GJIC渐弱剂18α-甘草次酸对兔脂肪细胞向成骨细胞横向分化的影响

Effects of GJIC diminuendo agent 18α-GA on transdifferentiation from adipocytes to osteoblasts in rabbits

目的探讨缝隙连接通讯渐弱剂18α-甘草次酸(GA)对脂肪细胞向成骨细胞横向分化及细胞间缝隙连接通讯的影响。方法选取3月龄新西兰大白兔腹股沟处脂肪组织,分离提取成熟脂肪细胞,使其去分化得到去分化脂肪细胞,取第三代去分化的脂肪细胞进行成骨诱导,加入GJIC渐弱剂18α-GA设为减弱组,并设立对照组,MTT法观察18α-GA对细胞倍增是否有影响,进行茜素红钙结节染色和I型胶原免疫组化染色对成骨细胞定性检测,Western blot技术和划痕标记染料示踪法检测缝隙连接蛋白43(Cx43)的表达。结果 MTT法检测结果显示18α-GA对细胞生长增殖无显著影响。茜素红染色显示均发现紫红色结节。免疫组化染色显示减弱组I型胶原表达减弱,SLDT法显示减弱组荧光染料扩散低于对照组,Western blot技术检测结果显示减弱组Cx43蛋白的表达下降。结论 18α-GA可以减弱缝隙连接通讯以降低脂肪细胞向成骨细胞分化的能力。

Objective It is to explore the effects of transdifferentiation from adipocytes to osteoblasts and gap junction intercellular communication（ GJIC） by gap junction Diminuendo agent 18α-GA.Methods Groin adipose tissue was taken from 3 months old New Zealand White Rabbit and collected mature adipocytes.Made the mature adipocytes dedifferentiate and obtained the dedifferentiated adipocytes.The 3rd generation of the dedifferentiated adipocytes were induced and differentiated into osteoblasts.There were two experimental groups that the diminuendo group with adding 18α-GA and the control group. The toxic action of 18α-GA in osteoblasts growth and proliferation were detected by MTT assay.Osteoblasts were qualitatively detected by alizarin red staining and collagen typeⅠimmunohistochemistry staining.The expressions of Cx43 were measured by western-blot and sorape-loading and dye transfet （ SLDT） method.Results The results of MTT assay showed that 18α-GA had no significant effect on cell proliferation.The results of Alizarin red staining showed that calcific nodules were dyed red in each group.Immunohistochemistry staining results showed that the expressions of collagen typeⅠwere weakened in the diminuendo group.The results of Western blot showed that the expressions of Cx43 were decreased in the diminuendo group. Conclusion 18α-GA diminuendo the capability of transdifferentiation from adipocytes to osteoblasts with enhancement of GJIC.