缺氧微环境中HIF1-α上调PLD2表达进而抑制结肠癌细胞凋亡的机制研究
发布时间:2018-05-09 01:42
本文选题:缺氧 + 凋亡 ; 参考:《重庆医科大学》2016年博士论文
【摘要】:结肠癌是消化系统常见的实体肿瘤,也是恶性肿瘤导致死亡的重要病因之一。过去几十年的研究表明结肠癌是多种基因异常表达共同作用的结果,但其发病的机制仍未完全阐明。作为一种常见的实体肿瘤,缺氧是其发生发展过程中的一个固有特点。研究表明HIF1-α在缺氧微环境中通过调控数百种靶基因,从而维持肿瘤细胞的存活。课题组前期通过干扰结肠癌细胞HIF1-α基因后进行蛋白质组学分析发现PLD2的变化明显。由此,我们推测在结肠癌中PLD2与HIF1-α可能具有相关性,并且受其调控,从而维持结肠癌细胞在缺氧的微环境得以存活。然而,目前尚未见在结肠癌中HIF1-α与PLD2关系及作用意义的文献报道,因此其具有重要的研究价值。本研究分以下三部分进行:第一部分HIF1-α与PLD2在结肠癌组织中的表达及临床意义目的:探讨HIF1-α与PLD2在结肠癌组织中的表达及相关性。方法:应用免疫组织化学染色法检测30例结肠癌组织及相对应的正常结肠粘膜组织中HIF1-α与PLD2蛋白的表达情况,探讨它们与临床病理参数之间的关系,并分析两者表达之间的相关性;分别用Western Blot和RT-PCR检测HIF1-α与PLD2在结肠癌及相对应的正常结肠粘膜中蛋白和mrna的表达水平。结果:免疫组织化学染色结果显示30例结肠癌组织中hif1-α与pld2蛋白的阳性表达率分别为76.67%(23/30)和73.33%(22/30),与两者在正常结肠粘膜组织中阳性表达率相比差异具有统计学意义(p0.05),两者共同表达与肿瘤的大少呈正相关,spearman相关性分析结果示结肠癌组织中hif1-α与pld2蛋白表达水平呈正相关(r=0.56,p0.05);rt-pcr和westernblot检查结果提示30例结肠癌组织中hif1-α与pld2mrna和蛋白均呈高表达,与正常结肠粘膜组织中表达水平相比差异具有统计学意义(p0.05)。结论:hif1-α与pld2在结肠癌组织中表达较正常结肠粘膜增高,两者共表达与患者的肿瘤大小有关,且两者表达水平呈正相关。第二部分缺氧微环境中hif1-α上调结肠癌细胞pld2表达的研究目的:从体内外实验探讨缺氧微环境中hif1-α对结肠癌细胞pld2表达水平的调控作用。方法:首先用westernblot和rt-pcr检测不同结肠粘膜起源细胞系(sw480,sw620,hct116,lovo及fhc)中hif1-α和pld2的mrna和蛋白的表达水平;再通过低氧培养模拟缺氧微环境并培养不同时间(0h,12h,24h及48h),用rt-pcr和westernblot检测缺氧对结肠癌细胞(sw480和sw620)中hif1-α和pld2mrna和蛋白表达水平的影响;用rt-pcr和westernblot检测干扰hif1-α基因后对缺氧培养24h或者不同氯化钴浓度(0,100μm,200μm)条件下培养的结肠癌细胞(sw480和sw620)中pld2mrna和蛋白的表达水平;最后建立结肠癌裸鼠皮下移植瘤模型,干扰hif1-α基因表达,观察裸鼠移植瘤生长情况,并用免疫组化和westernblot检测移植瘤中pld2蛋白的表达水平。结果:结肠癌细胞中(sw480,sw620,hct116及lovo)hif1-α与pld2mrna和蛋白的表达水平较fhc明显升高(p0.05);缺氧导致结肠癌细胞(sw480,sw620)中hif1-α与pld2的mrna和蛋白的表达水平较常氧升高(p0.05);成功构建携带人hif1-α基因sirna的腺病毒载体,转染结肠癌细胞(sw480,sw620),导致结肠癌细胞(sw480,sw620)中hif1-αmrna和蛋白的表达水平在常氧和缺氧条件下与各自对照组相比均降低(p0.05);干扰降低hif1-α基因的表达导致缺氧诱导的结肠癌细胞(sw480,sw620)中pld2蛋白和mrna的表达水平降低,与对照组相比差异具有统计学意义(p0.05);不同浓度氯化钴上调hif1-α的表达导致结肠癌细胞(sw480,sw620)中pld2蛋白和mrna表达水平随氯化钴浓度升高而升高,与对照组(0μm)相比差异具有统计学意义(p0.05);结肠癌裸鼠皮下移植瘤生长曲线显示hif1-α干扰组移植瘤从第21天开始肿瘤体积小于对照组(p0.05),免疫组化和westernblot结果均示,hif1-α干扰组移植瘤中的pld2蛋白表达的水平明显低于空白对照组(p0.01)。结论:hif1-α上调体内外缺氧微环境中结肠癌细胞pld2的mrna和蛋白的表达水平,阻断hif1-α和pld2的表达对裸鼠移植瘤的生长具有抑制作用。第三部分抑制pld2活性对缺氧微环境中结肠癌细胞凋亡的影响及机制研究目的:通过抑制pld2活性,探讨其对缺氧微环境中结肠癌细胞凋亡的影响及机制。方法:首先用磷脂酶d活性实验检测缺氧对结肠癌细胞(sw480、sw620、lovo和hct116)及fhc磷脂酶d活性的影响;再用磷脂酶d活性实验检测不同浓度(50nm,100nm)的pld2活性抑制剂(vu0364739)对缺氧诱导的结肠癌细胞(sw480和sw620)及fhc中磷脂酶d活性的影响,三种细胞株均分为:常氧组(basal组)、缺氧组(control组)、缺氧+vu0364739(50nm)及缺氧+vu0364739(100nm);利用流式细胞术检测抑制pld2活性对缺氧微环境中结肠癌细胞(sw480和w620)和fhc凋亡的影响,利用westernblot检测抑制pld2活性对缺氧环境中结肠癌细胞(sw480和sw620)和fhc中survivin、bcl2、p-pi3k/pi3k及p-akt/akt蛋白表达水平的影响,分组同上;sw620和sw480均分为:缺氧对照组、缺氧+vu0364739(100nm)组、缺氧+ly294002(10μm)组、缺氧+ly294002(10μm)+vu0364739(100nm)组,各组细胞的凋亡率用流式细胞检测,用westernblot检测各组细胞中survivin、bcl2、p-pi3k/pi3k及p-akt/akt蛋白的表达水平;最后用免疫组织化学染色检测50例结肠癌组织及相对应的正常结肠粘膜组织中pld2、survivin和bcl2的表达,并用spearman相关分别分析pld2与survivin和bcl2表达水平之间的相关性。结果:结肠癌细胞(sw480、sw620、lovo和hct116)及fhc中缺氧组的pld活性较常氧组升高,两组之间差异具有统计学意义(p0.05);vu0364739能显著抑制缺氧诱导升高的结肠癌细胞(sw620和sw480)及fhc中pld活性,缺氧+vu0364739(50nm)组或者缺氧+vu0364739(100nm)组与缺氧对照组相比差异具统计学意义(p0.05);缺氧导致结肠癌细胞(sw620和sw480)及fhc的凋亡率较basal组增加(p0.05),而抑制pld2活性后导致缺氧诱导的结肠癌细胞(sw480和sw620)凋亡增加更加明显,缺氧+vu0364739(50nm)组或者缺氧+vu0364739(100nm)组与缺氧对照组相比差异具统计学意义(p0.05),而对fhc凋亡无明显影响,缺氧+vu0364739(50nm)组或者缺氧+vu0364739(100nm)组与缺氧对照组相比差异无统计学意义(p0.05);缺氧导致结肠癌细胞(sw620和sw480)及fhc的survivin、bcl-2、p-pi3k/pi3k及p-akt/akt蛋白表达水平较basal组升高(p0.05),抑制pld2活性后导致缺氧诱导的结肠癌细胞(sw620和sw480)中survivin、bcl-2、p-pi3k/pi3k及p-akt/akt的蛋白表达水平又降低,缺氧+vu0364739(50nm)组或者缺氧+vu0364739(100nm)组与缺氧对照组相比差异具有统计学意义(p0.05),而对fhc中这些因子的蛋白表达水平无明显影响,缺氧+vu0364739(50nm)组或者缺氧+vu0364739(100nm)组与缺氧对照组相比差异无统计学意义(p0.05);抑制pi3k/akt信号通路、抑制pld2活性、或者两种方法相结合均导致缺氧诱导的结肠癌细胞(sw480和sw620)中survivin、bcl-2、p-pi3k/pi3k及p-akt/akt蛋白表达水平均降低,同时伴有这些细胞凋亡率增加,这三组细胞与缺氧对照组相比差异具有统计学意义(P0.05),而缺氧+LY294002(10μM)组与缺氧+VU0364739(100nM)组、缺氧+LY294002(10μM)组与缺氧+LY294002(10μM)+VU0364739(100nM)组以及缺氧+VU0364739(100nM)组与缺氧+LY294002(10μM)+VU0364739(100nM)组间相比这些因子蛋白表达和凋亡率变化的差异均不明显(P0.05);PLD2,Survivin及Bcl2在50例结肠癌组织中阳性表达率为分别为64%、68%和60%,与正常组织中这些因子的阳性表达率相比明显升高,差异具有统计学意义(P0.05),同时相关性分析结果显示PLD2与Survivin(r=0.379,P0.05),PLD2和Bcl2(r=0.405,P0.05)蛋白表达水平均呈正相关。结论:缺氧微环境中,PLD2可能通过激活PI3K/AKT信号通路上调Survivin和Bcl2的表达水平,从而抑制结肠癌细胞的凋亡。
[Abstract]:Colon cancer is a common solid tumor in the digestive system and one of the most important causes of death. Studies in the past few decades have shown that colon cancer is the result of the common effect of a variety of abnormal genes, but its pathogenesis is still not fully elucidated. As a common solid tumor, anoxia is one of its development processes. The study shows that HIF1- alpha maintains the survival of the tumor cells by regulating hundreds of target genes in the hypoxic microenvironment. By proteomic analysis after interfering with the HIF1- alpha gene in colon cancer cells, we found that the change of PLD2 is obvious. Therefore, we speculate that PLD2 and HIF1- alpha may have a correlation in colon cancer. Sex, and it is regulated to maintain the survival of colon cancer cells in the microenvironment of hypoxia. However, there is no literature on the relationship between HIF1- alpha and PLD2 in colon cancer and its significance. Therefore, it has important research value. This study is divided into three parts: the first part of the expression of HIF1- A and PLD2 in colon cancer tissue And clinical significance Objective: To investigate the expression and correlation of HIF1- alpha and PLD2 in colon cancer tissue. Methods: the expression of HIF1- alpha and PLD2 in 30 cases of colon cancer tissue and corresponding normal colonic mucosa were detected by immunohistochemical staining, and the relationship between them and pathological parameters was discussed and the expression of them was analyzed. Correlation between the protein and mRNA expression levels of HIF1- alpha and PLD2 in colon cancer and corresponding normal colon mucosa with Western Blot and RT-PCR. Results: immunohistochemical staining showed that the positive rates of hif1- alpha and PLD2 protein in 30 cases of colon cancer were 76.67% (23/30) and 73.33% (22/30), respectively. The positive expression rate in the normal colonic mucosa was statistically significant (P0.05), and the common expression was positively correlated with the large number of tumors. The results of Spearman correlation analysis showed that hif1- alpha was positively correlated with the expression level of PLD2 protein (r=0.56, P0.05) in colon cancer tissue (r=0.56, P0.05); the results of RT-PCR and Westernblot examination suggested 30 cases of colon cancer. The expression of hif1- alpha and pld2mrna and protein in the fabric were highly expressed, and the difference was statistically significant compared with the normal colonic mucosal tissue expression level (P0.05). Conclusion: the expression of hif1- alpha and PLD2 in colon cancer tissues is higher than that of normal colon mucosa. The co expression of the two is related to the tumor size of the patients, and the expression level of the two is positively correlated. Second parts are positively correlated. Study on the expression of PLD2 in colon cancer cells by hif1- - alpha in anoxic microenvironment. Objective: To investigate the regulation of hif1- alpha on the expression of PLD2 in colon cancer cells in the hypoxia microenvironment. Methods: first, Westernblot and RT-PCR were used to detect the hif1- alpha in different colonic mucosa origin cell lines (SW480, SW620, HCT116, LoVo and FHC). The expression level of mRNA and protein; then cultured hypoxia microenvironment through hypoxia and culture for different time (0h, 12h, 24h and 48h). The effects of hypoxia on hif1- A and pld2mrna and protein expression in colon cancer cells (SW480 and SW620) were detected by RT-PCR and Westernblot. The expression level of pld2mrna and protein in colon cancer cells (SW480 and SW620) was cultured under the condition of 24h or different cobalt chloride concentration (0100 mu m, 200 m). Finally, the model of subcutaneous transplantation tumor in nude mice was established, the expression of hif1- a gene was disturbed, the growth condition of the transplanted tumor in nude mice was observed, and the immunohistochemistry and Westernblot were used to detect PLD2 in the transplanted tumor. Expression level of protein. Results: the expression level of hif1- alpha and pld2mrna and protein in colon cancer cells (SW480, SW620, HCT116 and LoVo) is significantly higher than that of FHC (P0.05); hypoxia leads to the higher expression level of hif1- alpha and PLD2 in colon cancer cells (SW480, SW620), and a successful construction of the gland carrying human alpha gene. The expression of hif1- alpha mRNA and protein in colon cancer cells (SW480, SW620) decreased (P0.05) compared with the control group (P0.05) in SW480 (SW620), and the interference and reduction of the expression of hif1- a gene resulted in the PLD2 protein and mRNA table in the colon cancer cells (SW480, SW620) induced by oxygen deficiency (SW480, SW620). The difference was statistically significant (P0.05) compared with the control group (P0.05); the expression of hif1- alpha by different concentrations of cobalt chloride increased the expression of PLD2 protein and mRNA in colon cancer cells (SW480, SW620) and increased with the increase of cobalt chloride concentration. The difference was statistically significant (P0.05) compared with the control group (0 mu m); subcutaneous transplantation of colon cancer nude mice. The tumor growth curve showed that the tumor size of the transplanted tumor in the hif1- alpha interference group was less than the control group (P0.05) from twenty-first days, and the immunohistochemical and Westernblot results were all shown. The level of PLD2 protein expression in the hif1- alpha interference group was significantly lower than that of the blank control group (P0.01). Conclusion: hif1- alpha up regulation in the hypoxia microenvironment in and outside the body of the colon cancer cell PLD2 Mr The expression level of Na and protein, blocking the expression of hif1- alpha and PLD2 inhibits the growth of transplanted tumor in nude mice. Third the effect of inhibition of PLD2 activity on the apoptosis of colon cancer cells in anoxic microenvironment and its mechanism: To investigate the effect and mechanism of PLD2 activity on the apoptosis of colon cancer cells in anoxic microenvironment. Methods: first, the effects of hypoxia on the activity of colon cancer cells (SW480, SW620, LoVo and HCT116) and FHC phospholipase D activity were detected by phospholipase D activity, and the effects of PLD2 active inhibitor (vu0364739) on the activity of hypoxia induced colon cancer cells and phospholipase activity were detected with phospholipase D activity test. The three cell lines were divided into two groups: normal oxygen group (basal group), hypoxia group (control group), anoxic +vu0364739 (50nm) and hypoxia +vu0364739 (100nm). Flow cytometry was used to detect the effect of PLD2 activity on the apoptosis of colon cancer cells (SW480 and w620) and FHC in anoxic microenvironment and Westernblot detection of PLD2 activity to colon cancer in anoxic environment The effects of survivin, BCL2, p-pi3k/pi3k and p-akt/akt on the expression of survivin, BCL2, p-pi3k/pi3k and p-akt/akt in the cells (SW480 and FHC) were grouped together, SW620 and SW480 were equally divided into anoxia control group, hypoxia +vu0364739 (100nm) group, hypoxia +ly294002 (10 mu) group, hypoxia (10 mu) + group, and the cell apoptosis rate was detected by flow cytometry Blot was used to detect the expression of survivin, BCL2, p-pi3k/pi3k and p-akt/akt protein in each group of cells. Finally, the expression of PLD2, survivin and BCL2 in 50 cases of colon cancer tissue and corresponding normal colonic mucosa was detected by immunohistochemical staining, and the correlation between PLD2 and Survivin and BCL2 expression levels was analyzed with Spearman correlation. Results: the PLD activity of the colon cancer cells (SW480, SW620, LoVo and HCT116) and the hypoxia group was higher than that of the oxygen group, and the difference between the two groups was statistically significant (P0.05). Vu0364739 could significantly inhibit the activity of the colon cancer cells (SW620 and SW480) and FHC in the hypoxia induced (SW620 and SW480) and FHC, the hypoxia group or the hypoxia group. Compared with the hypoxia control group, the difference was statistically significant (P0.05); the apoptosis rate of colon cancer cells (SW620 and SW480) and FHC increased in comparison with basal group (P0.05), and the apoptosis of colon cancer cells (SW480 and SW620) induced by anoxia induced by PLD2 activity increased more clearly, and the hypoxia +vu0364739 (50nm) group or hypoxia +vu0364739 group was more significant. There was significant difference in the hypoxia control group (P0.05), but no significant effect on FHC apoptosis. There was no significant difference in the hypoxia +vu0364739 (50nm) group or the hypoxia +vu0364739 (100nm) group compared with the hypoxic control group (P0.05), and the hypoxia led to the survivin, Bcl-2, and protein expression of the colon cancer cells (SW620 and SW480) and FHC. The levels of survivin, survivin, Bcl-2, p-pi3k/pi3k and p-akt/akt in the colon cancer cells (SW620 and SW480) induced by hypoxia were also lower than those in the basal group (SW620 and SW480). The difference between the hypoxia +vu0364739 (50nm) group or the hypoxia group was statistically significant compared with the hypoxia control group. There was no significant effect on the protein expression levels of these factors. There was no significant difference in the hypoxia +vu0364739 (50nm) group or anoxic +vu0364739 (100nm) group compared with the hypoxic control group (P0.05); the inhibition of pi3k/akt signaling pathway, the inhibition of PLD2 activity, or two methods of phase junctions all led to the hypoxia induced colon cancer cells (SW480 and SW620) surviv. The expression levels of in, Bcl-2, p-pi3k/pi3k and p-akt/akt were all decreased, and the apoptosis rate of these cells increased. The difference between the three groups was statistically significant compared with the hypoxia control group (P0.05), while the hypoxia +LY294002 (10 M) group and the hypoxia +VU0364739 (100nM) group, the hypoxia +LY294002 (10 mu M) group and the hypoxia +LY294002 (10 micron) were the same. There was no significant difference in the expression and apoptosis rate of these factors in the group and the anoxic +VU0364739 (100nM) group and the anoxic +LY294002 (10 M) +VU0364739 (100nM) group, and the positive rates of PLD2, Survivin and Bcl2 in 50 cases of colon cancer were 64%, 68% and 60%, respectively, and the positive expression rate of these factors in normal tissues. The difference was statistically significant (P0.05), and the correlation analysis showed that PLD2 and Survivin (r=0.379, P0.05), PLD2 and Bcl2 (r=0.405, P0.05) protein expression level were all positively correlated. Conclusion: PLD2 may increase the level of expression by activating PI3K/ AKT signal pathway, thus inhibiting the colon and thus inhibiting the colon. Apoptosis of cancer cells.
【学位授予单位】:重庆医科大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R735.35
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