长波紫外线对无血清培养人角质形成细胞增殖的影响

Effect of UVA on the Proliferation of Keratinocyte in Serum Free Medium

目的建立人角质形成细胞无血清培养方法;观察长波紫外线对无血清培养的人角质形成细胞增殖活性的影响.方法采用两步消化法分离角质形成细胞,并用K-SFM无血清培养液培养,观察细胞形态并行角蛋白K19免疫组织化学染色鉴定;采用0、3.07、6.14、12.29、24.58J/cm2剂量的长波紫外线(UVA)照射细胞,以改良四甲基偶氮唑盐(MTT)法检测细胞增殖活性.结果免疫组织化学鉴定结果示,细胞阳性率接近100%;随UVA剂量的增加,其损伤率分别为0%、7.27%、32.73%、72.73%及95.45%,引起半数细胞损伤的UVA剂量为8.09J/cm2.结论体外无血清培养可获取较多高纯度的角质形成细胞,是一种理想的皮肤角质形成细胞分离培养的方法;在此体系中,UVA对角质形成细胞的损伤作用与剂量相关.

Objective To establish a method for serum-free culture of human keratinocyte （HKC） and to observe the effect of UVA on the proliferation of HKC. Methods We used a two-step digestion method to isolate the HKC and a K-SFM serum-free culture medium to culture. The morphology of HKC was observed and the expression of keratin 19 （K19） in HKC was detected with immunocytochemical methods. HKC were irradiated by UVA at 0 J/cm2, 3.07 J/cm2, 6.14 J/cm2, 12.29 J/cm2 and 24.58 J/cm2. The proliferation of HKC was detected with MTT method. Results Positive cells with expression of K19 accounted for nearly 100%. With the increasing of the UVA dosage, the damage ratios were from 0%, 7.27%, 32.73%, 72.73% to 95.45%, the UVA dosage caused half of cell damaged was 8.09 J/cm2. Conclusions Serum-free culture seems to be an ideal method for the culture of HKC. The UVA damage on HKC is related to dosage.