[Guilu Erxian Glue Induced Osteogenic Differentiation of BMMSCs and Its Effects on Wnt Signal Pathway]

Objective To observe the effects of Guilu Erxian Glue (GEG) containing serum on osteogenin differentiation of bone marrow mesenchymal stem cells (BMMSCs) and Wnt signal pathway related factors. Methods Totally 100 three months old female SD rats had their bilateral ovaries excised peritoneally They were divided into the low, middle, high GEG groups, and the blank control group by random digit table, 25 in each group. The dose of GEG was calculated according to body surface area, and GEG containing serum was administered by gastrogavage for 7 successive days. Blood was collect- ed by abdominal aorta to prepare drug containing serum. F3 passage BMMSCs of 1-month SD rats were i- solated by whole bone marrow adherent method, and cultured in vitro for 3 passages. The cell surface markers (CD45 and CD90) of F3 passage were detected by flow cytometry (FCM). BMMSCs were trea- ted with different concentrations GEG containing serum for 72 h. Then the cell cycle was determined by FCM, and the proliferation index calculated. The optimal intervention concentration was determined. Then F3 passage BMMSCs were divided into four groups, i.e., the fetal bovine serum (FBS) group, the blank control group, the GEG group, the classical induction group. After they were treated with corresponding medium for 21 days, BMMSCs were dyed with alizarin red staining (ARS) to observe their osteogenin dif- ferentiation. mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OC) of BMMSCs were detected by RT-PCR. mRNA and protein expressions of Wnt5a, β-catenin, and lymphoid enhancer factor- 1 (Lef-1) were detected by RT-PCR and Western blot. Results The ratio of CD45 positive expression was 1. 46% ?0. 23%, and the ratio of CD90 positive expression was 96. 97% ±3. 21%. Middle EGE contai- ning serum (10%) could significantly stimulate the proliferation of BMMSCs. In ARS citrus red calcium nodules could be seen in the GEG group and the classical induction group. Compared with the FBS group and the blank control group, mRNA expressions of OC and ALP were up-regulated, mRNA and protein expressions of Wnt5a and p-catenin were up-regulated in the GEG group and the classical induction group (P<0. 05). Compared with the FBS group, the blank control group, and the classical induction group, mRNA and protein expressions of Lef-1 were up-regulated in the EGE group (P <0. 05). Compared with the FBS group and the blank control group, protein expressions of Lef-1 increased in the classical induc- tion group (P <0. 05). Conclusions GEG containing serum had the functions of stimulating the prolifera- tion of BMMSCs, and inducing the osteogenic differentiation of BMMSCs. Its mechanism might be possi- bly related with regulating Wnt signal pathway related factors.