右归饮对模拟人体内应力环境中成骨细胞碱性磷酸酶分泌的影响

Influence of Yougui Yin on secretion of alkaline phosphatase of osteoblasts in simulated internal stress environment of human body

目的探究牵张应力作用下右归饮含药血清刺激大鼠成骨细胞后,对其碱性磷酸酶(ALP)分泌的影响,评价右归饮在模拟人体内应力环境中促进成骨细胞增殖分化的作用。方法随机将40只SD大鼠均分为右归饮组和生理盐水组,右归饮组每天按含生药量20 g/kg灌胃,生理盐水组灌以等量的生理盐水,连续灌胃1周;末次灌胃2 h后,常规麻醉,心脏取血,分离提取血清,-20℃保存2组血清;采用序列酶消化法培养成骨细胞,传至第4代将细胞等量接种为6组,每组6孔,分别标记为无血清培养基对照组、生理盐水血清组、右归饮含药血清组、无血清12%牵张形变组、生理盐水血清12%牵张形变组和右归饮含药血清12%牵张形变组,无血清培养基同步化培养24 h;将右归饮含药血清及生理盐水血清分别作用于右归饮含药血清组、右归饮含药血清12%牵张形变组和生理盐水血清组、生理盐水血清12%牵张形变组细胞,同时采用0.5 Hz频率、12%形变量的牵张力对相应无血清组细胞、右归饮血清组细胞及生理盐水血清组细胞进行力学加载。分别于12 h和24 h对各组细胞行ALP定量测定,检测各组细胞ALP活性;加力24 h后,行碱性磷酸酶染色(CAKP),显微镜镜检拍照,检测成骨细胞胞浆内CAKP颗粒数目及分布;并利用统计学软件对数据进行分析。结果 0.5 Hz、12%牵张应力条件下无血清组细胞与常规培养条件下无血清组细胞比较,前者ALP表达受抑制(P<0.05);牵张应力条件下右归饮血清培养液与生理盐水血清培养液,较常规培养条件下2种血清培养液比较,均能刺激成骨细胞ALP表达(P<0.05),且力学环境下右归饮组ALP值高于生理盐水血清组(P<0.05)。结论无血清条件下,牵张应力表现出抑制成骨细胞ALP表达现象;右归饮在模拟人体牵张应力环境下能促进成骨细胞的增殖分化。

Objective To study the influence of medicated serum of Yougui Yin （YY） on secretion of alkaline phosphatase （ALP）,and review the improving effect of YY on the proliferation and differentiation of osteoblasts in simulated internal stress environment of human body.Methods SD rats （n =40） were divided into YY group and normal saline （NS） group and given corresponding treatment for one week.After the last time treatment for 2 hours,serum samples were collected and in which osteoblasts were cultured to the fourth generation by using sequence enzyme digestion process,and then divided into 6 groups：non-serum group,NS serum group,YY serum group,non-serum 12％ stretch group,NS serum 12％ stretch group and YY serum 12％ stretch group.The content and activity of ALP were detected respectively after 12 hours and 24 hours.The number and distribution of CAKP were detected by using diazotypeprocess and microscope after 24 hours.All data was analyzed with statistical software.Results The expression of ALP was inhibited in non-serum group in the condition of 0.5 Hz and 12％ stretch compared with that in non-serum group in routine condition （P ＜ 0.05）.In stretch stress condition,YY serum 12％ stretch group and NS serum 12％ stretch group could stimulate the expression of ALP compared with them in routine condition （P ＜ 0.05）.ALP value was higher in YY serum 12％ stretch group than that in NS serum 12％ stretch group （P ＜ 0.05） in stretch stress condition.Conclusion In non-serum condition,expression of ALP is inhibited by stretch stress.Yougui Yin can improve the proliferation and differentiation of osteoblasts in simulated internal stress environment of human body.