2022, Vol. 26 ›› Issue (24): 3858-3864

Effect of paired box gene 6 protein on the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells

Wu Shuang, Zou Xingxing, Gao Jie, Hu Rong, Su Min   

Abstract: BACKGROUND: Keratopathy transplantation is faced with many problems, such as serious shortage of corneal donors, postoperative immune rejection and so on. While vigorously advocating corneal donation, there is an urgent need to find a new alternative treatment.  
OBJECTIVE: The effect of paired box gene 6 on the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells was investigated by optimizing the culture conditions of bone marrow mesenchymal stem cells.
METHODS:   Bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells overexpressing paired box gene 6 were identified according to cell morphology, osteogenic adipogenic differentiation ability, and cell surface antigen expression. According to different cultured cells and culture medium, they were divided into control group (bone marrow mesenchymal stem cells + complete medium), and experimental group A (bone marrow mesenchymal stem cells + conditioned medium), experimental group B (bone marrow mesenchymal stem cells + conditioned medium + cytokine), and experimental group C (bone marrow mesenchymal stem cells overexpressing paired box gene 6 + conditioned medium + cytokine). The expression levels of p63, k14 and k12 were detected by immunofluorescence and flow cytometry at 5 and 10 days after induction. The expression of Ki-67, an indicator of cell proliferation, and the rate of apoptosis, were measured by flow cytometry. Western blot assay and RT-PCR were used to determine the protein and mRNA expression of paired box gene 6.  
RESULTS AND CONCLUSION: (1) Before induction and culture, the two groups of cells had the characteristics of bone marrow mesenchymal stem cells. (2) After conditioned culture, spherical or oval cells appeared in the four groups after induction, which showed suspension growth. The expression of keratolimbal stem cell-like cells molecules p63 and k14 was positive in the four groups. The fluorescence expression in the experimental group was significantly higher than that in the control group (P < 0.05), but the expression of corneal epithelial molecule k12 was negative. With the optimization of the culture conditions, the cell proliferation of experimental group C was more obvious, and there were fewer apoptotic cells. After 5 days of induction, the expression of paired box gene 6 protein and mRNA in the experimental groups increased progressively at 5 days after induction. (3) Results suggested that conditioned medium combined with cytokine induction can accelerate the differentiation of bone marrow mesenchymal stem cells into keratolimbal stem cell-like cells. Bone marrow mesenchymal stem cells overexpressing paired box gene 6 are more superior in the speed and proportion of directional differentiation into keratolimbal stem cell-like cells, and it can promote cell proliferation and inhibit cell apoptosis.
Key words: stem cells, bone marrow mesenchymal stem cells, keratolimbal stem cell-like cells, paired box gene 6, cytokines, conditioned medium