Objective To investigate the effects of heat injured keratinocytes (KC) supernatant on the expressions of collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1) of dermal fibroblasts (Fb). Methods KC and Fb were isolated and cultured. Then the models of heat injured KC and Fb were reproduced in vitro, respectively. The heat injured and normal culture supernatant were collected respectively at 12 hours, and formulated as a 50% concentration of cell-conditioned medium. According to the culture medium, Fb at passage 3-5 was divided into 3 groups. Normal Fb was cultured with the conditioned medium containing 50% heat injured KC culture supernatant (group A), the conditioned medium containing 50% normal KC culture supernatant (group B), and DMEM (group C), respectively. The cells in 3 groups were collected at 24 hours. In addition， the cells in group A were collected at 0, 1, 2, 6, 12, 24, and 48 hours， respectively. Normal Fb was cultured with the conditioned medium containing 50% heat injured Fb culture supernatant. Then, the cells were collected at 0, 1, 2, 6, 12, 24, and 48 hours, respectively. The mRNA levels of the collagen type I, collagen type III, and MMP-1 of Fb were measured by real-time fluorescent quantitative PCR techniques. Results At 24 hours after cultured with supernatant of heat injured KC，mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A were significantly higher than those in groups B and C (P lt; 0.05). The mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A gradually increased with time going, showing significant differences between 0 hour and 2, 6, 12, 24, and 48 hours (P lt; 0.05); significant differences were found between different time points after 2 hours (P lt; 0.05). After Fb was treated with supernatant of heat injured Fb, the mRNA relative expression levels of MMP-1 gradually decreased with time going, showing significant differences between 0 hour and 1, 2, 6, 12, 24, and 24 hours (P lt; 0.05); after 2 hours of culture, significant differences were found among different time points (P lt; 0.05). Conclusion Heat injured KC supernatant may regulate the mRNA expressions of collagen type I, collagen type III, and MMP-1 of Fb.
【Abstract】 Objective To observe the effects of Angelica dahurica extracts on the biological characteristics of human keratinocytes (KC) in vitro and to explore the possible mechanism in promoting wound healing. Methods HaCaT cells of passage 5 from KC were used during the experiment. Different concentrations (5 × 10-2, 5 × 10-3, 5 × 10-4, and 5 × 10-5 g/L) of Angelica dahurica extracts, which was obtained by 95% ethanol from Angelica dahurica raw material, were prepared by DMEM containing 0.25% fetal bovine serum (FBS). After the extracts at different concentrations were respectively used for KC culture for 5 days, the cell proliferation activities were detected by MTT, and DMEM containing 0.25% FBS served as the negative control. According to the cell proliferation activity, the optimal concentration was determined. KC was further treated with Angelica dahurica extracts of the optimal concentration (experimental group) or with DMEM containing 0.25% FBS (control group) for 48 hours. The cell cycle was tested by flow cytometry. Cyclin D1 and Caspase-3 mRNA levels were also detected by real-time fluorescent quantitative PCR technique. Results Angelica dahurica extracts at concentrations of 5 × 10-4, 5 × 10-3,and 5 × 10-2 g/L could significantly enhance KC proliferation, showing significant differences in absorbance (A) values compared with that of control group (P lt; 0.05) with an optimal concentration of 5 × 10-3 g/L. At this concentration, an increased percentage of S and G2/M phase cells and a decreased percentage of G0/G1 phase cells were detected, showing significant differences when compared with control group (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that the cyclin D1 and Caspase-3 mRNA levels of experimental group was significantly down-regulated, showing significant differences when compared with control group (P lt; 0.05). Conclusion Angelica dahurica extracts can promote the proliferation of KC, accelerate the cell cycle of KC by down-regulating mRNA expressions of cyclin D1, and inhibit apoptosis by down-regulating mRNA expressions of Caspase-3. These effects might enhance the process of wound healing by expediting the process of epithelization.