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目的探讨角质形成细胞(keratinocytes,KC)热损伤后对真皮成纤维细胞(fibroblasts,Fb)Ⅰ、Ⅲ型胶原及基质金属蛋白酶1(matrix metalloproteinase 1,MMP-1)表达的影响。方法分离培养人正常Fb及KC,分别建立KC、Fb热损伤模型;收集正常及热损伤12 h后细胞培养上清,并配制成浓度为50%的细胞条件培养液。根据培养液不同将第3～5代Fb分为3组,分别采用含50%热损伤KC培养上清(A组)、含50%正常KC培养上清(B组)的条件培养液及单纯DMEM(C组)培养,24 h后收集3组细胞;另于培养0、1、2、6、12、24、48 h分别收集A组细胞。采用含50%热损伤Fb培养上清的条件培养液培养Fb,于0、1、2、6、12、24、48 h收集细胞。采用实时荧光定量PCR检测各时间点KC热损伤条件培养上清对FbⅠ、Ⅲ型胶原及MMP-1 mRNA表达影响,以及Fb热损伤条件培养上清对Fb MMP-1mRNA表达影响。结果 KC热损伤条件培养上清培养24 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均显著高于B、C组,差异有统计学意义(P<0.05)。培养2、6、12、24、48 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均高于0 h(P<0.05),1 h与0 h差异无统计学意义(P>0.05);随培养时间延长相对表达量逐渐增高,2 h后各时间点间比较差异均有统计学意义(P<0.05)。Fb热损伤条件培养上清培养1、2、6、12、24、48 h,MMP-1 mRNA相对表达量与0 h比较,差异均有统计学意义(P<0.05);培养2 h后相对表达量逐渐降低,各时间点间差异均有统计学意义(P<0.05)。结论热损伤后KC培养上清对FbⅠ、Ⅲ型胶原及MMP-1的表达具有调控作用。
Objective To investigate the effect of keratinocytes (KC) on the expression of collagen type Ⅰ and Ⅲ and the expression of matrix metalloproteinase 1 (MMP-1) in dermal fibroblasts (Fb). Methods Normal human Fb and KC were isolated and cultured to establish models of KC and Fb heat injury respectively. The supernatant of cell culture was collected after normal and thermal injury for 12 h, and the cell conditioned medium was prepared to 50% concentration. The 3rd to 5th passages of Fb were divided into 3 groups according to the different culture medium, and the conditioned medium containing 50% KC culture supernatant (group A), 50% normal KC culture supernatant (group B) DMEM (group C). Three groups of cells were collected after 24 h. Cells in group A were harvested at 0, 1, 2, 6, 12, 24 and 48 h respectively. Fb was cultured in a conditioned medium containing 50% heat-injured Fb culture supernatant and cells were harvested at 0, 1, 2, 6, 12, 24 and 48 h. Real-time fluorescent quantitative PCR was used to detect the expression of FbⅠ, Ⅲcollagen and MMP-1 mRNA in KC cells after heat injury at various time points, and the effect of Fb MMP-1mRNA expression on Fb thermal injury supernatant. Results The expression of collagen type Ⅰ, type Ⅲ and MMP-1 mRNA in group A were significantly higher than that in group B and C (P <0.05). The mRNA expression of type Ⅰ, type Ⅲ collagen and MMP-1 mRNA in group A were higher than that in 0 h (P <0.05) at 2, 6, 12, 24 and 48 h, but there was no significant difference between 1 h and 0 h > 0.05). The relative expression gradually increased with the prolongation of culture time, and the difference was statistically significant after 2 hours (P <0.05). The relative expression of MMP-1 mRNA at 1, 6, 6, 12, 24 and 48 h after cultured in Fb heat injury conditioned medium was significantly different from that at 0 h (P <0.05) The expression levels gradually decreased, and the differences among the time points were statistically significant (P <0.05). Conclusion The supernatant of KC after heat injury can regulate the expression of Fb Ⅰ, Ⅲ collagen and MMP-1.