Department of Histology and Embryology, Hebei North University, Zhangjiakou city, China.
Department of Histology and Embryology, Hebei North University, Zhangjiakou city, China.
Tissue Cell. 2021 Dec;73:101665. doi: 10.1016/j.tice.2021.101665. Epub 2021 Oct 14.
OBJECTIVE(S): To investigate and test the hypotheses that FGF-2 enhanced myocardial differentiation with rat bone marrow mesenchymal stem cells (BMSCs).
Lentiviral vectors carrying the FGF-2 gene were transfected into rat BMSCs firstly. According to the different inducing agents, they were divided into the following four groups: group A (BMSCs blank control group), group B (FGF-2 induction group), group C (Lenti-FGF-2-GFP lentivirus transfection group), and the group D (Lenti-control-GFP lentiviral transfer). Then several kinds of experimental methods such as real-time PCR, immunocytochemical staining, immunofluorescence staining, Western blot, and transmission electron microscopy were used to elucidate the effects by which FGF-2 adjusts myocardial differentiation in rat BMSCs.
The results of real-time PCR showed that GATA-4 and Nkx2.5 were expressed in all groups of cells. Compared with the experimental control group, the expression of GATA-4 and Nkx2.5 genes was the strongest after induction of 2 weeks in each induction group, and gradually decreased after induction of 4 weeks. Among them, the relative expression levels of GATA-4 and Nkx2.5 genes in Lenti-FGF-2-GFP were highest at all time points. The expressions of cTnI, cTnT, Cx43, and Desmin were detected by immunocytochemical staining and immunofluorescence staining. After 4 weeks of induction, cTnI, cTnT, Cx43, and Desmin were positively expressed in the cytoplasm of cells. Statistical analysis showed that the integrated optical density (IOD) values of the markers in the Lenti-FGF-2-GFP were the strongest. Cx43 and cTnI were weakly positive or negative in the experimental control group. There was a significant difference in the positive expression of each marker in each induction group and the experimental control group. Western blot analysis showed that Tromyosin (Tm) and Desmin were expressed in the blank group, FGF-2 drug-induced group, Lenti-FGF-2-GFP, and empty virus control transfection group after 4 weeks of induction, among which FGF-2 lentivirus transfected. The expression levels of Tm and Desmin were the highest in the staining induction group. Statistical analysis showed that the positive expressions of Tm and Desmin in each experimental group were statistically significant. Transmission electron microscopy showed that the nucleus of the cells transfected and induced by FGF-2 was located at the center of the cells. Myofilaments, rough endoplasmic reticulum, and mitochondria, and ribosomes were seen in the cytoplasm.
These results indicate that FGF-2 can transfect and induce differentiation of BMSCs into cardiomyocyte-like cells. Lentivirus-mediated FGF-2 transfection induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells better than FGF-2 direct induction.
研究并验证成纤维细胞生长因子 2（FGF-2）增强大鼠骨髓间充质干细胞（BMSCs）向心肌细胞分化的假说。
首先将携带 FGF-2 基因的慢病毒载体转染大鼠 BMSCs。根据不同的诱导剂，将其分为以下四组：A 组（BMSCs 空白对照组）、B 组（FGF-2 诱导组）、C 组（Lenti-FGF-2-GFP 慢病毒转染组）和 D 组（Lenti-control-GFP 慢病毒转染组）。然后采用实时 PCR、免疫细胞化学染色、免疫荧光染色、Western blot 和透射电镜等多种实验方法，探讨 FGF-2 对大鼠 BMSCs 向心肌细胞分化的调节作用。
实时 PCR 结果显示，各组细胞均有 GATA-4 和 Nkx2.5 的表达。与实验组相比，各诱导组在诱导 2 周时 GATA-4 和 Nkx2.5 基因的表达最强，诱导 4 周后逐渐减弱。其中，Lenti-FGF-2-GFP 组各时间点 GATA-4 和 Nkx2.5 基因的相对表达水平最高。免疫细胞化学染色和免疫荧光染色检测 cTnI、cTnT、Cx43 和 Desmin 的表达。诱导 4 周后，细胞胞质中 cTnI、cTnT、Cx43 和 Desmin 呈阳性表达。统计学分析显示，Lenti-FGF-2-GFP 组各标志物的积分光密度（IOD）值最强。实验组 Cx43 和 cTnI 弱阳性或阴性。各诱导组与实验组各标志物的阳性表达差异均有统计学意义。Western blot 分析显示，空白组、FGF-2 药物诱导组、Lenti-FGF-2-GFP 及空载病毒对照组在诱导 4 周后均表达 Troponin I（Tm）和 Desmin，其中 FGF-2 慢病毒转染组 Tm 和 Desmin 的表达水平最高。统计学分析显示，各实验组 Tm 和 Desmin 的阳性表达均有统计学意义。透射电镜观察到 FGF-2 转染和诱导后的细胞核位于细胞中央，细胞质中可见肌丝、粗面内质网、线粒体和核糖体。
这些结果表明，FGF-2 可转染诱导 BMSCs 向心肌细胞样细胞分化。慢病毒介导的 FGF-2 转染诱导骨髓间充质干细胞向心肌样细胞分化优于 FGF-2 直接诱导。
