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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 actinomycetemin actinomycetemcomitans (A.a) culture supernatant on osteoblast differentiation and cell gap junctional intercellular communication. Methods Osteoblasts were cultured in vitro in vitro. Osteoblasts were detected by neutral red. Osteoblast differentiation markers were detected by RT-PCR. Expression of collagen type Ⅰ was observed by immunohistochemistry. (SLDT) method and Western blot to detect the expression of Connexin43 and study the changes of osteoblast gap junctional intercellular communication. Results The culture supernatant of A.a at a concentration below 20% showed no obvious lethality to osteoblasts. RT-PCR results showed that the expression of osteoblast differentiation marker in A.a culture supernatant was decreased. The results of immunohistochemistry showed that the expression of type Ⅰ collagen in osteoblasts decreased in A.a culture supernatant. The result of SLDT showed that the proliferation of osteoblast fluorescent dye in A.a culture supernatant was lower than that in control group. The results of Western blot showed that the expression of Connexin43 in osteoblasts decreased in A.a culture supernatant. Conclusion A.a culture supernatant on osteoblast differentiation and cell gap junctional communication inhibited.