Rac1蛋白调控表皮干细胞迁移促进创面愈合的研究

Modulatory effect of Rac1 protein on epidermal stem cells migration during wound healing

目的观察Rac1蛋白对表皮干细胞（ESC）迁移运动的调控作用，为完善创面愈合的基础理论以及指导临床应用提供参考。方法将慢病毒空载体FUGW单独和分别与Rac1蛋白抑制型突变体Rac1T17N、Rac1蛋白活化型突变体Rac1Q61L融合后转染入ESC，按照随机数字表法分为3部分进行如下实验。（1）将ESC分别接种于Ⅰ型胶原溶液（20μg／mL）、Ⅳ胶原溶液（20μg／mL）或纤连蛋白溶液（10μg／mL）包被的24孔细胞培养板，采用CytoTox 96比色试剂盒检测ESC对不同基质的黏附率。（2）选取上述黏附于Ⅳ型胶原的1000个ESC，四甲基异硫氰酸罗丹明标记的鬼笔环肽染色，激光扫描共聚焦显微镜下观察黏附细胞的形态延展并比较面积大小。（3）选用Transwell小室，上室加入ESC、下室中加入含基质细胞衍生因子1（SDF-1）的限定性角质形成细胞无血清培养液（以不加SDF—1的培养液为对照），倒置相差显微镜下观察ESC的趋化能力，结果以细胞迁移变化率表示。（4）将ESC接种于6孔细胞培养板孵育12h，加入含4μg／mL丝裂霉素c的培养液继续孵育2h，于单层贴壁细胞划痕，6、12h后统计剩余划痕宽度百分率。对数据进行t检验。结果与转染FUGW的ESC比较，转染Rac1Q61L的ESC对Ⅰ型胶原的黏附率明显增加（t=5．302，P〈0．05），转染Rac1T17N的ESC对Ⅰ型胶原（t=13．741，P〈0．05）、Ⅳ型胶原（t=15．676，P〈0．05）及纤连蛋白（t=8．256，P〈0．05）的黏附率均明显下降。激光扫描共聚焦显微镜下观察，与转染FUGW的ESC比较，转染Rac1Q61L的ESC面积明显增大，细胞边缘有层板状伪足伸出；转染Rac1T17N的ESC面积显著缩小。在趋化因子SDF-1作用下，与转染FUGW的ESC比较，转染Rac1Q61L的ESC迁移变化率升高43．4％，转染Rac1T17N的ESC迁移变化率下降78．0％；无SDF—1作用时，与转染FUGW的ESC比较，转染Rac1T17N的ESC迁移变化率下降55．2％，转染Rac1Q61L的ESC迁移变化率未见明显变化（升高1．7％）。划痕后6、12h，转染Rac1Q61L的ESC剩余划痕宽度百分率分别为（39±9）％、（6±5）％，低于转染FUGW的ESC[（43±5）％，t=1．027，P〉0．05；（18±7）％，t=4．389，P〈0．05]；划痕后6、12h，转染Rac1T17N的ESC剩余划痕宽度百分率分别为（81±9）％、（71±11）％，明显高于转染FUGW的ESC（t值分别为11．386、11．726，P值均小于0．05）。结论Rac1蛋白可通过影响ESC的黏附、延展以及趋化能力调控细胞迁移，并可能因此参与ESC促进创面愈合的进程。

Objective To investigate modulatory role of Rac1 protein in epidermal stern cell （ESC） migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application. Methods 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 transfeeted 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 （20μg/mL） , collagen Ⅳ（20 μg/mL） or fibronectin （ 10 μg/mL）. Cells adhered to above matrices were quantitated using CytoTox 96 colorimetrie 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. Results 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 Rac1TlTN-transfected ESC was respectively larger and smaller as compared with that of hlank 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-trans- fected 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 atler 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）. Conclusions Rac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an aetive role in wound healing accelerated by ESC.