探索趋化因子CXC配体12（CXCL12）预处理骨髓间充质干细胞（BMSCs）对小鼠全层皮肤缺损创面愈合的影响及其机制。

选取第3~5代BMSCs，分为正常对照组、单纯CXCL12组、CXCL12+CXCL12受体阻断剂组并进行相应处理。培养48 h后，采用细胞计数试剂盒8（CCK-8）法检测细胞存活率，采用5-乙炔基-2′-脱氧尿嘧啶核苷（EdU）细胞增殖检测试剂盒染色观察细胞增殖活力；培养24 h后，采用蛋白质印迹法检测鸟苷酸交换因子1（Epac1）、细胞外调节蛋白激酶（Erk）、磷酸化Erk（p-Erk）的蛋白表达并计算p-Erk与Erk比值。以上实验样本数均为6。取18只成年雄性BALB/c小鼠，按照随机数字表法分为PBS对照组、BMSCs组、CXCL12预处理BMSCs组，每组6只，于小鼠背部建立全层皮肤缺损创面模型，3 d后通过创周组织皮下注射分别给予100 μl的PBS、100 μl含1×10⁶个BMSCs细胞悬液、100 μl含1×10⁶个CXCL12预处理BMSCs细胞悬液。伤后4、7、10 d，观察创面愈合情况并计算创面愈合率。伤后11 d，收集3组小鼠创面组织标本，采用苏木精-伊红染色法和Masson染色法分别检测创面组织缺损长度及胶原容积分数（CVF）。

培养48 h后，与正常对照组比较，单纯CXCL12组细胞存活率明显升高（P<0.05）；与单纯CXCL12组比较，CXCL12+CXCL12受体阻断剂组细胞存活率明显降低（P<0.05）。培养48 h后，与正常对照组比较，单纯CXCL12组细胞EdU染色数量增多，细胞增殖活力增强；与单纯CXCL12组比较，CXCL12+CXCL12受体阻断剂组细胞EdU染色数量减少，细胞增殖活力减弱。培养24 h后，与正常对照组比较，单纯CXCL12组细胞Epac1蛋白表达及p-Erk与Erk比值明显升高（P<0.05）；与单纯CXCL12组比较，CXCL12+CXCL12受体阻断剂组细胞Epac1蛋白表达及p-Erk与Erk比值均明显下降（P<0.05）。伤后4、7、10 d，CXCL12预处理BMSCs组小鼠创面愈合率（分别为56.7%±2.6%、70.4%±1.6%、84.5%±0.5%）均明显高于BMSCs组（分别为37.4%±5.2%、49.2%±3.7%、67.9%±1.1%），P值均<0.05。伤后11 d，CXCL12预处理BMSCs组创面组织缺损长度明显短于BMSCs组［（1.79±0.05） mm 比 （2.20±0.15） mm，P<0.05］。伤后11 d，与PBS对照组比较，BMSCs组小鼠创面组织CVF明显升高（P<0.05）；与BMSCs组比较，CXCL12预处理BMSCs组小鼠创面组织CVF明显降低（P<0.05）。

CXCL12预处理BMSCs能够明显促进小鼠全层皮肤缺损创面愈合，其机制可能为CXCL12通过激活Epac1/Erk信号通路改善BMSCs的生物学功能，从而提高BMSCs治疗皮肤缺损创面效果。

To investigate the effects and underlying mechanism of CXC chemokine ligand-12 （CXCL12） pretreated bone marrow mesenchymal stem cells （BMSCs） on the wound healing of full-thickness skin defects in mice.

The 3^rd to 5^th passages of BMSCs were divided into control group， CXCL12 group， CXCL12 + CXCL12 receptor antagonist group and treated accordingly. After 48 h of culture， the cell survival rate was detected by cell counting kit 8 （CCK-8） method， and the cell proliferation activity was observed by staining with 5-ethynyl-2'-deoxyuridine （EdU） cell proliferation test kit. After 24 h of culture， the protein expressions of exchange protein directly activated by cAMP 1 （Epac1）， extracellular regulated protein kinase （Erk）， phosphorylated Erk （p-Erk） were detected by Western blotting， and the ratio of p-Erk to Erk was calculated. The number of samples in above experiments was six. Eighteen adult male BALB/c mice were divided into PBS group， BMSCs group and CXCL12 pretreated BMSCs group （6 mice in each group） according to the random number table method. Full-thickness skin defect wound model was created on the back of all mice. On day 3 post-wounding， the mice in PBS group， BMSCs group and CXCL12 pretreated BMSCs group were topically， intra-dermally administered 100 μl of PBS， 100 μl of cell suspension containing 1×10⁶ BMSCs and 100 μl of cell suspension containing 1×10⁶ CXCL12 pretreated BMSCs， respectively. On day 4， 7 and 10 post-wounding， the wound healing was observed and the wound healing rate was calculated. On day 11 post-wounding， the samples of wound tissue from 3 groups of mice were collected. Hematoxylin-eosin staining and Masson staining were used to detect the length of wound tissue defect and the collagen volume fraction （CVF）， respectively.

After 48 h of culture， compared with control group， the cell survival rate of CXCL12 group was significantly increased （P<0.05）. Compared with CXCL12 group， the cell survival rate of CXCL12 + CXCL12 receptor antagonist group was significantly decreased （P<0.05）. After 48 h of culture， the number of EdU-positive cells was increased， and the cell proliferation activity was enhanced in CXCL12 group compared to those in control group. In CXCL12 + CXCL12 receptor antagonist group， the number of EdU-positive cells was decreased and the cell proliferation activity was attenuated compared to those in CXCL12 group. After 24 h of culture， compared with control group， the protein level of Epac1 and the ratio of p-Erk to Erk in CXCL12 group were significantly increased （P<0.05）. Compared with CXCL12 group， the protein level of Epac1 and the ratio of p-Erk to Erk in CXCL12 + CXCL12 receptor antagonist group were significantly decreased （P<0.05）. On day 4， 7 and 10 post-wounding， the wound healing rate of mice in CXCL12 pretreated BMSCs group （56.7%±2.6%，70.4%±1.6%，84.5%±0.5%，respectively） was significantly higher than that in BMSCs group （37.4%±5.2%，49.2%±3.7%，67.9%±1.1%，respectively），P<0.05. On day 11 post-wounding， the length of wound tissue defect in CXCL12 pretreated BMSCs group was significantly shorter than that in BMSCs group ［（1.79±0.05） mm vs. （2.20±0.15） mm，P<0.05）］. On day 11 post-wounding， the CVF of BMSCs group was significantly increased compared with PBS group （P<0.05）. Compared with BMSCs group， the CVF of CXCL12 pretreated BMSCs group was significantly decreased （P<0.05）.

Pretreatment of BMSCs with CXCL12 could significantly promote the wound healing of full-thickness skin defects in mice， and the mechanism may be ascribed to the activation of Epac1/Erk signaling pathway by CXCL12 in the improvement of BMSCs' biological function， which finally enhance the therapeutic effects of BMSCs on skin defect wounds.