Objective To find new ways for wound healing and tissue expansion by reviewing of progress in recent years in functional molecules which are used for signaling channels of mechanical stress perception and mechanotransduction of keratinocyte. Methods The domestic and international articles were reviewed to summarize the functional molecules and signaling channels of mechanical stress perception and mechanotransduction of keratinocytes. Results The mechanism of mechanical stress perception includes mechano-sensitive channels, growth factor receptor-mediated mechanical stress perception, and mechanical stress perception by protein deformation. The mechanism of mechanotransduction includes cell adhesion-mediated signaling, mitogen-activated protein kinase signaling, the cytoskeleton and extracellular matrix, and so on. Conclusion Keratinocytes can response to the mechanical stress and transfer the effective information to undergo shaping, migration, proliferation, differentiation, and other biological behavior in order to adjust itself to adapt to the new environment.
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.
Objective Dermal papillae cells are widely applied to reconstruction of tissue engineered hair foll icle and skin. To investigate the difference of the biological characteristics of dermal papillae cells cultured with keratinocyte medium (KM)and normal medium (NM), and to determin whether it is feasible for the reconstruction of tissue engineered hair foll icle using dermal papillae cells cultured in KM. Methods Scalp samples were obtained in rhytidectomy procedure. Dermal papillaes were isolated by two steps digestive treatment, then cultured with KM and NM in two groups. The time of dermal papillae adherence and cell outgrowth was recorded and the rate of dermal papillae adherence was determined after 5 days. As well as, the difference of cell morphology was observed through inverted phase contrast microscope. The maximum generations were determined in two groups and the cell sheets were observed by HE staining. In third-generation cells, the number of aggregates in every dish and the prol iferation by MTT were compared between two groups. Meanwhile, the expression of α-smooth muscle actin (α-SMA) and ALP were detected by immunofluorescence and specific staining in two groups. Results Dermal papillaes of KM group had a higher rate of adherence and fast outgrowth. The rates of adherence were 54.17% and 36.78% in KM group and in NM group, respectively. In KM group, cells adhered after 24 hours and outgrew after 64 hours. While, cells adhered after 48 hours and outgrew after 80 hours in NM group. The cells were bigger in NM group than in KM group. In third-generation cells, 3.06 ± 1.12 and 9.25 ± 1.73 aggregates formed in NM group and KM group, respectively, the difference was significant (P lt; 0.05). In addition, cells could form cell sheets which were muti-layers in KM group. Mostly 7 and 15 generations could been subcultured in NMgroup and KM group, respectively. The result of MTT indicated that cells prol iferated more actively in KM group; absorbance value of KM group was significantly higher than that of NM group after 7 days (P lt; 0.05). The positive of α-SMA were detected in the third-generation cells of both groups. Ocassionally a l ittle few cells expressed ALP with (987 ± 146) m2 positive area in the sixth-generation cells of NM group. However, the cells still expressed ALP with (8 757 ± 558) μm2 positive area in the fourteenthgeneration cells of KM group and the difference was significant (P lt; 0.05). Conclusion Cells proliferate actively and aggregate obviously and could been subcultured more generations in KM. Therefore, culturing dermal papillae cells with KM is feasible for the reconstruction of tissue engineered hair foll icle.
Objective To observe the change of the immunogenity of keratinocytes when cultured in vitro. Methods Health children foreskins were digested bydispase and trypase. The human keratinocytes were cultured in vitro and passaged in succession until the fifth passage. Different passage keratinobytes signed by SP method to show the percentage of langerhans cells and melanocytes. Every passage keratinocytes were respectively mixed with allogenic lymphocytes which isolated from peripheral blood, and then the proliferation degree of allogenic lymphocytes was tested. Results Keratinocytes were cultured well in KSFM medium. When keratinocytes conjugated, every passage cells grew like paving stone. After cryopreservation and then rewarming, the survival exceeded 80%. The percentages of langerhans cells and melanocytes in the primary passage were 5.8% and 8.1% respectively. In the 1st passage they were 2.1% and 2.8% respectively. They were not detected in the second passage. The values of cpm were respectively 482.13±46.61 (primary passage), 362.50±35.12(1st passage), 228.38±51.46(2nd passage), 171.86±34.63(3rd passage), 143.63±15.95(4th passage), and 123.25±14.39(5th passage), showing statistically significant differences when compared withcontrol (53.67±8.61) (Plt;0.05). There were statistically significant differences between the primary passage, the 1st passage respectively and the other passages(Plt;0.05). There were statistically significant differences between the4th passage, the 5th passage respectively and the 2nd passage (Plt;0.05). There was no statistically significant difference between the 2nd passageand the 3rd passages(Pgt;0.05). There was not statistically significant difference among the 3rd, the 4th and the 5th passages (Pgt;0.05). Conclusion Allogenic keratinocytes were cultured in vitro and passaged, and their immunogenity gradually decreased.
Objective To observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
目的 通过对白芍总苷治疗前后寻常型银屑病患者血清中白介素（IL）-22水平的研究，探讨其治疗寻常型银屑病的作用机制。 方法 2009年10月－2010年8月采用双抗体夹心酶联免疫吸附法，检测30例寻常型银屑病患者，经白芍总苷治疗前后及健康对照组20例外周血清中IL-22浓度的变化，分析其在治疗前、后与银屑病皮损面积和严重程度指数（PASI）评分的相关性。 结果 寻常型银屑病患者血清IL-22浓度[（90.50 ± 51.80）pg/mL]较对照组[（40.10 ± 17.20）pg/mL]升高，白芍总苷治疗后血清中IL-22水平[（48.70 ± 23.90）pg/mL]较治疗前降低（P＜0.05），并与对照组差异无统计学意义（P＞0.05）；治疗前、后患者血清IL-22水平与PASI评分呈正相关。结论 白芍总苷可能通过调节IL-22发挥治疗寻常型银屑病的作用。