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  

Actinobacillus actinomycetemcomitans induces differentiation and gap junctional intercellular communication in cultured osteoblasts

作　　者：陈允嘉[1] 王豫蓉[1] 邹林洪[1] 李闻颖[1] 郑雷蕾[1] 吴艳[1] 李艳[1] 

机构地区：[1]重庆医科大学附属口腔医院正畸科,重庆400015

出　　处：《第三军医大学学报》2010年第10期1059-1063,共5页Journal of Third Military Medical University

摘　　要：目的探讨放线共生放线杆菌(actinobacillus actinomycetemcom itans,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。Western blot检测结果显示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。Objective To investigate the effect of the supernatant of Actinobacillus actinomycetemcomitans （A.a） on the differentiation and gap junctional intercellular communication in osteoblasts in vitro.Methods Osteoblastic cells were maintained in BHI growth medium mixed with A.a culture supernatants.Neutral red was used to test cellular activity.Quantitative real-time PCR assay was used to detect the differentiation markers,including alkaline phosphatase （ALP）,osteocalcin （OCN） and bone sialoprotein （BSP）,and the immunohistochemistry for Ⅰ collagen.The change of gap junctional intercellular communication in osteoblastic cells was studied by scrape-loading and dye transfer technique （SLDT）,and the connexin43 expression was detected by Western blotting.Results Until concentration to 20% there was no changing on the viability of osteoblastic cells compared to BHI sample as control.SLDT showed the diffusion distance of fluorescent dye in cells cultured with A.a culture supernatants was shorter than those of control.Real-time PCR showed the mRNA expressions of ALP,BSP and OCN were decreased compared with those in control.Immunohistochemistry showed that Ⅰ collagen expression in the osteoblasts was decreased in A.a culture supernatants.The protein expression of Cx43 in sample treated with A.a culture supernatants was remarkable reduced than in sample treated with BHI.Conclusion A.a inhibits the differentiation and gap junctional intercellular communication in osteoblasts.

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 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.27[医药卫生—基础医学]