生肌玉红膏对人真皮成纤维细胞转分化和胶原产生的影响

摘要

目的 探讨生肌玉红膏对正常人真皮成纤维细胞(normal human dermal fibroblasts,NHDFs)增殖、转分化和胶原产生及TGF-β1/Smads通路的影响.方法 将第3～6代NHDFs按照随机数字表法分为对照组、5 μg/ml生肌玉红膏组、2 ng/ml TGF-β1组、2 ng/ml TGF-β1+5μg/ml生肌玉红膏组、5 ng/ml TGF-β1组、5 ng/ml TGF-β1 +5 μg/ml生肌玉红膏组、10 ng/ml TGF-β1组、10 ng/mlTGF-β1+5μg/ml生肌玉红膏组,每组6个样本,培养72 h后,CCK-8法检测NHDFs增殖,荧光定量PCR检测α-SMA和Ⅰ、Ⅲ型胶原的mRNA表达,消化法检测细胞培养上清液中羟脯氨酸含量,蛋白质印迹法检测α-SMA、Ⅰ、Ⅲ型胶原及Smad2、Smad3和P-Smad2、P-Smad3的蛋白表达.多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,P＜0.05为差异有统计学意义.结果 在2、5、10 ng/ml TGF-β1刺激下,吸光度A值、α-SMA和Ⅰ、Ⅲ型胶原的mRNA与蛋白表达、羟脯氨酸含量、Smad2/3及P-Smad2/3蛋白表达均较对照组显著升高(P＜0.05,P＜0.01).无TGF-β1刺激时,生肌玉红膏仅使吸光度A值由1.645±0.052升高至1.796±0.060(P＜0.05),α-SMA和Ⅰ、Ⅲ型胶原的mRNA与蛋白表达、羟脯氨酸含量、Smad2/3及P-Smad2/3蛋白表达,差异均无统计学意义(P＞0.05).在2、5 ng/ml TGF-β1刺激下,生肌玉红膏使吸光度A值分别由1.814±0.052、1.970±0.045升高至1.981±0.061、2.133±0.059(P ＜0.05),在10 ng/ml TGF-β1刺激下,生肌玉红膏使吸光度A值由2.130±0.050降至1.958±0.045(P ＜0.05).在2、5、10 ng/ml TGF-β1刺激下,生肌玉红膏使α-SMA mRNA和蛋白表达分别由1.04 ±0.06、2.42±0.07、7.17±0.11和0.48±0.05、1.17 ±0.09、2.04±0.09降至0.28±0.06、0.36±0.06、1.89±0.08和0.18±0.03、0.21±0.08、0.91±0.11(P＜0.01);Ⅰ型胶原的mRNA与蛋白表达分别由0.73±0.08、1.52±0.08、3.05±0.11和0.36±0.11、0.94±0.10、2.13±0.13降至0.45±0.07、0.46±0.05、1.28±0.09和0.21±0.13、0.24±0.08、0.87±0.09(P ＜0.01);Ⅲ型胶原的mRNA与蛋白表达分别由1.51±0.09、3.28±0.09、6.96±0.14和0.26±0.08、0.96±0.09、1.96±0.15降至0.66±0.08、0.69±0.08、2.23±0.10和0.08±0.02、0.12±0.02、0.43±0.06(P＜0.01);羟脯氨酸含量分别由(7.219±0.590) μg/ml、(8.745±0.514) μg/ml、(10.969±0.489) μg/ml降至(6.242±0.225) μg/ml、(6.603±0.336) μg/ml、(7.516±0.511) μg/ml (P＜0.05);在5 ng/ml TGF-β1刺激下,生肌玉红膏对Smad2、Smad3的蛋白表达差异无统计学意义(P＞0.05),使P-Smad2、P-Smad3蛋白表达分别由0.56±0.08、0.87±0.13降至0.31 ±0.07、0.46±0.05(P ＜0.01).结论 生肌玉红膏通过促进NHDFs增殖,抑制其向肌成纤维细胞转分化及胶原的产生和分泌,促进创面愈合并预防增生性瘢痕的形成.%Objective To explore the effects of Shengjiyuhong cream(SJYHC) on proliferation,transdifferentiation,collagen production and TGF-β1/Smads signaling of normal human dermal fibroblasts (NHDFs).Methods Primary cultured NHDFs between 3-6 passages derived from 6 hypertrophic scar samples were all treated with TGF-β1 (0,2,5,10 ng/ml)stimulation added 5 μg/ml SJYHC or not.After culturing 72 h,CCK-8 solution was added to record absorbance at 450 nm to test proliferation of NHDFs.Fluorescence quantitative PCR was used for testing mRNA expression of α-SMA and type Ⅰ and Ⅲ collagen.Digestion method was to test the hydroxyproline content in the supernatant liquor.Western Blot was used for testing protein expression of α-SMA,type Ⅰ and Ⅲ collagen and Smad2,Smad3,P-Smad2 and P-Smad3.One-way analysis of variance were uesd to analyze differences among more than two groups,while LSD-t test as post hoc test were uesd to make paired-comparisons among the groups.P ＜ 0.05 indicated significant difference.Results With the stimulation of 2,5,10 ng/ml TGF-β1,the absorbance values(A values),mRNA and protein expression of α-SMA and type Ⅰ and Ⅲ collagen,hydroxyproline content,and protein expression of Smad2,Smad3,P-Smad2 and P-Smad3 were all elevated contrasted with the control group (P ＜ 0.05,P ＜ 0.01).Without TGF-β1 stimulation,SJYHC only increased the absorbance values(A values) from 1.645 ±0.052 to 1.796 ±0.060(P ＜0.05),while mRNA and protein expression of α-SMA and type Ⅰ and Ⅲ collagen,hydroxyproline content,and protein expression of Smad2,Smad3,P-Smad2 and P-Smad3 were infinitely variable(P ＞ 0.05).With stimulation of 2,5 ng/ml TGF-β1,SJYHC elevated the the absorbance values (A values) from 1.814 ± 0.052,1.970 ± 0.045 to 1.981 ± 0.061,2.133 ± 0.059 (P ＜ 0.05).While stimulated with 10 ng/ml TGF-β1,SJYHC declined the absorbance values(A values) from 2.130 ± 0.050 to 1.958 ± 0.045 (P ＜ 0.05).With stimulation of 2,5,10 ng/ml TGF-β1,mRNA expression of α-SMA were declined by SJYHC from 1.04 ±0.06,2.42 ±0.07,7.17±0.11 to 0.28 ±0.06,0.36 ±0.06,1.89 ±0.08 respectively,protein expression from 0.48± 0.05,1.17 ±0.09,2.04 ±0.09 to 0.18 ±0.03,0.21 ±0.08,0.91 ±0.11 respectively (P＜0.01),mRNA expression of Col Ⅰ from 0.73 ± 0.08,1.52 ± 0.08,3.05 ± 0.11 to 0.45 ± 0.07 0.46 ± 0.05,1.28±0.09 respectively,protein expression from 0.36 ±0.11,0.94 ±0.10,2.13 ±0.13 to 0.21 ± 0.13,0.24 ±0.08,0.87 ±0.09 respectively (P ＜0.01),mRNA expression of Col Ⅲ from 1.51 ±0.09,3.28 ±0.09,6.96 ±0.14 to 0.66 ±0.08,0.69 ±0.08,2.23 ±0.10 respectively,protein expression from 0.26 ± 0.08,0.96 ±0.09,1.96 ±0.15 to 0.08 ±0.02,0.12 ±0.02,0.43 ±0.06 respectively (P ＜0.01),hydroxyproline content from (7.219 ±0.590) μg/ml,(8.745 ±0.514) μg/ml,(10.969 ± 0.489) μg/ml to (6.242 ±0.225) μg/ml,(6.603±0.336) μg/ml,(7.516±0.511) μg/ml (P＜ 0.05).Under stimulation of 5 ng/ml TGF-β1,SJYHC had no significant effect on protein expression of Smad2 and Smad3 (P ＞ 0.05),while protein expression of P-Smad2,P-Smad3 were all declined from 0.56±0.08,0.87 ±0.13 to 0.31 ±0.07,0.46 ± 0.05 (P ＜0.01).Conclusions SJYHC may accelerate wound healing and prevent HS by promoting proliferation,inhibiting transdifferation and collagen production and secretion of NHDFs.