ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
ObjectiveTo investigate the effect of daphnetin (DAP) combined with insulin-like growth factor 1 (IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) in rats.MethodsRat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP (0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8 (CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers (collagen type Ⅱ and Aggrecan) in each group; and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed.ResultsCCK-8 assay showed that with the increase of DAP concentration, the cell absorbance (A) value of the control group and the experimental group increased gradually (P<0.05). At the same DAP concentration, the cell A value of the experimental group was significantly higher than that of the control group (P<0.05). Real-time fluorescence quantitative PCR and Western blot showed that with the increase of DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the control group did not change significantly, and there was no significant difference among the different concentration groups (P>0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group (P<0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group (P<0.05). Toluidine blue staining showed that with the increase of DAP concentration, there was no significant difference in cell staining between the control group and the experimental group. At the same DAP concentration, the cells in the experimental group were slightly darker than those in the control group. Immunohistochemical staining of collagen type Ⅱ showed that with the increase of DAP concentration, there was no significant difference in the cytoplasmic brown-yellow coloring of the cells in the control group. The cytoplasmic brown-yellow coloring of the cells in the experimental group gradually deepened, with 60 and 90 μg/mL DAP concentration groups significantly deeper than 0 μg/mL DAP concentration group. At the same DAP concentration, the color of the cells in the experimental group was significantly deeper than that in the control group.ConclusionDAP can promote the proliferation of ADSCs in rats. The differentiation of ADSCs into chondrocytes induced by DAP alone was slightly, but DAP combined with IGF-1 gene transfection has obvious synergistic effect to promote chondrogenic differentiation of ADSCs.