The Induction Of Apigenin And Its Analogues For Bone Marrow Mesenchymal Stem Cells Into Osteogenic Differentiation And Efficacy Study

Objective:To compare the osteogenic effect of apigenin and its structural analogues(such as Kaba Kiso, chrysin, quercetin, baicalin) for acceleration of the related research,development of new drugs, the promotion of bone tissue engineering and clinical application.Materals and Methods:The experiment was carried out in 2013~2015 in medical experiment center of the Department of orthopedics disease institute. Experimental SD rats were purchased from the animal experimental center of Guangdong province. First, we extracted bone marrow cell by the whole marrow method, and conducted cell culture, examing cell growth curve using four methyl thiazolyl tetrazolium(MTT) and made flow cytometry analysis. Second, the third-generation of MSCs was divided into the following four groups:(1) the blank control group(negative control group, containing10% FBS L-DMEM);(2) classic bone induction group(10% FBS+L-DMEM + 50g/ml of vitamin +beta glycerophosphate + dexamethasone(10-8mol/L) group;(3) apigenin induction group(10%FBS+L-DMEM + 20 M ofapigenin);(4) apigenin structural analogues induction group(10%FBS+L-DMEM + 20 M chrysin or diosmetin or baicalein or quercetin). We measured the following indicators:(1) Alizarin red S staining to determine the extracellular mineralization;(2) after the 7d and 14 d, Real-time PCR detection of osteogenic related genes;(3) after the 7d and 14 d, Western blot detection of osteogenic related protein. We used correlation analysis of variance to compare the differences between groups, with P< 0.05 as the difference was significant, P< 0.01 as a very significant difference;(4) Western Blot detection of bone related proteins.Result:1. By cell culture, cell completely grew adhere to the cell wall after 7 days and14 days. Morphology of all cells was uniform and spindle shaped. Cell grew vigorously and can be stably passaged. No obvious changes were observed in cell morphology and growth speed.2. The first day of BMSCs being inoculated was cell potential adaptation period, growth curve from second to third days was linear curve, indicating that this period is the logarithmic growth phase of cells. Growth curve in the fourth day becomes smooth and in fifth days becomes sharp, the curve in sixth days becomes smooth again, which showed cell proliferation significantly appeared slow or even negative growth trend and the growth was into the platform period.3. The result of flow cytometry showed the third generation of cultured BMSCs expressed CD29, CD44 and CD90 uniformly, and the positive rate were95.65%, 87.09%, 93.51% respectively; the expression of CD34, CD45 were negative, and the positive rates were 0.96%, 1.19%.4. The result of Alizarin red S staining showed BMSCs of rats aggregated and promoted the extracellular deposition of calcium after induced by apigenin and classic osteoblast respectively for 7d and 14 d. In the center of the punctiform aggregation growth, salt staining positive calcified nodules can be seen.Among that, the classic osteoblast group has stronger effect in which the formation of calcified nodules not only has bigger quantity, and larger area.5. RT-PCR detection of osteogenic related genes Collagen I, OPN, ALP, Runx2,expression in general, research agreement with the front surface. Namely apigenin can promote expression of Collagen I, ALP, Runx2 and OPN m RNA.6. The gel imaging analysis system was used to detect the protein bands on PVDF membranes after the antibody were incubated, the photos showed that the expression of osteogenic related protein increased during osteogenic induce supplements group,Apigenin and Chrysin groups osteogenic induction, while there were no obvious changes in the control group.Conclusion: Comparison of the classic bone induction group, apigenin, chrysin treated group and MSCs into the relevantindexes of bone differentiation overall: Classic into the most powerful, bone induction group had the best effect, apigenin equal or close, apigenin analogues of apigenin and slightly lower than the control group, the effectiveness of the lowest osteogenic differentiation. This further demonstrates that the classical osteogenic inducer differentiation advantage to promote stem cells into, and apigenin in certain conditions may also be used instead of the traditional osteogenic inducer of pharmaceutical raw materials.