放线共生放线杆菌培养上清对成骨细胞分化及间隙连接通讯的影响

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1、放线共生放线杆菌培养上清对成骨细胞分化及间隙连接通讯的影响陈允嘉，王豫蓉，邹林洪，李闻颖，郑雷蕾，吴艳，李艳（400015重庆，重庆医科大学附属口腔医院正畸科）[摘要]目的探讨放线共生放线杆菌（actinobacillusactinomycetemcomitans，A.a）培养上清对成骨细胞分化以及细胞间隙连接通讯的影响。方法在A.a培养上清中体外培养成骨细胞，中性红检测成骨细胞活性，RT-PCR检测成骨细胞分化标记，免疫组化观察I型胶原蛋白表达，利用划痕标记荧光染料示踪(SLDT)法与Westernblot检测Connexin43的表达，研究成骨细胞间隙连接通讯的变化。结果20%以

2、下浓度的A.a培养上清对成骨细胞无明显致死性。RT-PCR结果显示A.a培养上清中成骨细胞分化标记的表达降低。免疫组化结果显示A.a培养上清中成骨细胞I型胶原蛋白表达降低。SLDT法结果显示A.a培养上清中成骨细胞荧光染料扩散低于对照组。Westernblot结果显示A.a培养上清中成骨细胞Connexin43表达降低。结论A.a培养上清对成骨细胞分化及细胞间隙连接通讯有抑制作用。[关键词]放线共生放线杆菌；成骨细胞；分化；细胞间隙连接通讯[中图法分类号]R783.5；R781.4+5[文献标志码]AActinobacillusactinomycetemcomitansinduces

3、differentiationandgapjunctionalintercellularcommunicationinculturedosteoblastsChenYunjia,WandYurong,ZouLinhong,LiWenying,ZhengLeilei,WuYan,LiYan(DepartmentofOrthodontics,AffiliatedHospitalofStomatology,ChongqingMedicalUniversity,Chongqing,400015,China)[Abstract]ObjectiveToinvestigatetheeffectof

4、thesupernatantofActinobacillusactinomycetemcomitans(Aa)onthedifferentiationandgapjunctionalintercellularcommunicationinosteoblastsinvitro.MethodsOsteoblasticcellwasmaintainedinBHIgrowthmediummixedwithAaculturesupernatants.Neutralredwasusedtotestcellularactivity.Quantitativerealtime-PCRassaywasu

5、sedtodetectthedifferentiationmarkers,includingalkalinephosphatase(ALP),osteocalcin(OCN)andbonesialoprotein(BSP),andtheimmunohistochemistryforIcollagen.Thechangeofgapjunctionalintercellularcommunicationinosteoblasticcellswasstudiedbyscrape-loadinganddyetransfertechnique(SLDT),andtheconnexin43exp

6、ressionwasdetectedWesternblotting.ResultsUntilconcentrationto20%therewasnochangingontheviabilityofosteoblasticcellscomparedtoBHIsampleascontrol.SLDTshowedthediffusiondistanceoffluorescentdyeincellsculturedwithAaculturesupernatantswasshorterthanthoseofcontrol.Realtime-PCRshowedthemRNAexpressions

7、ofALP,BSPandOCNweredecreasedcomparedwiththoseincontrol.ImmunohistochemistryshowedthatIcollagenexpressionintheosteoblastswasdecreasedinAaculturesupernatants.TheproteinexpressionofCx43insampletreatedwithAaculturesupernatantswasremar