【作者】 陈娜；

【导师】 冯泽国；

【作者基本信息】 中国人民解放军军医进修学院 ， 麻醉学， 2009， 硕士

【摘要】 N-型钙通道是治疗慢性神经痛的一个重要药理学靶点,目前临床上应用的N型钙通道阻断剂不仅影响伤害性感受传导通路的功能,也影响中枢神经系统及自主神经系统的功能。最近有研究发现N型钙通道Car2.2e[37a]亚型仅表达于背根神经节。本研究以CaV2.2e[37a]作为靶基因,设计筛选对该基因有特异性抑制作用的siRNA,并研究该siRNA对CaV2.2e[37a]基因的抑制效果,为进一步探讨用siRNA特异性抑制CaV2.2e[37a]产生的镇痛作用及研发以cav2.2e[37a]为靶点的镇痛药物提供实验依据。第一部分EGFP-e37a/e37b融合基因质粒的构建及e37a细胞株的建立目的:为筛选特异性抑制Car2.2e[37a】表达的siRNA,通过构建融合基因EGFP-e37a和EGFP-e37b作为模拟靶基因,建立筛选siRNA的技术平台。方法:①表达EGFP-e37a和EGFP-e37b融合基因质粒的构建:以pcDNA316-EGFP为模板,采用重叠延伸PCR的方法扩增EGFP-e37a/e37b靶基因片段。将目的片段与pMD18-T载体连接后测序,序列正确后用酶切法克隆入pAAV2-neo载体并鉴定,得到重组质粒pAAV2-neo-EGFP-e37a和pAAV2-neo-EGFP-e37b。②稳定表达EGFP-e37a融合基因的BHK细胞株的建立:将重组质粒pAAV2-neo-EGFP-e37a在脂质体作用下转染到BHK细胞表达,荧光显微镜检测;通过G418筛选后在荧光显微镜下用稀释法挑细胞单克隆,建立稳定表达EGFP-e37a融合蛋白的BHK细胞株,命名为e37a细胞株。结果:①将得到的DNA片段用PCR方法鉴定,可见在750～1000bp之间有一条清晰单一的扩增条带;对克隆到pMD18-T载体中的片段进行PCR鉴定并进行测序,将克隆入真核载体的目的基因酶切鉴定,得到与预期一致的结果;②重组质粒pAAV₂-neo-EGFP-e37a在BHK细胞中表达,荧光显微镜检测结果可见表达效率达到40-50%;挑取了7株阳性细胞克隆,经荧光显微镜检测可见BHK细胞100%表达融合蛋白EGFP-e37a。结论:利用重叠延伸PCR方法成功构建融合基因EGFP-e37a和EGFP-e37b;建立了针对Cav2.2基因外显子e37a进行RNAi的siRNA筛选平台。第二部分抑制CaV2.2e[37a]基因表达siRNA的设计与筛选目的:设计并构建以CaV2.2的外显子e37a为靶基因的siRNA质粒,筛选对EGFP-e37a有特异性抑制效果的siRNA。方法:①根据RNA干扰原理及siRNA设计原则设计siRNA,克隆至pGPU6/Neo-siRNA载体,分别命名为Ⅰ,Ⅱ,Ⅲ,Ⅳ;②质粒共转染进行RNAi实验:用质粒pGPU6/Neo siRNA分别与质粒pAAV₂-neo-EGFP-e37a共转染,转染质粒之比为10:1,并设质粒pAAV₂-neo-EGFP,pAAV2-neo-EGFP-e37b对照;并将pGPU6/Neo-siRNAⅡ与pAAV₂-neo-EGFP-e37a不同剂量共转染。利用荧光显微镜和流式细胞术检测干扰效果;③对e37a细胞株进行的RNAi实验:不同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株;相同剂量pGPU6/Neo-siRNAⅡ转染e37a细胞株后不同时间点（24h、48h、72h）检测干扰效果。④Western blotting检测pGPU6/Neo-siRNAⅡ对EGFP-e37a融合基因表达的干扰结果,并设EGFP-e37b、EGFP为对照。结果:①设计了4段长度为21bp的针对e37a基因的siRNA,酶切鉴定与预期一致。②质粒共转染进行RNAi抑制实验中筛选得到对EGFP-e37a有干扰作用而对EGFP-e37b无干扰作用的siRNAⅡ,干扰效率达到84-90%,siRNAⅡ与质粒pAAV2-neo-EGFP-e37a不同剂量共转染时流式细胞术检测平均荧光强度分别降低（%）:10:1时为86±6.1;5:1时为82±5.9;1:1时为80±4.2;1:5时为85±6.0;1:10时为79±4.6。③对e37a细胞株进行的RNAi抑制实验中,流式细胞术检测siRNAⅡ对EGFP-e37a的干扰效率（%）:3.6ug时为93±4.2,1.8ug时为95±4.9,0.9ug时为91±3.8,0.45ug时为86±5.4,0.225ug时为70±4.3.不同时间点（24h、48h、72h）的干扰效率荧光显微镜检测在24h不明显,48h抑制细胞数达到了74±4.8（%）,72h抑制细胞数82±3.3（%）。④Western blotting检测:siRNAⅡ对EGFP-e37a融合基因的表达明显抑制,蛋白条带明显减弱,而对于EGFP-e37b、EGFP基因的表达没有影响。结论:本研究在BHK细胞系中采用荧光显微镜观察和流式细胞术两种方法直观而简便地筛选出特异性干扰EGFP-e37a表达的siRNAⅡ,并对其RNAi的量效和时效进行探讨,为进一步在细胞和动物水平对Cav2.2基因的C末端外显子e37a进行RNAi的研究打下基础。第三部分特异性siRNA对CaV2.2e[37a]基因的RNAi研究目的:检测siRNAⅡ对于Car2.2e[37a]基因表达的干扰情况。方法:①设计测序引物对四质粒进行测序。②在293FT细胞中将pGPU6/Neo-siRNAⅡ与质粒Cav2.2e[37a]共转染,Western blotting检测siRNAⅡ对基因Car2.2e[37a]表达的抑制情况,并设转染Car2.2e[37b]组、空白293FT细胞组为对照组。结果:①四质粒测序结果与Genbank序列完全一致。②Western blotting检测质粒siRNAⅡ与基因Cav2.2e[37a]转染剂量在10:1时蛋白条带消失,而对于基因Cav2.2e[37b]、空白293FT细胞的蛋白表达没有影响。结论:siRNAⅡ对基因Cav2.2e[37a]表达有特异性抑制作用,为下一步在动物活体进行RNAi实验及为探讨CaV2.2 e[37a]亚型在慢性疼痛信号转导中的作用机制奠定了基础。更多还原

【Abstract】 N-type calcium channel is a key pharmacological target for treatment of pain, N-type calcium channel inhibitors always cause side effects.The present challenge lies in finding some ways to specifically inhibit N-type channels in the pain pathway,not only eliminating pathological pain but also maintaining normal nociception,as well as sensory and autonomic.N-type calcium channels have two isoforms,CaV2.2e[37a]and CaV2.2e[37b].All neurons express CaV2.2e[37b] but that a subset of nociceptive neurons,particularly in DRG,express CaV2.2e [37a].This study focused on specifically knockdown the expression of CaV2.2e [37a]gene by RNA interference to provide the base for exploring the N-type calcium channel isoform inhibitor.PartⅠConstruction of EGFP-e37a/e37b fusion gene plasmids and e37a cell lineObjective:To find out siRNAs specifically inhibiting CaV2.2e[37a]without inhibiting CaV2.2e[37b]gene expression Methods:①Construction of EGFP-37a/37b fusion gene plasmids as the mimic target genes.Full length cDNA fragment of EGFP was obtained from pcDNA316-EGFP,and amplified by high fidelity PCR Polymerase using sense primer with SOE- PCR.The fragment was inserted into eukaryotic reporter vector pAAV2 -neo by cutting with EcoRⅠand BglⅢ.The fusion gene vectors,named pAAV2 -neo-EGFP-37a/37b identified by restricted endonuclease digestion and DNA sequence analysis.②Establishment of stable cell line expressed EGFP-e37a fusion gene in BHK cells.The recombinant plasmids pAAV2 -neo-EGFP- e37a was transfect into BHK cells by Lipofectamine 2000.After transfection,cells were maintained with G418.pAAV2 -neo-EGFP-37a cell line was constructed with dilution.Result:①EGFP-37a/37b were characterized by PCR and digestion by EcoRⅠand BglⅡ.The length of fragments was about 829 bp.The positive clones were subjected to sequencing and the results were shown as expected.②The efficiency of recombinant plasmids pAAV2 -neo- EGFP- e37a transfection into BHK cells was about 40-50%.7 strains positive cell clones were selected by G418.Conclusions: Successfully construction of EGFP-37a/37b fusion gene plasmid as the mimic target genes by SOE-PCR.Establishment the screening platform to screen siRNA for inhibiting CaV2.2e[37a]gene expression.PartⅡDesigning and screening siRNA for inhibiting expression of CaV2.2e[37a]geneObjective:To design and construct plasmids siRNA according to CaV2.2e[37a] gene and find out siRNAs specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression.Methods:①Following siRNA design principles, four e37a siRNAs,namedⅠ,Ⅱ,Ⅲ,Ⅳ,were synthesized and clone into vector pGPU6/Neo.②Plasmids pAAV2- neo-EGFP-e37a were co-transfected into BHK cells with pGPU6 /Neo-siRNA at ratio 10:1.Plasmids pAAV2- neo- EGFP, pAAV2-neo-EGFP-e37b was used as control.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose.To assess the effect of RNAi on EGFP-37a fusion protein,microscopy and fluorescence activated cell sorting(FACS) were used.③RNAi in e37a cell line:Plasmids siRNAⅡwere transfected into e37a cell line at different dose and detected the effect of RNAi at different points of time.④Fusion protein expression was detected by western blotting analysis.Results:①Design 4 pairs of 21 bp siRNAs.The result of restricted endonuclease digestion results showed the inserted fragment was as expected.②In the 4 pairs of siRNA,siRNAⅡinduced a significant decrease of EGFP-e37a expression(84-90%),without interfering EGFP- 37b gene expression.Plasmids siRNAⅡwere co-transfected into BHK cells with pAAV2- neo-EGFP-e37a at different dose,and reducing the percentage of the mean fluorescence intensity(MFI) were:10:1,86±6.1(%);5:1,82±5.9 (%);1:1,80±4.2(%);1:5 85±6.0(%);1:10,79±4.6(%).③RNAi in e37a cell line:Plasmids siRNAⅡreducing the percentage of MFI were:3.6ug, 93±4.2(%);1.8ug,95±4.9(%);0.9ug,91±3.8(%);0.45ug,86±5.4(%);0.225ug, 70±4.3(%).24h after the transfection,the efficiency of RNAi was not obvious.At the time point of 48h,the efficiency of RNAi amount to 74±4.8(%);72h,82±3.3 (%).④siRNAⅡcould decrease the expression of EGFP-e37a protein level obviously,while not decrease the expression of EGFP-e37b and EGFR Conclusions:siRNAⅡthat specifically inhibiting EGFP-37a without inhibiting EGFP- 37b gene expression has been found out by fluorescence microscopy and FACS.So we provide a basis tbr studying of RNAi of Cav2.2e[37a]in vitro and in vivo.PartⅢStudy of the specific siRNA inhibiting CaV2.2e[37a]gene expressionObjective:To detect the effect of siRNAⅡinhibiting CaV2.2e[37a]gene expression by western blotting.Methods:①Design the primer for sequencing four plasmids.②Plasmid siRNAⅡco-transfected into 293FT cells with plasmids Cav2.2e[37a],Cav2.2e[37b],β3,α2σ1 respectively,and detected the expression of Cav2.2e[37a]gene by western blotting.Result:①The results of DNA sequencing showed the four plasmids were as Genebank sequences.②The protein on band 6 diminished obviously which indicate that siRNAⅡdecrease the generation of CaV2.2e[37a]expression protein in 293FT cells.Conclusions: SiRNAⅡcould inhibit Cav2.2e[37a]gene expression specifically without inhibiting CaV2.2e[37b]gene expression,and providing a potential agent for studying RNAi Cav2.2e[37a]isoform in noxious stimuli transmission..更多还原