【作者】 刘瑜；

【导师】 叶茂；

【作者基本信息】 重庆医科大学 ， 麻醉学， 2009， 硕士

【摘要】 背景与目的5-羟色胺5A受体是5-羟色胺受体家族中的一个亚型,是主要定位于中枢神经系统的一种G蛋白偶联受体。在对小鼠、大鼠和人mRNA的定位研究显示5-HT5A受体mRNA在脑组织中广泛表达,但在外周组织的表达很低甚至不表达。5-HT5A受体的分布区域与认知、焦虑的调节、感觉的形成、神经内分泌、运动的整合、昼夜节律的调节及疼痛的调控有关。虽然5-HT5A受体被发现已经超过10年,但是目前国内外少有对5-HT5A受体功能的深入研究。这主要是因为缺乏针对5-HT5A受体的高选择性配体和拮抗剂,因此很难界定5-HT5A受体在生理和病理过程中所发挥的作用。因此本实验构建了以5-HT5A受体基因为靶标的siRNA表达载体,并观察其在大鼠神经细胞中的表达,为在动物体内进一步研究该受体的功能提供了技术支持。研究方法1.设计并构建了针对5-HT5A受体mRNA的3条siRNA质粒表达载体pGenesil-2 Htr5a1、pGenesil-2 Htr5a2、pGenesil-2 Htr5a3和一条无关对照序列表达载体pGenesil-2 HK,同时还构建了一条携带有5-HT5A受体全长基因并同时表达红色和绿色荧光蛋白的质粒表达载体pDsRed-EGFP-Htr5a。分别将pGenesil-2 Htr5a1、pGenesil-2 Htr5a2、pGenesil-2 Htr5a3、pGenesil-2 HK质粒表达载体与pDsRed-EGFP-Htr5a质粒表达载体共转染293细胞,48小时后进行流式细胞筛选,通过用Red红色荧光做对照,EGFP绿色荧光为主要荧光,看干扰序列对EGFP的减弱程度,从而鉴定出相应的干扰序列对基因的干扰效果,对EGFP减弱程度越明显,说明干扰效果越明显。2.将筛选出的pGenesil-2 Htr5a2表达载体中的Htr5a2表达框亚克隆至pGenesil1.2构建pGenesil1.2 Htr5a2质粒表达载体,然后将pGenesil1.2 Htr5a2与pGSadeno腺病毒表达载体进行同源重组,构建pGSadeno Htr5a2重组腺病毒表达载体,将其转染293细胞进行包装出毒并进一步放大培养以获得需要的病毒量。将收获的pGSadeno Htr5a2重组腺病毒转染原代培养的大鼠神经细胞,48小时后用RT-PCR技术检测各组神经细胞内5-HT5A受体mRNA的表达情况。结果1.经酶切鉴定及DNA测序,重组质粒中已插入了目的基因片段。经流式细胞检测,通过比较GFP平均荧光强度与RFP平均荧光强度的比值,就能筛选出最有效的序列。其中pGenesil-2 Htr5a2的GFP平均荧光强度/RFP平均荧光强度的值为0.39,pGenesil-2 Htr5a1的值为0.539,pGenesil-2 Htr5a3的值为0.417,pGenesil-2 HK的值为1.626,由此可知pGenesil-2 Htr5a2能有效抑制5-HT5A受体基因的表达。2.通过酶切鉴定,证明了目的基因片段正确插入了pGSadeno腺病毒载体中。由于pGSadeno Htr5a2重组腺病毒中有绿色荧光蛋白真核表达框,因此在转染细胞后如果出现明显的绿色荧光蛋白表达,则说明有感染能力的重组腺病毒包装成功,而且经测定腺病毒滴度达到109 pfu/ml。将pGSadeno Htr5a2重组腺病毒转染神经细胞后24小时可以观察到绿色荧光蛋白的表达,48小时后经RT-PCR技术检测pGSadeno Htr5a2重组腺病毒能有效抑制5-HT5A受体mRNA的表达。结论1.成功构建了和鉴定了针对5-HT5A受体基因的RNA干扰重组质粒表达载体pGenesil-2 Htr5a1、pGenesil-2 Htr5a2、pGenesil-2 Htr5a3,并发现pGenesil2-Htr5a2能有效抑制外源性5-HT5A受体基因在293细胞中的表达,从而筛选出最有效的重组质粒表达载体,为下一步构建针对5-HT5A受体基因的siRNA重组腺病毒表达载体做好了准备。2.成功构建了和鉴定了针对5-HT5A受体基因的siRNA重组腺病毒表达载体pGSadeno Htr5a2,将其包装出毒,并且验证了pGSadeno Htr5a2重组腺病毒能有效抑制内源性5-HT5A受体基因在神经细胞中的表达,为以后的动物试验提供了技术支持。更多还原

【Abstract】 Background and Objective5-HT5A receptor is a subtype of 5-HT receptors’family, which is a kindof G-protein-coupled receptor located in central nervous system. mRNAlocalisation studies in mouse, rat and human brain have revealed that5-HT5A receptor mRNA is widely expressed. In the periphery 5-HT5Areceptor mRNA expression appears to be very low or absent. Thedistribution of 5-HT5A receptor mRNA have provided important pointers tothe potential functional roles of the receptor in central nervous system, suchas cognition regulation, anxiety regulation, formation of sensation,neuroendocrine regulation, locomotion integration, circadian regulationand pain regulation. 5-HT5A receptor has been found more than ten years, butthere are few studies about the function, which is due to the lack of selectiveligands and antagons, so it was difficult to define the function of 5-HT5Areceptor in physiological and pathological process. So we constructed rat5-HT5A receptor siRNA recombinant adenovirus expression vector, andobserved its expression in rat never cells to provide technique sustain to the studies about the function of 5-HT5A receptor in vivo.Methods1. Three 5-HT5A receptor siRNA plasmid expression vector ofpGenesil-2 Htr5a1, pGenesil-2 Htr5a2, pGenesil-2 Htr5a3, and onescrambled sequence of pGenesil-2 HK were designed and constructed. Atthe same time, another plasmid expression vector pDsRed-EGFP-Htr5awhich could express 5-HT5A receptor, EGFP and AFP was constructed either.In order to find out the most effective siRNA plasmid expression vector, therecombinant vectors of pGenesil-2 Htr5a1, pGenesil-2 Htr5a2, pGenesil-2Htr5a3 and pGenesil-2 HK were cotransfected respectively withpDsRed-EGFP-Htr5a into the 293 cells. Forty-eight hours aftercotransfection, fluorescence intensities of GFP and AFP were detected byflow cytometry on 293 cells. The weaker of the GFP intensity the moreeffective was the siRNA plasmid expression vector.2. The most effective siRNA expression frame Htr5a2 was subclonedinto pGenesil1.2 to construct pGenesil1.2 Htr5a2 plasmid expression vector.Then pGenesil1.2 Htr5a2 was homologous recombinated with pGSadenoexpression vector to construct recombinant adenovirus expression vector.Then the expression vector of pGSadeno-Htr5a2 was introduced into 293cells to product recombinant adenovirus. Subsequently, the recombinantadenovirus was transfected into 293 to generate a higher titer viral stock.Then the high titer recombinant adenovirus was transfected into primary cultured rat nerve cells. Forty-eight hours after transfection, the expressionof 5-HT5A at the level of mRNA was detected by RT-PCR.Results1. The siRNA expression vectors were successfully constructed, whichwas identificated by restriction endonuclease and DNA sequencinganalyzing. After cotransfection with pDsRed-EGFP-Htr5a into 293 cells,the most effective siRNA plasmid expression vector was ascertained byflow cytometry which could detect the fluorescence intensities of GFP andAFP. The ratio of the average fluorescence intensity of GFP to AFP was0.39 in 293 cells transfected with pGenesil-2 Htr5a2. The ratios were 0.539,0.417, and 1.626 in 293 cells transfected with pGenesil-2 Htr5a1,pGenesil-2 Htr5a3 and pGenesil-2 HK, respectively. So pGenesil-2 Htr5a2expression vector can suppress actively the expression of 5-HT5A.2. The recombinant adenovirus expression vector of pGSadeno Htr5a2was successfully constructed, which was identificated by restrictionendonuclease. Because there was a GFP expression frame in pGSadenoHtr5a2, GFP expression could be observed after that the vector wastransfected successfully into the 293 cells. The appearance of GFPexpression in 293 cells meant that recombinant adenovirus have beenpackaged successfully. And the titer of recombinant adenovirus was 109pfu/ml detected by TCID50. Then, pGSadeno Htr5a2 recombinantadenovirus was transfected into primary cultured rat nerve cells. Twenty-four hours after transfection, GFP expression could be observed innerve cells. And pGSadeno Htr5a2 recombinant adenovirus could suppressactively the expression of 5-HT5A in nerve cells, which was detected byRT-PCR after 48hrs.Conclusions1. 5-HT5A receptor siRNA plasmid expression vectors of pGenesil-2Htr5a1, pGenesil-2 Htr5a2 and pGenesil-2 Htr5a3 were constructedsuccessfully. And pGenesil-2 Htr5a2 was the most effective recombinantplasmid expression vector that could suppress actively the expression ofexogenous 5-HT5A in 293 cells, which was prepared for constructing5-HT5A siRNA recombinant adenovirus expression vector.2. 5-HT5A receptor siRNA recombinant adenovirus expression vectorof pGSadeno Htr5a2 was constructed and packaged successfully. AndpGSadeno Htr5a2 recombinant adenovirus could suppress actively theexpression of endogenous 5-HT5A in nerve cells, which provided techniquesustain to the studies about the function of 5-HT5A receptor in vivo.更多还原