富血小板血浆对股骨头坏死微环境下犬骨髓基质干细胞增殖与分化的影响

The effect of platelet-rich plasma on canine bone marrow stromal cells in the micro-environment offemoral head necrosis

目的初步探讨富血小板血浆（PRP）对股骨头坏死微环境下犬骨髓基质干细胞（BMSCs）增殖与成骨分化的影响。方法以含体积百分比为5％的股骨头坏死组织浸泡液的达尔伯克氏改良Eagle培养基（DMEM）模拟股骨头坏死微环境。采用全骨髓培养法培养成年杂种犬BMSCs，将第3代BMSCs分为5组：空白对照组（DMEM培养基培养）、A组（含5％浸泡液的DMEM条件培养基培养）、B组（含体积百分比为5％PRP、5％浸泡液的DMEM条件培养基培养）、C组（含10％PRP、5％浸泡液的DMEM条件培养基培养）、D组（含15％PRP、5％浸泡液的DMEM条件培养基培养）。于培养第3、6、8天检测各组（n=6）细胞的增殖活性。于培养第7大检测各组（n=6）细胞外碱性磷酸酶（ALP）活性。于培养第10天行细胞ALP染色，第19天行细胞钙结节染色。结果培养第3、6、8天B、C、D组BMSCs的OD值均较A组和空白对照组大，A组又较空白对照组大；培养第6天D组BMSCs的OD值较B、C组大；培养第8天B、C、D组BMSCs的OD值大小：D组〉C组〉B组，差异均有统计学意义（P〈0．05）。细胞外ALP活性和钙结节染色结果均显示：B组的成骨分化能力较空白对照组、A组、c组、D组强，差异均有统计学意义（P〈0．05）；但后4组之间两两比较差异均无统计学意义（P〉0．05）。细胞ALP染色结果也硅示B组的成骨分化能力较其他4组强。结论PRP对股骨头坏死微环境下犬BMSCs的增殖有促进作用。低浓度PRP对股骨头坏死微环境下犬BMSCs有促成骨分化作用。

Objective To observe the effect of platelet-rich plasma （PRP） on the proliferative and osteogenetie activity of canine bone marrow stromal cells （BMSCs） in the micro-environment of femoral heat] necrosis （FHN） . Methods The FHN mieroenviromnent was simulated with Dulbecco＇s modified Eagle medium （DMEM） containing 5% volume FHN soak solution. Whole bonc marrow cuhure was used to culture BMSCs from an adult mongrel. The 3rd BMSCs were divided intu 5 even groups： blank group （added with normal DMEM）, group A （added with DMEM with 5%, soak solution）, group B （added with DMEM with 5% soak solution and 5% PRP）, group C （added with DMEM with 5% soak solutiml and 10% PRP） and group D （added with DMEM with 5% soak solution and 15% PRP）. The proliferation of BMSCs in each group （ n = 6） was examined by the MTT assay at 3~ 6 and 8 days. Furthermore, extracellular alkaline phosphatase （ALP） assay （at 7 days）, ALP staining （at 10 days） and the calcium staining （at 19 days） were used to e- valuate the osteogenic differentiation of BMSCs. Results At 3, 6 and 8 days, the optical density （OD） was signifieantly larger in group A than in the blank group （ P 〈 0.05）, and significantly larger in groups B, C and D than in group A （ P 〈 O. 05）. The OD was significantly larger in group 1） than in groups B and C at 6 days （ P 〈 O. 05）. At 8 days, group D showed the largest OD, followed by group C and last by group D, with significant differences between groups （ P 〈 0.05）. Extracellular AI,P assay, ALP staining and calcium staining showed that the usteogenic differentiation in group B was significantly stronger than in the other groups （ P 〈 0.05） but there were no significant dift^rences between the other 4 groups （ P 〉 O. 05） . Conclu- sions In the FHN micro-environment, PRP promotes proliferation of canine BMSCs and low density PRP accelerates osteogenic differentiatinn of canine BMSCs.