[Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts].

Y W Wang, H Zhang, P Cao, W F Zhang, L Tong, S H Li, Y Chen, C Han, H Guan
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引用次数: 0

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 3rd to 5th 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 3rd 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 3rd 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.

[来自小鼠真皮乳头细胞的细胞外小泡对人类肥厚性疤痕成纤维细胞的影响和机制]。
研究目的研究小鼠真皮乳头细胞胞外小泡(DPC-EVs)对人类肥厚性瘢痕成纤维细胞(HSFs)的影响及其机制。研究方法本研究为实验研究。从 10 只 6 周大的雄性 C57BL/6J 小鼠身上提取胡须的原生真皮乳头细胞(DPCs)并成功鉴定。通过超速离心法从第3至第5周期的DPCs中提取DPC-EVs,培养24 h后用透射电子显微镜观察其形态,并用纳米颗粒跟踪分析仪检测其颗粒直径(n=3)。第 3 期 HSFs 分为 DPC-EV 组和磷酸盐缓冲液(PBS)组,分别用 DPC-EVs 和 PBS 培养。进行细胞划痕试验,计算划痕后24 h的细胞迁移率(n=5)。使用细胞计数试剂盒 8 检测培养后 0(饥饿处理 12 h 后,加入 DPC-EVs 或 PBS 前)、24、48、72 和 96 h 的细胞增殖水平(n=4)。免疫荧光法和 Western 印迹法检测培养 24 h 后细胞中α-平滑肌肌动蛋白(α-SMA)和胶原Ⅰ型(ColⅠ)的蛋白表达,Western 印迹法检测培养 24 h 后细胞中类克鲁珀尔因子 4(KLF4)的蛋白表达。用 DPC-EVs 培养第 3 期 HSFs 24 h 后,按随机数字表将细胞分为空白对照组、KLF4 敲除组和 KLF4 过表达组。KLF4敲除组和KLF4过表达组的细胞先用KLF4敲除病毒培养24小时,然后KLF4敲除组的细胞再常规培养24小时,KLF4过表达组的细胞再用KLF4过表达病毒培养24小时。培养48 h后,用Western印迹法检测细胞中KLF4、α-SMA和ColⅠ的蛋白表达。结果培养 24 h 后,提取的 DPC-EV 呈囊泡状结构,平均颗粒直径为 108.8 nm。搔抓后 24 h,PBS 组 HSFs 的迁移率为(54±10)%,显著高于 DPC-EV 组的(29±8)%(t=4.37,Pt 值分别为 4.06、5.76 和 6.41,PConclusions):小鼠DPC-EV可抑制人HSFs的增殖和迁移,并通过激活KLF4显著抑制人HSFs纤维化标志物α-SMA和ColⅠ的表达。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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