Effects and mechanisms of type Ⅲ collagen on wound healing in diabetic mice with full-thickness skin defect
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摘要:
目的 探讨Ⅲ型胶原蛋白（Col Ⅲ）对糖尿病小鼠全层皮肤缺损创面愈合的作用及其机制。 方法 该研究为包含成组设计、重复测量设计和析因设计的实验研究。取6只12周龄雄性db/db小鼠,采用Ⅱ型胶原酶消化法提取皮肤成纤维细胞（Fb）。将细胞分为采用终质量浓度分别为0.05、0.10、0.20 mg/mL的重组人Col Ⅲ处理的低浓度组、中浓度组、高浓度组及用磷酸盐缓冲液（PBS）处理的对照组。行细胞划痕试验后计算划痕后48 h划痕愈合率,行细胞Transwell试验后统计培养24 h后迁移细胞数,采用细胞计数试剂盒8检测培养3、5、7 d后细胞增殖活力,行免疫荧光染色检测培养24 h后细胞中α平滑肌肌动蛋白（α-SMA）、核因子κB1、p65的表达,行实时荧光定量反转录PCR检测培养24 h后细胞中增殖细胞核抗原（PCNA）、核因子κB1、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-6的mRNA表达。实验样本数均为5。取30只8周龄雄性C57BL/6J小鼠,成功制备糖尿病小鼠全层皮肤缺损创面后,按随机数字表法将小鼠分为分别于创缘皮下注射PBS和0.20 mg/mL Col Ⅲ溶液的PBS组、Col Ⅲ组,每组15只,计算术后7、14 d创面愈合率。取术后14 d创面组织,行Masson染色后量化胶原纤维沉积面积百分比,行免疫组织化学染色检测TNF-α、核因子κB1的表达,行免疫荧光染色检测CD31、PCNA的表达。 结果 与对照组比较,中浓度组和高浓度组细胞划痕后48 h划痕愈合率均明显升高（P<0.05）,培养24 h后迁移细胞数均明显增多（P<0.05）,培养3、5、7 d后增殖活力均明显升高（P<0.05）。培养24 h后,与对照组比较,高浓度组细胞中α-SMA的表达明显升高（P<0.05）,核因子κB1、p65的表达均明显降低（P<0.05）；中浓度组细胞中PCNA的mRNA表达明显升高（P<0.05）,IL-6的mRNA表达明显降低（P<0.05）；高浓度组细胞中PCNA的mRNA表达明显升高（P<0.05）,核因子κB1、TNF-α、IL-1β和IL-6的mRNA表达均明显降低（P<0.05）。术后7、14 d,Col Ⅲ组小鼠创面愈合率分别为（61.8±4.4）%、（95.1±2.7）%,均明显高于PBS组的（52.0±3.9）%、（76.0±7.2）%（t值分别为3.68、5.53,P<0.05）。术后14 d,与PBS组比较,Col Ⅲ组小鼠创面组织中胶原纤维沉积面积百分比明显升高（t=4.41,P<0.05）,TNF-α、核因子κB1的表达均明显降低（t值分别为4.10、3.07,P<0.05）,CD31和PCNA的表达均明显升高（t值分别为4.01、3.23,P<0.05）。 结论 Col Ⅲ可促进糖尿病小鼠皮肤Fb增殖和迁移,抑制核因子κB1介导的炎症反应,促进创面胶原纤维沉积和血管再生,从而加速糖尿病小鼠全层皮肤缺损创面愈合。 Abstract:Objective To investigate the effects and mechanisms of type Ⅲ collagen (Col Ⅲ) on wound healing in diabetic mice with full-thickness skin defect. Methods This study was an experimental study using a grouped design combined with repeated-measures and factorial designs. Skin fibroblasts (Fbs) were isolated from 6 male diabetic db/db mice aged 12 weeks by type Ⅱ collagenase digestion and divided into low, medium, and high concentration groups, which were treated with recombinant human Col Ⅲ at final mass concentrations of 0.05, 0.10, and 0.20 mg/mL, respectively, and control group treated with an equal volume of phosphate-buffered saline (PBS). The scratch healing rate was calculated after cell scratch assay at 48 h after scratch; the number of migrated cells was calculated after Transwell assay at 24 h after culture; the cell proliferative activity was detected by cell counting kit-8 at 3, 5, and 7 d after culture; the expressions of α-smooth muscle actin (α-SMA), nuclear factor-κB1 (NF-κB1), and p65 in cells were detected by immunofluorescence staining at 24 h after culture; the mRNA expressions of proliferating cell nuclear antigen (PCNA), NF-κB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in cells were detected by real-time fluorescence quantitative polymerase chain reaction at 24 h after culture. Full-thickness skin defect wounds were successfully created in 30 male C57BL/6J mice aged 8 weeks and with diabetes. The mice were then divided into PBS group and Col Ⅲ group by the random number table method, which were subcutaneously injected with 100 μL of PBS and 0.20 mg/mL Col Ⅲ solution at the wound margin, with 15 mice in each group. The wound healing rates at 7 and 14 d after surgery were calculated. The wound tissue was harvested at 14 d after surgery for Masson staining to quantify the percentage of collagen deposition area, for immunohistochemistry to detect the expressions of TNF-α and NF-κB1, and for immunofluorescence staining to detect the expressions of CD31 and PCNA. The sample size was 5 for all experiments. Results Compared with those in control group, the scratch healing rates in medium and high concentration groups of cells at 48 h after scratch were significantly increased (P<0.05), the number of migrated cells at 24 h after culture was significantly increased (P<0.05), and the proliferative activities at 3, 5, and 7 d after culture were significantly increased (P<0.05). After 24 h of culture, compared with those in control group, the expression of α-SMA in high concentration group of cells was significantly increased (P<0.05), while the expressions of NF-κB1 and p65 were significantly decreased (P<0.05); the mRNA expression of PCNA in medium concentration group of cells was significantly increased (P<0.05), while that of IL-6 was significantly decreased (P<0.05); the mRNA expression of PCNA in high concentration group of cells was significantly increased (P<0.05), while the mRNA expressions of NF-κB1, TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.05). At 7 and 14 d after surgery, the wound healing rates of mice in Col Ⅲ group were (61.8±4.4)% and (95.1±2.7)%, respectively, which were significantly higher than (52.0±3.9)% and (76.0±7.2)% in PBS group (with t values of 3.68 and 5.53, respectively, P<0.05). At 14 d after surgery, compared with those in PBS group, the percentage of collagen deposition area in the wound tissue of mice in Col Ⅲ group was significantly increased (t=4.41, P<0.05), the expressions of TNF-α and NF-κB1 were significantly decreased (with t values of 4.10 and 3.07, respectively, P<0.05), and the expressions of CD31 and PCNA were significantly increased (with t values of 4.01 and 3.23, respectively, P<0.05). Conclusions Col Ⅲ can promote the proliferation and migration of skin Fbs in diabetic mice, inhibit NF-κB1-mediated inflammatory response, and promote collagen deposition and angiogenesis in wounds, thereby accelerating the healing of full-thickness skin defect wounds in diabetic mice. -
Key words:
- Collagen type Ⅲ /
- Diabetes mellitus /
- Fibroblasts /
- Wound healing /
- NF-κB /
- Inflammation /
- Wound repair
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参考文献
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图 1 4组糖尿病小鼠皮肤成纤维细胞各时间点划痕愈合情况 倒置相差显微镜 ×50。1A、1B、1C、1D.分别为对照组、低浓度组、中浓度组、高浓度组细胞划痕后即刻；1E、1F、1G、1H.分别为对照组、低浓度组、中浓度组、高浓度组细胞划痕后48 h,图1E剩余划痕面积与图1F相近但明显大于图1G、1H
注：低浓度组、中浓度组、高浓度组细胞分别用终质量浓度为0.05、0.10、0.20 mg/mL的Ⅲ型胶原蛋白处理,对照组细胞用磷酸盐缓冲液处理
图 2 4组糖尿病小鼠皮肤成纤维细胞培养24 h后迁移情况 结晶紫 ×200。2A、2B、2C、2D.分别为对照组、低浓度组、中浓度组、高浓度组,图2A中迁移细胞数与图2B相近但明显少于图2C、2D
注：低浓度组、中浓度组、高浓度组细胞分别用终质量浓度为0.05、0.10、0.20 mg/mL的Ⅲ型胶原蛋白处理,对照组细胞用等体积的磷酸盐缓冲液处理；细胞阳性染色为紫色
图 3 4组糖尿病小鼠皮肤成纤维细胞各时间点增殖活力比较（样本数为5,
注：低浓度组、中浓度组、高浓度组细胞分别用终质量浓度为0.05、0.10、0.20 mg/mL的Ⅲ型胶原蛋白处理,对照组细胞用磷酸盐缓冲液处理；处理因素主效应,F=61.74,P<0.001；时间因素主效应,F=764.78,P<0.001；两者交互作用,F=11.92,P<0.001；与对照组比较,aP<0.05
图 4 4组糖尿病小鼠皮肤成纤维细胞培养24 h后细胞中α-SMA、核因子κB1和p65的表达比较。4A、4B、4C、4D.分别为对照组、低浓度组、中浓度组、高浓度组细胞中α-SMA的表达,图4A中α-SMA的表达与图4B、4C相近但明显低于图4D Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×200；4E、4F、4G、4H.分别为对照组、低浓度组、中浓度组、高浓度组细胞中核因子κB1和p65的表达,图4E中核因子κB1和p65的表达与图4F、4G相近但明显高于图4H Alexa Fluor 594-Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×200；4I.蛋白表达（以荧光强度表示）条图（样本数为5,
注：低浓度组、中浓度组、高浓度组细胞分别用终质量浓度为0.05、0.10、0.20 mg/mL的Ⅲ型胶原蛋白处理,对照组细胞用磷酸盐缓冲液处理；α平滑肌肌动蛋白（α-SMA）、p65阳性染色均为绿色,核因子κB1阳性染色为红色,细胞核阳性染色为蓝色；4组细胞中α-SMA、核因子κB1、p65的表达水平总体比较,差异均有统计学意义（F值分别为12.14、13.26、13.66,P<0.001）；与对照组比较,aP<0.05
图 5 4组糖尿病小鼠皮肤成纤维细胞培养24 h后细胞中PCNA和炎症因子的mRNA表达比较（样本数为5,
注：低浓度组、中浓度组、高浓度组细胞分别用终质量浓度为0.05、0.10、0.20 mg/mL的Ⅲ型胶原蛋白处理,对照组细胞用等体积的磷酸盐缓冲液处理；4组细胞中增殖细胞核抗原（PCNA）、核因子κB1、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-6的mRNA表达水平总体比较,差异均有统计学意义（F值分别为47.55、10.22、13.21、6.65、8.49,P值分别为<0.001、<0.001、<0.001、0.004、0.001）；与对照组比较,aP<0.05
图 6 2组糖尿病小鼠各时间点全层皮肤缺损创面愈合情况。6A、6B、6C.分别为PBS组术后0（即刻）、7、14 d；6D、6E、6F.分别为Col Ⅲ组术后0、7、14 d,图6B、6C创面面积分别大于图6E、6F
注：磷酸盐缓冲液（PBS）组、Ⅲ型胶原蛋白（Col Ⅲ）组小鼠分别于创缘皮下注射PBS和0.20 mg/mL Col Ⅲ溶液；硅胶垫片内径为8 mm
图 7 2组糖尿病小鼠术后14 d全层皮肤缺损创面组织中炎症、组织再生和血管再生情况。7A~7C.PBS组；7D~7F.Col Ⅲ组；7A、7D为TNF-α的表达,图7A中TNF-α的表达较图7D增多 二氨基联苯胺-苏木精 ×200；7B、7E为核因子κB1的表达,图7B中核因子κB1的表达较图7E增多 二氨基联苯胺-苏木精 ×200；7C、7F为CD31和PCNA的表达,图7C中CD31和PCNA的表达低于图7F Alexa Fluor 594-Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×100
注：磷酸盐缓冲液（PBS）组、Ⅲ型胶原蛋白（Col Ⅲ）组小鼠分别于创缘皮下注射100 μL PBS和0.20 mg/mL Col Ⅲ溶液；肿瘤坏死因子α（TNF-α）、核因子κB1阳性染色为棕褐色,CD31阳性染色为红色,增殖细胞核抗原（PCNA）阳性染色为绿色
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