本文關(guān)鍵詞：新型PDGFC剪接體在PDR纖維血管膜中的表達(dá)和功能　出處：《天津醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血小板源生長因子C 剪接體 纖維血管膜 增殖性糖尿病視網(wǎng)膜病變

【摘要】：背景:抗血管內(nèi)皮細(xì)胞生長因子(VEGF)療法已逐漸成為臨床上治療增殖性糖尿病視網(wǎng)膜病變(PDR)的一線藥物,但抗VEGF療法對相當(dāng)一部分PDR患者效果不理想。文獻(xiàn)報(bào)道,血小板源生長因子C(PDGFC)可經(jīng)不依賴于VEGF的通路在動(dòng)物模型中誘導(dǎo)視網(wǎng)膜和脈絡(luò)膜的新生血管生成,因而成為VEGF之外的拮抗新生血管生成的另一重要基因靶點(diǎn)。但PDGFC在PDR患者纖維血管膜中的表達(dá)和調(diào)控機(jī)制國內(nèi)外尚未見報(bào)道。目的:在PDR患者纖維血管膜中檢測PDGFC的表達(dá)狀況,分離并克隆新型PDGFC剪接體,并研究其在視網(wǎng)膜血管內(nèi)皮細(xì)胞中的表達(dá)和功能。方法:在玻璃體切割術(shù)中,收集8名PDR患者的纖維血管膜。從這些纖維血管膜中提取總RNA并逆轉(zhuǎn)成cDNA。以cDNA為模板和針對全長PDGFC的引物行PCR反應(yīng),可見多個(gè)PDGFC擴(kuò)增條帶。PCR擴(kuò)增產(chǎn)物克隆到TOPO-TA PCR2.1載體后進(jìn)行測序。將測序結(jié)果進(jìn)行blast search,以證實(shí)陽性克隆分別是兩個(gè)新型剪接體和全長的PDGFC。繼而將編碼全長PDGFC和兩個(gè)剪接體的cDNA序列亞克隆入pcDNA表達(dá)載體,并命名為p-PDGFCfull p-PDGFC1,和p-PDGFC2。為了便于檢測,在蛋白質(zhì)編碼序列的C末端加上了 FLAG標(biāo)簽。將去內(nèi)毒素的質(zhì)粒p-PDGFCfull,p-PDGFC1,p-PDGFC2和pcDNA載體單獨(dú)轉(zhuǎn)染或共轉(zhuǎn)染入猴視網(wǎng)膜血管內(nèi)皮細(xì)胞。同時(shí)共轉(zhuǎn)染表達(dá)綠色熒光蛋白(GFP)的質(zhì)粒以估算轉(zhuǎn)染效率,或用實(shí)時(shí)基因組PCR檢測轉(zhuǎn)染效率。轉(zhuǎn)染后,用免疫熒光檢測PDGFCfull,PDGFC1和PDGFC2的表達(dá)和細(xì)胞內(nèi)定位。功能性檢測用細(xì)胞計(jì)數(shù)試劑盒檢測質(zhì)粒轉(zhuǎn)染后視網(wǎng)膜微血管內(nèi)皮細(xì)胞的增殖狀況,并用實(shí)時(shí)定量PCR檢測轉(zhuǎn)染后細(xì)胞內(nèi)總PDGFC(內(nèi)源性和外源性)的mRNA含量。結(jié)果:瓊脂糖凝膠電泳可見不同長度的PCR產(chǎn)物。PCR產(chǎn)物測序結(jié)果經(jīng)序列比對證實(shí)陽性克隆與人PDGFC序列同源,進(jìn)一步分析結(jié)果表明這些PCR產(chǎn)物包含全長PDGFC和兩個(gè)新型剪接體。經(jīng)開放讀碼框架分析,這兩個(gè)剪接體均導(dǎo)致了截短的蛋白。進(jìn)而將這兩個(gè)剪接體和全長PDGFC克隆入pcDNA表達(dá)載體并用限制性內(nèi)切酶驗(yàn)證克隆的正確性。質(zhì)粒p-PDGFCfull,p-PDGFC1,和p-PDGFC2轉(zhuǎn)染入視網(wǎng)膜微血管內(nèi)皮細(xì)胞,全長和新型剪接體的蛋白質(zhì)主要分布在內(nèi)皮細(xì)胞的胞漿。從功能上來講,在相似的轉(zhuǎn)染效率下,轉(zhuǎn)染PDGFC1和PDGFC2的細(xì)胞較單純轉(zhuǎn)染pcDNA載體的細(xì)胞,數(shù)目顯著減少(p0.001 p-PDGFC1轉(zhuǎn)染的細(xì)胞,p0.05 p-PDGFC2轉(zhuǎn)染的細(xì)胞);而轉(zhuǎn)染p-PDGFCfull的細(xì)胞較轉(zhuǎn)染pcDNA載體的細(xì)胞表現(xiàn)出顯著增加的增殖趨勢(p0.001),而且,PDGFC1或p-PDGFC2可抵消PDGFCfull引起的血管內(nèi)皮細(xì)胞增殖(p均0.05)。實(shí)時(shí)PCR結(jié)果顯示各組轉(zhuǎn)染細(xì)胞內(nèi)總PDGFC的mRNA含量與細(xì)胞增殖檢測的結(jié)果有相似的變化趨勢。結(jié)論:在PDR病人的纖維血管膜中發(fā)現(xiàn)兩個(gè)PDGFC的新型剪接體。這兩個(gè)剪接體在猴視網(wǎng)膜微血管內(nèi)皮細(xì)胞中過表達(dá)后可引起視網(wǎng)膜血管內(nèi)皮細(xì)胞數(shù)量的減少,并可拮抗全長PDGFC誘導(dǎo)的細(xì)胞增殖。這些結(jié)果提示在PDR纖維血管膜中PDGFC的表達(dá)可能存在一種負(fù)反饋調(diào)節(jié)機(jī)制,即在PDR病理?xiàng)l件下,產(chǎn)生全長PDGFC的同時(shí),也產(chǎn)生了兩個(gè)新型剪接體來拮抗全長PDGFC的促新生血管的作用,其作用機(jī)制可能是在轉(zhuǎn)錄水平上降調(diào)節(jié)PDGFC,從而拮抗全長PDGFC的促新生血管的作用。更重要的是,新型剪接體的發(fā)現(xiàn)、克隆和表達(dá)為進(jìn)一步研發(fā)干預(yù)PDR中新生血管生成的新型藥物奠定了理論和實(shí)驗(yàn)基礎(chǔ)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R774.1

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