摘要
背景:氯化锂可以通过Wnt通路激活来治疗骨质疏松,并能激活诱导骨细胞自噬,推测提高氯化锂在Akt,mTOR磷酸化表达可以作为提高成骨细胞分化的作用机制之一。目的:分析雌激素受体抑制下氯化锂对大鼠成骨细胞AKT-mTOR通路作用机制。方法:SPF级出生3 d的SD乳鼠,购买于黑龙江中医药大学动物实验中心。麻醉下分离乳鼠颅骨成骨细胞,采用酶消化法和茜素红染色法获得和鉴定成骨细胞。将成骨细胞随机分为空白组(正常培养细胞)、抑制剂组(ICI182780)、治疗组(氯化锂+ICI182780)。倒置显微镜下观察细胞形态;加入不同浓度的氯化锂(0,1,2,5 nmol/L)处理细胞24 h,采用CCK-8检测各组细胞增殖情况;采用茜素红染色及碱性磷酸酶活性检测成骨细胞分化情况;Western blot法测定成骨细胞中p-AKT,p-mTOR的表达。结果与结论:①倒置显微镜下观察细胞多为单核、多边形及梭形,局部有细胞密集的细胞团;②不同浓度的氯化锂都能对成骨细胞增殖产生促进作用,在一定范围内随着氯化锂浓度的增加促进作用逐渐增强;③与空白组比较,抑制剂组成骨细胞矿化能力明显降低,与抑制剂组比较,治疗组成骨细胞矿化结节能力明显增加;④与空白组比较,抑制剂组中碱性磷酸酶活性明显降低,与抑制剂组比较,治疗组中碱性磷酸酶活性明显增加;⑤与空白组比较,抑制剂组中细胞p-AKT,p-mTOR蛋白表达明显降低,与抑制剂组比较,治疗组中p-AKT,p-mTOR蛋白表达显著增加;⑥结果说明,雌激素受体抑制下氯化锂可以通过激活AKT-mTOR通路促进成骨细胞增殖分化。
BACKGROUND: Lithium chloride treats osteoporosis by activating Wnt signaling pathway, and can induce autophagy. Thereafter, improving the expression of lithium chloride in Akt and mTOR phosphorylation can promote osteoblast differentiation. OBJECTIVE: To analyze the effect of lithium chloride on AKT-mTOR signal pathway in osteoblasts after estrogen receptor inhibition.METHODS: Sprague-Dawley newly born 3-day rats, SPF grade(provided by Laboratory Animal Center of Heilongjiang University of Chinese Medicine) were selected. The osteoblasts were isolated from rat cranial bone by enzyme digestion, and then identified by alizarin red staining. The osteoblasts were randomized into blank control(normal culture), inhibitor(ICI182780), and treatment(lithium chloride plus ICI182780) groups. The cell morphology was observed under inverted microscope. Different concentrations of lithium chloride(0, 1, 2, 5 nmol/L) were added and cultured for 24 hours, and the cell proliferation was detected by cell counting-kit 8 assay. The osteoblast differentiation was detected by alizarin red staining and alkaline phosphatase activity. The contents of p-AKT and p-mTOR in osteoblasts were determined by western blot assay. RESULTS AND CONCLUSION:(1) Inverted microscope showed that most of cells were mononuclear, polygonal and fusiform, with local cell-dense cell clusters.(2) Different concentrations of lithium chloride could promote osteoblast proliferation in a concentration-dependent manner.(3) Compared with the blank control group, mineralization ability of osteoblasts in the inhibitor was significantly decreased. Compared with the inhibitor group, the mineralization ability of osteoblasts in the treatment group was significantly increased.(4) The alkaline phosphatase activity in the inhibitor group was significantly lower than that in the blank control and treatment groups.(5) The expression levels of p-AKT and p-mTOR in the inhibitor group were significantly lower than those in the blank control and treatment groups.(6) To conclude, lithium chloride can promote the proliferation and differentiation of osteoblasts by activating the AKT-mTOR pathway with estrogen receptor inhibition.
作者
徐轶尔
孙贵才
陈水林
樊祥伟
Xu Yier;Sun Guicai;Chen Shuilin;Fan Xiangwei(Technology Center of Harbin Pharmaceutical Group Co.,Ltd.,Harbin 150000,Heilongjiang Province,China;Department of Orthopedics,the Fourth Affiliated Hospital of Nanchang University,Nanchang 330003,Jiangxi Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第19期3002-3006,共5页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81473503)
项目负责人:孙贵才~~
关键词
氯化锂
成骨细胞
雌激素受体
骨质疏松
成骨细胞自噬
成骨细胞分化
lithinm chloride
osteoblasts
estrogen receptor
osteoporosis
osteoblast autophagy
osteoblast differentiation
作者简介
徐轶尔,男,1985年生,黑龙江省哈尔滨市人,汉族,2012黑龙江中医药大学毕业,硕士,研究员,主要从事中药药理及分子生物学研究;通讯作者:孙贵才,博士,主任中医师,南昌大学第四附属医院骨科,江西省南昌市330003。
