CAJ | 학술논문

目的构建可转染神经胶质细胞和神经元的增强型绿色荧光蛋白-神经标识(EGFP-ID)真核表达载体,为神经突起损伤活体研究提供技术方法. 方法采用PCR方法分别从质粒pEGFP-N1载体和大鼠脑组织基因组DNA中扩增EGFP和ID序列.通过T4 DNA连接酶将纯化后的PCR产物EGFP与经过BamH Ⅰ和EcoR Ⅰ双酶切的pcDNA4.1-hisB载体连接.将经克隆测序确定的pcDNA4.1-EGFP经由NotⅠ和Xba Ⅰ双酶切并与纯化的PCR产物ID连接,再次克隆测序确定为pcDNA4.1-EGFP-ID.将pcDNA4.1-EGFP与pcDNA4.1-EGFP-ID在转染试剂Fugene6 介导下转染人神经胶质瘤U87细胞和小鼠原代培养神经元,通过倒置荧光显微镜观察EGFP在2种细胞内的分布.此外,用50 μmol/L核苷类似物司他夫定处理神经元8d,第8天末观察EGFP-ID 示踪细胞突起发育情况,并与免疫荧光检测结果作比较. 结果成功扩增得到EGFP和ID基因片段,重组得到EGFP-ID真核表达载体,该载体携带的EGFP基因在U87细胞和神经元内表达并布满细胞突起,示踪神经元突起形态变化的结果与免疫荧光观察结果一致. 结论 pcDNA4.1-EGFP-ID能够介导EGFP基因在神经元和胶质细胞突起内表达,从而直观再现神经突起形态特征.
목적 구건가전염신경효질세포화신경원적증강형록색형광단백-신경표식(EGFP-ID)진핵표체재체,위신경돌기손상활체연구제공기술방법. 방법 채용PCR방법분별종질립pEGFP-N1재체화대서뇌조직기인조DNA중확증EGFP화ID서렬.통과T4 DNA련접매장순화후적PCR산물EGFP여경과BamH Ⅰ화EcoR Ⅰ쌍매절적pcDNA4.1-hisB재체련접.장경극륭측서학정적pcDNA4.1-EGFP경유NotⅠ화Xba Ⅰ쌍매절병여순화적PCR산물ID련접,재차극륭측서학정위pcDNA4.1-EGFP-ID.장pcDNA4.1-EGFP여pcDNA4.1-EGFP-ID재전염시제Fugene6 개도하전염인신경효질류U87세포화소서원대배양신경원,통과도치형광현미경관찰EGFP재2충세포내적분포.차외,용50 μmol/L핵감유사물사타부정처리신경원8d,제8천말관찰EGFP-ID 시종세포돌기발육정황,병여면역형광검측결과작비교. 결과 성공확증득도EGFP화ID기인편단,중조득도EGFP-ID진핵표체재체,해재체휴대적EGFP기인재U87세포화신경원내표체병포만세포돌기,시종신경원돌기형태변화적결과여면역형광관찰결과일치. 결론 pcDNA4.1-EGFP-ID능구개도EGFP기인재신경원화효질세포돌기내표체,종이직관재현신경돌기형태특정.
Objective To construct the enhanced green fluorescent protein-neuronal identifier (EGFP-ID) eukaryotic expression vector to in vivo trace the neurite morphology of neurons and glioblastoma cells,and provide study methods for neurite damage.Methods EGFP and ID sequences were amplified by PCR from DNAs in the pEGFP-N1 plasmids and genomes of rat brain tissues,respectively.Purified PCR product EGFP was linkaged with revised pcDNA4.1-hisB vector digested by restriction enzymes BamHI and EcoRI via T4 DNA ligase.Then,the recombined product pcDNA4.1-EGFP was further digested by restriction enzymes NotI and XbaI and linkaged with purified PCR product ID,resulting in recombined plasmids pcDNA4.1-EGFP-ID.The pcDNA4.1-EGFP and pcDNA4.1-EGFP-ID were identified by sequencing,and then,transfected into human glioma U87 cell line and mouse cortical neurons by fugene 6 oligfectamine reagent.The distributions of pcDNA4.1-EGFP and pcDNA4.1-EGFP-ID in cells were detected by reverse fluorescence microscopy.Furthermore,50 μmol/L nucleoside analogue stavudine was kept in the culture media for 8 days,and EGFP-ID vector was traced the damage of stavudine to mouse cortical neurons,which was compared with those results by immunofluorescence test.Results The gene fragments of EGFP and ID were successfully amplified,and eukaryotic expression vector of pcDNA4.1-EGFP-ID,carried EGFP gene,was successfully constructed and expressed in U87 cells,nerve cells and their neuritis; the results of tracing morphological changes of the cells were consistent between the immunofluorescence and the vector.Conclusion The pcDNA4.1-EGFP-ID can in vivo mediate EGFP gene expressing into neurite of neuron and astrocytes and directly reveal the morphological characteristics ofneurite.