摘要：
目的 探讨变性Ⅰ型胶原在人成纤维细胞(Fb)向肌Fb分化中的作用. 方法 取烧伤瘢痕手术患者捐献的少量正常皮肤,组织块培养法获得人Fb,并进行传代培养,取第4代细胞进行以下实验.(1)取Fb,按随机数字表法分为正常胶原组和变性胶原组,每组10孔.正常胶原组Fb接种于正常Ⅰ型胶原包被的盖玻片,变性胶原组Fb接种于变性Ⅰ型胶原包被的盖玻片.免疫组织化学法检测增殖细胞核抗原(PCNA)表达,计算PCNA表达阳性细胞百分比.(2)另取Fb,同(1)进行分组处理,每组12孔,噻唑蓝比色法检测细胞增殖活性.(3)另取Fb,同(1)进行分组处理,罗丹明-鬼笔环肽染色观察细胞微丝形态.(4)另取Fb,同(1)进行分组处理,免疫组织化学法检测细胞中α平滑肌肌动蛋白(α-SMA)表达,免疫荧光法检测细胞中OB钙黏蛋白表达.(5)另取Fb,按随机数字表法分为正常胶原组、变性胶原组和普通玻片组,每组6孔.正常胶原组和变性胶原组Fb处理同(1),普通玻片组Fb接种于无胶原包被的盖玻片.蛋白质印迹法检测细胞中α-SMA和OB钙黏蛋白表达.(6)另取Fb,同(5)进行分组处理,实时荧光定量反转录-聚合酶链反应法检测细胞中Ⅰ型胶原和Ⅲ型胶原及α-SMA的mRNA表达.对数据行t检验、单因素方差分析、LSD检验. 结果 (1)变性胶原组PCNA表达阳性细胞百分比为(83±9)％,明显高于正常胶原组的(29±9)％(t=13.53,P＜0.01).(2)变性胶原组Fb的增殖活性为0.32 ±0.06,明显高于正常胶原组的0.25 ±0.05(t=3.06,P＜0.01).(3)正常胶原组Fb微丝均纵向平行排列,贯穿细胞长轴.变性胶原组Fb微丝更为密集、粗大.(4)正常胶原组Fb基本呈典型的长梭形;变性胶原组Fb形态发生变化,细胞明显铺展.变性胶原组Fb的α-SMA、OB钙黏蛋白表达较正常胶原组强.(5)变性胶原组、正常胶原组、普通玻片组Fb的α-SMA表达量分别为1.69 ±0.41、0.89 ±0.27、1.46 ±0.42,变性胶原组Fb的α-SMA表达量明显高于正常胶原组(P＜0.01).变性胶原组、正常胶原组、普通玻片组Fb的OB钙黏蛋白表达量分别为5.17 ±0.28、2.21 ±0.10、4.01 ±0.56,变性胶原组Fb的OB钙黏蛋白表达量明显高于正常胶原组(P＜0.01).(6)正常胶原组、变性胶原组与普通玻片组Fb中Ⅰ型胶原的mRNA表达量相近(F =2.71,P＞0.05).正常胶原组Fb中Ⅲ型胶原及α-SMA的mRNA表达量明显低于变性胶原组(p＜0.01). 结论 变性Ⅰ型胶原影响Fb活性,诱导Fb向肌Fb分化.%Objective To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts.Methods A small amount of normal skin donated by burn patients undergoing scar surgery was collected.Human fibroblasts were obtained by method of explant culture and then sub-cultured.The fourth passage of cells were used in the following experiments.(1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table,with 10 wells in each group.Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips.Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips.Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method,and the percentage of PCNA positive cells was calculated.(2) Another batch of fibroblasts were grouped and treated as in (1),with 12 wells in each group.Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method.(3) Another batch of fibroblasts were grouped and treated as in (1),and the microfilament morphology of cells was observed by rhodamine-phalloidin staining.(4) Another batch of fibroblasts were grouped and treated as in (1).Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method,and expression of OB-cadherin of cells was detected by immunofluorescence method.(5) Another batch of fibroblasts were divided into normal collagen,denatured collagen,and common coverslips groups according to the random number table,with 6 wells in each group.Fibroblasts in normal collagen and denatured collagen groups were treated as in (1),while fibroblasts in common coverslips group were cultured on coverslips without collagen coating.Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting.(6) Another batch of fibroblasts were grouped and treated as in (5),and then the mRNA expressions of collagen type Ⅰ,collagen type Ⅲ,and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction.Data were processed with t test,one way analysis of variance,and least-significant difference test.Results (1) The percentage of PCNA positive cells in denatured collagen group was (83 ± 9)％,significantly higher than (29 ± 9)％ in normal collagen group (t =13.53,P ＜ 0.01).(2) The proliferation activity of fibroblasts in denatured collagen group was 0.32 ± 0.06,significantly higher than 0.25 ± 0.05 in normal collagen group (t =3.06,P ＜ 0.01).(3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way,paralleling the long axis of cells.The microfilament of fibroblasts in denatured collagen group was denser and thicker.(4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically.Morphology of fibroblasts in denatured collagen group changed,and cells were obviously spreading.Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group.(5) Expressions of α-SMA of fibroblasts in denatured collagen,normal collagen,and common coverslips groups were respectively 1.69 ± 0.41,0.89 ±0.27,and 1.46 ±0.42.Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P ＜ 0.01).Expressions of OB-cadherin of fibroblasts in denatured collagen,normal collagen,and common coverslips groups were respectively 5.17 ± 0.28,2.21 ±0.10,and 4.01 ±0.56.Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P ＜ 0.01).(6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen,normal collagen,and common coverslips groups (F =2.71,P ＞ 0.05).The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P ＜ 0.01).Conclusions Denatured collagen type Ⅰ may influence the activity of fibroblasts,thus inducing fibroblasts differentiating into myofibroblasts.
