Chai Lin-lin, Cao Chuan, Zhao Shu-wen, Li Shi-rong, Bi Sheng, Gan Lu
Department of Plastic Surgery, Southwest Hospital, the Third Military Medical University, Chongqing 400038, China.
Zhonghua Shao Shang Za Zhi. 2011 Jun;27(3):205-9.
To investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application.
Constitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW, and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First, equal numbers of cells were inoculated into 24-well plates coated with collagen I (20 µg/mL), collagen IV (20 µg/mL) or fibronectin (10 µg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second, 1000 cells adhered to collagen IV, after being stained with tetramethyl rhodamine isothiocyanate-phalloidin, were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third, ESC with density of 2 × 10(5) cells per well were placed in upper compartment of Transwell chamber, DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope, and the result was denoted as migration rate. Lastly, ESC with density of 7.5 × 10(5) cells per well was inoculated into 6-well plates for 12 hours, and treated with 4 µg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.
Compared with that of blank control, the number of Rac1Q61L-transfected cells adhered to collagen I was significantly increased (t = 5.302,P < 0.05), while the number of Rac1T17N-transfected cells adhered to collagen I, IV, and fibronectin were all obviously decreased (with t value respectively 13.741, 15.676, 8.256, P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%, while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching, the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control [(39 ± 9)% vs. (43 ± 5)%, (6 ± 5)% vs. (18 ± 7)%, with t value respectively 1.027, 4.389, with P value respectively above and below 0.05], while that in Rac1T17N-transfected ESC [(81 ± 9)%, (71 ± 11)%, respectively] was obviously higher as compared with that in blank control (with t value respectively 11.386, 11.726, P values all below 0.05).
Rac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an active role in wound healing accelerated by ESC.
探讨Rac1蛋白在伤口愈合过程中对表皮干细胞（ESC）迁移的调节作用，为丰富伤口愈合基础理论及指导临床应用提供参考。
采用逆转录病毒载体FUGW将Rac1蛋白的组成型活性突变体（Rac1Q61L）或显性负性异构体（Rac1T17N）转染至ESC，以单独转染逆转录病毒载体FUGW的ESC作为空白对照。根据随机数字表将细胞分为3组并进行如下处理。首先，将等量细胞接种于包被有I型胶原（20 μg/mL）、IV型胶原（20 μg/mL）或纤连蛋白（10 μg/mL）的24孔板中。使用细胞毒性96比色试剂盒对黏附于上述基质的细胞进行定量。其次，1000个黏附于IV型胶原的细胞经异硫氰酸四甲基罗丹明-鬼笔环肽染色后，收集用于在共聚焦激光扫描显微镜下观察细胞形态并比较铺展面积。第三，将密度为2×10⁵个细胞/孔的ESC置于Transwell小室的上室，在Transwell小室的下室中加入单独的DK-SFM培养基或含基质细胞衍生因子1（SDF-1）的培养基。使用倒置相差显微镜观察ESC的迁移情况，结果以迁移率表示。最后，将密度为7.5×10⁵个细胞/孔的ESC接种于6孔板中培养12小时，并用4 μg/mL丝裂霉素C处理2小时。划痕后6小时或12小时分别测量单层剩余划痕宽度，计算剩余划痕宽度百分比。数据采用t检验进行处理。
与空白对照相比，转染Rac1Q61L的细胞黏附于I型胶原的数量显著增加（t = 5.302，P < 0.05），而转染Rac1T17N的细胞黏附于I型、IV型胶原及纤连蛋白的数量均明显减少（t值分别为13.741、15.676、8.256，P值均低于0.05）。共聚焦激光扫描显微镜显示，与空白对照相比，转染Rac1Q61L的ESC（边缘有层状伪足）和转染Rac1T17N的ESC的铺展面积分别更大和更小。在SDF-1作用下，与空白对照相比，转染Rac1T17N的ESC迁移率降低了78.0%，转染Rac1Q61L的ESC迁移率增加了43.4%。在无SDF-1作用时，转染Rac1T17N的ESC迁移率降低了55.2%，而转染Rac1Q61L的ESC迁移率与空白对照接近。划痕后6小时或12小时，与空白对照相比，转染Rac1Q61L的ESC剩余划痕宽度百分比更低[（39±9）%对（43±5）%，（6±5）%对（18±7）%，t值分别为1.027、4.389，P值分别大于和小于0.05]，而转染Rac1T17N的ESC的剩余划痕宽度百分比[分别为（81±9）%、（71±11）%]明显高于空白对照（t值分别为11.386、11.726，P值均低于0.05）。
Rac1蛋白可能通过调节ESC的黏附、铺展和趋化性来控制其迁移，在ESC加速伤口愈合过程中发挥积极作用。
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