摘要
目的探讨丹参对系统性硬化病患者皮损高胶原合成成纤维细胞克隆Ⅰ型前胶原基因转录的调控。方法采用系统性硬化病患者皮损和正常人皮肤胶原合成异质性成纤维细胞克隆,通过四甲基偶氮唑盐比色法,检测1g/L丹参注射液及其主要水溶性单体(20mg/L丹参素钠、5mg/L丹酚酸B、5mg/L原儿茶醛)和脂溶性单体(5mg/L丹参酮ⅡA)对系统性硬化病和正常人高、低胶原合成克隆增殖活性(A490)的影响。利用瞬时转染、双荧光素酶报告基因技术,检测上述药物对克隆Ⅰ型前胶原仅1链(COL1A1)基因近端启动区活性的调控。结果在早期(≤3d),丹参注射液及单体对成纤维细胞克隆增殖的抑制不显著(P〉0.05)。但随作用时间延长,它们对克隆增殖的抑制作用多逐渐增强,第5天,丹参注射液组与水溶性阴性对照组比较,差异有统计学意义(q’=3.22,P〈0.01);第7天,丹参注射液组、丹酚酸B组和原儿茶醛组与水溶性阴性对照组比较,差异均有统计学意义(q’分别为4.74、3.03、2.56,P值均〈0.05)。第5天和第7天,丹参酮ⅡA组与脂溶性阴性对照组比较,差异均有统计学意义(t值分别为2.22和2.15,P值均〈0.05)。丹参注射液、丹参酮ⅡA和原儿茶醛抑制系统性硬化病和正常人成纤维细胞克隆中COL1A1近端启动区的活性(P〈0.01),前两种药优先下调系统性硬化病高胶原合成克隆中COL1A1近端启动区的活性,COL1A1近端启动区在系统性硬化病高、低胶原合成克隆中的活性水溶性阴性对照组为12.019±0.830和5.388±0.480,丹参注射液组为4.445±1.061和2.856±0.597,F=31.78,P〈0.01;脂溶性阴性对照组为14.155±0.672和4.299±0.252,丹参酮ⅡA组为9.638±0.854和3.192±0.450,F=24.10,P〈0.01。结论丹参可抑制系统性硬化病高胶原合成成纤维细胞克隆Ⅰ型前胶原基因的转录,其单体丹参酮ⅡA和原儿茶醛可能发挥主要作用。
Objective To study the transcriptional regulation of type Ⅰ procollagen gene in systemic seleroderma (SS)-derived high collagen-producing fibroblast clones by Radix Salviae Miltiorrhizae (RSM). Methods Fibroblast clones with different collagen-producing capacity were previously obtained from patients with SS and normal human controls, and divided into 5 groups to be treated with RSM (1 g/L) injection, its water-soluble active monomers including sodium danshensu (20 mg/L), salvianolie acid B (5 mg/L) and protoeatechuic aldehyde (5 mg/L), and lipid-soluble active monomer (tanshinone ⅡA, 5mg/L) respectively. The fibroblast clones incubated with no drugs served as the water soluble negative control group, and those with dimethyl sulfoxide (DMSO) as the lipid soluble negative control group. MTT assay was performed to evaluate the proliferation of the fibroblast clones after 1-, 3-, 5-, and 7-day treatment, transient transfection and duallueiferase reporter assay system to quantify the relative activity of collagen type Ⅰ, alpha 1 (COL1A1) proximal promoter in these fibroblast clones. Results The inhibitory effect of RSM and its active monomers on the proliferation of fibroblast clones was inapparent within the initial 3 days (P 〉 0.05 ), but was enhanced with incubation time. A significant difference was observed in the proliferation level of fibroblast clones between RSM group and water-soluble negative control group on day 5 (q' = 3.22, P 〈 0.01 ), between RSM, salvianolic acid B, protocatechuic aldehyde groups and the water-soluble negative control group (q' =4.74, 3.03, 2.56, all P 〈 0.05) on day 7, and between tanshinone ⅡA and lipid-soluble negative control group on day 5 and 7 (t =2.22, 2.15, both P 〈 0.05). RSM injection, tanshinone ⅡA and protocatechuic aldehyde significantly inhibited COL1A1 proximal promoter activity in SS-derived and normal control fibroblast clones (all P 〈 0.01 ), and the former two drugs preferentially downregulated COL1A1 proximal promoter activity in SS-derived high collagenproducing fibroblast clones. Significantly different COL1A1 proximal promoter activity was observed in SS-derived high and low collagen-producing fibroblast clones between water-soluble negative control group and RSM injection group (12.019 ± 0.830 vs. 4.445 ± 1.061, 5.388 ± 0.480 vs. 2.856 ± 0.597, F = 31.78, P 〈 0.01), and between lipid-soluable negative control group and tanshinone ⅡA group (14.155± 0.672 vs. 9.638 ± 0.854, 4.299 ± 0.252 vs. 3.192 ± 0.450, F = 24.10, P 〈 0.01). Conclusions RSM inhibits the transcription of COL1A1 gene in SS-derived high collagen-producing fibroblast clones, which may be mainly attributed to tanshinone ⅡA and protocatechuic aldehyde.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2011年第10期693-696,共4页
Chinese Journal of Dermatology
基金
国家自然科学基金(30471561)
关键词
硬皮病
系统性
成纤维细胞
克隆细胞
胶原Ⅰ型
丹参
Scleroderma, systemic
Fibroblasts
Clone cells
Collagen type Ⅰ
Salvia miltiorrhiza