基于雌激素受体研究氯化锂干预成骨细胞的分化及自噬

Lithium chloride increases differentiation and autophagy of osteoblasts inhibited by estrogen receptor

背景:临床应用锂治疗造血系统疾病和双极性疾病已经有数十年,并且发现锂可以直接刺激骨髓间充质干细胞的增殖。目的:研究雌激素受体抑制下氯化锂对大鼠成骨细胞增殖分化及自噬的影响。方法:实验选取10只出生72 h的SPF级SD乳鼠(由黑龙江中医药大学提供)。采用酶消化法和茜素红染色法获得和鉴定成骨细胞。取对数生长期成骨细胞,于培养基中加入不同浓度的氯化锂(0,1,2,5 nmol/L)处理24 h,采用CCK-8法检测各组细胞增殖情况。另取成骨细胞随机分为空白组(正常培养细胞)、抑制剂组(10^(-7) mol/L ICI182780)、治疗组(5 nmol/L氯化锂+10^(-7) mol/L ICI182780)。采用钙化结节染色及碱性磷酸酶活性检测各组成骨细胞分化情况;Western blot法测定各组成骨细胞中Beclin1和Runx2蛋白表达情况。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)CCK-8法检测结果显示不同浓度的氯化锂都能对成骨细胞增殖产生促进作用,呈浓度依赖性;(3)茜素红染色结果显示与空白组比较,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(P <0.05);活性测定结果显示与空白组比较,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(P <0.05);(4)Western blot结果显示与空白组比较,抑制剂组中Beclin1和Runx2蛋白表达明显降低,与抑制剂组比较,治疗组中Beclin1和Runx2蛋白表达显著增加(P <0.05);(5)结果提示,雌激素受体抑制下氯化锂可以提高大鼠成骨细胞增殖分化及自噬。

BACKGROUND:Lithium has been used in the treatment of hematopoietic diseases and bipolar diseases for decades,and it can directly stimulate the proliferation of bone marrow mesenchymal stem cells.OBJECTIVE:To investigate the effects of lithium chloride on the proliferation,differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.METHODS:Ten neonatal Sprague-Dawley rats,72days old,SPF grade,were provided by the Heilongjiang University of Chinese Medicine.(1)Osteoblasts were isolated by enzymatic digestion and alizarin red staining,and logarithmically growing osteoblasts were then cultured in media containing different concentrations of lithium chloride(0,1,2,5nmol/L)for24hours.Cell proliferation was measured by cell counting kit-8.(2)Osteoblasts at logarithmical growth phase were randomly divided into blank group(normal cultured cells),inhibitor group(10-7mol/L ICI182780),treatment group(5nmol/L lithium chloride+10-7mol/L ICI182780).Calcified nodule staining and alkaline phosphatase activity were used to detect the differentiation of osteoblasts.Expression of Beclin1and Runx2in osteoblasts were determined by western blot.RESULTS AND CONCLUSION:Under the inverted microscope,the cells were mostly mononuclear,polygonal and fusiform,with local cell-dense cell clusters.Results from the cell counting kit-8assay showed that different concentrations of lithium chloride promoted the proliferation of osteoblasts in a concentration-dependent manner.Alizarin red staining results showed that the mineralization ability of osteoblasts was significantly reduced in the inhibitor group compared with the blank group,while compared with the inhibitor group,the mineralization ability of osteoblasts was increased significantly in the treatment group(P<0.05).The activity of alkaline phosphatase was significantly reduced in the inhibitor group compared with the blank group,while compared with the inhibitory group,the activity of alkaline phosphatase increased significantly in the treatment group(P<0.05).Western blot results showed that the expression of Beclin1and Runx2proteins in the inhibitor group decreased significantly compared with blank group,while compared with the inhibitor group,the expression of Beclin1and Runx2protein in the treatment group increased significantly(P<0.05).In conclusion,lithium chloride can increase the proliferation,differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.