自体富血小板血浆诱导兔脂肪干细胞成软骨分化的实验研究

An experimental study to determine whether autologous platelet- rich plasma induces chondrogenic differentiation of rabbit adipose- derived stem cells

[目的]体外分离、培养、鉴定兔脂肪干细胞,探讨富血小板血浆体外诱导脂肪干细胞成软骨分化潜能。[方法]取Ⅰ型胶原酶消化兔脂肪后,贴壁法分离培养脂肪干细胞,取第3代细胞分别予以成脂、成骨诱导,证实其多向分化潜能;同时取第3代细胞予以富血小板血浆诱导,2周后倒置显微镜观察细胞形态,行Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色和实时荧光定量PCR检测Ⅱ型胶原和聚集蛋白聚糖的表达。[结果]可以从兔脂肪中培养出脂肪干细胞,成脂、成骨诱导证实其多向分化潜能。经自体富血小板血浆诱导的脂肪干细胞,其Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色均为阳性。实时荧光定量PCR检测发现经自体富血小板血浆诱导的兔脂肪干细胞Ⅱ型胶原α1链基因和聚集蛋白聚糖基因表达明显高于对照组未经诱导的兔脂肪干细胞(P<0.01)。[结论]自体富血小板血浆可以有效诱导兔脂肪干细胞表达II型胶原和蛋白聚糖,可以诱导兔脂肪干细胞向软骨细胞方向分化。

[ Objective] To isolate, culture, and identify adipose- derived stem cells in vitro and to explore the potential of platelet -rich plasma （PRP） on the chondrogenic differentiation of adipose -derived stem cells. [ Methods] Rabbit adipose - derived stem ceils were isolated using the type I coUagenase digestion method. Passage 3 adipose - derived stem cells were in- duced to undergo adipogenic and osteogenic differentiation in vitro. Further, these passage 3 cells were cultured with or without 10% PRP. Two weeks after PRP induction, we observed the morphology of these ceils under an inverted micro- scope. Immunofluorescence staining, toluidine blue staining, and RT - PCR were used to detect the expression of type lI collagen and aggrecan. [ Results] Rabbit adipose - derived stem ceils were successfully isolated and had differentiated into adipocytes and osteocytes. When cultured with PRP, these cells showed chondrogenic differentiation. The cells were positive for type Ⅱ colla- gen on immunofluorescence and toluidine blue staining. The expressions of type II collagen and aggrecan mRNA were significantly upregnlated in the PRP group compared to the control group （P 〈0. 01 ） . [ Conclusion] Autologous PRP could induce adi- pose - derived stem cells manifesting type Ⅱ collagen and aggrecan expression; this indicates that PRP can promote chondrogenic differentiation of rabbit adipose - derived stem cells in vitro.