种新的肿瘤治疗方向。p53和雌激素受体(ER)都是乳腺癌发生发展重要的因子,研究发现,p53能促进ER表达,反过来,ER可拮抗p53的促凋亡作用,抑制p53介导的细胞凋亡反应。氟维司群(fulvestrant)是全新的乳腺癌内分泌治疗药物,称为选择性雌激素受体下调剂(Selective Estrogen Receptor Downregulators, SERDs),能够高亲和力地结合阻断并下调雌激素受体(ER)的数量,使激素受体上的转录活性区域AF1和AF2均失活,并加速了雌激素受体功能的丧失。我们通过联合应用重组人p53腺病毒和氟维司群,观察乳腺癌细胞系MCF-7的形态学变化、p53蛋白和ER蛋白的表达水平、MCF-7细胞的凋亡情况以及增殖抑制率等,探讨氟维司群是否能阻断并下调ER,解除其对p53的抑制作用,观察两者是否具有协同作用,为基础研究与临床乳腺癌治疗的新方法开拓新的方向。研究方法1. 比较各组细胞的生长及形态学变化：设对照组(空载病毒组)、氟维司群(1μmol/1)组、Ad-p53 (50MOI)组、Ad-p53 (50MOI)+氟维司群(1μmol/l)组作用于MCF-7细胞72h后,倒置显微镜观察不同实验组细胞的生长情况以及形态学变化。2. 比较各组细胞的增殖抑制率：设对照组(空载病毒组)、氟维司群(1μmol/l)组、Ad-p53 (50MOI)组、Ad-p53 (50MOI)+氟维司群(1μmol/l)组作用于MCF-7细胞72h后,比较各实验组细胞的增殖抑制率。3. 比较各组细胞的凋亡率：设对照组(空载病毒组)、氟维司群(1μmol/l)组、Ad-p53 (50MOI)组、Ad-p53 (50MOI)+氟维司群(1μmol/l)组作用于MCF-7细胞72h后,用流式细胞仪AnnexinV-FITC/PI双标法检测分析不同处理组MCF-7细胞的凋亡情况。4. 比较各组细胞p53和ER蛋白的表达水平：设对照组(空载病毒组)、氟维司群(1μmol/l)组、Ad-p53 (50MOI)组、Ad-p53 (50MOI)+氟维司群(1μmol/l)组作用于MCF-7细胞72h后,检测各实验组细胞p53和ER蛋白的表达情况。研究结果1.倒置显微镜观察细胞生长状态：对照组MCF-7细胞正常贴壁生长,培养基清澈无浑浊,细胞生长状态佳,氟维司群组细胞贴壁生长可,培养基稍浑浊,出现一定的悬浮细胞；Ad-p53组可见贴壁细胞生长状态不佳,细胞密度较低,细胞形态不规则,出现细胞碎片、细胞脱落等细胞凋亡的改变；Ad-p53与氟维司群联合作用于MCF-7细胞后,高倍镜下可见散在生长细胞,密度较低,贴壁细胞数量大幅减少,细胞出现皱缩,悬浮细胞数目更多,细胞培养液出现浑浊。2.流式细胞术检测细胞凋亡结果：对照组、氟维司群组、Ad-p53组、 Ad-p53+氟维司群组中细胞凋亡率分别是(6.3±1.5)%、(13.3±1.2)%、(14.5±2.9)%和(38.1±5.9)%。3.MTS法检测细胞增殖抑制率结果：50 MOI Ad-p53和氟维司群分别处理MCF-7细胞24"96 h后,增殖抑制率分别为(8.21±0.54)%、(28.5±1.42)%、(50.14±0.78)%、(58.25±2.92)%和(9.73±1.68)%、(25.26±0.82)%、(35.25±3.94)%、(46.37±2.56)%；氟维司群与Ad-p53联合作用24～96h后,细胞的增殖抑制率为(12.42±1.76)%、(35.20±0.58)%、(62.08±2.56)%及(75.43%±3.56)%。4.蛋白印迹法(Western blot)检测结果：对照组、氟维司群组中均有p53蛋白的低表达；Ad-p53组、Ad-p53+氟维司群组中p53蛋白高表达；与对照组相比,Ad-p53组细胞的ER蛋白表达增加,但是联合用药组ER蛋白表达降低。研究结论1.Ad-p53感染乳腺癌MCF-7后,细胞生长缓慢且形态学发生明显改变,且当与氟维司群联合使用时细胞生长几乎停止,细胞密度极低,调亡细胞更多,细胞碎片更多,细胞凋亡现象更明显。2.Ad-p53或氟维司群单独作用乳腺癌MCF-7细胞后,MTS法检测细胞的各时期增殖抑制率均增加；当Ad-p53与氟维司群联合作用于MCF-7细胞,24～96 h的各时期MCF-7细胞的增殖抑制率均高于各单药组。3.Ad-p53或氟维司群单药均可明显诱导乳腺癌MCF-7细胞发生凋亡增加,联合用药组诱导细胞凋亡作用更加明显。4.对照组、氟维司群组中均有p53蛋白的低表达；Ad-p53组、Ad-p53+氟维司群组中p53蛋白高表达,说明腺病毒介导的p53基因成功转入MCF-7细胞中并有效表达,但是Ad-p53组和联合组蛋白表达没有明显差异；与对照组相比,Ad-p53组细胞的ER蛋白表达增加,但是联合用药组ER蛋白表达降低。
[Abstract]:Its structure and function have been widely recognized since the discovery of the p53 gene in 1979. The post transcriptional encoded p53 protein has become an important participant in the development of tumor cells. In more than 100 human tumors, more than 60% of the tumors are associated with the mutation of the p53 gene. At present, the mutation rate of p53 gene in human breast cancer is known. For 20%, the other 80% breast cancers all contain wild type p53, which are different from cancer types such as gastric cancer, large intestine cancer and lung cancer. The cause of breast cancer is due to the gene mutation of tumor suppressor gene p53, most of which is caused by the normal p53 (wild type p53) in the gene sequence. The tumor suppressor gene (p53 gene) is introduced into the tumor, and through the apoptosis, bystander effect, inhibition of multidrug resistance gene expression and other anti-tumor mechanisms, inhibiting the growth of tumor tissue and promoting the apoptosis of tumor cells, it has become a new tumor treatment direction.P53 and estrogen receptor (ER) are the important causes of the development of breast cancer. The study found that p53 can promote the expression of ER, in turn, ER can antagonize the apoptotic effect of p53 and inhibit the apoptosis response mediated by p53. The fluavinic group (fulvestrant) is a new endocrine therapy drug for breast cancer, known as the selective estrogen receptor (Selective Estrogen Receptor Downregulators, SERDs), and is capable of high affinity. Combined with the number of blocking and down-regulation of estrogen receptor (ER), both AF1 and AF2 were inactivated in the transcriptional active region of the hormone receptor and the loss of the function of estrogen receptor was accelerated. We observed the morphological changes of MCF-7 in the breast cancer cell line, the expression of p53 protein and ER protein by combining the recombinant human p53 adenovirus and the fluinosus group, MCF, MCF. -7 cell apoptosis and proliferation inhibition rate and so on, to explore whether the fluvia group can block and downregulate ER, relieve its inhibitory effect on p53, observe the synergistic effect between the two, and explore new directions for the new methods of basic research and clinical breast cancer treatment. Method 1. compare the growth and morphological changes of cells in each group: set up Control group (unloaded virus group), fluinus group (1 mol/1) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluidus group (1 mol/l) group after MCF-7 cell 72h, inverted microscope observation of the growth of cells in different experimental groups and morphological changes.2. compared each group of cell proliferation inhibition rate: set control group (empty virus group), fluinus Group (1 mol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluflund group (1 mu) group acted on MCF-7 cell 72h, compared the proliferation inhibition rate of each experimental group, compared the apoptosis rate of each group: the control group (empty virus group), the fluflex group (1 mu mol/l) group, Ad-p53 (50MOI) group, 1 micron group (1 mu) group. After MCF-7 cell 72h, the apoptosis of MCF-7 cells in different treatment groups was detected and analyzed by flow cytometry AnnexinV-FITC/PI double standard.4. comparison of the expression level of p53 and ER protein in each group: the control group (empty virus group), the fluflex group (1 mu mol/l) group, Ad-p53 (50MOI) group, Ad-p53 (50MOI) + fluflex group (1 mu) group acted on the After F-7 cell 72h, the expression of p53 and ER protein in all the experimental groups was detected. Results 1. inverted microscope was used to observe the cell growth state: the MCF-7 cells in the control group were normally adhered to the wall, the culture medium was clear and no turbidity, the cell growth state was good, the cell wall growth of the fluorine group group, the medium turbidity in the culture medium, and the certain suspended cells; A In group d-p53, the growth of adherent cells was poor, cell density was low, cell morphology was irregular, cell debris, cell fall and other cell apoptosis changes. After the combination of Ad-p53 and fluinisus group, the growth cells were scattered under the high magnification microscope, the density was low, the number of adherent cells decreased significantly, and the cell crinkled, and the cells crinkled and suspended. The number of floating cells was more, and the apoptotic results were detected by turbid.2. flow cytometry in the cell culture solution: the apoptosis rates in the control group, the flufir group, the Ad-p53 group and the Ad-p53+ flock group were (6.3 + 1.5)%, (13.3 + 1.2)%, (14.5 + 2.9)% and (38.1 + 5.9)%.3.MTS method to detect the cell proliferation inhibition rate: 50 MOI Ad-p53 and fluorine. The proliferation inhibition rate of MCF-7 cells was (8.21 + 0.54)%, (28.5 + 1.42)%, (50.14 + 0.78)%, (58.25 + 2.92)%, (58.25 + 2.92)% and (9.73 + 1.68)%, (25.26 + 1.68)%, (58.25 + 0.78)%, (58.25 + 2.92)%, (50.14 +%)%, and (28.5 + 1.42)%. % and (75.43% + 3.56)%.4. blot method (Western blot) test results: the control group, the low expression of p53 protein in the control group, and the high expression of p53 protein in the group Ad-p53 and the Ad-p53+ fluovers group; and the ER protein expression in the Ad-p53 group was increased compared with the control group, but the expression of ER protein in the combined group was reduced. The conclusion 1.Ad-p of the study was 1.Ad-p. 53 after MCF-7 infection of breast cancer, the cell growth is slow and the morphology changes obviously. And when combined with the fluinfill group, the cell growth almost stops, the cell density is very low, the cell number is more, the cell debris is more, the apoptosis phenomenon is more obvious that.2.Ad-p53 or the fluinfill group acts on the MCF-7 cells of breast cancer alone, and the MTS method is used to detect the cells. The proliferation inhibition rate increased at all times. When Ad-p53 and fluvia were combined with MCF-7 cells, the proliferation inhibition rate of MCF-7 cells in each period of 24~96 h was higher than that of the single drug group.3.Ad-p53 or the single drug of fluodex group, which could induce the increase of apoptosis of MCF-7 cells in breast cancer, and the combined drug group could induce apoptosis more obviously. In the.4. control group, the low expression of p53 protein in the fluovers group and the high expression of p53 protein in the group Ad-p53 and the Ad-p53+ fluovers group showed that the adenovirus mediated p53 gene was successfully transferred into MCF-7 cells and expressed effectively, but there was no significant difference in the expression of the protein in the Ad-p53 group and the combined histone. Compared with the control group, the ER protein table of the Ad-p53 group cells was compared with the control group. The expression of ER decreased in combination group.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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