组织蛋白酶B在光老化皮肤中的表达及意义

Expressions of cathepsin B in pbotoaging skin and its significance

目的探讨组织蛋白酶B在光老化皮肤中的表达意义。方法6例成人曝光和非曝光皮肤标本，采用免疫组化法定位及对比组织蛋白酶B的表达。体外培养原代人皮肤成纤维细胞，甲氧沙林＋UVA法体外诱导培养细胞光老化。衰老相关-β-半乳糖苷酶染色证明老化诱导成功。Western印迹技术及RT-PCR对比检测光老化成纤维细胞及正常成纤维细胞组织蛋白酶B蛋白及基因表达。结果6例成人曝光和非曝光活体皮肤均见组织蛋白酶B阳性染色，和非曝光部位相比，曝光部位皮肤阳性染色A值降低。Western印迹结果示，成纤维细胞光老化诱导组蛋白表达较UVA诱导组、甲氧沙林孵育组及空白组明显下调。光老化成纤维细胞诱导后1周，组织蛋白酶B与内参的灰度比由28．099±0．054下降为25．103±0．102，诱导后3周灰度值进一步下降为17．693±0．099。实时定量RT—PCR结果示，光老化细胞组织蛋白酶BmRNA表达下调为正常组的64％（P〈0．05）。结论组织蛋白酶B在光老化皮肤及老化成纤维细胞中表达降低，且有时间依赖性，与光老化皮肤自我修复能力下降有关。

Objective To investigate the expression of cathepsin B in photoaging skin and its significance. Methods Skin specimens were obtained from the right forearm （sun-exposed sites） and buttock （unexposed sites） of 6 healthy volunteers with informed consent and subjected to immunohistochemistry for the detection of eathepsin B expression. Primary human fibroblasts derived from the prepuce of children aged 3 to 6 years were cultured in vitro; after 10 to 15 passages, cells were divided into four groups to be treated with methoxsalen of 50 mg/L for 24 hours followed by ultraviolet A （UVA） exposure （premature senescence group）, phosphate buffered saline （PBS） only （control group）, UVA exposure only （UVA group）, methoxsalen only （methoxsalen group）. Then, the protein and mRNA expressions of cathepsin B were detected by Western blot and RT-PCR, respectively, in these fibroblasts 1, 2, 3 weeks after the treatment. Results Cathepsin B was observed in both exposed and unexposed sites of all volunteers, and the average absorbence of cathepsin B was significantly lower in exposed sites than in unexposed sites （0.2130 ± 0.7997 vs 0.4520 ± 0.5921, t = 5.37, P 〈 0.05）. Decreased protein expression of cathepsin B was also noted in the premature senescence group compared with the other three groups. Moreover, the gray ratio between cathepsin B protein and glyceraldehyde phosphate dehydrogenase （GAPDH） in premature senescence group reduced from 28.099 ± 0.054 before treatment to 25.103 ± 0.102 in week 1 and 17.693 ± 0.099 in week 3 after UVA exposure. RT-PCR revealed that the mRNA expression level of cathepsin B in fibroblasts of premature senescence group decreased by 36 percent compared with that in the control group. Conclusions The expression of cathepsin B decreases in photoaged skin as well as in UVA-exposed fibroblasts in a time-dependent manner, which may be associated with the self-repair of photoaged skin.