Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts
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摘要:
目的 探讨小鼠真皮毛乳头细胞外囊泡（DPC-EV）对人增生性瘢痕成纤维细胞（HSF）的影响及其机制。 方法 该研究为实验研究。取10只6周龄雄性C57BL/6J小鼠，提取触须的原代真皮毛乳头细胞（DPC）并成功鉴定。取第3~5代DPC，于培养24 h采用超高速离心法提取DPC-EV，采用透射电子显微镜观察形态、纳米颗粒跟踪分析仪检测粒径（样本数为3）。将第3代HSF分为DPC-EV组和磷酸盐缓冲液（PBS）组，分别加入DPC-EV、PBS培养，行细胞划痕试验并计算划痕后24 h细胞迁移率（样本数为5），采用细胞计数试剂盒8检测培养0（行饥饿处理12 h后、加入DPC-EV或PBS前）、24、48、72、96 h细胞增殖水平（样本数为4），采用免疫荧光法、蛋白质印迹法检测培养24 h细胞中α平滑肌肌动蛋白（α-SMA）及Ⅰ型胶原蛋白（ColⅠ）的蛋白表达情况，采用蛋白质印迹法检测培养24 h细胞中Krüppel样因子4（KLF4）的蛋白表达情况。取第3代HSF，加入DPC-EV培养24 h后，采用随机数字表法将HSF分为空白对照组、KLF4敲低组和KLF4过表达组，其中空白对照组细胞仅常规培养48 h，KLF4敲低组和KLF4过表达组细胞均先加入KLF4敲低病毒培养24 h，而后KLF4敲低组细胞常规培养24 h，KLF4过表达组细胞加入KLF4过表达病毒培养24 h。于培养48 h，采用蛋白质印迹法检测细胞中KLF4、α-SMA、ColⅠ的蛋白表达情况。 结果 培养24 h，提取的DPC-EV为囊泡状结构，平均粒径108.8 nm。划痕后24 h，PBS组HSF的迁移率为（54±10）%，明显高于DPC-EV组的（29±8）%（ t=4.37， P<0.05）。培养48、72、96 h，DPC-EV组HSF的增殖水平均明显低于PBS组（ t值分别为4.06、5.76、6.41， P<0.05）。培养24 h，DPC-EV组HSF中α-SMA和ColⅠ的蛋白表达均明显低于PBS组，而KLF4的蛋白表达明显高于PBS组。培养48 h，与空白对照组比较，KLF4敲低组HSF中KLF4的蛋白表达下调，α-SMA及ColⅠ的蛋白表达均上调；与KLF4敲低组比较，KLF4过表达组HSF中的KLF4的蛋白表达上调，ColⅠ和α-SMA的蛋白表达均下调。 结论 小鼠DPC-EV可抑制人HSF的增殖和迁移，并通过激活KLF4抑制人HSF中纤维化标志物α-SMA和ColⅠ的表达。 -
关键词:
- 瘢痕 /
- 胞外囊泡 /
- 成纤维细胞 /
- Krüppel样因子4 /
- 真皮毛乳头细胞
Abstract:Objective To investigate the influences and mechanism of extracellular vesicles from dermal papilla cells (DPC-EVs) of mice on human hypertrophic scar fibroblasts (HSFs). Methods The study was an experimental research. The primary dermal papilla cells (DPCs) of whiskers were extracted from 10 6-week-old male C57BL/6J mice and identified successfully. The DPC-EVs were extracted from the 3 rd to 5 th passage DPCs by ultracentrifugation, and the morphology was observed through transmission electron microscope and the particle diameter was detected by nanoparticle tracking analyzer ( n=3) at 24 h after culture. The 3 rd passage of HSFs were divided into DPC-EV group and phosphate buffer solution (PBS) group, which were cultured with DPC-EVs and PBS, respectively. The cell scratch test was performed and cell migration rate at 24 h after scratching was calculated ( n=5). The cell proliferation levels at 0 (after 12 h of starvation treatment and before adding DPC-EVs or PBS), 24, 48, 72, and 96 h after culture were detected by using cell counting kit 8 ( n=4). The protein expressions of α-smooth muscle actin (α-SMA) and collagen typeⅠ (ColⅠ) in cells at 24 h after culture were detected by immunofluorescence method and Western blotting, and the protein expression of Krüppel-like factor 4 (KLF4) in cells at 24 h after culture was detected by Western blotting. After the 3 rd passage of HSFs were cultured with DPC-EVs for 24 h, the cells were divided into blank control group, KLF4 knockdown group, and KLF4 overexpression group according to the random number table. The cells in blank control group were only routinely cultured for 48 h. The cells in KLF4 knockdown group and KLF4 overexpression group were incubated with KLF4 knockdown virus for 24 h, then the cells in KLF4 knockdown group were routinely cultured for 24 h while the cells in KLF4 overexpression group were incubated with KLF4 overexpression virus for 24 h. The protein expressions of KLF4, α-SMA, and ColⅠ in cells were detected by Western blotting at 48 h after culture. Results At 24 h after culture, the extracted DPC-EVs showed vesicular structure with an average particle diameter of 108.8 nm. At 24 h after scratching, the migration rate of HSFs in PBS group was (54±10)%, which was significantly higher than (29±8)% in DPC-EV group ( t=4.37, P<0.05). At 48, 72, and 96 h after culture, the proliferation levels of HSFs in DPC-EV group were significantly lower than those in PBS group (with t values of 4.06, 5.76, and 6.41, respectively, P<0.05). At 24 h after culture, the protein expressions of α-SMA and ColⅠ of HSFs in DPC-EV group were significantly lower than those in PBS group, while the protein expression of KLF4 was significantly higher than that in PBS group. At 48 h after culture, compared with those in blank control group, the protein expression of KLF4 of HSFs in KLF4 knockdown group was down-regulated, while the protein expressions of α-SMA and ColⅠ were both up-regulated; compared with those in KLF4 knockdown group, the protein expression of KLF4 of HSFs in KLF4 overexpression group was up-regulated, while the protein expressions of ColⅠ and α-SMA were down-regulated. Conclusions The DPC-EVs of mice can inhibit the proliferation and migration of human HSFs and significantly inhibit the expressions of fibrosis markers α-SMA and ColⅠ in human HSFs by activating KLF4. -
Key words:
- Cicatrix /
- Extracellular vesicles /
- Fibroblasts /
- Krüppel-like factor 4 /
- Dermal papilla cells
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参考文献
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1 小鼠真皮毛乳头细胞外囊泡（DPC-EV）的鉴定。1A.DPC-EV为囊泡状结构 透射电子显微镜×40 000；1B.DPC-EV平均粒径为108.8 nm，符合细胞外囊泡粒径
2 2组人增生性瘢痕成纤维细胞划痕后24 h的迁移情况 倒置相差显微镜×100。2A、2B.分别为PBS组在划痕后0（即刻）、24 h的划痕情况；2C、2D.分别为DPC-EV组在划痕后0、24 h的划痕情况，图2D的细胞迁移较图2B少
注：图中竖/斜线为细胞迁移的边缘线；磷酸盐缓冲液（PBS）组加入PBS，真皮毛乳头细胞外囊泡（DPC-EV）组加入DPC-EV
3 免疫荧光法检测的2组人增生性瘢痕成纤维细胞培养24 h后α-SMA和ColⅠ的蛋白表达 Alexa Fluor 488-4'，6-二脒基-2-苯基吲哚×10。3A、3B、3C.分别为PBS组细胞核染色、α-SMA染色、细胞核与α-SMA染色重叠图片，α-SMA的蛋白表达较多；3D、3E、3F.分别为DPC-EV组的细胞核染色、α-SMA染色、细胞核与α-SMA染色重叠图片，图3E细胞质中α-SMA的蛋白表达较图3B明显减少；3G、3H、3I.分别为PBS组细胞核染色、ColⅠ染色、细胞核与ColⅠ染色重叠图片，ColⅠ的蛋白表达较多；3J、3K、3L.分别为DPC-EV组细胞核染色、ColⅠ染色、细胞核与ColⅠ染色重叠图片，图3K细胞质中ColⅠ的蛋白表达较图3H明显减少
注：α-SMA为α平滑肌肌动蛋白，其阳性染色为绿色；ColⅠ为Ⅰ型胶原蛋白，其阳性染色为绿色；细胞核阳性染色为蓝色；磷酸盐缓冲液（PBS）组加入PBS，真皮毛乳头细胞外囊泡（DPC-EV）组加入DPC-EV
4 蛋白质印迹法检测的2组人增生性瘢痕成纤维细胞培养24 h后α-SMA和ColⅠ的蛋白表达
注：α-SMA为α平滑肌肌动蛋白，ColⅠ为Ⅰ型胶原蛋白，GAPDH为3-磷酸甘油醛脱氢酶，PBS为磷酸盐缓冲液，DPC-EV为真皮毛乳头细胞外囊泡；条带上方1、2分别指示加入PBS的PBS组和加入DPC-EV的DPC-EV组
5 蛋白质印迹法检测的2组人增生性瘢痕成纤维细胞培养24 h后KLF4的蛋白表达
注：KLF4为Krüppel 样因子4，GAPDH为3-磷酸甘油醛脱氢酶，PBS为磷酸盐缓冲液，DPC-EV为真皮毛乳头细胞外囊泡；条带上方1、2分别指示加入DPC-EV的DPC-EV组和加入PBS的PBS组
6 蛋白质印迹法检测的3组人增生性瘢痕成纤维细胞培养48 h后KLF4、ColⅠ、α-SMA的蛋白表达
注：KLF4为Krüppel样因子4，ColⅠ为Ⅰ型胶原蛋白，α-SMA为α平滑肌肌动蛋白，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3分别指空白对照组、KLF4敲低组、KLF4过表达组；空白对照组细胞仅常规培养，KLF4敲低组细胞先加入KLF4敲低病毒培养再常规培养，KLF4过表达组细胞先加入KLF4敲低病毒培养再加入KLF4过表达病毒培养
表1 2组人增生性瘢痕成纤维细胞培养各时间点细胞增殖水平比较（
表1. Comparison of cell proliferation levels of human hypertrophic scar fibroblasts at different time points of cell culture between the two groups
组别 样本数 0 h 24 h 48 h 72 h 96 h PBS组 4 0.213±0.012 0.607±0.100 0.907±0.088 1.287±0.065 1.504±0.064 DPC-EV组 4 0.216±0.018 0.444±0.051 0.672±0.075 1.033±0.059 1.195±0.073 t值 0.26 2.89 4.06 5.76 6.41 P值 0.999 0.181 0.035 0.006 0.004 注：0 h为行饥饿处理12 h后、真皮毛乳头细胞外囊泡（DPC-EV）组加入DPC-EV或磷酸盐缓冲液（PBS）组加入PBS前；处理因素主效应， F=80.93， P<0.001；时间因素主效应， F=391.40， P<0.001；两者交互作用， F=6.73， P<0.001 
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