浓缩生长因子与1,25二羟基维生素D3对骨髓间充质干细胞增殖与成骨分化的影响

目的:研究浓缩生长因子(concentrated growth factor,CGF)及其与1,25二羟基维生素D3(1,25(OH)_2VD_3)联合应用时,对骨髓间充质干细胞(BMSCs)增殖及成骨分化的影响。方法:全骨髓培养法提取、分离SD大鼠股骨BMSCs,分别采用6种不同培养液而分为6组。使用SD大鼠血液在Medifuge离心机中制备CGF,并收集CGF浓缩液。实验A、B、C组,分别为采用含5%、10%和20%浓度CGF的L-DMEM培养液组,单独使用1,25(OH)_2VD_3(10~(-10)mol/L)的为D组。B组与D组混合为E组。F组为空白对照组。采用上述6组培养基孵育BMSCs 7 d。采用CCK-8实验评价大鼠BMSCs在不同条件下的生长增殖情况;ALP活性染色实验检测各组ALP的生成,实时定量PCR检测成骨分化标志物基因OCN、Col-1和Runx2共3种成骨分化标志物基因的表达情况。结果:5%和10%的CGF浓缩液能促进大鼠BMSCs增殖,且10%浓度优于5%浓度。而20%CGF和1,25(OH)_2VD_3(10~(-10)mol/L)都抑制了大鼠BMSCs的增殖。1,25(OH)_2VD_3(10~(-10)mol/L)与CGF(10%)浓缩液联合应用时,细胞增殖介于两者之间。A、B、C组ALP活性及OCN、Col-1和Runx2的mRNA表达均受到了抑制。D组和E组均促进了大鼠BMSCs的ALP活性,并提高了OCN、Col-1和Runx2的mRNA表达水平。结论:CGF浓缩液(10%)与1,25(OH)_2VD_3(10~(-10)mol/L)的联合应用,可促进大鼠BMSCs的增殖,并对其成骨分化具有促进作用。

Concentrated Growth Factor and 1, 25 Dihydroxy Vitamin D3 Enhance Proliferation and Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem cells

Objective: The goal of this study was to gain insight into whether the cytokine concentrated growth factor (CGF) and vitamin D3(1, 25(OH)2VD3), alone or in combination are associated with the proliferation and osteogenic differ-entiation of bone marrow mesenchymal stem cells (BMSCs). Methods:The BMSCs were isolated from SD rats' femur bone. Medifuge centrifuge machine was used to prepare CGF from rats blood and concentrates were collected. CGF concentrate were desolved in Low-Dulbcco's Modifed Eagle Medium with different ratios, in order to get CGF 5%, 10%, and 20% in three different concentrations, which was named as group A, B and C. 1, 25 (OH)2VD3 (10-10 mol/L) alone was named as group D. Group B combined with group D were named as group E. Group F was the blank control . BMSCs were placed into 96-well culture plates with different concentrations of CGF alone or in combination with 1, 25(OH)2VD3. Cell prolifer-ation was assessed with the use of a CCK-8 kit assay according to the manufacturer's instructions. ALP activity was mea-sured with the use of an ALP Activity Assay Kit. We performed q-PCR to analyze the expression of the osteoblastic differ-entiation markers OCN, CoL-I, and Runx2. Results: 5%and 10%CGF concentrates enhanced the proliferation of BMSCs, and 10%is superior to 5%. While CGF in 20%concentration, and 1, 25(OH) 2VD3(10-10 mol/L) alone inhibited the prolifer-ation process repectively. 10% CGF concentrate combined with 1, 25(OH) 2VD3(10-10 mol/L) enhanced proliferation of BM-SCs in a moderate degree. Osteoblastic differentiation markers ALP activity, OCN, Col-1, and Runx2 of BMSCs were sub-dued in groups A, B and C, but enhanced in group D and E. Conclusion: The combination of CGF concentrate and 1, 25(OH)2VD3 (10-10 mol/L) can promote the proliferation of BMSCs and have a promoting effect on the osteogenetic differentiation.