脂肪干细胞向血管平滑肌细胞诱导的实验研究

EXPERIMENT OF ADIPOSE DERIVED STEM CELLS INDUCED INTO SMOOTH MUSCLE CELLS

目的研究人脂肪干细胞(adipose derived stem cells,ADSCs)在体外单层培养条件下诱导为血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的可行性。方法应用酶消化法消化人脂肪组织获得ADSCs,基础培养液培养传代,取第1代细胞用于诱导分化实验。实验分两组,ADSCs以含5ng/mLTGF-β1及50ng/mLPDGF-BB的M-199诱导液联合诱导,作为诱导组;以含10%FBS的M-199培养液培养ADSCs作为未诱导组。镜下观察细胞生长情况;免疫荧光和RT-PCR方法检测平滑肌细胞特异标记的表达情况;流式细胞仪检测诱导阳性率。结果诱导组细胞形态形成平滑肌细胞特有的"峰-谷"生长模式,未诱导组细胞形态未发生改变,与原代ADSCs相似呈成纤维细胞样生长。诱导培养14d,诱导组细胞体外扩增能力较未诱导组显著降低(P<0.01)。免疫荧光检测:诱导组表达平滑肌特异标记α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、平滑肌肌球蛋白重链(smooth muscle-myosin heavy chain,SM-MHC)和Calponin,未诱导组无表达。细胞诱导前α-SMA、SM-MHC及Calponin阳性表达率分别为3.26%±1.31%、3.55%±1.60%、4.02%±1.81%;诱导组阳性表达率分别为48.13%±8.31%、45.33%±10.68%、39.13%±9.42%;诱导前、后差异有统计学意义(P<0.01)。RT-PCR检测:诱导组表达平滑肌特异标记α-SMA、SM-MHC、Calponin和SM-22α,未诱导组无表达。结论脂肪干细胞经体外培养及诱导后,呈明显的VSMCs特性,有可能成为血管组织工程新的种子细胞来源。

Objective To study the feasibility of human adipose derived stem cells （ADSCs） in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering. Methods The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 × 10^5/cm^2. Cells were cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02% EDTA treatment, PDGF-BB （50 ng/mL） and TGF-β1 （5 ng/mL） were added at the passage 1 to enhance the smooth muscle cells＇ phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry （FACs） was used to examine the positive rate of induced team. Results Cocultured in M-199 media including TGF-β1 and PDGF-BB, the proliferating capability of the induced cells was significantly downregulated compared with the uninduced cells（P 〈 0.01）. The induced cells exhibited ＂Hill and Valley＂ morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-like morphology. The results of immunofluorescence indicated that the induced cells expressed smooth muscle （SM） cell- specific markers including α-smooth muscle actin （α-SMA）, SM-myosin heavy chain （SM-MHC） and Calponin. The results of RT-PCR revealed that the induced cells also expressed α-SMA, SM-MHC, Calponin and SM-22α.The positive rates of α-SMA, SM-MHC and Calponin in FACs were 3.26% ± 1.3196, 3.55% ± 1.6% and 4.02% ± 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% ±8.31%, 45.33%±10.68% and 39.13%± 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells（P 〈 0.01）. Conclusion The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.