二甲雙胍聯(lián)合索拉非尼抑制小鼠肝癌原位瘤術(shù)后的復(fù)發(fā)和轉(zhuǎn)移
發(fā)布時(shí)間：2018-10-31 18:53
【摘要】：目的:手術(shù)切除是目前治療原發(fā)性肝癌的主要手段,但術(shù)后較高的復(fù)發(fā)和轉(zhuǎn)移率仍然是影響患者預(yù)后的一個(gè)重要因素。索拉非尼是晚期肝癌患者的標(biāo)準(zhǔn)用藥,然而,由于其潛在的毒副作用,許多患者不得不減少劑量或者被迫停止用藥。某些患者對(duì)索拉非尼的治療存在一定的抵抗作用;特別是減量用藥的患者可能增加轉(zhuǎn)移風(fēng)險(xiǎn),影響總體療效。因此,深入研究索拉非尼的耐藥機(jī)制以及尋找可能的方法來(lái)增加索拉非尼的療效顯得尤為重要。二甲雙胍是目前用于II型糖尿病治療的一線藥物,有文獻(xiàn)報(bào)道指出二甲雙胍能夠降低糖尿病患者某些腫瘤的發(fā)生率。本研究旨在觀察二甲雙胍能否增敏索拉非尼的療效,進(jìn)而抑制荷瘤小鼠肝癌手術(shù)切除后的轉(zhuǎn)移與復(fù)發(fā),探討其作用機(jī)制。內(nèi)容:研究二甲雙胍聯(lián)合索拉非尼對(duì)肝癌細(xì)胞增殖和凋亡的影響以及對(duì)小鼠肝癌手術(shù)切除后復(fù)發(fā)和轉(zhuǎn)移的影響;探討二甲雙胍增效索拉非尼的作用機(jī)制。方法:應(yīng)用CCK8(Cell Counting Kit-8)細(xì)胞活力實(shí)驗(yàn)檢測(cè)二甲雙胍和索拉非尼單獨(dú)用藥以及二者聯(lián)合用藥對(duì)MHCC97H肝癌細(xì)胞增殖的影響;采用Annexin V-FITC細(xì)胞凋亡實(shí)驗(yàn)檢測(cè)二甲雙胍和索拉非尼單獨(dú)用藥以及二者聯(lián)合用藥對(duì)細(xì)胞凋亡的影響;應(yīng)用Western blot技術(shù)在MHCC97H細(xì)胞中檢測(cè)二甲雙胍和索拉非尼單獨(dú)用藥以及聯(lián)合用藥后,HIF2α,TIP30,E-Cadherin,N-Cadherin,pAMPK等在蛋白水平的表達(dá)情況;采用RNA敲減以及核染色質(zhì)免疫共沉淀等實(shí)驗(yàn)進(jìn)一步研究HIF-2α與TIP30之間的關(guān)系。體內(nèi)實(shí)驗(yàn),手術(shù)切除小鼠肝癌原位瘤后,給予小鼠口服二甲雙胍和索拉非尼,觀察不同治療組復(fù)發(fā)的腫瘤大小以及肺內(nèi)轉(zhuǎn)移灶的數(shù)目;應(yīng)用免疫組化染色的方法對(duì)不同治療組中與細(xì)胞增殖相關(guān)的Ki67、凋亡相關(guān)的TUNEL以及微血管密度相關(guān)的CD31等指標(biāo)進(jìn)行檢測(cè);應(yīng)用Western blot技術(shù)檢測(cè)小鼠肝癌組織中HIF2α,TIP30,E-Cadherin,N-Cadherin及pAMPK等蛋白水平的表達(dá)情況。結(jié)果:1.低劑量索拉非尼通過(guò)上調(diào)HIF-2α來(lái)抑制TIP30的表達(dá)進(jìn)而促進(jìn)腫瘤的侵襲和轉(zhuǎn)移。2.聯(lián)合應(yīng)用二甲雙胍和索拉非尼抑制HIF-2α蛋白的表達(dá)。3.聯(lián)合應(yīng)用二甲雙胍和索拉非尼促進(jìn)肝癌細(xì)胞的凋亡、抑制肝癌細(xì)胞的增殖及降低微血管密度。4.二甲雙胍通過(guò)增敏索拉非尼抑制荷瘤小鼠肝癌術(shù)后的復(fù)發(fā)和轉(zhuǎn)移。結(jié)論:二甲雙胍通過(guò)調(diào)控HIF-2α及TIP30的表達(dá)來(lái)增敏索拉非尼的療效,進(jìn)而抑制小鼠肝癌手術(shù)切除后的復(fù)發(fā)和轉(zhuǎn)移。
[Abstract]:Objective: surgical resection is the main method for the treatment of primary liver cancer, but the high recurrence and metastasis rate is still an important factor affecting the prognosis of the patients. Solafenib is the standard drug for patients with advanced liver cancer. However, because of its potential side effects, many patients have to reduce doses or be forced to stop. Some patients have some resistance to the treatment of Solafenib, especially the patients with reduced dosage may increase the risk of metastasis and affect the overall efficacy. Therefore, it is very important to study the drug resistance mechanism of sorafenib and to find possible ways to increase the efficacy of sorafenib. Metformin is a first-line drug used in the treatment of type II diabetes. It has been reported that metformin can reduce the incidence of certain tumors in patients with diabetes. The purpose of this study was to observe whether metformin could increase the sensitivity of solafenil and to inhibit the metastasis and recurrence of liver cancer in mice after resection, and to explore its mechanism. Content: to study the effects of metformin combined with Solafenib on the proliferation and apoptosis of hepatoma cells and on the recurrence and metastasis after hepatectomy in mice, and to explore the mechanism of metformin combined with solafenil. Methods: the effects of metformin and Solafenib alone and their combination on the proliferation of MHCC97H hepatoma cells were detected by CCK8 (Cell Counting Kit-8 cell viability test. The effect of metformin and solafenib on apoptosis was detected by Annexin V-FITC cell apoptosis assay. Western blot technique was used to detect the expression of HIF2 偽, TIP30,E-Cadherin,N-Cadherin,pAMPK and other proteins in MHCC97H cells treated with metformin and solafenib alone or in combination. The relationship between HIF-2 偽 and TIP30 was further studied by RNA knockout and chromatin immunoprecipitation. In vivo experiment, mice were given metformin and solafenib orally after surgical resection of hepatoma in situ. The size of recurrent tumor and the number of metastatic foci in lung were observed in different treatment groups. Immunohistochemical staining was used to detect TUNEL associated with Ki67, apoptosis and microvessel density (CD31) in different treatment groups. The expression of HIF2 偽, TIP30,E-Cadherin,N-Cadherin and pAMPK in mouse liver cancer was detected by Western blot technique. The result is 1: 1. Low dose sorafenil inhibits the expression of TIP30 by up-regulating HIF-2 偽 and promotes tumor invasion and metastasis. 2. The expression of HIF-2 偽 protein was inhibited by metformin combined with solafenil. Combined use of metformin and Solafenil promoted apoptosis of hepatoma cells, inhibited proliferation of hepatoma cells and decreased microvessel density. Metformin inhibits the recurrence and metastasis of tumor-bearing mice after operation by sensitizing solafenil. Conclusion: metformin enhances the efficacy of solafenib by regulating the expression of HIF-2 偽 and TIP30, and then inhibits the recurrence and metastasis after hepatectomy in mice.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7
，
本文編號(hào)：2303219
[Abstract]:Objective: surgical resection is the main method for the treatment of primary liver cancer, but the high recurrence and metastasis rate is still an important factor affecting the prognosis of the patients. Solafenib is the standard drug for patients with advanced liver cancer. However, because of its potential side effects, many patients have to reduce doses or be forced to stop. Some patients have some resistance to the treatment of Solafenib, especially the patients with reduced dosage may increase the risk of metastasis and affect the overall efficacy. Therefore, it is very important to study the drug resistance mechanism of sorafenib and to find possible ways to increase the efficacy of sorafenib. Metformin is a first-line drug used in the treatment of type II diabetes. It has been reported that metformin can reduce the incidence of certain tumors in patients with diabetes. The purpose of this study was to observe whether metformin could increase the sensitivity of solafenil and to inhibit the metastasis and recurrence of liver cancer in mice after resection, and to explore its mechanism. Content: to study the effects of metformin combined with Solafenib on the proliferation and apoptosis of hepatoma cells and on the recurrence and metastasis after hepatectomy in mice, and to explore the mechanism of metformin combined with solafenil. Methods: the effects of metformin and Solafenib alone and their combination on the proliferation of MHCC97H hepatoma cells were detected by CCK8 (Cell Counting Kit-8 cell viability test. The effect of metformin and solafenib on apoptosis was detected by Annexin V-FITC cell apoptosis assay. Western blot technique was used to detect the expression of HIF2 偽, TIP30,E-Cadherin,N-Cadherin,pAMPK and other proteins in MHCC97H cells treated with metformin and solafenib alone or in combination. The relationship between HIF-2 偽 and TIP30 was further studied by RNA knockout and chromatin immunoprecipitation. In vivo experiment, mice were given metformin and solafenib orally after surgical resection of hepatoma in situ. The size of recurrent tumor and the number of metastatic foci in lung were observed in different treatment groups. Immunohistochemical staining was used to detect TUNEL associated with Ki67, apoptosis and microvessel density (CD31) in different treatment groups. The expression of HIF2 偽, TIP30,E-Cadherin,N-Cadherin and pAMPK in mouse liver cancer was detected by Western blot technique. The result is 1: 1. Low dose sorafenil inhibits the expression of TIP30 by up-regulating HIF-2 偽 and promotes tumor invasion and metastasis. 2. The expression of HIF-2 偽 protein was inhibited by metformin combined with solafenil. Combined use of metformin and Solafenil promoted apoptosis of hepatoma cells, inhibited proliferation of hepatoma cells and decreased microvessel density. Metformin inhibits the recurrence and metastasis of tumor-bearing mice after operation by sensitizing solafenil. Conclusion: metformin enhances the efficacy of solafenib by regulating the expression of HIF-2 偽 and TIP30, and then inhibits the recurrence and metastasis after hepatectomy in mice.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7
，
本文編號(hào)：2303219
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