【摘要】：背景： 肝細胞癌（HCC）是常見的惡性腫瘤之一，其惡性程度高，預后較差，每年約有50萬-100萬人死于肝癌。手術切除是世界公認的治療肝癌最好的方式，，但是手術后總的5年生存率較低，大肝癌為34.6%，小肝癌為62.9%。近年來，隨著科學技術的不斷發(fā)展，一些新的治療方法逐漸進入人們的視野。μ-阿片受體（MOR）是阿片受體超家族成員之一，廣泛分布于體內(nèi)。研究發(fā)現(xiàn)，MOR激活可促進多種細胞增殖，包括腫瘤細胞（如肺癌，乳腺癌，神經(jīng)母細胞瘤和結腸癌等）的生長，這意味著MOR在多種細胞的增殖和分化中發(fā)揮重要作用。以往實驗表明，MOR在肝癌細胞及組織中表達。然而，MOR如何影響肝細胞癌的進展尚不明確。除此之外，MOR促進腫瘤生長的相關機制也鮮少報道。 目的： 以往實驗表明，MOR在肝癌組織及細胞中表達，但MOR影響肝癌進程的機制尚不明確。本實驗的目的是研究MOR在肝癌組織及細胞的表達水平并探討MOR下調(diào)對人類肝癌進展的影響及可能的機制。 方法： 1.選取人肝癌細胞系HepG2、BEL7402及人正常肝臟細胞系LO2為研究對象，利用RT-PCR及Western blot檢測各細胞系中MOR mRNA及蛋白質(zhì)的表達水平。另外，收集2009至2010年間，吉林大學第一醫(yī)院手術切除的病理證實為肝癌并且未曾治療過的60例肝癌組織和20例因肝臟創(chuàng)傷、肝內(nèi)膽管結石等手術而獲得的正常肝組織。將上述組織經(jīng)石蠟包埋切片后，利用免疫組化分析MOR在肝癌組織與正常肝組織中的表達情況。 2.對人肝癌HepG2細胞進行不同濃度的MOR siRNA轉(zhuǎn)染后，采用RT-PCR和Western blot檢測siRNA的干擾效率；不同濃度的納洛酮和不同濃度的MORsiRNA分別處理HepG2細胞24、48及72小時后，采用MTT法測定細胞活力，分析MOR下調(diào)對HepG2細胞生長的影響；用Hoechst33342染色后，在熒光顯微鏡下觀察細胞凋亡情況；用Annexin Ⅴ-FITC/PI染色，采用流式細胞儀檢測細胞凋亡情況；用PI染色，采用流式細胞儀檢測細胞周期的變化；采用RT-PCR和Western blot分別檢驗JNK mRNA、MKK7mRNA、JNK蛋白和MKK7蛋白表達及其磷酸化水平，研究MOR下調(diào)影響人肝癌細胞生長的分子機制；利用RNA干擾來沉默MKK7表達并觀察人肝癌細胞的生長，進一步驗證MKK7在MOR下調(diào)抑制人肝癌細胞生長中是否起到關鍵作用。 3.體內(nèi)實驗中，分別利用HepG2細胞、MOR沉默后的HepG2細胞建立裸鼠腫瘤模型，觀察各組的腫瘤生長情況；飼養(yǎng)四周后處死，切除皮下移植瘤組織，采用游標卡尺測量腫瘤體積。 結果： 1.通過分析RT-PCR及Western blot檢測結果，發(fā)現(xiàn)在HepG2及BEL7402兩種肝癌細胞系中，MOR mRNA及MOR蛋白表達水平均高于正常肝細胞系LO2。另外，通過免疫組化結果，發(fā)現(xiàn)人類肝癌組織中MOR表達明顯高于正常肝臟組織。 2.利用RNA干擾技術可有效沉默MOR基因進而影響MOR表達，45nM的MOR siRNA轉(zhuǎn)染HepG2細胞24小時，可以顯著降低MOR mRNA的表達水平，MOR蛋白的表達水平也相應地下降，可持續(xù)至少72小時。隨著納洛酮和MORsiRNA濃度的增加，HepG2細胞的活力下降，表明MOR下調(diào)能夠抑制人肝癌細胞的生長。經(jīng)納洛酮及MOR siRNA處理過的HepG2細胞，Hoechst33342染色后發(fā)現(xiàn)細胞染色質(zhì)固縮，亮度明顯高于正常對照組；流式細胞儀檢測結果示其細胞凋亡率分別為18.36％和22.99％，均高于對照組（6.6％），并且停滯在G0/G1期的細胞數(shù)顯著增加。通過納洛酮和MOR siRNA處理HepG2細胞使其MOR表達下調(diào)后，發(fā)現(xiàn)MKK7mRNA和MKK7的蛋白表達水平?jīng)]有明顯變化，而MKK7磷酸化水平顯著增加；此外，我們發(fā)現(xiàn)JNK及其磷酸化水平增加。為了進一步驗證MKK7在MOR下調(diào)抑制肝癌進程中的關鍵性作用，我們利用RNA干擾技術沉默NKK7基因，發(fā)現(xiàn)與單純MOR下調(diào)組相比，MKK7被沉默后，肝癌細胞的A490nm的吸光度值明顯增加，這說明MOR下調(diào)對細胞增殖的抑制作用減弱。同時，Western blot結果顯示MKK7被沉默后JNK及其磷酸化水平下降。 3.體內(nèi)研究發(fā)現(xiàn)，正常對照組的5只裸鼠均發(fā)生腫瘤，成瘤率達到100％，MOR siRNA組有4只裸鼠發(fā)生腫瘤，成瘤率80％。MOR siRNA組與正常對照組相比，腫瘤的生長受到明顯抑制。 結論： 1. MOR在人肝癌細胞和肝組織中的表達水平均高于正常肝細胞和組織。 2.阿片受體拮抗劑納洛酮及siRNA沉默MOR均可引起人肝癌細胞的MOR下調(diào)；MOR下調(diào)會促進肝癌細胞凋亡、將細胞周期阻滯在G0/G1期; MOR的下調(diào)會促進MKK7磷酸化，JNK及其磷酸化水平增加，抑制人肝癌細胞增殖。 3. MOR下調(diào)后的肝癌細胞降低了裸鼠成瘤率、抑制腫瘤生長、進展，這可能與人肝癌細胞的MOR下調(diào)有關，具體的機制有待進一步研究。
[Abstract]:Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, with high degree of malignancy and poor prognosis. Surgical resection is the best way to treat the liver cancer in the world, but the overall 5-year survival rate after the operation is low, the large liver cancer is 34.6%, and the small liver cancer is 62.9. In recent years, with the development of science and technology, some new methods of treatment have gradually entered people's view The wild. mu.-opioid receptor (MOR) is one of the superfamily members of the opioid receptor and is widely distributed in the body. The study found that the activation of MOR can promote the proliferation of multiple cells, including the growth of tumor cells (e.g., lung cancer, breast cancer, neuroblastoma and colon cancer, etc.), which means that MOR plays an important role in the proliferation and differentiation of multiple cells The results of the previous experiments show that MOR is in the cells and tissues of the liver cancer. Da. However, how the MOR affects the progression of hepatocellular carcinoma is unknown In addition, the relevant mechanism of MOR to promote the growth of the tumor is also less reported. Road. Objective: In the past, the expression of MOR in the tissues and cells of the liver cancer is shown, but the MOR affects the process of the liver cancer. The purpose of this experiment is to study the expression level of MOR in liver cancer tissues and cells and to explore the effect of the reduction of MOR on the progression of human liver cancer. possible Methods:1. The human liver cancer cell line HepG2, BEL7402 and human normal liver cell line LO2 were selected as the research object, and the MOR mRNA in each cell line was detected by RT-PCR and Western blot. In addition, in the period of 2009 to 2010, the pathology of the operation of the first hospital in Jilin University was confirmed to be liver cancer and 60 cases of liver cancer and 20 cases of liver injury and hepatolithiasis were not treated. and the normal liver tissue obtained by the method comprises the following steps of: embedding the tissue into a paraffin embedded section, and using the immunohistochemical method to analyze the MOR in the liver cancer tissue and the normal liver tissue; 2. After transfection of human liver cancer HepG2 cells with MOR siRNA of different concentration, the interference efficiency of siRNA was detected by RT-PCR and Western blot. The different concentrations of naloxone and MORsiRNA at different concentrations were used to treat HepG2 cells for 24,48 and 72 hours respectively. The cell viability was determined by MTT method, and the effect of the down-regulation of MOR on the growth of HepG2 cells was analyzed. After staining with Hoechst33342, the apoptosis of HepG2 cells was observed under a fluorescence microscope. The apoptosis of the cells was detected by flow cytometry with Annexin V-FITC/ PI staining. The expression of JNK mRNA, MKK7mRNA, JNK protein and MKK7 protein and its phosphorylation were tested by RT-PCR and Western blot. The molecular mechanism of the regulation of MOR down-regulation on the growth of human liver cancer cells was studied. The expression of MKK7 with RNA interference was used to silence the expression of MKK7. and the growth of human liver cancer cells is observed, the MKK7 is further verified to inhibit the down-regulation of the human liver cancer cell in the MOR, In vivo, HepG2 cells and MOR-silent HepG2 cells were used to establish a model of tumor in nude mice, and the growth of tumor in each group was observed. , adopt Results:1. The results of RT-PCR and Western blot were used to detect the MOR mRNA and MOR eggs in HepG2 and BEL7402 cell lines. The level of white expression was higher than that of normal liver cell line LO2. In addition, the human liver cancer group was found by immunohistochemistry. 2. The expression level of MOR mRNA and the expression of MOR protein can be significantly reduced by using the RNA interference technique to effectively silence the MOR gene and to influence the expression of the MOR. The expression level of the MOR mRNA and the expression of the MOR protein can be significantly reduced. As the concentration of naloxone and MORsiRNA increased, the activity of HepG2 cells decreased, The results showed that the regulation of MOR down-regulation could inhibit the growth of human liver cancer cells. The cells of HepG2 cells treated with naloxone and MOR siRNA, Hoechst33342, found that the cell chromatin was fixed and the brightness was higher than that of the normal control group. The results of flow cytometry showed that the cell apoptosis rate was 18.36%, respectively. 22.99%, higher than the control group (6.6%). And the number of cells in the G0/ G1 phase was significantly increased, and the expression level of MKK7mRNA and MKK7 was not significantly changed after the HepG2 cells were treated with naloxone and MOR siRNA, and the phosphorylation level of MKK7 was increased significantly. In addition, we found that the level of JNK and its phosphorylation were increased. To further verify the pivotal role of MKK7 in the reduction of the MOR down-regulation of the liver cancer, we used the RNA interference technique to silence the NKK7 gene and found that, compared to the simple MOR down-regulation group, MKK7 was silent and the liver the absorbance value of the a490nm of the cancer cell was significantly increased, The inhibitory effect of the down-regulation of MOR on the proliferation of cells was described, and the results of Western blot showed that M. KK7 was silent and the level of JNK and its phosphorylation decreased.3. In vivo studies,5 nude mice in the normal control group had a tumor, and the tumor-forming rate was 100%, and 4 nude mice in the MOR siRNA group had tumors, and the tumor-forming rate was 80%. MOR siRNA group The growth of the tumor was significantly inhibited in comparison with the control group. Conclusion:1. MOR is in human The expression levels in both the liver cancer cells and the liver tissues were higher than those of the normal liver cells and tissues.2. Both the opioid receptor antagonist naloxone and the siRNA-silent MOR can cause the regulation of the MOR of the human liver cancer cells, the regulation of the MOR down-regulation can promote the apoptosis of the liver cancer cells, block the cell cycle in the G0/ G1 phase, and the regulation of the MOR will promote the MKK. 3. After the down-regulation of MOR, the tumor rate of nude mice was decreased, and the growth and progress of the tumor were inhibited.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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相關期刊論文 前2條

1 Bernardino Rampone;Beniamino Schiavone;Antonio Martino;Carmine Viviano;Giuseppe Confuorto;;Current management strategy of hepatocellular carcinoma[J];World Journal of Gastroenterology;2009年26期

2 Wanqing Chen;Rongshou Zheng;Siwei Zhang;Ping Zhao;Hongmei Zeng;Xiaonong Zou;Jie He;;Annual report on status of cancer in China,2010[J];Chinese Journal of Cancer Research;2014年01期

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