外周血单个核细胞向平滑肌祖细胞分化的体外培养

In vitro differentiaion of peripheral blood mononuclear cells into smooth muscle progenitor cells

目的探讨外周血单个核细胞向平滑肌祖细胞分化的培养方法。方法分离人外周血单个核细胞,接种于加有牛垂体提取物的M-199培养基中,第8天予以血小板源性生长因子-BB(PDGF-BB)刺激促进细胞分化,刺激15 d后运用RT-PCR、免疫荧光法和Western blot检测平滑肌细胞特异性α-辅肌动蛋白(α-SMA)、平滑肌肌球蛋白重链(SMMHC)、调宁蛋白(Calponin)、CD34、Tie-2和Flk-1的表达,并以流式细胞技术检测培养细胞α-SMA阳性表达率,对培养细胞进行鉴定。结果在PDGF-BB刺激培养15 d后细胞表达平滑肌细胞的标记α-SMA、SM MHC和Calponin,同时表达干细胞的标记CD34,并发现其Flk-1阳性表达,Tie-2阴性表达。PDGF-BB刺激后细胞α-SMA阳性表达率为(90.57±5.63)%,与阴性对照细胞之间有统计学差异(P<0.05)。结论外周血单个核细胞分离后第8天,使用PDGF-BB刺激可使其向平滑肌前体细胞分化,提出了一种新的外周血单个核细胞向平滑肌细胞分化的培养方法。

Objective To optimize methods of culturing smooth muscle progenitor cells （SPCs） from mononuclear cells （MNCs） of peripheral blood. Methods Human MNCs isolated from buffy coat were seeded on M-199 with bovine pituitary extraction. On the eighth day outgrowth cells were stimulated with platelet-derived growth factor-BB （PDGF-BB）. Fifteen days later, immunofluorescence, Western blot or RT-PCR was used to analyzed the expression of smooth muscle cell specific α-actin （α-SMA）, smooth muscle myosin heavy chain（SM MHC）, Calponin, CD34, Tie-2 and Flk-1, and fluorescence activated cell sorter was employed to examine α-SMA positive cells ratio. Results The cells stimulated by PDGF-BB for 15 d were positive for α-SMA, SM MHC, Calponin, CD34 and Flk-1, but negative for Tie-2. The α-SMA positive cells ratio was （90.57 ± 5.63 ）% , significantly different from that of the control（ P 〈 0.05 ）. Conclusion Circulating MNCs stimulated by PDGF-BB from the eighth day may differentiate into SPCs in vitro. A new method of culturing SPCs from MNCs of peripheral blood is established.