新型PDGFC剪接体在PDR纤维血管膜中的表达和功能
本文关键词:新型PDGFC剪接体在PDR纤维血管膜中的表达和功能 出处:《天津医科大学》2013年博士论文 论文类型:学位论文
更多相关文章: 血小板源生长因子C 剪接体 纤维血管膜 增殖性糖尿病视网膜病变
【摘要】:背景:抗血管内皮细胞生长因子(VEGF)疗法已逐渐成为临床上治疗增殖性糖尿病视网膜病变(PDR)的一线药物,但抗VEGF疗法对相当一部分PDR患者效果不理想。文献报道,血小板源生长因子C(PDGFC)可经不依赖于VEGF的通路在动物模型中诱导视网膜和脉络膜的新生血管生成,因而成为VEGF之外的拮抗新生血管生成的另一重要基因靶点。但PDGFC在PDR患者纤维血管膜中的表达和调控机制国内外尚未见报道。目的:在PDR患者纤维血管膜中检测PDGFC的表达状况,分离并克隆新型PDGFC剪接体,并研究其在视网膜血管内皮细胞中的表达和功能。方法:在玻璃体切割术中,收集8名PDR患者的纤维血管膜。从这些纤维血管膜中提取总RNA并逆转成cDNA。以cDNA为模板和针对全长PDGFC的引物行PCR反应,可见多个PDGFC扩增条带。PCR扩增产物克隆到TOPO-TA PCR2.1载体后进行测序。将测序结果进行blast search,以证实阳性克隆分别是两个新型剪接体和全长的PDGFC。继而将编码全长PDGFC和两个剪接体的cDNA序列亚克隆入pcDNA表达载体,并命名为p-PDGFCfull p-PDGFC1,和p-PDGFC2。为了便于检测,在蛋白质编码序列的C末端加上了 FLAG标签。将去内毒素的质粒p-PDGFCfull,p-PDGFC1,p-PDGFC2和pcDNA载体单独转染或共转染入猴视网膜血管内皮细胞。同时共转染表达绿色荧光蛋白(GFP)的质粒以估算转染效率,或用实时基因组PCR检测转染效率。转染后,用免疫荧光检测PDGFCfull,PDGFC1和PDGFC2的表达和细胞内定位。功能性检测用细胞计数试剂盒检测质粒转染后视网膜微血管内皮细胞的增殖状况,并用实时定量PCR检测转染后细胞内总PDGFC(内源性和外源性)的mRNA含量。结果:琼脂糖凝胶电泳可见不同长度的PCR产物。PCR产物测序结果经序列比对证实阳性克隆与人PDGFC序列同源,进一步分析结果表明这些PCR产物包含全长PDGFC和两个新型剪接体。经开放读码框架分析,这两个剪接体均导致了截短的蛋白。进而将这两个剪接体和全长PDGFC克隆入pcDNA表达载体并用限制性内切酶验证克隆的正确性。质粒p-PDGFCfull,p-PDGFC1,和p-PDGFC2转染入视网膜微血管内皮细胞,全长和新型剪接体的蛋白质主要分布在内皮细胞的胞浆。从功能上来讲,在相似的转染效率下,转染PDGFC1和PDGFC2的细胞较单纯转染pcDNA载体的细胞,数目显著减少(p0.001 p-PDGFC1转染的细胞,p0.05 p-PDGFC2转染的细胞);而转染p-PDGFCfull的细胞较转染pcDNA载体的细胞表现出显著增加的增殖趋势(p0.001),而且,PDGFC1或p-PDGFC2可抵消PDGFCfull引起的血管内皮细胞增殖(p均0.05)。实时PCR结果显示各组转染细胞内总PDGFC的mRNA含量与细胞增殖检测的结果有相似的变化趋势。结论:在PDR病人的纤维血管膜中发现两个PDGFC的新型剪接体。这两个剪接体在猴视网膜微血管内皮细胞中过表达后可引起视网膜血管内皮细胞数量的减少,并可拮抗全长PDGFC诱导的细胞增殖。这些结果提示在PDR纤维血管膜中PDGFC的表达可能存在一种负反馈调节机制,即在PDR病理条件下,产生全长PDGFC的同时,也产生了两个新型剪接体来拮抗全长PDGFC的促新生血管的作用,其作用机制可能是在转录水平上降调节PDGFC,从而拮抗全长PDGFC的促新生血管的作用。更重要的是,新型剪接体的发现、克隆和表达为进一步研发干预PDR中新生血管生成的新型药物奠定了理论和实验基础。
[Abstract]:Background: vascular endothelial growth factor (VEGF) therapy has become the treatment of proliferative diabetic retinopathy (PDR) of the first-line drugs, but the anti VEGF therapy for a considerable part of the effect is not ideal. PDR patients reported in the literature, platelet-derived growth factor C (PDGFC) angiogenesis via the pathway is not dependent on the induction of VEGF in the retina and choroid in animal models, and thus become the new generation of VEGF vascular antagonist is another important target gene. But the expression and regulation mechanism of PDGFC in PDR patients with vascular membrane in the fiber has not been reported. Objective: the expression of PDGFC in the detection of vascular membrane in patients with PDR fiber, isolate and clone novel PDGFC splicing, and the study on retinal vascular endothelial cells in expression and function. Methods: in the vitreous body incision, 8 patients with PDR were collected from these fiber fibrovascular membranes. Vascular membrane total RNA was extracted and reversed into cDNA. using cDNA as template and primers for full-length PDGFC for PCR reaction, visible multiple amplified bands of PDGFC.PCR by sequencing the PCR products were cloned into TOPO-TA PCR2.1 vector. The sequencing results were confirmed by BLAST search, the positive clones respectively are two new splicing and the full-length PDGFC. and cDNA sequences encoding the full-length PDGFC and two splicing was subcloned into pcDNA expression vector, named p-PDGFCfull p-PDGFC1, and p-PDGFC2. in order to facilitate the detection, at the end of the protein sequence of C encoding with FLAG tags. The plasmid p-PDGFCfull will go to p-PDGFC1 p-PDGFC2 and pcDNA endotoxin, transfected with vector alone or co transfection into monkey retinal vascular endothelial cells. The expression of green fluorescent protein (GFP) and co transfected the plasmid to evaluate the transfection efficiency, or by real-time PCR analysis of genome transfection efficiency. Stained by immunofluorescence detection of PDGFCfull, PDGFC1 and PDGFC2 expression and cell proliferation in localization of retinal microvascular endothelial cells by plasmid transfection assay cell counting kit after functional testing, and using PDGFC real-time quantitative PCR detection of transfected cells (endogenous and exogenous) the content of mRNA. Agarose gel electrophoresis showed different PCR products.PCR sequencing length by sequence confirmed positive clones with homologous PDGFC sequences, further analysis showed that these PCR products contain full-length PDGFC and two novel splicing. The open reading frame analysis, the two variants have led to a truncated protein then. The two splicing and full-length PDGFC cloned into pcDNA expression vector by restriction endonuclease and correctness verification clones. Plasmid p-PDGFCfull, p-PDGFC1, and p-PDGFC2 were transfected into retinal micro Vascular endothelial cells, and the full-length model splicing protein mainly distributed in the cytoplasm of endothelial cells. Functionally speaking, in similar transfection efficiency, transfection of PDGFC1 and PDGFC2 cells compared with transfection of pcDNA vector cells, significantly reduce the number of (p0.001 cells p-PDGFC1 transfected cells, P0.05 transfected with p-PDGFC2); and transfection of p-PDGFCfull cells transfected with pcDNA vector cells showed a significant increase in the growth trend (p0.001), and PDGFC1 or p-PDGFC2 can counteract vascular endothelial cell proliferation induced by PDGFCfull (P 0.05). Real time PCR results showed that mRNA content and cell proliferation detection of total PDGFC transfected cells within each group the results have the same trend. Conclusion: the discovery of new splicing two PDGFC body in fibrovascular membranes of PDR patients. The two isoform expression in rhesus retinal vascular endothelial cells can cause Reduce the number of retinal vascular endothelial cells, and can antagonize the full-length PDGFC induced cell proliferation. These results suggest that the expression of PDGFC in vascular membrane in PDR fibers may have a negative feedback mechanism, namely PDR in pathological conditions, the production of full-length PDGFC at the same time, also produced the angiogenesis effect of two novel splicing the body to antagonize the length of PDGFC, its mechanism may be that PDGFC is down regulated at the transcriptional level, thereby antagonizing the full-length PDGFC role in promoting neovascularization. More importantly, the discovery of new splicing, cloning and expression laid the theoretical and experimental basis for new drug neovascularization further research of intervention in the PDR generation.
【学位授予单位】:天津医科大学
【学位级别】:博士
【学位授予年份】:2013
【分类号】:R774.1
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