骨髓间充质干细胞向成骨细胞分化中CXCR4蛋白表达的研究
The study on the expression of CXCR4 protein in the differentiation of osteoblasts by bone marrow mesenchymal stem cells
  
DOI:10.3969/j.issn.1006.7108.2023.03.002
中文关键词:  骨髓间充质干细胞  CXCR4  BMP2  补肾、健脾  骨质疏松症
英文关键词:bone marrow mesenchymal stem cells  CXCR4  BMP2  nourishing the kidney  strengthening the spleen  osteoporosis
基金项目:国家自然科学基金(81973719);沈阳市中青年科技创新人才计划(RC200160)
作者单位
邸贵鑫 杨芳* 林庶茹 杨铠宁 付夜平 安勇 辽宁中医药大学辽宁 沈阳 110847 
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中文摘要:
      目的 探究中医补肾阴、补肾阳、健脾、补肾健脾不同治法对骨髓间充质干细胞向成骨细胞分化过程中CXCR4蛋白表达的影响,从而为中药防治骨质疏松症提供依据。方法 体外分离培养大鼠BMSCs,将SPF雌性SD大鼠用随机数字表法随机分成6组,依次分别为:正常组、诱导液组、补肾阴组、补肾阳组、健脾组、补肾健脾组。对大鼠进行7 d灌胃,制备含药血清,各组成骨诱导18 d后,进行茜素红染色检测矿化情况,采用酶联免疫吸附法(ELISA)检测上述每组细胞上清液CXCR4、BMP2蛋白表达水平。结果 诱导18 d后,与正常组相比,补肾阳组与补肾健脾组BMP-2含量均显著升高(P<0.01),诱导组、补肾阴组BMP-2含量有所升高,但差异不具有统计学意义(P>0.05);与诱导组相比,补肾健脾组BMP-2含量均显著升高(P<0.01),补肾阴组与补肾阳组BMP-2含量有所升高,但差异无统计学意义(P>0.05),各含药组BMP-2蛋白浓度比较依次为:补肾健脾组>补肾阳组>补肾阴组>健脾组;与正常组相比,各组CXCR4含量均显著升高(P<0.01),且补肾健脾组上升最高(P<0.01);与诱导组相比,补肾阴组CXCR4含量明显升高(P<0.05),补肾阳组与补肾健脾组CXCR4含量均显著升高(P<0.01),健脾组CXCR4含量也有所上升,但差异无统计学意义(P>0.05),各含药组CXCR4蛋白含量比较依次为:补肾健脾组>补肾阳组>补肾阴组>健脾组;与正常组和诱导液组以及其他中药组相比较,补肾健脾组矿化结节数目最多,且体积也比其他组大。结论 补肾阴、补肾阳、健脾、补肾健脾不同治法对大鼠骨髓间充质干细胞向成骨细胞分化起到正向促进作用,这一作用可能是通过调控CXCR4蛋白水平而转变的。且本研究中补肾健脾法调控效果优于其他中药组,印证了中医“肾主骨,生髓;脾主肌肉,四肢”理论,进而证实肾虚是骨质疏松症发病根本,脾虚是骨质疏松症发病关键,治疗应该注重补肾健脾。
英文摘要:
      Objective To explore the effects of traditional Chinese medicine (TCM) invigorating kidney yin and kidney yang, invigorating spleen, and invigorating kidney and spleen on the expression of CXCR4 protein during the differentiation of osteoblasts by bone marrow mesenchymal stem cells, so as to provide evidence for the prevention and treatment of osteoporosis with traditional Chinese medicine. Methods Rat BMSCs were isolated and cultured in vitro. SPF female SD rats were randomly divided into 6 groups by random number table method. They were normal group, induction group, Bushen-Yang group, Bushen-Yin group, tonifying spleen group, and tonifying kidney and spleen group. The rats received intragastric administration of prepare drug-containing serum for 7 days. After 18 days of bone induction in each group, Alizarin red staining was used to detect the mineralization, and enzyme-linked immunosorbent assay (ELISA) was used to detect CXCR4 and BMP2 in the supernatant of each group of cells. Results After 18 days of induction, compared with the normal group, the content of BMP-2 in the Bushen-Yang group and the Bushen-Jianpi group increased significantly (P<0.01). There was no significant difference in the induction group and Bushen-Yin group (P>0.05). The content of BMP-2 in the tonifying kidney and spleen group increased significantly (P<0.01). The content of BMP-2 in the Bushen-Yang group and Bushen-Yin group increased, but it was not significant (P>0.05). The comparison of BMP-2 protein concentration in each drug-containing group was in the following order: Bushen and spleen group> kidney-yang group> kidney-yin group> spleen-invigorating group. Compared with the normal group, CXCR4 increased in all the groups, and it was the highest in the tonifying kidney and spleen group. Compared with the induction group, the content of CXCR4 in the Bushen-Yin group increased significantly (P<0.05). The content of CXCR4 in the Bushen-Yang group and tonifying kidney and spleen group increased significantly (P<0.01). The content of CXCR4 in the spleen-invigorating group also increased, but it was not significant (P>0.05). Compared with the normal group, the induction group, and other traditional Chinese medicine groups, the number of mineralized nodules in the kidney-invigorating and spleen-invigorating group were the largest, and the volume was larger than other groups. Conclusion The different treatment methods of tonifying kidney yin, tonifying kidney yang, strengthening spleen, and tonifying kidney and spleen positively promote the differentiation of rat bone marrow mesenchymal stem cells into osteoblasts. The function may be through the regulating of CXCR4 protein level. In addition, in this experiment, the regulation effect of the method of tonifying the kidney and strengthening the spleen is better than other traditional Chinese medicine groups. The deficiency of the kidney is the key to the pathogenesis of osteoporosis. The treatment should focus on tonifying the kidney and strengthening the spleen.
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