CAJ | 학술논문

目的确定TAT-tCNTF融合蛋白能够透过细胞膜进入细胞内,并研究其对Aβ诱导SH-SY5Y细胞损伤的保护作用.方法重组PCR获得人截短型CNTF基因后,与pBV 220-TAT载体连接,在大肠杆菌中表达,细胞免疫荧光方法检测TAT蛋白运载融合蛋白穿过SH-SY5Y细胞膜的作用,Aβ_(25-35)诱导SH-SY5Y细胞产生神经毒性,采用MTT(四甲基偶氮唑盐)法检测融合蛋白对细胞存活率的影响,Heochst 33342/PI荧光双染法进行细胞凋亡和坏死的形态学观察,并进行LDH释放分析进一步检测TAT-tCNTF对细胞损伤后的保护作用.结果成功构建pBV220-TAT-tCNTF表达载体,Western blot结果表明重组融合蛋白可以与CNTF抗体特异性结合,细胞免疫荧光方法显示TAT-tCNTF能大量进入细胞,而rhCNTF只有微量进入细胞.对Aβ_(25-35)诱导损伤的SH-SY5Y细胞MTT、Heochst 33342/PI荧光双染法及LDH分析都表明TAT-tCNTF能明显提高细胞的存活率.结论 TAT-tCNTF融合蛋白可以跨越细胞膜,能够保护Aβ_(25-35)诱导损伤的SH-SY5Y细胞,具有促进细胞生存生长的活性.
목적 학정TAT-tCNTF융합단백능구투과세포막진입세포내,병연구기대Aβ유도SH-SY5Y세포손상적보호작용.방법 중조PCR획득인절단형CNTF기인후,여pBV 220-TAT재체련접,재대장간균중표체,세포면역형광방법검측TAT단백운재융합단백천과SH-SY5Y세포막적작용,Aβ_(25-35)유도SH-SY5Y세포산생신경독성,채용MTT(사갑기우담서염)법검측융합단백대세포존활솔적영향,Heochst 33342/PI형광쌍염법진행세포조망화배사적형태학관찰,병진행LDH석방분석진일보검측TAT-tCNTF대세포손상후적보호작용.결과 성공구건pBV220-TAT-tCNTF표체재체,Western blot결과표명중조융합단백가이여CNTF항체특이성결합,세포면역형광방법현시TAT-tCNTF능대량진입세포,이rhCNTF지유미량진입세포.대Aβ_(25-35)유도손상적SH-SY5Y세포MTT、Heochst 33342/PI형광쌍염법급LDH분석도표명TAT-tCNTF능명현제고세포적존활솔.결론 TAT-tCNTF융합단백가이과월세포막,능구보호Aβ_(25-35)유도손상적SH-SY5Y세포,구유촉진세포생존생장적활성.
Aim To determine TAT-Tcntf penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by β-amyloid peptide 25-35(Aβ_(25-35) ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by Aβ_(25-35).And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusion sTAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after Aβ25-35 exposure.