μ-阿片受体下调抑制人类肝癌进程及其相关机制的研究

发布时间：2019-05-27 19:21

【摘要】：背景：肝细胞癌（HCC）是常见的恶性肿瘤之一，其恶性程度高，预后较差，每年约有50万-100万人死于肝癌。手术切除是世界公认的治疗肝癌最好的方式，，但是手术后总的5年生存率较低，大肝癌为34.6%，小肝癌为62.9%。近年来，随着科学技术的不断发展，一些新的治疗方法逐渐进入人们的视野。μ-阿片受体（MOR）是阿片受体超家族成员之一，广泛分布于体内。研究发现，MOR激活可促进多种细胞增殖，包括肿瘤细胞（如肺癌，乳腺癌，神经母细胞瘤和结肠癌等）的生长，这意味着MOR在多种细胞的增殖和分化中发挥重要作用。以往实验表明，MOR在肝癌细胞及组织中表达。然而，MOR如何影响肝细胞癌的进展尚不明确。除此之外，MOR促进肿瘤生长的相关机制也鲜少报道。目的：以往实验表明，MOR在肝癌组织及细胞中表达，但MOR影响肝癌进程的机制尚不明确。本实验的目的是研究MOR在肝癌组织及细胞的表达水平并探讨MOR下调对人类肝癌进展的影响及可能的机制。方法： 1.选取人肝癌细胞系HepG2、BEL7402及人正常肝脏细胞系LO2为研究对象，利用RT-PCR及Western blot检测各细胞系中MOR mRNA及蛋白质的表达水平。另外，收集2009至2010年间，吉林大学第一医院手术切除的病理证实为肝癌并且未曾治疗过的60例肝癌组织和20例因肝脏创伤、肝内胆管结石等手术而获得的正常肝组织。将上述组织经石蜡包埋切片后，利用免疫组化分析MOR在肝癌组织与正常肝组织中的表达情况。 2.对人肝癌HepG2细胞进行不同浓度的MOR siRNA转染后，采用RT-PCR和Western blot检测siRNA的干扰效率；不同浓度的纳洛酮和不同浓度的MORsiRNA分别处理HepG2细胞24、48及72小时后，采用MTT法测定细胞活力，分析MOR下调对HepG2细胞生长的影响；用Hoechst33342染色后，在荧光显微镜下观察细胞凋亡情况；用Annexin Ⅴ-FITC/PI染色，采用流式细胞仪检测细胞凋亡情况；用PI染色，采用流式细胞仪检测细胞周期的变化；采用RT-PCR和Western blot分别检验JNK mRNA、MKK7mRNA、JNK蛋白和MKK7蛋白表达及其磷酸化水平，研究MOR下调影响人肝癌细胞生长的分子机制；利用RNA干扰来沉默MKK7表达并观察人肝癌细胞的生长，进一步验证MKK7在MOR下调抑制人肝癌细胞生长中是否起到关键作用。 3.体内实验中，分别利用HepG2细胞、MOR沉默后的HepG2细胞建立裸鼠肿瘤模型，观察各组的肿瘤生长情况；饲养四周后处死，切除皮下移植瘤组织，采用游标卡尺测量肿瘤体积。结果： 1.通过分析RT-PCR及Western blot检测结果，发现在HepG2及BEL7402两种肝癌细胞系中，MOR mRNA及MOR蛋白表达水平均高于正常肝细胞系LO2。另外，通过免疫组化结果，发现人类肝癌组织中MOR表达明显高于正常肝脏组织。 2.利用RNA干扰技术可有效沉默MOR基因进而影响MOR表达，45nM的MOR siRNA转染HepG2细胞24小时，可以显著降低MOR mRNA的表达水平，MOR蛋白的表达水平也相应地下降，可持续至少72小时。随着纳洛酮和MORsiRNA浓度的增加，HepG2细胞的活力下降，表明MOR下调能够抑制人肝癌细胞的生长。经纳洛酮及MOR siRNA处理过的HepG2细胞，Hoechst33342染色后发现细胞染色质固缩，亮度明显高于正常对照组；流式细胞仪检测结果示其细胞凋亡率分别为18.36％和22.99％，均高于对照组（6.6％），并且停滞在G0/G1期的细胞数显著增加。通过纳洛酮和MOR siRNA处理HepG2细胞使其MOR表达下调后，发现MKK7mRNA和MKK7的蛋白表达水平没有明显变化，而MKK7磷酸化水平显著增加；此外，我们发现JNK及其磷酸化水平增加。为了进一步验证MKK7在MOR下调抑制肝癌进程中的关键性作用，我们利用RNA干扰技术沉默NKK7基因，发现与单纯MOR下调组相比，MKK7被沉默后，肝癌细胞的A490nm的吸光度值明显增加，这说明MOR下调对细胞增殖的抑制作用减弱。同时，Western blot结果显示MKK7被沉默后JNK及其磷酸化水平下降。 3.体内研究发现，正常对照组的5只裸鼠均发生肿瘤，成瘤率达到100％，MOR siRNA组有4只裸鼠发生肿瘤，成瘤率80％。MOR siRNA组与正常对照组相比，肿瘤的生长受到明显抑制。结论： 1. MOR在人肝癌细胞和肝组织中的表达水平均高于正常肝细胞和组织。 2.阿片受体拮抗剂纳洛酮及siRNA沉默MOR均可引起人肝癌细胞的MOR下调；MOR下调会促进肝癌细胞凋亡、将细胞周期阻滞在G0/G1期; MOR的下调会促进MKK7磷酸化，JNK及其磷酸化水平增加，抑制人肝癌细胞增殖。 3. MOR下调后的肝癌细胞降低了裸鼠成瘤率、抑制肿瘤生长、进展，这可能与人肝癌细胞的MOR下调有关，具体的机制有待进一步研究。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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