摘要目的 观察骨碎补总黄酮(total flavonoidsin drynaria fortunei,TFDF)对大鼠成骨细胞增殖、分化及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)表达的影响.方法 选择10月龄SD雄性大鼠,按体质量采用随机数字表法分为5组:空白对照组,阳性对照组,低、中、高剂量组,每组8只.空白对照组给予等溶生理盐水灌胃;阳性对照组给予蒸馏水配制的骨松宝溶液,灌胃剂量为5.710 g·kg-1· d-1;低、中、高剂量组给予蒸馏水配制的TFDF溶液,灌胃剂量分别为0.054、0.108、0.216 g·kgq·d-1.5组连续灌胃8d,末次灌胃后1.5 h,大鼠麻醉处死,无菌心脏取血,提取上清,同组混匀,56℃水浴30 min灭活补体,滤膜除菌分装血清用于成骨细胞培养.成骨细胞来自3～5日龄SD乳鼠股骨,DMEM培养液冲洗骨髓腔,吹打制备单细胞悬液,采用三维细胞培养系统体外培养大鼠成骨细胞.在无血清DMEM培养后,分别用含上述血清的培养基培养,采用倒置相差显微镜观察成骨细胞形态学变化,噻唑蓝(MTT)法检测成骨细胞增殖情况,对硝基苯磷酸盐(PNPP)法测定碱性磷酸酶(ALP)活性,PCR法测定ERK1、ERK2 mRNA表达.结果 成骨细胞增殖能力组间比较差异有统计学意义(F=8.850,P＜ 0.05),其中阳性对照组,低、中、高剂量组(0.329±0.004、0.302±0.038、0.342±0.010、0.353±0.006)明显高于空白对照组(0.272±0.005,P均＜0.05),中、高剂量组高于低剂量组(P均＜0.05).成骨细胞ALP活性组间比较差异有统计学意义(F=13.406,P＜0.05),其中阳性对照组,中、高剂量组[(15.60±0.38)、(14.62±0.36)、(13.48±0.18)U/mg pro]明显高于空白对照组、低剂量组[(6.34±0.16)、(8.68±0.35)U/mg pro,P均＜0.05],低剂量组高于空白对照组(P＜0.05).成骨细胞ERK1、ERK2 mRNA表达组间比较,差异有统计学意义(F=19.452、15.306,P均＜0.05),其中阳性对照组(2.407±0.022、2.026±0.014)、中剂量组(2.385±0.018、2.018±0.020)明显高于空白对照组(1.032±0.016、0.968±0.018,P均＜0.05).结论 TFDF可促进成骨细胞的分化成熟,其作用机制可能与ERK通路相关.

abstractsObjective To observe the effects of total flavonoidsin drynaria fortunei (TTDF) on growth,differentiation of rat osteoblast and expression of extracellular signal-regulated kinase (ERK).Methods Ten months old male SD rats were divided to 5 groups by body mass with random number table:blank control group,positive control group,low dose group,middle dose group and high dose group,eight rats in each group.Blank control animals were given 0.9％ normal saline by oral garage;positive control animals were given Gusongbao by oral garage at a dose of 5.710 g·kg-1·d-1;low dose,middle dose and high dose animals were given TFDF by oral gavage at doses of 0.054,0.108,0.216 g·kg-1·d-1,respectively.All animals were treated for 8 days,1.5 hours after the last oral gavage,animals were anesthesia to death.Heart blood was collected under sterile conditions,serum was separated,the serum of the same group of rats was mixed,complement was inactivated in a 56 ℃ water bath,and the serum was filtered through a aseptic packed membrane filter for osteoblast culture.Osteoblasts were separated from femoral of 3-5 days old rats,the marrow cavity was rinsed with DMEM culture medium,and then blended to prepare single cell suspension,and osteoblasts were in vitro cultured in a 3 dimensional cell culture system.After cultured in serum free DMEM culture medium,the cells were cultured with above-mentioned serum.Changes of cell morphology were observed,methyl thiazolyl tetrazolium (MTT) was used to test proliferation of cells,P-nitrophenyl phosphate (PNPP) was used for determining alkaline phosphatase (ALP) activity,and PCR was used for determining the mRNA expression of ERK1,ERK2.Results Compared between the 5 groups,the differences of osteoblasts proliferation [absorbance (A)] were statistically significant (F =8.850,P ＜ 0.05);positive control group (0.329 ± 0.004),low dose group (0.302 ± 0.038),middle dose group (0.342 ± 0.010) and high dose group (0.353 ± 0.006) were significantly higher than that of blank control group (0.272 ± 0.005,all P ＜ 0.05);middle dose group and high dose group were higher than low dose group (all P ＜ 0.05).The differences of osteoblasts ALP activity between groups were statistically significant (F =13.406,P ＜ 0.05);the positive control group [(15.60 ± 0.38) U/mg pro],middle dose group [(14.62 ± 0.36) U/mg pro] and high dose group [(13.48 ± 0.18) U/mg pro] were significantly higher than the blank control group [(6.34 ± 0.16) U/mg pro] and low dose group [(8.68 ± 0.35) U/mg pro,all P ＜ 0.05];low dose group was higher than the blank control group (P ＜ 0.05).The expression differences of ERK1,ERK2 mRNA between groups were statistically significant (F =19.452,15.306,all P ＜ 0.05);the positive control group (2.407 ± 0.022,2.026 ± 0.014),middle dose group (2.385 ± 0.018,2.018 ± 0.020) were significantly higher than the blank control group (1.032 ± 0.016,0.968 ± 0.018,all P ＜ 0.05).Conclusion TFDF can promote differentiation and mature of osteoblast,and the mechanism of action may be achieved through activation of ERK pathway.