本文選題：缺氧 + 凋亡　； 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：結(jié)腸癌是消化系統(tǒng)常見(jiàn)的實(shí)體腫瘤,也是惡性腫瘤導(dǎo)致死亡的重要病因之一。過(guò)去幾十年的研究表明結(jié)腸癌是多種基因異常表達(dá)共同作用的結(jié)果,但其發(fā)病的機(jī)制仍未完全闡明。作為一種常見(jiàn)的實(shí)體腫瘤,缺氧是其發(fā)生發(fā)展過(guò)程中的一個(gè)固有特點(diǎn)。研究表明HIF1-α在缺氧微環(huán)境中通過(guò)調(diào)控?cái)?shù)百種靶基因,從而維持腫瘤細(xì)胞的存活。課題組前期通過(guò)干擾結(jié)腸癌細(xì)胞HIF1-α基因后進(jìn)行蛋白質(zhì)組學(xué)分析發(fā)現(xiàn)PLD2的變化明顯。由此,我們推測(cè)在結(jié)腸癌中PLD2與HIF1-α可能具有相關(guān)性,并且受其調(diào)控,從而維持結(jié)腸癌細(xì)胞在缺氧的微環(huán)境得以存活。然而,目前尚未見(jiàn)在結(jié)腸癌中HIF1-α與PLD2關(guān)系及作用意義的文獻(xiàn)報(bào)道,因此其具有重要的研究?jī)r(jià)值。本研究分以下三部分進(jìn)行:第一部分HIF1-α與PLD2在結(jié)腸癌組織中的表達(dá)及臨床意義目的:探討HIF1-α與PLD2在結(jié)腸癌組織中的表達(dá)及相關(guān)性。方法:應(yīng)用免疫組織化學(xué)染色法檢測(cè)30例結(jié)腸癌組織及相對(duì)應(yīng)的正常結(jié)腸粘膜組織中HIF1-α與PLD2蛋白的表達(dá)情況,探討它們與臨床病理參數(shù)之間的關(guān)系,并分析兩者表達(dá)之間的相關(guān)性;分別用Western Blot和RT-PCR檢測(cè)HIF1-α與PLD2在結(jié)腸癌及相對(duì)應(yīng)的正常結(jié)腸粘膜中蛋白和mrna的表達(dá)水平。結(jié)果:免疫組織化學(xué)染色結(jié)果顯示30例結(jié)腸癌組織中hif1-α與pld2蛋白的陽(yáng)性表達(dá)率分別為76.67%(23/30)和73.33%(22/30),與兩者在正常結(jié)腸粘膜組織中陽(yáng)性表達(dá)率相比差異具有統(tǒng)計(jì)學(xué)意義(p0.05),兩者共同表達(dá)與腫瘤的大少呈正相關(guān),spearman相關(guān)性分析結(jié)果示結(jié)腸癌組織中hif1-α與pld2蛋白表達(dá)水平呈正相關(guān)(r=0.56,p0.05);rt-pcr和westernblot檢查結(jié)果提示30例結(jié)腸癌組織中hif1-α與pld2mrna和蛋白均呈高表達(dá),與正常結(jié)腸粘膜組織中表達(dá)水平相比差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:hif1-α與pld2在結(jié)腸癌組織中表達(dá)較正常結(jié)腸粘膜增高,兩者共表達(dá)與患者的腫瘤大小有關(guān),且兩者表達(dá)水平呈正相關(guān)。第二部分缺氧微環(huán)境中hif1-α上調(diào)結(jié)腸癌細(xì)胞pld2表達(dá)的研究目的:從體內(nèi)外實(shí)驗(yàn)探討缺氧微環(huán)境中hif1-α對(duì)結(jié)腸癌細(xì)胞pld2表達(dá)水平的調(diào)控作用。方法:首先用westernblot和rt-pcr檢測(cè)不同結(jié)腸粘膜起源細(xì)胞系(sw480,sw620,hct116,lovo及fhc)中hif1-α和pld2的mrna和蛋白的表達(dá)水平;再通過(guò)低氧培養(yǎng)模擬缺氧微環(huán)境并培養(yǎng)不同時(shí)間(0h,12h,24h及48h),用rt-pcr和westernblot檢測(cè)缺氧對(duì)結(jié)腸癌細(xì)胞(sw480和sw620)中hif1-α和pld2mrna和蛋白表達(dá)水平的影響;用rt-pcr和westernblot檢測(cè)干擾hif1-α基因后對(duì)缺氧培養(yǎng)24h或者不同氯化鈷濃度(0,100μm,200μm)條件下培養(yǎng)的結(jié)腸癌細(xì)胞(sw480和sw620)中pld2mrna和蛋白的表達(dá)水平;最后建立結(jié)腸癌裸鼠皮下移植瘤模型,干擾hif1-α基因表達(dá),觀(guān)察裸鼠移植瘤生長(zhǎng)情況,并用免疫組化和westernblot檢測(cè)移植瘤中pld2蛋白的表達(dá)水平。結(jié)果:結(jié)腸癌細(xì)胞中(sw480,sw620,hct116及l(fā)ovo)hif1-α與pld2mrna和蛋白的表達(dá)水平較fhc明顯升高(p0.05);缺氧導(dǎo)致結(jié)腸癌細(xì)胞(sw480,sw620)中hif1-α與pld2的mrna和蛋白的表達(dá)水平較常氧升高(p0.05);成功構(gòu)建攜帶人hif1-α基因sirna的腺病毒載體,轉(zhuǎn)染結(jié)腸癌細(xì)胞(sw480,sw620),導(dǎo)致結(jié)腸癌細(xì)胞(sw480,sw620)中hif1-αmrna和蛋白的表達(dá)水平在常氧和缺氧條件下與各自對(duì)照組相比均降低(p0.05);干擾降低hif1-α基因的表達(dá)導(dǎo)致缺氧誘導(dǎo)的結(jié)腸癌細(xì)胞(sw480,sw620)中pld2蛋白和mrna的表達(dá)水平降低,與對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(p0.05);不同濃度氯化鈷上調(diào)hif1-α的表達(dá)導(dǎo)致結(jié)腸癌細(xì)胞(sw480,sw620)中pld2蛋白和mrna表達(dá)水平隨氯化鈷濃度升高而升高,與對(duì)照組(0μm)相比差異具有統(tǒng)計(jì)學(xué)意義(p0.05);結(jié)腸癌裸鼠皮下移植瘤生長(zhǎng)曲線(xiàn)顯示hif1-α干擾組移植瘤從第21天開(kāi)始腫瘤體積小于對(duì)照組(p0.05),免疫組化和westernblot結(jié)果均示,hif1-α干擾組移植瘤中的pld2蛋白表達(dá)的水平明顯低于空白對(duì)照組(p0.01)。結(jié)論:hif1-α上調(diào)體內(nèi)外缺氧微環(huán)境中結(jié)腸癌細(xì)胞pld2的mrna和蛋白的表達(dá)水平,阻斷hif1-α和pld2的表達(dá)對(duì)裸鼠移植瘤的生長(zhǎng)具有抑制作用。第三部分抑制pld2活性對(duì)缺氧微環(huán)境中結(jié)腸癌細(xì)胞凋亡的影響及機(jī)制研究目的:通過(guò)抑制pld2活性,探討其對(duì)缺氧微環(huán)境中結(jié)腸癌細(xì)胞凋亡的影響及機(jī)制。方法:首先用磷脂酶d活性實(shí)驗(yàn)檢測(cè)缺氧對(duì)結(jié)腸癌細(xì)胞(sw480、sw620、lovo和hct116)及fhc磷脂酶d活性的影響;再用磷脂酶d活性實(shí)驗(yàn)檢測(cè)不同濃度(50nm,100nm)的pld2活性抑制劑(vu0364739)對(duì)缺氧誘導(dǎo)的結(jié)腸癌細(xì)胞(sw480和sw620)及fhc中磷脂酶d活性的影響,三種細(xì)胞株均分為:常氧組(basal組)、缺氧組(control組)、缺氧+vu0364739(50nm)及缺氧+vu0364739(100nm);利用流式細(xì)胞術(shù)檢測(cè)抑制pld2活性對(duì)缺氧微環(huán)境中結(jié)腸癌細(xì)胞(sw480和w620)和fhc凋亡的影響,利用westernblot檢測(cè)抑制pld2活性對(duì)缺氧環(huán)境中結(jié)腸癌細(xì)胞(sw480和sw620)和fhc中survivin、bcl2、p-pi3k/pi3k及p-akt/akt蛋白表達(dá)水平的影響,分組同上;sw620和sw480均分為:缺氧對(duì)照組、缺氧+vu0364739(100nm)組、缺氧+ly294002(10μm)組、缺氧+ly294002(10μm)+vu0364739(100nm)組,各組細(xì)胞的凋亡率用流式細(xì)胞檢測(cè),用westernblot檢測(cè)各組細(xì)胞中survivin、bcl2、p-pi3k/pi3k及p-akt/akt蛋白的表達(dá)水平;最后用免疫組織化學(xué)染色檢測(cè)50例結(jié)腸癌組織及相對(duì)應(yīng)的正常結(jié)腸粘膜組織中pld2、survivin和bcl2的表達(dá),并用spearman相關(guān)分別分析pld2與survivin和bcl2表達(dá)水平之間的相關(guān)性。結(jié)果:結(jié)腸癌細(xì)胞(sw480、sw620、lovo和hct116)及fhc中缺氧組的pld活性較常氧組升高,兩組之間差異具有統(tǒng)計(jì)學(xué)意義(p0.05);vu0364739能顯著抑制缺氧誘導(dǎo)升高的結(jié)腸癌細(xì)胞(sw620和sw480)及fhc中pld活性,缺氧+vu0364739(50nm)組或者缺氧+vu0364739(100nm)組與缺氧對(duì)照組相比差異具統(tǒng)計(jì)學(xué)意義(p0.05);缺氧導(dǎo)致結(jié)腸癌細(xì)胞(sw620和sw480)及fhc的凋亡率較basal組增加(p0.05),而抑制pld2活性后導(dǎo)致缺氧誘導(dǎo)的結(jié)腸癌細(xì)胞(sw480和sw620)凋亡增加更加明顯,缺氧+vu0364739(50nm)組或者缺氧+vu0364739(100nm)組與缺氧對(duì)照組相比差異具統(tǒng)計(jì)學(xué)意義(p0.05),而對(duì)fhc凋亡無(wú)明顯影響,缺氧+vu0364739(50nm)組或者缺氧+vu0364739(100nm)組與缺氧對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05);缺氧導(dǎo)致結(jié)腸癌細(xì)胞(sw620和sw480)及fhc的survivin、bcl-2、p-pi3k/pi3k及p-akt/akt蛋白表達(dá)水平較basal組升高(p0.05),抑制pld2活性后導(dǎo)致缺氧誘導(dǎo)的結(jié)腸癌細(xì)胞(sw620和sw480)中survivin、bcl-2、p-pi3k/pi3k及p-akt/akt的蛋白表達(dá)水平又降低,缺氧+vu0364739(50nm)組或者缺氧+vu0364739(100nm)組與缺氧對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(p0.05),而對(duì)fhc中這些因子的蛋白表達(dá)水平無(wú)明顯影響,缺氧+vu0364739(50nm)組或者缺氧+vu0364739(100nm)組與缺氧對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05);抑制pi3k/akt信號(hào)通路、抑制pld2活性、或者兩種方法相結(jié)合均導(dǎo)致缺氧誘導(dǎo)的結(jié)腸癌細(xì)胞(sw480和sw620)中survivin、bcl-2、p-pi3k/pi3k及p-akt/akt蛋白表達(dá)水平均降低,同時(shí)伴有這些細(xì)胞凋亡率增加,這三組細(xì)胞與缺氧對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而缺氧+LY294002(10μM)組與缺氧+VU0364739(100nM)組、缺氧+LY294002(10μM)組與缺氧+LY294002(10μM)+VU0364739(100nM)組以及缺氧+VU0364739(100nM)組與缺氧+LY294002(10μM)+VU0364739(100nM)組間相比這些因子蛋白表達(dá)和凋亡率變化的差異均不明顯(P0.05);PLD2,Survivin及Bcl2在50例結(jié)腸癌組織中陽(yáng)性表達(dá)率為分別為64%、68%和60%,與正常組織中這些因子的陽(yáng)性表達(dá)率相比明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),同時(shí)相關(guān)性分析結(jié)果顯示PLD2與Survivin(r=0.379,P0.05),PLD2和Bcl2(r=0.405,P0.05)蛋白表達(dá)水平均呈正相關(guān)。結(jié)論:缺氧微環(huán)境中,PLD2可能通過(guò)激活PI3K/AKT信號(hào)通路上調(diào)Survivin和Bcl2的表達(dá)水平,從而抑制結(jié)腸癌細(xì)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.35

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