支架材料对棕色脂肪源性干细胞向起搏细胞诱导分化的影响

目的观察不同三维支架材料对棕色脂肪来源干细胞（BADSCs）诱导分化成起搏细胞的效果，为构建生物起搏器提供实验依据。 方法将培养7 d的原代BADSCs分别种植到胶原海绵、明胶海绵和透明质酸水凝胶3种不同的材料中，在不同时间用光镜和扫描电镜观察细胞-支架复合体中细胞形态学的变化，免疫荧光染色检测心肌细胞、起搏细胞相关蛋白的表达。采用单因素方差分析。 结果细胞在3种支架上均能存活、增殖，LIVE/DEAD检测显示，培养3 d的胶原海绵、明胶海绵和透明质酸水凝胶3种细胞-支架复合物死细胞率分别为（46.35±1.50）%、（47.00±1.60）%和（1.76±1.08）%，其中细胞在透明质酸水凝胶中死亡率最低，并且细胞-透明质酸水凝胶复合物可自发性地搏动，三组比较差异具有统计学意义（F = 37.56，P < 0.05）。培养至2周时，胶原海绵、明胶海绵和透明质酸水凝胶中Connexin45细胞阳性率分别为（10.67±1.25）%、（13.67±1.25）%和（21.00±1.60）%，差异有统计学意义（F = 9.435，P < 0.01），HCN2细胞阳性率分别为（11.00±1.60）%、（14.00±2.16）%和（34.33±3.68）%，差异有统计学意义（F = 17.52，P < 0.01），HCN4细胞阳性率分别为（18.67±2.05）%、（13.00±1.60）%和（66.00±2.94）%，差异有统计学意义（F = 27.96，P < 0.01），Sr细胞阳性率分别为（13.00±1.63）%、（14.33±1.24）%和（75.33±3.30）%，差异有统计学意义（F = 36.40，P < 0.01），水凝胶中Connexin45、HCN2、HCN4和Sr的细胞阳性率均高于胶原海绵和明胶海绵，差异均具有统计学意义（P < 0.05）。 结论BADSCs在胶原海绵、明胶海绵和透明质酸水凝胶中均能很好地生长和分化，但透明质酸水凝胶更适用于组织工程化起搏器的构建。

Effect of brown adipose-derived stem cells differentiating into pacemaker cells induced by scaffold materials

ObjectiveThe effects of brown adipose-derived stem cells (BADSCs) differentiating into pacemaker cells induced by three dimensional scaffold materials were observed to provide experimental evidence for the construction of tissue engineered pacemaker in vitro. MethodsBADSCs, cultivated for 7 days, were planted into the collagen sponge, gelatin sponge and hydrogel separately. Cell morphology changes in the cell-scaffold complex were observed with the light microscope and scanning electron microscope at different time. Immunofluorescence staining was used to detect the expression of myocardial and pacemaker cell-related protein. The values were analyzed using one-way ANOVA. ResultsBADSCs survived and proliferated in all three kinds of scaffold. According to the LIVE/DEAD detection, the cell death rate in collagen sponges, gelatin sponges and hyaluronic acid hydrogels was (46.35±1.50) %, (47±1.60) % (1.76±1.08) %, respectively. The cells in the hyaluronic acid hydrogel had the lowest mortality (F = 37.56, P?< 0.05) , while the cell-hyaluronic acid hydrogel complex spontaneously pulsated. Two weeks after planting, the positive rate of Connexin45 cells in collagen sponges, gelatin sponges and hyaluronic acid hydrogels was (10.67±1.25) %, (13.67±1.25) %and (21±1.60) %respectively. The positive rate of HCN2 cells was (11±1.60) %, (14±2.16) % and (34.33±3.68) %, the positive rate of HCN4 cells was (18.67±2.05) %, (13±1.60) % and (66±2.94) %respectively and the positive rate of Sr cells was (13±1.63) %, (14.33±1.24) % and (75.33±3.30) %respectively. Positive rates of Connexin45, HCN2, HCN4 and Sr cells in hydrogels were significantly higher than that in collagen sponge and gelatin sponge (P < 0.05) . ConclusionBADSCs could grow and differentiate well in collagen sponges, gelatin sponges and hyaluronic acid hydrogels, and hyaluronic acid hydrogels was more suitable for the construction of tissue engineered pacemaker.