本文選題：肝細(xì)胞癌 + BTG2　； 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：研究背景肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是世界上發(fā)病率排名第六的癌癥,排在癌癥導(dǎo)致人類(lèi)死亡的第三位。由于缺乏明確的早期診斷標(biāo)志物、明顯的臨床癥狀等,肝癌患者就診時(shí)通常已是晚期,不少患者失去了手術(shù)機(jī)會(huì),總的5年生存期較短。因此,如何提高肝細(xì)胞癌患者的臨床診斷率和治療療效是提高其生存期的關(guān)鍵。臨床上肝細(xì)胞癌的治療主要以手術(shù)切除癌癥病灶、肝移植、化療、射頻消融、放射治療和分子靶向藥物如索拉菲尼等為主。放射治療主要用于和其它治療手段相結(jié)合的綜合治療,目前為止,由于肝癌細(xì)胞較低的放療敏感性及肝臟較低的耐受劑量等原因,肝細(xì)胞癌的放射治療效果仍不能令人滿(mǎn)意。因此,研究如何增加肝細(xì)胞癌的放射治療效應(yīng)在臨床上有著重要意義。B細(xì)胞易位基因2(B cell translocation gene 2,BTG2)是一個(gè)瞬時(shí)早期反應(yīng)基因,屬于抗增殖基因家族,BTG2在多種組織和器官中均有表達(dá),如肺組織、脾、胸腺、胃腸組織等。其參與了細(xì)胞分化、生長(zhǎng)、凋亡和DNA損傷修復(fù)等多種生物學(xué)功能。既往研究表明,BTG2被公認(rèn)為腫瘤抑制基因,其在不少癌癥中表達(dá)減少或者缺失,從而在腫瘤的發(fā)生發(fā)展中發(fā)揮了重要的作用。放療射線(xiàn)對(duì)細(xì)胞的損傷可通過(guò)直接作用于DNA分子,導(dǎo)致DNA鏈斷裂,研究表明p53可以增強(qiáng)口腔癌和胰腺癌等腫瘤的放療敏感性,而B(niǎo)TG2是DNA損傷細(xì)胞反應(yīng)通路的p53依賴(lài)性的成分之一,提示BTG2可能參與放療射線(xiàn)對(duì)細(xì)胞損傷的過(guò)程。我們課題組近年來(lái)致力于BTG2的研究,發(fā)現(xiàn)BTG2參與了肝癌的發(fā)生、肝細(xì)胞的再生以及肝損傷的修復(fù)等一系列復(fù)雜的病理生理過(guò)程,本研究將繼續(xù)通過(guò)相關(guān)的實(shí)驗(yàn)探索BTG2與肝細(xì)胞癌放療敏感性的關(guān)系,以求能夠?qū)Ω渭?xì)胞癌患者的放射治療提供新的思路和理論依據(jù)。目的通過(guò)臨床肝細(xì)胞癌患者的腫瘤組織樣本評(píng)估BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后的相關(guān)性;并通過(guò)體外細(xì)胞實(shí)驗(yàn)及裸鼠成瘤模型體內(nèi)實(shí)驗(yàn)等方法檢測(cè)BTG2對(duì)肝細(xì)胞癌放射治療敏感性的影響,以期為肝細(xì)胞癌的放射治療相關(guān)靶基因的研究提供一定的理論依據(jù)。方法1.BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后的相關(guān)性:肝細(xì)胞癌患者腫瘤組織樣本收集、組織芯片的制作及免疫組化分析,44名肝細(xì)胞癌患者全部來(lái)自重慶市第三軍醫(yī)大學(xué)大坪醫(yī)院,這些患者依據(jù)世界衛(wèi)生組織標(biāo)準(zhǔn)均被病理學(xué)診斷為HCC,所有的患者在手術(shù)前都沒(méi)有進(jìn)行過(guò)化療或者放射治療。選擇新鮮手術(shù)標(biāo)本進(jìn)行固定和預(yù)處理,按照步驟制作組織芯片。常規(guī)的免疫組織化學(xué)方法檢測(cè)各組織標(biāo)本BTG2的表達(dá)情況,統(tǒng)計(jì)分析BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后的相關(guān)性。2.體外細(xì)胞實(shí)驗(yàn)檢測(cè)BTG2對(duì)肝細(xì)胞癌放療敏感性的影響:細(xì)胞株的選擇、培養(yǎng)及轉(zhuǎn)染,選用人的肝細(xì)胞癌細(xì)胞株Huh7進(jìn)行常規(guī)細(xì)胞培養(yǎng),用Lipofectamine 2000(Invitrogen)轉(zhuǎn)染真核表達(dá)載體p EGFP-N1-BTG2及空載體作為對(duì)照。轉(zhuǎn)染細(xì)胞經(jīng)蛋白提取及免疫蛋白印跡分析各組BTG2蛋白的表達(dá)情況,RT-PCR分析各組BTG2的RNA水平,篩選并鑒定出BTG2穩(wěn)定表達(dá)的細(xì)胞株。對(duì)BTG2穩(wěn)定轉(zhuǎn)染的細(xì)胞株進(jìn)行放射線(xiàn)處理(8Gy,6MV的放射線(xiàn)),細(xì)胞繼續(xù)培養(yǎng)24小時(shí)后用流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡及細(xì)胞周期,并進(jìn)行細(xì)胞增殖實(shí)驗(yàn)。3.裸鼠成瘤模型檢測(cè)BTG2對(duì)肝細(xì)胞癌放療敏感性的影響:購(gòu)買(mǎi)一般情況較好、性狀基本一致的免疫缺陷裸鼠12只,隨機(jī)分為兩組:BTG2過(guò)表達(dá)組和空載體對(duì)照組,每組6只。待BTG2穩(wěn)定表達(dá)的Huh7細(xì)胞培養(yǎng)到較好的細(xì)胞狀態(tài),以每只裸鼠1×107個(gè)的細(xì)胞量注射入裸鼠右上腋皮膚下,每天觀(guān)察裸鼠的一般情況及成瘤情況。實(shí)驗(yàn)中注射第10天開(kāi)始成瘤,之后每隔一天測(cè)量裸鼠腫瘤體積并記錄。在兩組裸鼠腫瘤直徑大約1cm左右(注射后第21天)行放射線(xiàn)處理(8Gy,6MV的放射線(xiàn)),以后每隔2天照射裸鼠一次,共照射3次。照射期間注意觀(guān)察裸鼠的一般情況及腫瘤體積并記錄。在第30天時(shí)處死裸鼠,取腫瘤標(biāo)本進(jìn)行后續(xù)實(shí)驗(yàn)。腫瘤組織分別進(jìn)行Ki67免疫組織化學(xué)染色、TUNEL細(xì)胞凋亡及蛋白印跡法凋亡相關(guān)蛋白Bax等檢測(cè)。結(jié)果1.BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后呈正相關(guān):將44例肝細(xì)胞癌患者組織標(biāo)本進(jìn)行組織芯片制作及分析。根據(jù)免疫組化染色結(jié)果將患者分為BTG2高表達(dá)組和低表達(dá)組,并進(jìn)行生存期分析。結(jié)果發(fā)現(xiàn)BTG2核高表達(dá)組的患者較BTG2核低表達(dá)組的患者具有較好的生存率。提示BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后呈顯著正相關(guān)。2.體外細(xì)胞實(shí)驗(yàn)證實(shí)BTG2增強(qiáng)肝細(xì)胞癌放療敏感性:人肝細(xì)胞癌細(xì)胞株Huh7成功轉(zhuǎn)染p EGFP-N1-BTG2真核表達(dá)載體,并經(jīng)蛋白免疫印記證實(shí)成功構(gòu)建BTG2穩(wěn)定過(guò)表達(dá)的細(xì)胞株(BTG2過(guò)表達(dá)轉(zhuǎn)染組是空載體轉(zhuǎn)染組的3.6倍,P0.05)。各組細(xì)胞經(jīng)放射線(xiàn)處理后,BTG2過(guò)表達(dá)組的細(xì)胞增殖能力較空載體組細(xì)胞的增值能力明顯降低,空載體組和未轉(zhuǎn)染組之間無(wú)顯著性差異。進(jìn)一步研究發(fā)現(xiàn),BTG2過(guò)表達(dá)組細(xì)胞的凋亡率較空載體組細(xì)胞的凋亡率增加約2.5倍(55.9%:24.6%,P0.05),空載體組和未轉(zhuǎn)染組的細(xì)胞凋亡率無(wú)顯著性差異(24.6%:17.2%,P0.05)。電離輻射后,各組細(xì)胞均出現(xiàn)不同程度的G1期細(xì)胞阻滯,BTG2過(guò)表達(dá)組G1期細(xì)胞百分率較空載體組和空白對(duì)照組均有所提高,但是無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。以上結(jié)果證實(shí)BTG2可增強(qiáng)肝細(xì)胞癌的放療敏感性。3.裸鼠成瘤模型證實(shí)BTG2促進(jìn)肝細(xì)胞癌放射治療敏感性:在成功構(gòu)建裸鼠移植瘤模型的基礎(chǔ)上,我們發(fā)現(xiàn),與空載體組相比,BTG2過(guò)表達(dá)組的肝癌細(xì)胞腫瘤生長(zhǎng)并未受到明顯影響。在放射線(xiàn)照射后(實(shí)驗(yàn)中觀(guān)察發(fā)現(xiàn),裸鼠在注射腫瘤細(xì)胞10天后開(kāi)始成瘤,分別在第21天、24天和27天進(jìn)行裸鼠麻醉后的放射線(xiàn)處理8Gy每次),BTG2過(guò)表達(dá)組的裸鼠腫瘤體積較空載體組明顯縮小。經(jīng)TUNEL檢測(cè)發(fā)現(xiàn),BTG2同樣影響了腫瘤細(xì)胞的凋亡,結(jié)果顯示:BTG2轉(zhuǎn)染組的凋亡細(xì)胞數(shù)量較空載體組明顯增多(BTG2組較對(duì)照組增加39.8%,P0.05)。進(jìn)一步經(jīng)Western blot檢測(cè)發(fā)現(xiàn)BTG2過(guò)表達(dá)組的凋亡相關(guān)蛋白Bax的表達(dá)是轉(zhuǎn)染空載體組的1.5倍。以上結(jié)果進(jìn)一步證實(shí)BTG2可提高肝細(xì)胞癌對(duì)電離輻射的敏感性。結(jié)論1.BTG2核表達(dá)高的肝細(xì)胞癌患者的生存率明顯高于BTG2核表達(dá)低的肝細(xì)胞癌患者,提示BTG2的表達(dá)與肝細(xì)胞癌患者預(yù)后呈顯著正相關(guān)。進(jìn)一步證實(shí)BTG2可能作為一種抑癌基因在肝細(xì)胞癌的發(fā)生發(fā)展中發(fā)揮重要作用。2.電離輻射后,BTG2過(guò)表達(dá)的肝細(xì)胞癌細(xì)胞株Huh7的增殖減慢,BTG2過(guò)表達(dá)組的細(xì)胞凋亡明顯增加。提示BTG2可以增強(qiáng)肝細(xì)胞癌對(duì)電離輻射的敏感性。3.電離輻射后,BTG2過(guò)表達(dá)組裸鼠移植瘤對(duì)電離輻射敏感性顯著高于對(duì)照組,且其腫瘤組織中細(xì)胞凋亡程度也明顯強(qiáng)于對(duì)照組。結(jié)果進(jìn)一步證實(shí)BTG2能夠增強(qiáng)肝細(xì)胞癌的放療敏感性。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.7

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