【作者】 卢强；

【导师】 王云杰； 韩勇；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 研究一人食管平滑肌细胞中GIRK表达的实验研究目的:探讨食管下段平滑肌中有无GIRK的表达以及各亚型之间表达的差异性,同时明确食管环形平滑肌(CM)与纵行平滑肌(LM)之间表达的区别,从食管自节律入手深入研究食管运动功能的深层机制,为进一步研究食管功能的调节打下基础。方法:根据24h食管pH值检测、食管镜检查及HE染色结果,分辨、筛选正常人食管平滑肌,按人GIRK 1-4序列设计并合成各自的高效引物,利用RT-PCR、Western Blotting方法检测人食管平滑肌细胞中GIRK 1-4的表达。结果:RT-PCR及Western Blotting检测证明人食管平滑肌细胞仅表达GIRK 1-4中的GIRK2、3、4;其中GIRK4表达最为丰富;但GIRK2与GIRK3的表达无明显差异性(P>0.05);而GIRK2与GIRK4,GIRK3与GIRK4的表达却差异均显著( P<0.05); LM和CM组的表达亦无明显差异(P>0.05)。结论:人食管平滑肌细胞不表达GIRK1,仅表达GIRK 2、3、4;其中GIRK4的表达最丰富;GIRK2、GIRK3的表达相对较少;人食管LM和CM GIRK 2、3、4的表达无显著差异性。研究二筛选高效人GIRK4基因RNA干扰序列、构建重组腺病毒载体实验一:RNA干扰质粒的构建目的:构建编码人GIRK4的shRNA真核表达载体质粒,为利用RNA干扰技术从转录后水平进行GIRK4的研究做准备。方法:根据人GIRK4序列设计并合成四组shRNA寡核苷酸片段,退火形成双链并克隆进入载体pGCsi-U6,采用PCR进行鉴定,并使用377型测序仪进行测序分析。结果:PCR证明构建的shRNA已插入载体,经测序证明与与设计的相同,获得了人GIRK4的shRNA表达载体质粒(pGCsi-GIRK4-shRNA-U6)。结论:构建GIRK4的shRNA真核表达载体可进入哺乳动物细胞内表达短发夹RNA,通过Dicer酶裂解成siRNA并降解靶基因mRNA,而达到RNA干扰的作用,为利用其进行GIRK4功能研究奠定基础。实验二:GIRK4基因融合蛋白表达载体的构建目的:构建编码人GIRK4真核表达载体质粒,为进行人GIRK4的shRNA序列的筛选作准备。方法:以购买的人GIRK4基因为模板,PCR其cDNA编码序列克隆进入载体pEGFP-C1,进行PCR鉴定、测序及转染293细胞在荧光显微镜下观察。结果:PCR证明cDNA已插入载体,经测序显示与与设计的序列相同,获得人GIRK4融合蛋白的表达载体质粒(pEGFP-GIRK4-C1),并能转染293细胞表达融合蛋白。结论:构建的人GIRK4真核表达载体可进入哺乳细胞内表达GIRK4-EGFP融合蛋白,为进行针对GIRK4基因RNA干扰序列筛选打下基础。实验三:RNA干扰有效靶点的筛选目的:确定外源表达系统中(HEK293 cell)GIRK4-shRNA沉默GIRK4基因表达的有效性及选择高效的干扰序列。方法:将已构建的四个靶向GIRK4基因不同区域GIRK4的shRNA表达载体质粒(pGCsi-GIRK4-shRNA-U6)、对照质粒分别与人GIRK4的真核表达载体质粒(pEGFP-GIRK4-C1)共转染HEK293细胞,收集转染48h后的细胞,进行Western blotting检测。结果:转染48小时后与对照组相比,四个不同序列的shRNA表达载体质粒(pGCsi-GIRK4-shRNA-U6)使GIRK4表达在48小时分别减少78.1%、85.3%、88.3%、94.3%,其中以4#载体GGCTGAGCAGAATGAAGAA呈最有效的干扰序列。结论:成功地在外源表达系统筛选出了针对人GIRK4基因的高效RNA干扰序列GGCTGAGCAGAATGAAGAA。实验四:腺病毒表达载体构建目的:利用AdMax腺病毒载体系统构建重组腺病毒载体Ad-GIRK4-shRNA并在293细胞中扩增制备重组腺病毒载体。方法:将合成的针对GIRK4的4#高效干扰序列,克隆到pDC316-EGFP-U6载体中,再将pDC316-GIRK4-shRNA-EGFP-U6载体和骨架病毒pBHGlox_E1,3Cre共转染293细胞,包装成重组的病毒颗粒,并行PCR鉴定和测序。结果:经PCR鉴定和测序,证实构建的4#干扰序列重组腺病毒载体(Ad-GIRK4-shRNA)正确并制备出高滴度重组病毒。结论:成功构建了携带人GIRK4 shRNA片段的高效重组腺病毒载体(Ad-GIRK4-shRNA),为深入研究人食管平滑肌GIRK4基因奠定了基础。研究三RNA干扰人食管平滑肌细胞GIRK4基因的实验研究实验一:人食管平滑肌细胞的培养目的:培养出一定数量状态良好的人食管平滑肌细胞备实验二用。方法:联合运用Ⅰ型胶原酶和ⅩⅣ型蛋白酶消化人下段食管标本,利用差速贴壁法消除大部分非食管平滑肌细胞,采用α-actin染色,Elivision二步法进行免疫组化鉴定。结果:经actin抗体鉴定,培养出的细胞90%以上均为食管平滑肌细胞。结论:成功培养出人食管平滑肌细胞,可供进一步行RNA干扰人食管平滑肌细胞GIRK4基因实验所用。实验二:RNA干扰人食管平滑肌细胞GIRK4基因的实验研究目的:探讨GIRK4 shRNA导入人食管平滑肌细胞进行RNA干扰GIRK4表达的可行性,观察对迷走神经通路上的M2AChR表达及IKACh电流密度的影响。方法:用重组腺病毒载体Ad-GIRK4-shRNA转染人食管平滑肌细胞,于转染后不同时间收集细胞,利用RT-PCR、蛋白印迹来检测GIRK4、M2AChR的表达,全细胞膜片钳技术检测IKACh、IK1电流密度的变化。结果:Ad-GIRK4-shRNA在MOI 20对人食管平滑肌细胞转染效率达到95%以上,呈现剂量依赖性和时间依赖性: MOI 5 , 20 , 50时,Ad-GIRK4-shRNA干扰效率分别为15.1%, 35.2%, 51.1%(P<0.05);与空病毒感染组比较,人食管平滑肌细胞感染Ad-GIRK4-shRNA后GIRK4 mRNA表达在24th、48 th、96 th分别为50.9%、14.7%、98.1%;Ads-GIRK4-shRNA在mRNA和蛋白水平使M2AChR的表达增加了19.6%,18.6%(P<0.05)。Ad-GIRK4-shRNA对IK1的电流密度无影响(P>0.05),使IKACh的电流密度下降了53%(P<0.05)。结论:Ad-GIRK4-shRNA可高效、成功转染人食管平滑肌细胞,其可能在mRNA和蛋白水平降低了GIRK4的表达,呈现剂量依赖和时间依赖性关系;提示人食管平滑肌细胞行RNA干扰GIRK4的表达是可能的;GIRK4的表达下降进而导致M2AChR表达的增高,IKACh的电流密度下降;表明GIRK4在食管M2受体-G蛋白-IKACh通路和IKACh通道中起重要作用,干扰GIRK4的表达具有潜在的治疗效应;为将RNA干扰技术引入食管运动功能障碍机制的研究、基因治疗提供了新思路。更多还原

【Abstract】 Background and objective :Disoerder of the esophageal function frequently results in an impotence of esophageal smooth muscles contraction [1], impermanency relaxation of lower esophageal sphinctor[2], inefficacy of the esophagus elimination [3; 4] that consequently leads to the gastroesophageal reflux diseases, such as reflux esophagitis, Barrett esophagus and esophageal carcinoma[5; 6; 7]. Contraction of the esophageal muscles is dominated and regulated by Ach which is abele to active M2AChR and IKACh. GIRK channels, or acetylcholine-sensitive potassium channels (IKACh), were constructed by GIRK family including GIRK1, GIRK2, GIRK3 and GIRK4 subunits [8; 9; 10]. GIRK subunits are expressed in human various tissues. Thereinto, GIRK1, GIRK2 and GIRK3 subunits are expressed in various regions of the CNS, such as the olfactory bulb, cerebral cortex, amygdala, hippocampus, thalamus, cerebellum, substantia nigra, ventral tegmental area, locus coeruleus, some nuclei of brainstem and spinal cord [11; 12; 13; 14], indicating their possible involvement in various CNS functions such as cognition, memory, emotions and motor coordination. In contrast, GIRK4 subunits are expressed in only a few regions of the brain [12; 14; 15]. Neuronal GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK2 subunits in most brain regions [13; 16] or homomultimers composed of GIRK2 subunits in the substantia nigra[17]. GIRK1 subunits do not form functional homomeric channels [18; 19]. In the heart, atrial GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK4 subunits [18]. GIRK4 is the functional subunit of IKACh and ablation of GIRK4 should result in the functional elimination of IKACh. GIRK1 could not form a functional ion channel without GIRK4. Humman esophageal IKACh was activated by various receptors coupled to G-proteins of pertussis toxin-sensitive class. Among these receptors, muscarinic 2 acetylcholine receptor was the most important one. However, it was still unclear whether GIRK channels exist in esophageal SMCs.RNA interference (RNAi) by short double stranded RNA represents an efficient and frequently used way to study gene function. RNAi utilizes sequence-specific double-stranded small interfering RNA to silence gene expression in mammalian cells. RNAi in human esophageal smooth muscles (SMCs) would increase the value for applications, however the related experiments were seldom performed in human SMCs. To investigate the expression of GIRK gene and its subunits in esophageal smooth muscles, a series of experiments was designed in this study.Methods GIRK gene expression and was detected in human esophageal SMCs with the methods of reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The human esophageal SMCs were cultured for experiment models. The plasmid was constructed and then rearranged into adenovirus to achive vector Ad-GIRK4-shRNA.The human esophageal smooth muscles were transfected by Ad-GIRK4-shRNA and the expression of GIRK4、M2AChR was detected. The density of IKACh was observed using electrophysiological recording system.Results (1) The mRNA and protein expression of GIRK 2, 3, 4 subunits except for GIRK 1 were detected in human esophageal either longitudinal muscle or circular muscle cells.(2) A high effective RNAi sequence (GGCTGAGCAGAATGAAGAA) for GIRK4 was sccessfully achieved. (3) The plasmid pGCsi-GIRK4-shRNA-U6 with expression of GIRK4-shRNA was constructed successfully. (4)The adenoviral vector (Ad-GIRK4-shRNA) to express GIRK4-shRNA was constructed successfully. (5)The efficiency of knockdown in GIRK4 expression was different between the exogenous systems in 48.9% and endogenous in 94.3%. (6) RNA interference to GIRK4 expression was demonstrated both in time-dependent and dose-dependent model. Comparing the efficiency of knockdown in experimental and control group, the valiue of ratio from two groups was 15.1%, 35.2% and 51.1% in MOI 5, 20, 50 respectively in protein level. It was 49.1%, 85.3%, 1.7% at 24th, 48 th, 96 th hours in MOI 50 respectively in mRNA level. (7) Adenovirus was a sutiable vector for transferring shRNA into the human esophageal SMCs with more than 95% of efficiency. (8) Expression of M2AChR in sh-GIRK4 group was 0.609±0.027 which was more 19.6% than 0.509±0.028 in the control in mRNA level (p<0.05). It was 0.531±0.022 in sh-GIRK4 group which was more 18.6% than 0.448±0.023 in the control in protein level (p<0.05). (9)The current density of IKACh in Ad-GIRK4-shRNA group was–0.93±0.28pA/pF which was 53% less tha–1.95±0.51pA/pF in the control (p<0.01).Conclusion: Expression of GIRK 2-4 subunits was found in human esophageal SMCs.That indicated there would be functional GIRK channels in the SMCs.The expression of GIRK4 in human esophageal SMCs was not only decreased by adenovirus-delivered short hairpin RNA targeting GIRK4, but also the expression of M2AChRand the amplitude of IKACh were affected. GIRK4 might play an important role in the pathway(Ach-M2AChR-G protein- IKACh) and IKACh channel.更多还原