论文部分内容阅读
目的观察龟鹿二仙胶含药血清对SD大鼠骨髓基质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化的作用及其对Wnt信号通路相关因子的影响。方法 100只3月龄雌性SD大鼠经腹腔入路切除双侧卵巢,按随机数字表法分为低剂量组、中剂量组、高剂量组和空白组,每组25只。按体表面积换算龟鹿二仙胶剂量并灌胃给药,连续7天后腹主动脉采血制备含药血清。1月龄SD大鼠全骨髓贴壁法分离BMMSCs,流式细胞法(flow cytometry,FCM)法检测F3代细胞CD45和CD90表达;不同浓度龟鹿二仙胶含药血清干预F3代BMMSCs 72 h后,FCM法检测细胞周期的影响并计算增殖指数,确定最佳干预浓度。F3代细胞分为FBS组、空白组、龟鹿二仙胶组和经典诱导组,诱导培养21天后,茜素红(alizarin red staining,ARS)染色法观察BMMSCs成骨分化,RT-PCR检测成骨分化相关基因碱性磷酸酶(alkaline phosphatase,ALP)和骨钙素(osteocalcin,OC)mRNA的表达;RT-PCR和Western blot检测Wnt5a、β-连环蛋白(β-catenin)、淋巴样增强因子-1(lymphoid enhancer factor-1,Lef-1)mRNA和蛋白的表达。结果 F3代细胞CD45阳性表达率为1.46%±0.23%,CD90阳性表达率为96.97%±3.21%;浓度为10%的龟鹿二仙胶中剂量组含药血清能显著促进BMMSCs增殖;龟鹿二仙胶组和经典诱导组ARS染色可见橘红色钙结节。与空白组及FBS组比较,龟鹿二仙胶组和经典诱导组OC和ALP mRNA表达上调(P<0.05),Wnt5a、β-catenin mRNA和蛋白表达上调(P<0.05);与空白组、FBS组和经典诱导组比较,龟鹿二仙胶组Lef-1 mRNA和蛋白表达均升高(P<0.05);与FBS组和空白组比较,经典诱导组Lef-1蛋白表达上调(P<0.05)。结论龟鹿二仙胶含药血清具有促进BMMSCs增殖,诱导BMMSCs向成骨细胞分化的作用,其机制可能与调控Wnt信号通路相关因子的表达有关。
Objective To observe the effect of serum containing Guilu Eryng sebum on osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) of SD rats and its effect on related factors of Wnt signaling pathway. Methods 100 female 3-month-old SD rats were divided into two groups: the low-dose group, middle-dose group, high-dose group and blank group by random number table. According to the body surface area converted Guilu Erxian glue dose and intragastric administration, continuous abdominal blood aorta seven days after the preparation of drug-containing serum. BMMSCs were isolated from bone marrow of 1-month old SD rats by bone marrow adherence method. Flow cytometry (FCM) was used to detect the expression of CD45 and CD90 on F3 generation cells. After the FCM method to detect the impact of cell cycle and calculate the proliferation index to determine the best intervention concentration. The F3 generation cells were divided into the FBS group, the blank group, the Guilu Eryngsian Group and the classical induction group. After induced for 21 days, alizarin red staining (ARS) staining was used to observe the osteogenic differentiation of BMMSCs. RT- The expression of alkaline phosphatase (ALP) and osteocalcin (OC) mRNA were detected by RT-PCR and Western blot. The expressions of Wnt5a, β-catenin and lymphoid enhancer -1 (lymphoid enhancer factor-1, Lef-1) mRNA and protein expression. Results The positive rate of CD45 in F3 cells was 1.46% ± 0.23% and the positive rate of CD90 was 96.97% ± 3.21%. The drug-containing serum of Guilu Erxian at a concentration of 10% could significantly promote the proliferation of BMMSCs. Ershen gum group and the classic induction group ARS staining visible orange-red calcium nodules. Compared with blank group and FBS group, the expressions of OC and ALP mRNA in Guilu Eryngsian and classical induction groups were up-regulated (P <0.05), while the expressions of Wnt5a and β-catenin mRNA and protein were up-regulated (P <0.05) The expression of Lef-1 mRNA and protein in Guilu Erxian group was higher than that in the normal group (P <0.05). Compared with the FBS group and the blank group, the expression of Lef-1 protein was up-regulated in the classical induction group (P < 0.05). Conclusions The serum of Gulu Erxianjiao can promote the proliferation of BMMSCs and induce the differentiation of BMMSCs into osteoblasts. The mechanism may be related to the regulation of Wnt signaling pathway related factors.