摘要
目的 探讨祛湿健骨方对MC3T3-E1细胞的作用机理.方法 将祛湿健骨方配成不同浓度溶液(0.005、0.025、0.05、0.1、0.2、0.4 mg/mL),用不同浓度祛湿健骨方对细胞进行干预,干预48 h后,采用CCK-8法检测细胞增殖率,筛选最佳药物浓度.将MC3T3-E1细胞制成细胞悬液,随机分为对照组、地塞米松组、低剂量组、中剂量组、高剂量组,接种至六孔板.待细胞贴壁达到80%后进行造模(对照组不添加药物;地塞米松组使用地塞米松干预;低剂量组、中剂量组、高剂量组分别用相应浓度的祛湿健骨方+地塞米松进行干预)48 h后更换为成骨诱导培养基进行培养.诱导15 d后,测定各组细胞的碱性磷酸酶(Alkaline phosphatase,ALP)含量和Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨细胞特异性转录因子(Osterix,OSX)蛋白的表达量.结果 经CCK-8实验结果对比,药物筛选前3位存活率浓度为0.1 mg/mL(低浓度)、0.2 mg/mL(中浓度)、0.4 mg/mL(高浓度).与对照组相比,地塞米松组、低剂量组、中剂量组、高剂量组细胞ALP活性均降低差异有统计学意义(P<0.05);与地塞米松组相比,低剂量组、中剂量组差异无统计学意义(P>0.05),高剂量组细胞ALP活性升高,差异有统计学意义(P<0.05).与对照组比较,中剂量组、低剂量组、地塞米松组细胞的RUNX2、OSX蛋白表达均降低,差异有统计学意义(P<0.05),高剂量组细胞OSX和RUNX2蛋白表达差异无统计学意义(P>0.05);与地塞米松组比较,高剂量组 OSX和 RUNX2蛋白表达差异均有统计学意义(P<0.05).结论 祛湿健骨方能够促进 MC3T3-E1细胞的成骨分化能力,其可能是通过调节RUNX2、OSX等骨形成相关蛋白,以此纠正骨形成与骨吸收的平衡,达到治疗骨质疏松症的作用.
Abstract
Objective To explore the mechanism of action on MC3T3-E1 cells.Methods The solution were mixed with different concentrations(0.005 mg/mL,0.025 mg/mL,0.05 mg/mL,0.1 mg/mL,0.2 mg/mL,0.4 mg/mL),and the cells were intervened with different concentrations.After 48 hours of intervention,the cell proliferation rate was detected by Cell Counting Kit-8(CCK-8)method,and the best drug concen-tration was screened.MC3T3-E1 cells were made into cell suspension and randomly divided into blank control,dexamethasone,low dose drug(D),medium dose drug(Z),high dose drug(G)and seeded to a six-well plate.The mold was made after the cell adhesion reached 80%(no drug was added in the blank group;DEX intervention with DEX;D,Z and G intervention with corresponding concentration of bone prescription + DEX)for 48 hours,it replaced with osteogenic induction medium for culture.After 15 days of induction,the alkaline phosphatase(ALP)content and the expression levels of RUNX 2 and OSX protein were measured in each group.Results Compared with the results of CCK-8 experiment,the first three survival concentrations were the drug was selected as 0.1 mg/mL(low concentration),0.2 mg/mL(middle concentration)and 0.4 mg/mL(high concentration).Compared with control group,the ALP ac-tivity in the dexamethasone group(P<0.05),the low-dose group,the middle-dose group and the high-dose group was decreased significantly ALP,there was no significant difference between low-dose and mid-dle-dose groups(P>0.05),but the ALP activity in high-dose groups were increased significantly(P<0.05).Compared with the control group,the expression of RUNX2 and OSX protein in the middle-dose group,low-dose group and dexamethasone group was decreased significantly ALP,there was no signifi-cant difference in the expression of OSX and RUNX2 protein between the high-dose group and the control group(P<0.05),but there was significant difference in the expression of OSX and RUNX2 protein be-tween the high-dose group(P<0.05).Conclusion Through the study,it can be found that the bone pre-scription can promote the osteogenic differentiation ability of MC3T3-E1 cells.Meanwhile,it can be in-ferred that it may regulate bone formation related proteins such as RUNX 2,OSX to correct the balance between bone formation and bone resorption and achieve the treatment of osteoporosis.
基金项目
国家自然科学基金(81660831)
新疆医科大学研究生创新创业训练计划(CXCY2023010)
NETL
NSTL
万方数据