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目的骨髓间充质干细胞(MSC)移植治疗已经成为治疗心肌梗死的主要途径,但是MSC在体内微环境中是否会被诱导分化为其他细胞而产生副效应如加重再狭窄尚不十分清楚。实验模拟MSC与血管平滑肌细胞(VSMC)混合的微环境,探讨氧化修饰型低密度脂蛋白(ox-LDL)和转化生长因子β1(TGFβ1)对共培养体系中MSC增殖和向平滑肌方向分化的影响。方法建立MSC与VSMC直接共培养体系;利用24孔板进行细胞爬片的培养,MSC先行4,6联脒2苯基吲哚(DAPI)5μg/L标记,然后再与VSMC进行直接避光共培养,分别给予ox-LDL(5 mg/L)和TGFβ1(5μg/L)干预及10%胎牛血清DMEM培养液作为对照,5 d后终止实验,在相同放大倍数高倍视野下计数DAPI阳性细胞核数量,用免疫荧光法检测MSC平滑肌细胞质肌丝蛋白(α-SM-actin)的表达。结果ox-LDL干预显著升高DAPI标记MSC数量[(115.7±23.1)比对照组[(68.3±25.0)/高倍视野,P<0.05],但MSC表达α-SM-actin的阳性率反而较对照组明显下降(13.5%比对照组26.6%,P<0.05);TGFβ1干预组作用相反,DAPI标记MSC阳性细胞核数量[(42.0±12.9)/高倍视野]较对照组[(68.3±25.0)/高倍视野]下降(P<0.05),而MSCα-SM-actin阳性率(40.7%)较对照组(26.6%)明显升高(P<0.05)。结论ox-LDL(5 mg/L)促进直接共培养体系中MSC增殖,但是抑制MSC向平滑肌方向分化,而TGFβ1(5μg/L)抑制MSC增殖,促进MSC向平滑肌方向分化。
OBJECTIVE: Bone marrow mesenchymal stem cells (MSCs) transplantation has become the main approach for the treatment of myocardial infarction. However, whether MSC is induced to differentiate into other cells in the in vivo microenvironment to produce side effects such as aggravating restenosis is not yet clear. The effects of ox-LDL and TGFβ1 on MSC proliferation and differentiation toward smooth muscle in co-culture system were investigated by simulating the microenvironment mixed with vascular smooth muscle cells (VSMCs) . Methods The direct co-culture system of MSC and VSMC was established. The cell slides were cultured in 24-well plates. MSCs were labeled with 5μg / L DAPI and then directly with VSMC The cells were treated with ox-LDL (5 mg / L) and TGFβ1 (5 μg / L) and 10% fetal bovine serum (DMEM) as control. After 5 d, the experiment was terminated. DAPI positive nuclei were counted at the same magnification , And the expression of α-SM-actin in MSC smooth muscle cells was detected by immunofluorescence. Results Compared with the control group [(68.3 ± 25.0) / high power field, P <0.05], ox-LDL significantly increased the number of DAPI-labeled MSCs (115.7 ± 23.1), but the positive rate of α-SM- (P <0.05). Compared with the control group (68.3 ± 25.0) / high magnification, the number of DAPI-labeled MSC positive nuclei (42.0 ± 12.9) / high power field was significantly lower than that of the control group (P <0.05), while the positive rate of MSCα-SM-actin (40.7%) was significantly higher than that of the control group (26.6%) (P <0.05). Conclusion Ox-LDL (5 mg / L) promoted the proliferation of MSCs in direct co-culture system, but inhibited the differentiation of MSCs to smooth muscle. TGFβ1 (5μg / L) inhibited the proliferation of MSCs and promoted the differentiation of MSCs to smooth muscle.