BTG2与肝细胞癌放疗敏感性的相关研究

发布时间：2018-05-12 19:06

本文选题：肝细胞癌 + BTG2　；参考：《第三军医大学》2015年博士论文

【摘要】：研究背景肝细胞癌(Hepatocellular carcinoma,HCC)是世界上发病率排名第六的癌症,排在癌症导致人类死亡的第三位。由于缺乏明确的早期诊断标志物、明显的临床症状等,肝癌患者就诊时通常已是晚期,不少患者失去了手术机会,总的5年生存期较短。因此,如何提高肝细胞癌患者的临床诊断率和治疗疗效是提高其生存期的关键。临床上肝细胞癌的治疗主要以手术切除癌症病灶、肝移植、化疗、射频消融、放射治疗和分子靶向药物如索拉菲尼等为主。放射治疗主要用于和其它治疗手段相结合的综合治疗,目前为止,由于肝癌细胞较低的放疗敏感性及肝脏较低的耐受剂量等原因,肝细胞癌的放射治疗效果仍不能令人满意。因此,研究如何增加肝细胞癌的放射治疗效应在临床上有着重要意义。B细胞易位基因2(B cell translocation gene 2,BTG2)是一个瞬时早期反应基因,属于抗增殖基因家族,BTG2在多种组织和器官中均有表达,如肺组织、脾、胸腺、胃肠组织等。其参与了细胞分化、生长、凋亡和DNA损伤修复等多种生物学功能。既往研究表明,BTG2被公认为肿瘤抑制基因,其在不少癌症中表达减少或者缺失,从而在肿瘤的发生发展中发挥了重要的作用。放疗射线对细胞的损伤可通过直接作用于DNA分子,导致DNA链断裂,研究表明p53可以增强口腔癌和胰腺癌等肿瘤的放疗敏感性,而BTG2是DNA损伤细胞反应通路的p53依赖性的成分之一,提示BTG2可能参与放疗射线对细胞损伤的过程。我们课题组近年来致力于BTG2的研究,发现BTG2参与了肝癌的发生、肝细胞的再生以及肝损伤的修复等一系列复杂的病理生理过程,本研究将继续通过相关的实验探索BTG2与肝细胞癌放疗敏感性的关系,以求能够对肝细胞癌患者的放射治疗提供新的思路和理论依据。目的通过临床肝细胞癌患者的肿瘤组织样本评估BTG2的表达与肝细胞癌患者预后的相关性;并通过体外细胞实验及裸鼠成瘤模型体内实验等方法检测BTG2对肝细胞癌放射治疗敏感性的影响,以期为肝细胞癌的放射治疗相关靶基因的研究提供一定的理论依据。方法1.BTG2的表达与肝细胞癌患者预后的相关性:肝细胞癌患者肿瘤组织样本收集、组织芯片的制作及免疫组化分析,44名肝细胞癌患者全部来自重庆市第三军医大学大坪医院,这些患者依据世界卫生组织标准均被病理学诊断为HCC,所有的患者在手术前都没有进行过化疗或者放射治疗。选择新鲜手术标本进行固定和预处理,按照步骤制作组织芯片。常规的免疫组织化学方法检测各组织标本BTG2的表达情况,统计分析BTG2的表达与肝细胞癌患者预后的相关性。2.体外细胞实验检测BTG2对肝细胞癌放疗敏感性的影响:细胞株的选择、培养及转染,选用人的肝细胞癌细胞株Huh7进行常规细胞培养,用Lipofectamine 2000(Invitrogen)转染真核表达载体p EGFP-N1-BTG2及空载体作为对照。转染细胞经蛋白提取及免疫蛋白印迹分析各组BTG2蛋白的表达情况,RT-PCR分析各组BTG2的RNA水平,筛选并鉴定出BTG2稳定表达的细胞株。对BTG2稳定转染的细胞株进行放射线处理(8Gy,6MV的放射线),细胞继续培养24小时后用流式细胞仪检测细胞的凋亡及细胞周期,并进行细胞增殖实验。3.裸鼠成瘤模型检测BTG2对肝细胞癌放疗敏感性的影响:购买一般情况较好、性状基本一致的免疫缺陷裸鼠12只,随机分为两组:BTG2过表达组和空载体对照组,每组6只。待BTG2稳定表达的Huh7细胞培养到较好的细胞状态,以每只裸鼠1×107个的细胞量注射入裸鼠右上腋皮肤下,每天观察裸鼠的一般情况及成瘤情况。实验中注射第10天开始成瘤,之后每隔一天测量裸鼠肿瘤体积并记录。在两组裸鼠肿瘤直径大约1cm左右(注射后第21天)行放射线处理(8Gy,6MV的放射线),以后每隔2天照射裸鼠一次,共照射3次。照射期间注意观察裸鼠的一般情况及肿瘤体积并记录。在第30天时处死裸鼠,取肿瘤标本进行后续实验。肿瘤组织分别进行Ki67免疫组织化学染色、TUNEL细胞凋亡及蛋白印迹法凋亡相关蛋白Bax等检测。结果1.BTG2的表达与肝细胞癌患者预后呈正相关:将44例肝细胞癌患者组织标本进行组织芯片制作及分析。根据免疫组化染色结果将患者分为BTG2高表达组和低表达组,并进行生存期分析。结果发现BTG2核高表达组的患者较BTG2核低表达组的患者具有较好的生存率。提示BTG2的表达与肝细胞癌患者预后呈显著正相关。2.体外细胞实验证实BTG2增强肝细胞癌放疗敏感性:人肝细胞癌细胞株Huh7成功转染p EGFP-N1-BTG2真核表达载体,并经蛋白免疫印记证实成功构建BTG2稳定过表达的细胞株(BTG2过表达转染组是空载体转染组的3.6倍,P0.05)。各组细胞经放射线处理后,BTG2过表达组的细胞增殖能力较空载体组细胞的增值能力明显降低,空载体组和未转染组之间无显著性差异。进一步研究发现,BTG2过表达组细胞的凋亡率较空载体组细胞的凋亡率增加约2.5倍(55.9%:24.6%,P0.05),空载体组和未转染组的细胞凋亡率无显著性差异(24.6%:17.2%,P0.05)。电离辐射后,各组细胞均出现不同程度的G1期细胞阻滞,BTG2过表达组G1期细胞百分率较空载体组和空白对照组均有所提高,但是无统计学差异(P0.05)。以上结果证实BTG2可增强肝细胞癌的放疗敏感性。3.裸鼠成瘤模型证实BTG2促进肝细胞癌放射治疗敏感性:在成功构建裸鼠移植瘤模型的基础上,我们发现,与空载体组相比,BTG2过表达组的肝癌细胞肿瘤生长并未受到明显影响。在放射线照射后(实验中观察发现,裸鼠在注射肿瘤细胞10天后开始成瘤,分别在第21天、24天和27天进行裸鼠麻醉后的放射线处理8Gy每次),BTG2过表达组的裸鼠肿瘤体积较空载体组明显缩小。经TUNEL检测发现,BTG2同样影响了肿瘤细胞的凋亡,结果显示:BTG2转染组的凋亡细胞数量较空载体组明显增多(BTG2组较对照组增加39.8%,P0.05)。进一步经Western blot检测发现BTG2过表达组的凋亡相关蛋白Bax的表达是转染空载体组的1.5倍。以上结果进一步证实BTG2可提高肝细胞癌对电离辐射的敏感性。结论1.BTG2核表达高的肝细胞癌患者的生存率明显高于BTG2核表达低的肝细胞癌患者,提示BTG2的表达与肝细胞癌患者预后呈显著正相关。进一步证实BTG2可能作为一种抑癌基因在肝细胞癌的发生发展中发挥重要作用。2.电离辐射后,BTG2过表达的肝细胞癌细胞株Huh7的增殖减慢,BTG2过表达组的细胞凋亡明显增加。提示BTG2可以增强肝细胞癌对电离辐射的敏感性。3.电离辐射后,BTG2过表达组裸鼠移植瘤对电离辐射敏感性显著高于对照组,且其肿瘤组织中细胞凋亡程度也明显强于对照组。结果进一步证实BTG2能够增强肝细胞癌的放疗敏感性。
[Abstract]:Background Hepatocellular carcinoma (HCC) is the sixth largest incidence of cancer in the world, which is ranked third in the death of cancer. Due to the lack of clear early diagnosis markers and obvious clinical symptoms, patients with liver cancer are usually late, and many patients have lost the opportunity to operate for a total of 5 years. It is short. Therefore, how to improve the clinical diagnosis and therapeutic effect of hepatocellular carcinoma is the key to improve the survival time. The main clinical treatment of hepatocarcinoma is surgical resection of cancer focus, liver transplantation, chemotherapy, radiofrequency ablation, radiofrequency therapy and molecular targeting drugs such as Suo Rafini. Combined therapy, so far, the effect of radiotherapy on hepatocellular carcinoma is still not satisfactory due to low radiation sensitivity of liver cancer cells and low tolerance dose of liver. Therefore, it is important to study how to increase the radiation therapy effect of hepatocellular carcinoma (.B cell translocation gene 2 (B CEL). L translocation gene 2, BTG2) is a transient early response gene, belonging to the antiproliferative gene family. BTG2 is expressed in many tissues and organs, such as lung tissue, spleen, thymus and gastrointestinal tissue. It participates in a variety of biological functions, such as cell differentiation, growth, apoptosis and repair of DNA damage. Previous studies have shown that BTG2 is recognized as tumor suppressor. Gene, whose expression is reduced or missing in many cancers, plays an important role in the development of the tumor. The damage to the cells by radiation radiation can be directly acted on the DNA molecule, causing DNA strand breaks. The study shows that p53 can enhance the radiation sensitivity of the tumors such as oral and pancreatic cancer, and BTG2 is a DNA damaged cell. One of the p53 dependent components of the reaction pathway suggests that BTG2 may be involved in the process of cell damage by radiation radiation. Our research group has recently devoted to the study of BTG2, and found that BTG2 has been involved in a series of complex pathophysiological processes, such as the occurrence of liver cancer, the regeneration of hepatocytes and the repair of liver damage. This study will continue to pass through the relevant research. The relationship between BTG2 and radiosensitivity of hepatocellular carcinoma was investigated in order to provide new ideas and theoretical basis for the radiotherapy of patients with hepatocellular carcinoma. Objective to evaluate the correlation between the expression of BTG2 and the prognosis of patients with hepatocellular carcinoma through the tumor tissue samples of the patients with clinical hepatocellular carcinoma, and through in vitro cell experiments and nude mice model. The effects of BTG2 on radiosensitivity of hepatocellular carcinoma were detected in order to provide a theoretical basis for the study of target genes related to radiation therapy for hepatocellular carcinoma. Method the correlation between the expression of 1.BTG2 and the prognosis of the patients with hepatocellular carcinoma: the collection of tissue samples, the fabrication and exemptions of the tissue microarray in the patients with hepatocellular carcinoma All 44 patients with hepatocellular carcinoma were from the Daping Hospital of Third Military Medical University in Chongqing. These patients were diagnosed as HCC by pathology according to the WHO standards. All patients had no chemotherapy or radiotherapy before the operation. Tissue microarray. Routine immunohistochemical method was used to detect the expression of BTG2 in tissue specimens, the correlation between the expression of BTG2 and the prognosis of patients with hepatocellular carcinoma.2. in vitro cell test was used to detect the effect of BTG2 on the sensitivity of radiotherapy for hepatocellular carcinoma: cell line selection, culture and transfection, and the selection of human hepatocellular carcinoma cell line Huh7 Routine cell culture was performed with Lipofectamine 2000 (Invitrogen) transfected with eukaryotic expression vector p EGFP-N1-BTG2 and empty carrier as control. The transfected cells were extracted and immunoblotting was used to analyze the expression of BTG2 protein in each group. The RNA level of BTG2 in each group was analyzed by RT-PCR, and the stable expression of BTG2 cell line was screened and identified. The stability of BTG2 was stable. The transfected cell lines were treated with radiation (8Gy, 6MV radiation). Cell apoptosis and cell cycle were detected by flow cytometry for 24 hours after continuous culture, and cell proliferation experiment was carried out to detect the effect of BTG2 on the sensitivity of radiotherapy for hepatocellular carcinoma in.3. nude mice. 12 nude mice were randomly divided into two groups: the BTG2 overexpression group and the empty carrier control group, 6 in each group. The stable expression of Huh7 cells in each group was cultured to the better cell status, and 1 x 107 cells in nude mice were injected into the right upper armpit of nude mice, and the general situation and tumor formation of nude mice were observed every day. The experiment was injected for tenth days. The tumor was measured and recorded every other day in nude mice. In the two groups of nude mice, the diameter of the tumor was about 1cm (twenty-first days after the injection). The radiographic treatment (8Gy, 6MV radiation) was performed. The nude mice were irradiated once every 2 days and were irradiated for 3 times. The general situation and tumor volume of nude mice were observed and recorded during the irradiation. The naked mice were killed at thirtieth days. The tumor specimens were taken for follow-up experiments. The tumor tissues were stained with Ki67 immunohistochemical staining, TUNEL cell apoptosis and Western blot apoptosis related protein Bax. Results the expression of 1.BTG2 was positively correlated with the prognosis of patients with hepatocellular carcinoma: 44 cases of hepatocellular carcinoma tissue specimens were made and analyzed by tissue chip. The results of immunohistochemical staining were divided into BTG2 high expression group and low expression group, and the survival time analysis was carried out. The results showed that the patients with high expression of BTG2 nucleus had better survival rate than those in the BTG2 nuclear low expression group. It was suggested that the expression of BTG2 was positively correlated with the prognosis of the patients with hepatocellular carcinoma and.2. in vitro cell experiment confirmed that BTG2 enhanced the liver cells. Cancer radiation sensitivity: human hepatocellular carcinoma cell line Huh7 successfully transfected P EGFP-N1-BTG2 eukaryotic expression vector, and confirmed the successful construction of BTG2 stably overexpressed cell line by protein immuno imprint (3.6 times of BTG2 overexpression transfection group as empty carrier transfection group, P0.05). Cell proliferation ability of BTG2 overexpression group after radiation treatment in each group There was no significant difference between the cells in the more empty carrier group and no significant difference between the unloaded group and the untransfected group. Further studies found that the apoptosis rate of the BTG2 overexpressed group was 2.5 times higher than that of the empty carrier group (55.9%: 24.6%, P0.05), and there was no significant difference in the apoptosis rate between the unloaded group and the untransfected group (24.6%: 17.2). %, P0.05). After the ionizing radiation, all the cells in each group had different degrees of G1 phase cell block. The percentage of G1 cells in the BTG2 overexpression group was higher than that in the empty carrier group and the blank control group, but there was no statistical difference (P0.05). The results confirmed that BTG2 could enhance the radiation sensitivity of the hepatocellular carcinoma with.3. nude mice model to confirm that BTG2 promotes the liver. Radiosensitivity of cell carcinoma: Based on the successful construction of a nude mouse xenograft model, we found that the tumor growth of the hepatoma cells in the BTG2 overexpressed group was not significantly affected by the unloaded body group. After radiation (the experiment was observed, nude mice began to become tumor after 10 days of injection of tumor cells, at twenty-first days, 24 days respectively. " The tumor volume in the BTG2 overexpressed group was significantly reduced in the nude mice after the 27 day of the nude mice after the anaesthesia of the nude mice. The TUNEL detection showed that BTG2 also affected the apoptosis of the tumor cells. The results showed that the number of apoptotic cells in the BTG2 transfected group was significantly higher than that in the empty carrier group (BTG2 group was more than the control group, and increased by 39.8%, P0.05). Further Western blot detection showed that the expression of apoptosis related protein Bax in the BTG2 overexpressed group was 1.5 times more than that of the empty carrier group. The above results further confirmed that BTG2 could improve the sensitivity of hepatocellular carcinoma to ionizing radiation. Conclusion the survival rate of the patients with high 1.BTG2 nuclear expression is significantly higher than that of the hepatocellular carcinoma with low expression of BTG2 nucleus. There is a significant positive correlation between the expression of BTG2 and the prognosis of patients with hepatocellular carcinoma. It is further confirmed that BTG2 may play an important role in the development of hepatocellular carcinoma, which may play an important role in the development of hepatocellular carcinoma. After.2. ionizing radiation, the proliferation of BTG2 overexpressed hepatocellular carcinoma cell line Huh7 is slowed down, and the apoptosis of BTG2 overexpressed group is significantly increased. BTG2 The sensitivity of hepatocellular carcinoma to ionizing radiation could be enhanced by.3. ionizing radiation. The sensitivity of BTG2 overexpressed nude mice to ionizing radiation was significantly higher than that of the control group, and the degree of apoptosis in the tumor tissues was significantly stronger than that of the control group. The results further confirmed that BTG2 could enhance the radiation sensitivity of hepatocellular carcinoma.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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