CAJ | 학술논문

目的构建rAAV2-hGAD65重组载体,观察其体内外功能.方法应用RT-PCR的方法克隆hGAD65基因,与AAV载体连接得到重组载体(rAAV2-hGAD65).包装重组腺相关病毒并检测病毒的滴度.体外感染成纤维细胞后,用免疫组织化学的方法检测GAD65在细胞中的表达水平,用高效液相色谱(HPLC)法检测培养上清中γ-氨基丁酸(GABA)的含量.在体实验中,向丘脑底核(STN)立体定位注射rAAV2-hGAD65后,用HPLC方法检测黑质网状部(SNr)中的GABA含量.结果应用RT-PCR方法成功地从人胚胎端脑组织中克隆出GAD65基因的cDNA,基因测序显示与基因库中人GAD65基因序列一致,亚克隆入AAV载体并包装后所得的病毒颗粒的滴度达到4.5×10~(11)/ml.组织化学检测感染大鼠肺成纤维细胞的效率约为80%,HPLC检测培养上清中GABA的含量为(45.66±6.07)nmol/L.STN立体注射rAAV2-hGAD65后,在STN可以检测到hGAD65的表达,SNr区GABA的含量由原来的(5.66±1.07)nmol/g升高到(12.66±2.59)nmol/g.结论成功地克隆出了人GAD65基因,并构建了AAV重组载体.AAV病毒颗粒在体外能感染成纤维细胞并具有催化谷氨酸合成GABA的功能.在体内实验中,向STN注射rAAV2-hGAD65后,可以增加黑质网状部(SNr)中的GABA含量.
목적 구건rAAV2-hGAD65중조재체,관찰기체내외공능.방법 응용RT-PCR적방법극륭hGAD65기인,여AAV재체련접득도중조재체(rAAV2-hGAD65).포장중조선상관병독병검측병독적적도.체외감염성섬유세포후,용면역조직화학적방법검측GAD65재세포중적표체수평,용고효액상색보(HPLC)법검측배양상청중γ-안기정산(GABA)적함량.재체실험중,향구뇌저핵(STN)입체정위주사rAAV2-hGAD65후,용HPLC방법검측흑질망상부(SNr)중적GABA함량.결과 응용RT-PCR방법성공지종인배태단뇌조직중극륭출GAD65기인적cDNA,기인측서현시여기인고중인GAD65기인서렬일치,아극륭입AAV재체병포장후소득적병독과립적적도체도4.5×10~(11)/ml.조직화학검측감염대서폐성섬유세포적효솔약위80%,HPLC검측배양상청중GABA적함량위(45.66±6.07)nmol/L.STN입체주사rAAV2-hGAD65후,재STN가이검측도hGAD65적표체,SNr구GABA적함량유원래적(5.66±1.07)nmol/g승고도(12.66±2.59)nmol/g.결론 성공지극륭출료인GAD65기인,병구건료AAV중조재체.AAV병독과립재체외능감염성섬유세포병구유최화곡안산합성GABA적공능.재체내실험중,향STN주사rAAV2-hGAD65후,가이증가흑질망상부(SNr)중적GABA함량.
Objective To construct the recombinant rAAV2-hGAD65 vector and detect its function both in vitro and in vivo. Methods The cDNA of human glutamic acid decarboxylase 2 (hGAD65) gene, which was one of gamma-aminobutyric acid (GABA) synthetase, cloned by the method of RT-PCR, was subcloned into the adeno-associated virus (AAV) vector and formed the recombinant vector of AAV-hGAD65 (rAAV2-hGAD65). The recombinant vector was packaged by the AAV Helper-Free System and its titer was detected. The primary fibroblast, cultured from the rat lung, was transfected by the rAAV2-hGAD65. The expression of the hGAD65 in fibroblast was detected by immunohistochemical method and the level of GABA in the media was assayed by high performance liquid chromatograph (HPLC). In vivo, the hGAD65 was detected by immunohistochemical method in STN and the concentration of the GABA in the reticular part of substantia nigra (SNr) was assayed by HPLC after the rAAV2-hGAD65 was injected into the subthalamic nucleus (STN) by the stereotaxic method. Results The sequence of hGAD65 cDNA was in accordance with that in the Genebank. The amino acid sequence of hGAD65 had no mutation and the titer of rAAV2-hGAD65 was reached 4.5 ×10~(11) per milliliter. The efficiency of infection to the rat primary firoblasts was 80%, and the concentration of GABA in the media of fibroblasts was (45.66±6.07)nmol/L per liter. In vivo, hGAD65 could be detected in STN, and the concentrateion of the GABA in the SNr was increased from (5.66±1.07)nmol/g to (12.66±2.59)nmol/g.Conclusion The cDNA of hGAD65 was cloned by RT-PCR and the recombinant vector of rAAV2-hGAD65 was constructed. The AAV can infect the primary fibroblast in vitro and the hGAD65 can catalyse the glutamic acic to GABA. In vivo, the concentration of GABA in the SNr was heighten after the rAAV2-hGAD65 was injected into the STN.