香烟烟雾提取物致体外皮肤成纤维细胞损伤和对胶原合成的影响

Effect of Cigarette Smoke Extract on Cell Biological Activities and Collagen Synthesis of Primary Culture of Skin Fibroblast

目的探讨香烟烟雾提取物(CSE)对体外培养的皮肤成纤维细胞的损伤作用,观察CSE对成纤维细胞Ⅰ型胶原合成的影响作用。方法在光镜下观察染毒后细胞形态和结构的变化;采用四甲基偶氮唑盐(MTT)比色法检测不同浓度的CSE在不同的时间对体外皮肤成纤维细胞存活力的影响,并绘制不同浓度下细胞的生长曲线;β-半乳糖苷酶染色方法检测CSE连续作用5代后成纤维细胞β-半乳糖苷酶的变化情况;流式细胞仪检测不同浓度的CSE作用下和不同染毒时间的细胞周期变化情况;RT-PCR检测不同浓度的CSE对成纤维细胞Ⅰ型前胶原mRNA表达的影响。结果染毒后光镜下呈典型的损伤细胞形态;随着CSE染毒浓度的增大和时间的延长,细胞存活力明显下降;细胞生长曲线示各染毒组细胞生长速度较对照组明显减慢;染毒后细胞经5次传代后β-半乳糖苷酶染色呈明显的阳性,对照组染色为阴性;细胞周期动力学显示,随着作用浓度的增加和作用时间的延长,G1、G2期细胞数增加,S期细胞数减少;CSE作用后,成纤维细胞的Ⅰ型前胶原mRNA表达降低。结论CSE对体外成纤维细胞具有明显的损伤作用,对细胞的增殖和生长有一定的抑制作用;低浓度CSE长期作用的积累,使多次传代的成纤维细胞具有衰老细胞的特点;CSE能够抑制细胞活性,引起细胞DNA复制和有丝分裂障碍,对各期有不同程度的阻滞。CSE对成纤维细胞的胶原合成有一定抑制作用。

Objective To study the biological changes and the collagen synthesis of the primary cultured skin fibroblast treated with Cigarette Smoke Extract （CSE）. Methods The morphological changes of fibroblasts after 24 hours＇ treatment with CSE were observed with invert microscope. The inhibitory effect at different concentrations of CSE on fibroblast activities was determined by the tetrazolium dye colorimetric test （MTT Test）. The growth curves of fibroblasts treated with CSE were drawn with MTT method. Cell aging was observed with β-galactosidase, which was the biological marker of senescence. Flow cytometry （FCM） was used to estimate cell cycle phases after the fibroblasts were treated at different concentrations of CSE and different time. The mRNA expression of type Ⅰ procollagen was detected by RT-PCR. Results After the treatment, the fibroblasts displayed morphological changes and the growth of fibroblasts was apparently slowed down by CSE. The positive 8 galactosidase staining was observed in the treated fibroblasts, which were affected by CSE for 5 passages. FCM analysis demonstrated that CSE decreased the cells in S phase and increased the cells in G1 and G2 phase. The result of RT-PCR showed that type Ⅰ procollagen was decreased after the treatment with CSE. Conclusion CSE can not only inhibit the growth and proliferation of the skin fibroblasts, but also decrease collagen syn thesis of dermal fibroblast which is very important to the skin health.