己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子-β1表达的影响北大核心CSCD

目的探讨己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子（TGF）-β1表达的影响。方法取人瘢痕疙瘩及正常皮肤组织培养的第5～8代成纤维细胞,在含有0．1—3g／L己酮可可碱的环境中培养。应用噻唑蓝（M1Tr）法检测成纤维细胞增殖,双抗体夹心-ELISA法测定TGF—β1表达,RT—PCR检测I、Ⅲ型前胶原mRNA表达。结果0．1～2g／L己酮可可碱能明显抑制瘢痕疙瘩和正常皮肤成纤维细胞的增殖,呈明显的量效一时效关系,抑制作用在浓度2g／L时达到最高。浓度为0．5～2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞TGF—B1表达,1或2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞I、Ⅲ型前胶原mRNA表达。结论己酮可可碱对瘢痕疙瘩和正常皮肤成纤维细胞的增殖、TGF—β1以及I、Ⅲ型前胶原表达均有明显的抑制作用。     

己酮可可碱对兔耳增生性瘢痕纤维化的影响

目的探索己酮可可碱对兔耳瘢痕成纤维细胞增殖及Ⅰ、Ⅲ型胶原纤维合成的影响。方法建立60个兔耳增生性瘢痕模型,随机分为10组,以0. 1～16 g/L不同浓度的己酮可可碱溶液注射瘢痕,1次/周,共4周,用药后4周进行成纤维细胞计数,确定己酮可可碱抑制成纤维细胞增殖的最适浓度。另取54个瘢痕,随机分为3组即实验组(注射最适浓度的己酮可可碱)、阳性对照组(注射曲安奈德)和阴性对照组(注射蒸馏水),1次/周,共4周,用药后2、4、6周分别进行成纤维细胞计数和Ⅰ、Ⅲ型胶原含量测定。结果己酮可可碱抑制成纤维细胞增殖的最适浓度为1 g/L。用药后2周,实验组成纤维细胞数量较阴性对照组少,差异有统计学意义(P 0. 05)。用药后4、6周,实验组成纤维细胞数量均较阳性对照组及阴性对照组少,差异均有统计学意义(P 0. 05)。用药后2、4、6周,实验组比阴性对照组Ⅰ型胶原含量少,Ⅰ/Ⅲ型胶原比例低,差异有统计学意义(P 0. 05)。结论局部注射己酮可可碱可抑制兔耳增生性瘢痕内成纤维细胞增殖,最适浓度为1 g/L。己酮可可碱亦可抑制Ⅰ型胶原合成,降低Ⅰ/Ⅲ型胶原比例。     

氧化应激所致的人皮肤成纤维细胞衰老中β-连环蛋白的表达研究

目的 观察β-连环蛋白在氧化应激所致的人皮肤成纤维细胞衰老中的变化,探讨其在应激诱导的皮肤衰老中可能的作用.方法 从儿童包皮分离培养原代成纤维细胞,H2O2:处理第2～4代人皮肤成纤维细胞,显微镜观察其形态学改变,β-半乳糖苷酶细胞衰老试剂盒检测β-半乳糖苷酶活性,RT-PCR、Western印迹检测β连环蛋白mRNA和蛋白表达水平.结果 150 μmol/L H2O2:处理2 h后可以诱导人皮肤成纤维细胞衰老.在应激诱导的衰老(SIPS)细胞中β-半乳糖苷酶阳性率达到(37.67±1.53)%.而对照细胞中只有(2.97±0.25)%,两组差异有统计学意义(P<0.01).RT-PCR 结果显示,在对照细胞组β连环蛋白/GAPDH mRNA比值为0.59±0.04,SIPS细胞组为0.29±0.30,对照细胞组明显高于SIPS细胞组,两组差异有统计学意义(t=10.06,P<0.01).Western印迹结果显示,对照细胞组β连环蛋白/GAPDH蛋白比值为0.62±0.03,SIPS细胞组为0.31±0.01,对照细胞组明显高于SIPS细胞组,两组差异有统计学意义(t=14.97,P<0.0).结论 150 μmol/L H2O2:处理2 h可以诱导人皮肤成纤维细胞发生衰老改变,SIPS细胞中β连环蛋白表达水平明显降低,初步推测β连环蛋白可能是调控皮肤衰老的重要靶基因.

Abstract：

Objective To observe the changes of β-catenin expression in human skin fibroblasts (HSFs) after induced by oxidative stress, and to explore its possible roles in oxidative stress-induced premature senescence (SIPS) of HSFs. Methods Fibroblasts were isolated from the foreskin of a child and subjected to a primary culture. The fibroblasts of second to fourth passage were treated with various concentrations of H2O2 for 2 hours to establish an optimized model of stress-induced premature senescence, β-galactosidase assay kit was used to detect the activity of β-galactosidase in H2O2rinduced HSFs, RT-PCR and Western blot to measure the mRNA and protein expressions of β-catenin in control and senescent HSFs. Results Premature senescence of HSFs could be induced by the treatment with H2O2 of 150 μmol/L for 2 hours. The proportion of β-galactosidase-positive cells was (2.97 ± 0.25)% in control HSFs and (37.67 ± 1.53)% in senescent HSFs (P< 0.01). A significant increase was observed in the β-catenin/GAPDH protein ratio and β-catenin/GAPDH mRNA ratio in control HSFs compared with the senescent HSFs (0.62 ± 0.03 vs. 0.31 ± 0.01, t = 14.97, P < 0.01; 0.59 ± 0.04 vs. 0.29 ± 0.30, t = 10.06, P < 0.01). Conclusions The two-hour treatment with H2O2 of 150 μmol/L could induce the premature senescence of HSFs, and there is a notable decrease in the expression of β-catenin in prematurely senescent HSFs induced by oxidative stress, implying that β-catenin is an important target gene for the regulation of skin aging.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

黄芪甲苷对光老化小鼠皮肤中转化生长因子-β信号通路的影响

目的 探讨黄芪甲苷对皮肤光老化的保护机制.方法 BALB/c小鼠分为模型组、模型+基质组、模型+黄芪甲苷组、正常对照组.RT-PCR测定转化生长因子β受体Ⅱ(TGF-βRⅡ)、Smad 7的mRNA表达水平,免疫组化观察TGF-βR Ⅱ、Smad 7在小鼠皮肤组织中的蛋白表达情况.结果 正常对照组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.5688±0.0439、0.5900 ±0.0585,阳性表达率分别为(53.00 ±4.72)%、(47.50±3.81)%;模型组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.2588±0.0283、0.8637±0.0514,阳性表达率分别为(28.20±5.24)%、(82.06±2.18)%;模型+基质组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.2653±0.0456、0.8553±0.0575,阳性表达率分别为(28.74±2.28)%、(82.62±4.02)%;模型+黄芪甲苷组中TGF-βRⅡ、Smad 7的灰度值比值分别为0.3767±0.0374、0.7131±0.0410,阳性表达率分别为:(41.64±2.59)%、(64.36±2.62)%.皮肤组织中TGF-βRⅡ和Smad灰度值比值4组小鼠间比较,F值分别为80.98和736.80,TGF-βRⅡ和Smad 7的阳性表达率4组小鼠间比较,F值分别为45.36和132.25,P值均<0.01.与正常对照组相比,模型组TGF-βR Ⅱ mRNA和蛋白表达明显降低,Smad 7 mRNA和蛋白表达显著升高(P值均<0.01).与模型组和模型+基质组比较,模型+黄芪甲苷组TGF-βRⅡ mRNA和蛋白表达明显升高,Smad 7 mRNA和蛋白表达显著下调(P值均<0.01).模型+基质组TGF-βR Ⅱ、Smad 7的mRNA和蛋白表达与模型组相比差异无统计学意义(P值均>0.01).结论 黄芪甲苷可以通过上调TGF-βRⅡ表达和下调Smad 7表达而改变TGF-β通路的信号转导参与抗光老化.

Abstract：

Objective To study the protective mechanism of astragaloside on skin photoaging. Methods BALB/c mice were randomly divided into four groups: model group irradiated with ultraviolet rays (UV), model plus matrix group pretreated with the matrix before UV irradiation, model plus astragaloside group pretreated with astragaloside 0.08% cream before UV irradiation, normal control group received no irradiation or pretreatment. After 4-week irradiation, the mice were sacrificed, and skin tissues were resected from the back of these mice. Then, reverse transeription PCR (RT-PCR) and immunohistochemistry were performed to detect the mRNA and protein expression of TGF-βR Ⅱ and Smad 7, respectively. Gray scale ratio was used to represent the mRNA levels of TGF-βR Ⅱ and Smad 7. Results There was a significant difference in the mRNA level (F = 80.98, 736.80, respectively, both P < 0.01) and protein positivity rate (F = 45.36,132.25, respectively,both P < 0.01) of TGF-βR Ⅱ and Smad 7 among the 4 groups. The mRNA level and protein positivity rate of TGF-βR Ⅱwere 0.2588±0.0283 and (28.20 ± 5.24)% respectively in the model group, significantly lower than those in the normal control group[0.5688 ± 0.0439, (53.00 ± 4.72)%, both P < 0.01] and model plus astragaloside group [0.3767 ± 0.0374, (41.64 ± 2.59)%, both P< 0.01]; on the contrary, the mRNA level and protein positivity rate of Smad 7 in the model group [0.8637 ± 0.0514, (82.06 ± 2.18)%] were significantly higher than those in the normal control group [0.5900 ± 0.0585, (47.50±3.81)%, both P < 0.01] and model plus astragaloside group [0.7131 ± 0.0410, (64.36 ± 2.62)%, both P< 0.01]. In the model plus astragaloside group, the mRNA level and protein positivity rate of TGF-βR Ⅱ were significantly higher than in the model plus matrix group [0.2653 ± 0.0456, (28.74 ± 2.28)%, both P < 0.01], while those of Smad 7 were statistically lower than in the model plus matrix group [0.8553 ± 0.0575, (82.62 ± 4.02)%, both P < 0.01]. However,no significant difference was observed in the mRNA level or protein positivity rate of TGF-βR Ⅱ or Smad 7 between the model group and model plus matrix group (all P > 0.01). Conclusion Astragaloside can prevent skin photoaging by the alteration of TGF-β pathway via up-regulating TGF-βR Ⅱ expression and down-regulating Smad 7 expression.     

槲皮素对瘢痕疙瘩成纤维细胞胶原合成的影响

目的研究槲皮素对瘢痕疙瘩成纤维细胞胶原合成的影响,探讨其作用机理。方法体外培养瘢痕疙瘩及正常皮肤成纤维细胞,应用羟脯氨酸比色法对不同药物浓度处理后成纤维细胞胶原合成量进行分析;RT-PCR及Real-timePCR检测Ⅰ、Ⅲ型胶原及TGFβ-1基因表达水平。结果槲皮素可抑制体外培养瘢痕疙瘩成纤维细胞胶原合成,该作用呈剂量依赖效应;槲皮素可降低Ⅰ、Ⅲ型胶原和TGFβ-1基因mRNA水平。结论槲皮素对于瘢痕疙瘩成纤维细胞胶原合成有显著抑制效应,其作用机制之一为通过抑制TGFβ-1基因在转录水平降低了前胶原mRNA水平,因而使成纤维细胞胶原合成减少。