二甲双胍联合索拉非尼抑制小鼠肝癌原位瘤术后的复发和转移
发布时间：2018-10-31 18:53
【摘要】：目的:手术切除是目前治疗原发性肝癌的主要手段,但术后较高的复发和转移率仍然是影响患者预后的一个重要因素。索拉非尼是晚期肝癌患者的标准用药,然而,由于其潜在的毒副作用,许多患者不得不减少剂量或者被迫停止用药。某些患者对索拉非尼的治疗存在一定的抵抗作用;特别是减量用药的患者可能增加转移风险,影响总体疗效。因此,深入研究索拉非尼的耐药机制以及寻找可能的方法来增加索拉非尼的疗效显得尤为重要。二甲双胍是目前用于II型糖尿病治疗的一线药物,有文献报道指出二甲双胍能够降低糖尿病患者某些肿瘤的发生率。本研究旨在观察二甲双胍能否增敏索拉非尼的疗效,进而抑制荷瘤小鼠肝癌手术切除后的转移与复发,探讨其作用机制。内容:研究二甲双胍联合索拉非尼对肝癌细胞增殖和凋亡的影响以及对小鼠肝癌手术切除后复发和转移的影响;探讨二甲双胍增效索拉非尼的作用机制。方法:应用CCK8(Cell Counting Kit-8)细胞活力实验检测二甲双胍和索拉非尼单独用药以及二者联合用药对MHCC97H肝癌细胞增殖的影响;采用Annexin V-FITC细胞凋亡实验检测二甲双胍和索拉非尼单独用药以及二者联合用药对细胞凋亡的影响;应用Western blot技术在MHCC97H细胞中检测二甲双胍和索拉非尼单独用药以及联合用药后,HIF2α,TIP30,E-Cadherin,N-Cadherin,pAMPK等在蛋白水平的表达情况;采用RNA敲减以及核染色质免疫共沉淀等实验进一步研究HIF-2α与TIP30之间的关系。体内实验,手术切除小鼠肝癌原位瘤后,给予小鼠口服二甲双胍和索拉非尼,观察不同治疗组复发的肿瘤大小以及肺内转移灶的数目;应用免疫组化染色的方法对不同治疗组中与细胞增殖相关的Ki67、凋亡相关的TUNEL以及微血管密度相关的CD31等指标进行检测;应用Western blot技术检测小鼠肝癌组织中HIF2α,TIP30,E-Cadherin,N-Cadherin及pAMPK等蛋白水平的表达情况。结果:1.低剂量索拉非尼通过上调HIF-2α来抑制TIP30的表达进而促进肿瘤的侵袭和转移。2.联合应用二甲双胍和索拉非尼抑制HIF-2α蛋白的表达。3.联合应用二甲双胍和索拉非尼促进肝癌细胞的凋亡、抑制肝癌细胞的增殖及降低微血管密度。4.二甲双胍通过增敏索拉非尼抑制荷瘤小鼠肝癌术后的复发和转移。结论:二甲双胍通过调控HIF-2α及TIP30的表达来增敏索拉非尼的疗效,进而抑制小鼠肝癌手术切除后的复发和转移。
[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 伪, 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 伪 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 伪, 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 伪 and promotes tumor invasion and metastasis. 2. The expression of HIF-2 伪 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 伪 and TIP30, and then inhibits the recurrence and metastasis after hepatectomy in mice.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
，
本文编号：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 伪, 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 伪 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 伪, 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 伪 and promotes tumor invasion and metastasis. 2. The expression of HIF-2 伪 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 伪 and TIP30, and then inhibits the recurrence and metastasis after hepatectomy in mice.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
，
本文编号：2303219
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