miRNA-22抑制肝星状细胞中galectin-1表达及其促肝细胞性肝癌免疫抑制作用的研究

发布时间：2018-05-21 14:14

本文选题：肝细胞性肝癌 + 肝星状细胞　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景肝细胞性肝癌(Hepatocellular carcinoma,HCC)是一类全球范围高发病率和高死亡率的恶性肿瘤。肝星状细胞(Hepatic stellate cells,HSCs)已被证实可以通过促进肿瘤免疫抑制来促进HCC的进展,然而其具体机制尚不完全清楚。半乳凝集素-1(galectin-1)是半乳凝集素家族的重要成员之一,已被发现可以通过促进免疫抑制来促进肿瘤进展,在HCC中亦有着显著的促癌活性,然而其相关机制仍有待深入探讨。mi RNA-22是mi RNA中的一种,其在HCC中的显著抑癌作用已经得到证实,然而相关机制仍不明确。鉴于HSC和galectin-1在HCC中相似的促肿瘤进展机制以及前期研究中发现的galectin-1和mi RNA-22间可能存在的靶向调控关系,我们推测mi RNA-22可能通过调控HSC中galectin-1表达发挥其在HCC中的抑癌作用。因此,我们通过研究HSC和galectin-1在促免疫抑制以及促HCC肿瘤进展作用中的相关性,并寻找其与mi RNA-22靶向调控的相关证据,为探索以mi RNA-22-HSC-galectin-1为靶点的HCC治疗提供新的理论基础和实验依据。研究方法和结果1、半乳凝集素-1在人肝星状细胞中的表达及作用研究从西南医院肝胆外科收集肝脏局部良性疾病手术患者正常肝脏组织,提取人原代肝星状细胞并鉴定。采用与CD3+T淋巴细胞共培养的方式观察到HSC拥有促T细胞凋亡以及促Th1/Th2免疫平衡偏移的能力。采用RT-PCR、Western blot、免疫组化以及ELISA等方式证实人HSC中有galectin-1的表达和分泌。构建galectin-1抑制和过表达载体,干预HSC中galectin-1表达后发现其促T细胞凋亡以及促Th1/Th2免疫平衡偏移的能力与其galectin-1表达情况呈正向相关。以上结果证实(1)活化人肝星状细胞中确有galectin-1的表达和分泌;(2)活化人肝星状细胞可以促CD3+T淋巴细胞凋亡及Th1/Th2免疫平衡偏移,且此能力与其galectin-1表达密切相关。2、半乳凝集素-1在肝星状细胞中的表达与肝细胞性肝癌肿瘤进程的相关性研究从西南医院肝胆外科收集肝细胞性肝癌手术患者肿瘤肝组织,提取人原代肝星状细胞。比较不同来源HSC(HCC源:Ca-HSCs,正常肝组织源:N-HSCs)中galectin-1表达及免疫调节能力差异,发现Ca-HSCs拥有明显更高的galectin-1表达以及更强的促T细胞凋亡和促Th1/Th2免疫平衡偏移能力。收集临床HCC患者肿瘤组织样本和病例资料,通过免疫荧光染色证实了活化HSC标志物α-SMA和galectin-1在肝组织中的共表达情况,通过连续石蜡切片免疫组化染色发现了活化HSC标志物α-SMA和galectin-1在HCC组织间质中的高度共表达情况。通过统计学分析发现了galectin-1表达以及CD3表达与HCC患者临床病理特征参数的显著相关性,进一步通过生存分析发现galectin-1高表达以及CD3低表达都是预示HCC患者不良预后的独立危险因素,且这两者间呈明显负性相关。以上结果表明,肝星状细胞可以通过galectin-1促进肝细胞性肝癌中免疫抑制环境的形成并促进肿瘤进展。3、mi RNA-22与肝星状细胞中半乳凝集素-1表达及促免疫抑制功能的相关性研究通过RT-PCR检测不同组织源肝星状细胞中mi RNA-22的表达情况后发现,在HSC中mi RNA-22的表达与galectin-1表达呈显著负相关,且癌组织源HSC中mi RNA-22表达明显低于正常肝组织源HSC。通过双荧光素酶实验证实了mi RNA-22与galectin-1的靶向调控关系。通过mi RNA-22 mimic转染HSC发现,HSC中mi RNA-22的表达升高可以显著下调其galectin-1表达,并抑制HSC促免疫抑制功能的发挥。以上结果表明,mi RNA-22可以抑制肝星状细胞中galectin-1的表达以及其参与诱导的促T细胞凋亡作用和促Th1/Th2免疫平衡偏移作用,而这种负性调控可能还影响了肝星状细胞在肝细胞性肝癌中的促免疫抑制及促肿瘤进展作用。结论肝星状细胞可以通过galectin-1促进肝细胞性肝癌中免疫抑制环境的形成进而促进肿瘤进展,而这一作用机制受到mi RNA-22的抑制和调控。
[Abstract]:Hepatocellular carcinoma (HCC) is a kind of malignant tumor with high global incidence and high mortality. Hepatic stellate cells (HSCs) has been proved to promote the progression of HCC by promoting tumor immunosuppression, but its specific mechanism is not completely clear. Galactin -1 (Gal) -1 (Gal). Ectin-1) is one of the most important members of the galactoglutpins family, which has been found to promote tumor progression by promoting immunosuppression and also has significant cancer promoting activity in HCC. However, its mechanism remains to be discussed in depth of.Mi RNA-22 as one of MI RNA, and its significant tumor suppressor effect in HCC has been confirmed, however, related machines The system is still unclear. In view of the similar tumor promoting mechanism of HSC and galectin-1 in HCC and the possible targeting regulation relationship between galectin-1 and MI RNA-22 found in previous studies, we speculate that MI RNA-22 may play its role in inhibiting cancer by regulating galectin-1 expression in HSC. The correlation of n-1 in promoting immunosuppression and promoting the progression of HCC tumor, and looking for evidence related to the targeting regulation of MI RNA-22, providing a new theoretical basis and experimental basis for exploring HCC therapy targeting mi RNA-22-HSC-galectin-1. Methods and results 1, the expression and role of galactocoagulin -1 in human hepatic stellate cells The primary hepatic stellate cells were extracted from the patients with benign liver diseases in the Department of hepatobiliary surgery of Southwest Hospital, and the human primary hepatic stellate cells were extracted and identified. The ability of HSC to promote T cell apoptosis and promote Th1/Th2 immune balance migration was observed by co culture with CD3+T lymphocyte. RT-PCR, Western blot, immunohistochemistry were used. The expression and secretion of galectin-1 in human HSC were confirmed by ELISA and other methods. The expression of galectin-1 inhibition and overexpression vector was constructed. After the intervention of galectin-1 expression in HSC, the ability to promote the apoptosis of T cells and the ability to promote the migration of Th1/Th2 immune balance were positively correlated with the galectin-1 expression. The results confirmed (1) the activation of human hepatic stellate cells was confirmed. The expression and secretion of galectin-1; (2) activation of human hepatic stellate cells can promote CD3+T lymphocyte apoptosis and Th1/Th2 immune balance migration, and this ability is closely related to the expression of galectin-1 and.2. The relationship between the expression of galactoagglutinin -1 in hepatic stellate cells and the progression of hepatocellular carcinoma in the Department of hepatobiliary surgery is collected from the Southwest Hospital The human primary hepatic stellate cells were extracted from the liver tissues of the patients with hepatoma hepatoma, and the expression of galectin-1 and the immune regulation were compared in different sources of HSC (HCC source: Ca-HSCs, normal liver tissue source: N-HSCs). It was found that Ca-HSCs had a significantly higher galectin-1 expression and stronger T cell apoptosis and Th1/Th2 immune balance bias. The tumor tissue samples and case data of HCC patients were collected and the co expression of the activated HSC marker alpha -SMA and galectin-1 in the liver tissues was confirmed by immunofluorescence staining. The high co expression of the activated HSC marker alpha -SMA and galectin-1 in the interstitial tissue of HCC was found by continuous paraffin immunohistochemical staining. The significant correlation between the expression of galectin-1 and the expression of CD3 and the clinicopathological parameters of HCC patients was found by statistical analysis. Further through survival analysis, the high expression of galectin-1 and the low expression of CD3 were independent risk factors for the poor prognosis of HCC patients, and there was a significant negative correlation between these two factors. It is clear that hepatic stellate cells can promote the formation of immunosuppressive environment in hepatocellular carcinoma by galectin-1 and promote tumor progression.3. The correlation between the expression of MI RNA-22 and the expression of galactoagglutinin -1 in hepatic stellate cells and the function of promoting immunosuppression by RT-PCR detection of the expression of MI RNA-22 in the hepatic stellate cells of different tissues by RT-PCR The expression of MI RNA-22 in HSC was significantly negatively correlated with the expression of galectin-1, and the expression of MI RNA-22 in HSC of cancer tissue was significantly lower than that of normal liver tissue. The relationship between MI RNA-22 and galectin-1 was confirmed by the experiment of HSC. through the double luciferase experiment. The results showed that MI RNA-22 could inhibit the expression of galectin-1 in hepatic stellate cells and the effect of its involvement in inducing apoptosis of T cells and promoting the migration of Th1/Th2 immune balance in hepatic stellate cells, and this negative regulation may also affect hepatic stellate cells in liver cell liver. The results suggest that MI RNA-22 can inhibit the expression of galectin-1 in hepatic stellate cells. Conclusion hepatic stellate cells can promote the formation of immunosuppressive environment in hepatocellular carcinoma and promote tumor progression through galectin-1, and the mechanism of this action is inhibited and regulated by Mi RNA-22.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

【相似文献】