淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究  

The experimental study of the effects of icariin on increasing Smad4 mRNA level in MC3T3-E1 cell in vitro

作　　者：雪原[1] 王沛[1] 齐清会[2] 倪虹[3] 马信龙[1] 郭世绂[1] 

机构地区：[1]天津医科大学总医院骨科,300052 [2]中西医结合外科 [3]南开大学生命科学院分子生物学二室

出　　处：《中华骨科杂志》2005年第2期119-123,共5页Chinese Journal of Orthopaedics

基　　金：天津市自然科学基金(033609511)

摘　　要：目的检测淫羊藿甙对体外培养的成骨细胞MC3T3-E1中Smad4mRNA的影响。方法用0、10、20、40ng/ml的淫羊藿甙刺激MC3T3-E1细胞。用倒置相差显微镜观察细胞生长情况。通过四甲基噻唑蓝(MTT)法测绘细胞生长曲线和计算细胞群体倍增时间;描述刺激MC3T3-E1细胞的增殖能力。用速率分光光度法测定ALP的含量,免疫组织化学法观察Ⅰ型胶原的产生,用以判断细胞的分化情况。用RT-PCR测定Smad4mRNA的数量。结果10、20ng/ml浓度淫羊藿甙可使MC3T3-E1细胞的生长曲线明显上移(P<0.01);0、10、20和40ng/ml组群体倍增时间分别为(39.37±2.35)h、(26.76±1.78)h、(29.30±2.12)h和(36.35±2.34)h,各组间比较差异有统计学意义(P<0.01);0、10ng/ml可使MC3T3-E1细胞产生ALP和Ⅰ型胶原量增加,刺激ALP作用呈现时间和剂量的依赖性(P<0.01);RT-PCR显示10、20ng/ml浓度淫羊藿甙可以刺激MC3T3-E1细胞Smad4mRNA的数量增加。上述作用以10ng/ml浓度最强(P<0.01)。结论淫羊藿甙刺激成骨细胞MC3T3-E1增殖与分化,可能是通过提高MC3T3-E1细胞Smad4mRNA的量而产生作用。Objective To explore the effect of icariin on stimulating Smad4 mRNA level in MC3T3-E1 cells. Methods MC3T3-E1 cells were treated by 0 ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml icariin respectively. MTT method and population diploid time were applied to observe the cell proliferation. The cell ALP level was measured by atomic absorptiometry and the type Ⅰ collagen was observed by immunohisto-chemistry. RT-PCR was used to find the Smad4 mRNA level in the MC3T3-E1 cells. Results Under the inversion phase contrast microscopy, MC3T3-E1 cells were oligodendrocytes-shaped rich with cell organs. The cells density in 10 ng/ml, 20 ng/ml were higher than those in 0 ng/ml,40 ng/ml. Cell growth curve in 10 ng/ml was higher than other 3 groups and the 20ng/ml was higher than 0 ng/ml, 40 ng/ml groups( P<0.01). The population diploid time in the 10 ng/ml group was shorter than all other three groups (P<0.01). Two days later ALP value in 10 ng/ml and 20 ng/ml were higher than 40 ng/ml and 0 ng/ml with significant dif-ference (P<0.01). There were significant differences among groups (P<0.01). Also there was a time depen-dent relationship between icariin and ALP value in the MC3T3-E1 cell. Immunohistochemistry assay showed that the activity of type Ⅰ collagen in 0 ng/ml,10 ng/ml,20 ng/ml,40 ng/ml was ++, +++, +++, ++ stained respectively. RT-PCR showed that Smad4 mRNA OD value in 0 ng/ml,10 ng/ml,20 ng/ml,40 ng/ml was 1.5×104, 6.4×104, 4.5×104, 1.3×104 respectively and the differences among groups were significant(P<0.01). Conclusion Elevation in growth curve and shortened population diploid time of MC3T3-E1 cells cultured with icariin medium indicates that the icariin can stimulate MC3T3-E1 cell proliferation. The increase of ALP value and the actively stained type Ⅰ collagen after using icariin reveal the promoting MC3T3-E1 cell differentiation role of icariin. The icariin has the function to stimulate Smad4 mRNA level in MC3T3-E1 cells and 10 ng/ml concentration of icariin shows the most potential effects.

关 键 词：淫羊藿甙 SMAD4 MC3T3-E1 成骨细胞 RNA 刺激 Ⅰ型胶原 浓度 显微镜观察 组群 

分 类 号：R285[医药卫生—中药学]