CAJ | 학술논문

目的：确定问号钩端螺旋体（简称钩体） LB361基因产物磷脂酰肌醇特异性磷脂酶C （ phosphatidylinositol phospholipase C ，L-PI-PLC）功能及其诱导巨噬细胞凋亡的作用与机制。方法采用生物信息学技术分析问号钩体赖型赖株LB361基因序列中PI-PLC结构功能域。采用原核表达系统表达该基因产物（ rL-PI-PLC）。采用IP3荧光偏振试验了解rL-PI-PLC水解PIP2底物产生IP3的活性。采用实时荧光定量RT-PCR及Western blot法分别检测问号钩体赖株感染人THP-1巨噬细胞时LB361基因转录、表达及分泌情况。构建LB361基因转染THP-1细胞株，分别采用激光共聚焦显微镜法和流式细胞术，检测LB361基因产物THP-1通过IP3引起内质网钙释放，从而导致细胞胞内游离钙离子浓度（[ Ca2＋] i）升高并诱导细胞凋亡的作用。结果 rL-PI-PLC能水解PIP2产生IP3，其Km和Kcat值分别为199μmol/L和8．566×10-5 S-1。问号钩体赖株感染THP-1细胞后，LB361-mRNA及L-PI-PLC蛋白表达水平显著升高并外分泌。与未转染正常细胞比较，LB361基因转染THP -1细胞中IP3浓度及[ Ca2＋] i明显升高，从而引起部分 THP-1细胞发生[ Ca2＋] i 依赖性凋亡。结论问号钩体LB361基因产物是磷脂酰肌醇特异性磷脂酶C，该酶在问号钩体感染巨噬细胞过程中可引起[ Ca2＋] i升高而导致细胞凋亡。
목적：학정문호구단라선체（간칭구체） LB361기인산물린지선기순특이성린지매C （ phosphatidylinositol phospholipase C ，L-PI-PLC）공능급기유도거서세포조망적작용여궤제。방법채용생물신식학기술분석문호구체뢰형뢰주LB361기인서렬중PI-PLC결구공능역。채용원핵표체계통표체해기인산물（ rL-PI-PLC）。채용IP3형광편진시험료해rL-PI-PLC수해PIP2저물산생IP3적활성。채용실시형광정량RT-PCR급Western blot법분별검측문호구체뢰주감염인THP-1거서세포시LB361기인전록、표체급분비정황。구건LB361기인전염THP-1세포주，분별채용격광공취초현미경법화류식세포술，검측LB361기인산물THP-1통과IP3인기내질망개석방，종이도치세포포내유리개리자농도（[ Ca2＋] i）승고병유도세포조망적작용。결과 rL-PI-PLC능수해PIP2산생IP3，기Km화Kcat치분별위199μmol/L화8．566×10-5 S-1。문호구체뢰주감염THP-1세포후，LB361-mRNA급L-PI-PLC단백표체수평현저승고병외분비。여미전염정상세포비교，LB361기인전염THP -1세포중IP3농도급[ Ca2＋] i명현승고，종이인기부분 THP-1세포발생[ Ca2＋] i 의뢰성조망。결론문호구체LB361기인산물시린지선기순특이성린지매C，해매재문호구체감염거서세포과정중가인기[ Ca2＋] i승고이도치세포조망。
Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.