本文選題：乳腺癌 + MCF-7細胞　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景目的近年來,我國乳腺癌的發(fā)病率和死亡率均呈持續(xù)上升趨勢,其中雌激素依賴型乳腺癌發(fā)病率約占50%~70%。內(nèi)分泌治療對雌激素依賴型乳腺癌具有良好療效,但其治療后期轉(zhuǎn)移率高,導(dǎo)致治療效果不佳。作為腫瘤細胞有氧糖酵解途徑的關(guān)鍵酶,磷酸葡萄糖異構(gòu)酶(phosphoglucose isomerase,PGI)在糖酵解代謝過程中調(diào)控葡萄糖-6-磷酸酶與果糖-6-磷酸酶之間的互相轉(zhuǎn)化。PGI分泌到細胞外后被稱為自分泌因子(autocrine motility factors,AMF),AMF與自分泌因子受體相結(jié)合,促進腫瘤的增殖、浸潤和轉(zhuǎn)移。在乳腺癌、肺癌等多種腫瘤中均可見PGI的異常表達,而PGI的異常表達可能還與腫瘤惡性程度密切相關(guān)。缺氧誘導(dǎo)因子(hypoxia inducible factor-1,HIF-1)是低氧條件下腫瘤細胞的中心調(diào)節(jié)因子,參與了腫瘤血管形成、細胞增殖和細胞粘附等一系列過程。HIF-1包括HIF-1α和HIF-1β,HIF-1α主要調(diào)控腫瘤的生長和轉(zhuǎn)移,此外,還可以調(diào)控HKII、PGI、PKM2和LDHA等基因的轉(zhuǎn)錄和表達。新近研究表明:糖酵解關(guān)鍵酶中的PKM2可反向調(diào)控HIF-1α的表達。PGI是否也有類似的作用?PGI與HIF-1α二者相關(guān)性如何?PGI是怎樣進行調(diào)控的?目前尚未見報道。因此,本研究對乳腺癌中的PGI和HIF-1α進行檢測,探究兩者在乳腺癌中的表達情況,通過RNAi技術(shù)干擾PGI的表達,進而檢測干擾PGI對HIF-1α的表達以及對MCF-7細胞增殖、凋亡和糖酵解的影響,并對其相關(guān)調(diào)控機制進行初步探究。方法第一部分收集30例乳腺癌患者的癌組織及其相應(yīng)癌旁組織,通過PCR、免疫組化檢測癌組織與癌旁組織中PGI及HIF-1α的表達;用PCR和Western blot檢測乳腺癌細胞MCF-7及正常乳腺細胞MCF-10A中PGI以及HIF-1α的表達。第二部分采用慢病毒質(zhì)粒進行感染,構(gòu)建干擾PGI的MCF-7細胞并篩選穩(wěn)轉(zhuǎn)株,采用免疫熒光、RT-PCR和Western blot檢測感染效果,并采用Western blot技術(shù)檢測干擾PGI對MCF-7細胞中HIF-1α蛋白表達的影響。光學(xué)顯微鏡下觀察干擾PGI對細胞生長的影響;CCK-8法檢測干擾PGI對細胞增殖的抑制作用;Hochest染色以及FCM檢測干擾PGI對細胞凋亡的影響;同時通過FCM檢測干擾PGI對MCF-7細胞周期的影響;隨后,采用Western blot檢測干擾PGI對細胞周期以及凋亡通路蛋白的影響。通過乳酸含量檢測法檢測干擾PGI對MCF-7細胞乳酸產(chǎn)量的影響,并采用Western blot檢測干擾PGI對腫瘤糖酵解相關(guān)酶表達的影響。結(jié)果1.乳腺癌組織中的PGI和HIF-1α均明顯高于癌旁組織。2.乳腺癌細胞MCF-7中PGI和HIF-1α的含量高于正常乳腺細胞MCF-10A。3.乳腺癌細胞MCF-7中干擾PGI后HIF-1α的表達量隨之減少。4.干擾PGI明顯抑制MCF-7細胞的增殖能力。5.干擾PGI可將MCF-7細胞的周期阻滯在G0/G1期,各組G0/G1期所占比例如下:Control組(43.98±0.45)%、Mock組的(45.57±1.16)%、PGI-si RNA組(66.12±1.12)%,差異均具有統(tǒng)計學(xué)意義(P0.01)。6.FCM檢測結(jié)果顯示:Control組、Mock組和PGI-si RNA組凋亡率分別為(3.45±0.12)%、(4.35±0.08)%和(11.89±0.65)%,PGI-si RNA組細胞凋亡率高于Control組和Mock組(P0.01)。7.Hochest 33258染色結(jié)果顯示:Control組和Mock組細胞呈均勻的藍色熒光;PGI-si RNA組細胞可見不同程度的染色質(zhì)凝集、核濃縮且發(fā)出較強的藍白色熒光等典型的凋亡形態(tài)學(xué)改變。8.乳酸含量檢測結(jié)果顯示:Control組、Mock組和PGI-si RNA組乳酸濃度分別為(3.801±0.346)mmol/L、(3.863±0.194)mmol/L和(1.133±0.194)mmol/L,PGI-si RNA組乳酸濃度低于Control組和Mock組(P0.01)。結(jié)果提示:干擾PGI可使MCF-7細胞的乳酸總產(chǎn)量降低。9.Western blot檢測:干擾PGI后凋亡相關(guān)蛋白Bax和Cleaved Caspase-3表達增加,抑凋亡蛋白Bcl-2表達減少;細胞周期相關(guān)蛋白Cyclin D1和CDK4及糖酵解相關(guān)酶HKII、PKM2和LDHA的表達均減少;PI3K、AKT和m TOR蛋白的表達亦減少。結(jié)論1.乳腺癌中HIF-1α與PGI均異常增高。2.干擾PGI后HIF-1α的表達量減少,同時可以抑制MCF-7細胞的糖酵解和細胞增殖并促進細胞的凋亡,其相關(guān)調(diào)控機制可能與HIF-1α介導(dǎo)的PI3K/AKT/m TOR信號通路有關(guān)。
[Abstract]:Background objective in recent years, the incidence and mortality of breast cancer in China are on the upward trend, and the incidence of estrogen dependent breast cancer is about 50%~70%. endocrine therapy for estrogen dependent breast cancer, but its late stage of treatment is high, resulting in poor treatment effect. As a tumor cell aerobic glycolysis way The key enzyme, phosphoglucose isomerase, PGI, regulates the transformation of glucose -6- phosphatase from fructose -6- phosphatase between glucose -6- phosphatase and fructose -6- phosphatase during the secretion of.PGI to the cell and is called the autocrine (autocrine motility factors, AMF), and the AMF is combined with the autocrine receptor to promote the tumor. Proliferation, infiltration and metastasis. The abnormal expression of PGI in all kinds of tumors such as breast cancer and lung cancer, and the abnormal expression of PGI may also be closely related to the malignancy of the tumor. The hypoxia inducible factor (hypoxia inducible factor-1, HIF-1) is the central regulatory factor of the tumor cells under the hypoxic condition, and participates in the angiogenesis and cell proliferation of the tumor. A series of processes, such as cell adhesion,.HIF-1 including HIF-1 alpha and HIF-1 beta, HIF-1 alpha mainly regulate the growth and metastasis of tumor, in addition, it can regulate the transcription and expression of HKII, PGI, PKM2 and LDHA genes. What is the correlation of alpha two? How is PGI regulated? So far, there is no report on the expression of PGI and HIF-1 alpha in breast cancer. The expression of both in breast cancer and the expression of PGI are investigated by RNAi technique, and then the expression of HIF-1 alpha by interfering PGI and the proliferation, apoptosis and glycolysis of MCF-7 cells are detected. In the first part, we collected 30 cases of breast cancer tissues and their corresponding para cancerous tissues, and detected the expression of PGI and HIF-1 alpha in the cancer tissues and para cancerous tissues by PCR, and used PCR and Western blot to detect MCF-7 in mammary gland cancer cells and PGI in MCF-10A of normal breast cells. And the expression of HIF-1 alpha. The second part was infected by the lentivirus plasmid, constructed the MCF-7 cells that interfered with PGI and screened the stable transgenic plants. The effect of the infection was detected by immunofluorescence, RT-PCR and Western blot, and the effect of PGI on the expression of HIF-1 alpha protein in MCF-7 cells was detected by Western blot technique. The interference PGI was observed under the optical microscope. The effect of cell growth; CCK-8 assay was used to detect the inhibitory effect of interfering PGI on cell proliferation; Hochest staining and FCM detected the effect of PGI on cell apoptosis; meanwhile, the effect of PGI on the cell cycle of MCF-7 was detected by FCM; and then Western blot was used to detect the effect of interference PGI on the cell cycle and apoptosis pathway protein. The effect of interference PGI on the output of lactic acid in MCF-7 cells was detected by the content detection method, and the effects of PGI on the expression of glycolysis related enzymes were detected by Western blot. Results 1. the PGI and HIF-1 alpha in the breast cancer tissues were significantly higher than those of.2. breast cancer cells in the Para cancerous tissue, and the PGI and HIF-1 alpha levels were higher than those of normal mammary gland cell MCF-10A.3. milk. The expression of HIF-1 alpha in the adenocarcinoma cell MCF-7 decreased after the interference of the.4. interference PGI significantly inhibited the proliferation of MCF-7 cells and inhibited the MCF-7 cell proliferation ability.5. interference PGI could block the MCF-7 cell cycle in G0/G1 period. The proportion of G0/G1 phase in each group was as follows: Control group (43.98 + 0.45)%, (45.57 + 1.16)%, 66.12 + 1.12%, and the difference was all The results of P0.01.6.FCM detection showed that the apoptosis rate of group Control, Mock and PGI-si RNA group was (3.45 + 0.12)%, (4.35 + 0.08)% and (11.89 + 0.65)%, and the apoptosis rate of PGI-si RNA group was higher than that of Control group and Mock group (P0.01).7.Hochest 33258 staining results showed that the cells and cells showed uniform blue fluorescence. The group cells showed different degrees of chromatin agglutination, nuclear concentration and strong blue white fluorescence and other typical morphological changes of.8. lactic acid. The results of.8. lactic acid content in Control group, Mock group and PGI-si RNA group were (3.801 + 0.346) mmol/L, (3.863 + 0.194) mmol/L and (1.133 + 0.194) mmol/L, and PGI-si RNA group was strong lactic acid. The results were lower than that of group Control and group Mock (P0.01). The results suggest that interference of PGI can reduce the total output of lactic acid in MCF-7 cells by.9.Western blot detection: the expression of Bax and Cleaved Caspase-3, and the decrease of apoptotic protein Bcl-2 expression after PGI, decrease the expression of apoptosis protein Bcl-2 expression. The expression of PI3K, AKT and m TOR also decreased. Conclusion 1. breast cancer, HIF-1 alpha and PGI all increase the decrease of HIF-1 alpha expression after.2. interference PGI, and can inhibit the glycolysis and cell proliferation of MCF-7 cells and promote cell apoptosis. The related regulatory mechanism may be associated with HIF-1 alpha mediated PI3K/AKT/m signaling. The road is related.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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