DMSO在诱导绵羊脐带间充质干细胞为成肌细胞中的作用

The role of DMSO in inducing sheep umbilical cord mesnchymal stem cells into myoblasts

为了探明二甲基亚砜(DMSO)在绵羊脐带间充质干细胞(MSCs)分化为成肌细胞的作用,研究将绵羊MSCs用含有DMSO的F12/DMEM合成培养基进行诱导培养后观察其细胞形态、免疫荧光并利用流式细胞仪检测成肌细胞特异因子,如肌分化因子(Myo D)、肌间线蛋白(Desmin)、肌细胞生成素(Myo G)等的表达及RT-PCR检测成肌细胞特异因子的相对表达量。结果表明:与对照组(不含DMSO的F12/DMEM培养基)细胞形态不发生变化相比,用含DMSO的F12/DMEM培养基诱导培养21 d后,其细胞由长梭形变为具有成肌细胞特点的细长管状;在开始诱导培养后的第16天进行成肌细胞标志性蛋白Myo D、Desmin、Myo G抗体免疫荧光检测,与对照组呈现阴性相比,所诱导细胞呈阳性。用流式细胞仪的检测结果显示,诱导细胞的表达成肌细胞特异因子Myo D、Desmin、Myo G的阳性细胞率分别为99.3%、99.5%、86.6%;RT-PCR的检测结果显示,与对照组(表达量为1)相比,诱导细胞的Myo D、Myo G、Desmin相对表达量均升高,分别为(3.71±0.01)倍、(1.86±0.01)倍、(5.27±0.01)倍。表明DMSO具有诱导绵羊MSCs分化为成肌细胞的功效。

To ascertain the role of dimethyl sulfoxide （DMSO） in sheep umbilical cord mesenchymal stem cells（MSCs） to differentiate into myo- blasts, this study aims to add DMSO F12/DMEM synthetic medium culture, sheep umbilical cord MSCs were used for induction culture using a synthetic medium containing DMSO F12/DMEM, and then the cell morphology was observed. The immunofluorescence and flow cytometry were used to detect the specific factors of myoblasts, such as the expressions of^yogenic differentiation antigen （MyoD）, desmin, and myoge- nin（ MyoG）, as well as the relative expression levels of specific factors of myoblasts using RT- PCR detection. The results showed that the cell morphology changed from long fusiform to slender tube with the characteristics of myoblasts after 21 d of the induction culture using the synthetic medium containing DMSO F12/DMEM, compared with the control group without any changes in the cell morphology, which was cultured in a medium without containing DMSO FI 2/DMEM; the immunofluorescence test was used for the detection of antibodies of MyoD, desmin and MyoG as the iconic proteins of myoblasts on the 16 d after the induction culture, and the induced cells were positive compared with negative control group. The results detected by flow cytometry showed that the positive cells rates of induced cells expressing myoblast - specific factor MyoD, Desmin and MyoG were 99.3%, 99.5% and 86.6%, respectively. The results detected by RT - PCR showed that the relative expres- sion levels of MyoD, MyoG and Desmin of induced ceils were increased by 3.71 ±0.01, 1.86 ±0.01, and 5.27±0.01 times compared with the control group with 1 of expression level, respectively. The results indicate that DMSO has an effect on inducing sheep umbilical cord MSCs to differentiate into myoblasts.