Objective: To preliminarily explore the effects of human dermis derived mesenchymal stem cells (hDMSCs) on expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in hypertrophic scars fibroblasts (HSFB) at different periods,and to explore the feasibility of MSCs in prevention and treatment of HSFBs.
Methods: hDMSCs were cultured with mechanical method combined with enzyme digestion.The cells of the third generation which were well grown were taken,and flow cytometry (FCM) was used to detect CD molecules in hDMSCs.Immunocytochemistry was used to detect cytokeratin 19 (CK19) and vimentin and identify the separated cells.The cells were differentiated into lipoblasts,chondroblasts and osteoblasts.According to the formation course of hypertrophic scar,the scar specimens were divided into 6-month,l-year,and 2-year group with three cases in each group.HSFBs from different groups were co-cultured with well-adherent hDMSCs of the third generation in non-contact transwell co-culture system for 21 days. And HSFBs from the corresponding groups were cultured in normal six-well plate as the controls.Real-time fluorescent-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expressions of mRNA and proteins of α-SMA and DCN in HSFBs from different groups.
Results: hDMSCs highly expressed the surface markers including CD73,CD105,CD44 and CD90,etc.,but did not express hematopoietic stem cell surface markers including CD 14,CD34 and CD45.They positively expressed vimentin but not CK19.The cells can be differentiated into lipoblasts,chondroblasts and osteoblasts,which was in line with the minimum identification standards of mesenchymal stem cells.For HSFB cultured in normal six-well plates,α-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 198.20 ± 15.46/0.29 ± 0.070,175.24 ± 17.04/0.38 ± 0.110,and 125.73 ± 6.99/0.33 ±0.085,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 61.30 ± 9.79/0.015 ± 0.003,70.89 ± 11.29/0.020 ± 0.007,and 77.31 ± 4.80/0.023 ± 0.003,respectively.For HSFB co-cultured with 5 × 104 hDMSCs, oα-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 48.40 ± 6.42/ 0.100 ± 0.020,192.16 ± 11.37/0.110 ± 0.014,and 73.33 ± 6.29/0.110 ± 0.016,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 156.92 ± 14.91/0.049 ±0.015,154.42 ± 18.17/0.033 ± 0.008,and 140.82 ± 7.32/0.030 ± 0.004,respectively.Compared with the control group(Cultured in normal six-well plates), mRNA and protein expressions of α-SMA in HSFBs were decreased after co-culture with 5 × 104 hDMSCs, mRNA and protein expressions of DCN were increased. Furthermore, it suggested that hypertrophic scar changed significantly in the early formation stage namely in the 6-month group.
Conclusions: hDMSCs can down-regulate α-SMA mRNA and protein expressions of HSFBs and up-regulate mRNA and protein expressions of DCN in the in-vitro culture system.Those effects were particularly obvious on fibroblasts at the early formation of hyperplastic scar.Anti fibrosis role of hDMSCs is expected to be used in increasing the healing quality of the wound and in the prevention and treatment of pathological scars.