【作者】 乌云格日乐；

【导师】 阿拉坦高勒；

【作者基本信息】 内蒙古大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 背景及研究内容：溶血磷脂酸(lysophosphatidic acid,LPA)是体内生理活性脂质,通过其G蛋白偶联受体(LPAR1-7)激活多条信号通路而诱导下产生诸多的细胞生理反应,参与调控细胞的运动、增值,凋亡、分化和分泌等多种细胞机能。目前,研究最多的是溶血磷脂酸受体1-3,属于G-蛋白偶联受体,内皮分化基因家族(endothelial differentiation gene EDG family)成员。LPA受体,在人体正常组织中普遍表达,随后又期研究发现与许多恶性肿瘤及一些相关疾病的发生和发展密切相关。我们在前期研究中将LPA1为在动脉硬化相关基因(未发表),同时也发现LPA2受体在胃肠肿瘤中参与LPA诱导的细胞迁移。然而该研究中却发现除LPA2以外LPA3在人胃癌细胞BGC-803中高表达,并参与LPA诱导的细胞迁移中起重要作用。所以,该研究中探索性研究了LPA1, LPA2和LPA3在人胃癌细胞BGC-803中如何进行协同作用,分别与哪一类型的G偶联蛋白结合促进细胞的迁移活动的。我们研究结果表明,LPA2和LPA3分别与不同的G偶联蛋白结合,促进胃癌细胞BGC-803的迁移活性,而LPA1与上述功能为无关,提示LPA2和LPA3受体均为治疗胃肠肿瘤的潜在的靶位点。方法：培养人胃癌BGC-803细胞株作为细胞模型。迁移检测用transwell法、Boydenchamber法；法测定LPA受体表达格式用实时定量PCR；分别用SiRNA干扰技术和药理学方法,研究LPA受体在LPA诱导的细胞迁移参与情况。结果：1)胃癌BGC-803细胞株中,LPA1, LPA2和LPA3均有表达,而LPA3受体表最高；2)LPA能够诱导的细胞迁移被LPA1和LPA3受体的拮抗Kil6425、LPA2和LPA3受体特异性SiRNA所抑制。3)LPA诱导的细胞迁移被LPA1和LPA3受体特异性SiRNA所抑制。结论：LPA诱导的人胃癌BGC-803细胞迁移中参与的主要受体是LPA3,其次是LPA2,而LPA1不参与LPA诱导的细胞迁移。LPA3受体与Gi偶联蛋白结合。LPA2和LPA3受体均为治疗胃肠肿瘤的潜在的靶位点。更多还原

【Abstract】 Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator, which elicits a variety of biological functions mainly through G-protein coupled receptors. Although LPA is shown to stimulate proliferation and motility via LPA receptors, LPAR1and LPAR3in several cancer cell lines, but the role of LPA receptors in gastric cancer cells is still being unknown. However, several researches reported that LPAR2play an important role in the carcinogenesis of gastric cancer, but there is no report to show the LPAR3involvement in the carcinogenesis. For this reason, we examined LPA receptors (LPAR1, LPAR2and LPAR3) in BGC-803cells along with real time PCR method.Among these receptors, LPAR2and LPAR3were shown to be highly expressed in BGC-803cells, a human gastric cancer cell line. Transient transfection with LPAR2and LPAR3siRNAs were observed to reduce LPAR3mRNA in BGC-803cells and eliminate the LPA-induced cell migration. The results suggest that the LPAR3involves LPA-induced BGC-803cell migration. Our main findings were as follows:1. LPA significantly increased cellular migration.2. Ki-16425, antagonist of LPA1and LPA3receptors suppressed the LPA-induced migration of BGC-803cells. LPA-induced migration of BGC-803cell depends on LPA1and LPA3receptors.3. We discovered that LPA-induced cell migration is reduced by adding pertussis toxin (PTX). This result shows that Gi protein more involved in LPA-induced cell migration than Gq protein (Fig.3.4). 4. Results of real time-PCR method shown that LPAR3receptor predominantly expressed in human gastric cell line BGC-803cells among other receptors.5. LPAR2and LPAR3play an important roles in the LPA-induced cell migration, their expression was silenced by LPAR2and LPAR3-specific shRNAs in the BGC-803cells (Figures4.6,4.7and4.8). RT-PCR analysis showed that shLPAR3expression was decreased by53%compared with the control (Fig.4.4). However shLPAR2expression was decreased less than shLPAR3(Fig.4.5). As mentioned, shLPAR3expression was more decreased in the BGC-803cells, suggesting that LPAR3may be important in LPA-induced cell migration.6. Silencing LPAR2-3expression by shLPAR2and shLPAR3the LPA-induced cell migration of BGC-803were completely inhibited. Silencing LPAR3expression significantly decreased the LPA-induced migration of BGC-803cells compared with silencing LPAR2in BGC-803cells (Fig.4.9). The results demonstrated that LPA-induced migration of BGC-803gastric cancer cells was mainly mediated by LPAR3.7. The cell migration induced by LPA in shLPAR2-transfected BGC-803cells was significantly inhibited by PTX. However, shLPAR3-transfected cell migrating activity in the presence of PTX is not significant (Fig.4.10). These results suggest that PTX-sensitive G proteins involved in BGC-803cell migration.更多还原