Influence and mechanism of bone marrow mesenchymal stem cells overexpressing growth arrest specific 6 on full-thickness skin defect wounds in diabetic mice
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摘要:
目的 探讨过表达生长停滞特异性蛋白6（GAS6）的骨髓间充质干细胞（BMSC）,即GAS6/BMSC对糖尿病小鼠全层皮肤缺损创面的影响及其机制。 方法 该研究为实验研究。将12只8周龄雄性C57BL/6J小鼠按照随机数字表法分为仅造成全层皮肤缺损的对照创面组与造成糖尿病全层皮肤缺损的糖尿病创面组,每组6只小鼠。伤后3、7、14、21 d,计算创面愈合率。伤后21 d,收集创面组织标本,行苏木精-伊红染色观察组织病理学情况；行Masson染色检测胶原沉积情况；行免疫组织化学染色检测增殖细胞核抗原（PCNA）阳性细胞数和CD31阳性细胞数,分别表示细胞增殖情况和毛细血管密度；行免疫荧光染色检测F4/80和髓过氧化物酶（MPO）双阳性细胞数,表示胞葬情况。取2只4周龄雄性C57BL/6J小鼠,提取BMSC,通过腺病毒转染构建GAS6/BMSC并成功鉴定。取18只8周龄雄性C57BL/6J小鼠制成糖尿病全层皮肤缺损创面模型后,按照随机数字表法将其分为磷酸盐缓冲液（PBS）组、BMSC组和GAS6/BMSC组（每组6只小鼠）,伤后即刻于3组小鼠创面局部分别注射PBS、BMSC单细胞悬液、GAS6/BMSC单细胞悬液,于前述实验相同时间点计算创面愈合率、检测细胞增殖情况和毛细血管密度及胞葬情况。 结果 伤后3、7、14、21 d,糖尿病创面组小鼠创面愈合率均显著低于对照创面组（t值分别为7.99、8.62、9.80、5.85,P<0.05）。与对照创面组相比,糖尿病创面组小鼠伤后21 d的创面组织中大量炎症细胞浸润,胶原沉积减少。伤后21 d,糖尿病创面组小鼠创面组织中PCNA阳性细胞数和CD31阳性细胞数均显著少于对照创面组（t值分别为6.61、5.38,P<0.05）。伤后21 d,糖尿病创面组小鼠创面组织中F4/80和MPO双阳性细胞数为（3.3±0.8）个,较对照创面组的（12.7±1.8）个显著减少（t=11.00,P<0.05）。伤后14、21 d,BMSC组小鼠创面愈合率均显著高于PBS组（P<0.05）；伤后3、7、14、21 d,GAS6/BMSC组小鼠创面愈合率均显著高于BMSC组（P<0.05）。伤后21 d,BMSC组小鼠创面组织中PCNA阳性细胞数显著多于PBS组（P<0.05）,GAS6/BMSC组小鼠创面组织中PCNA阳性细胞数与CD31阳性细胞数均显著多于BMSC组（P<0.05）。伤后21 d,BMSC组小鼠创面组织中F4/80和MPO双阳性细胞数为（4.2±1.2）个,与PBS组的（3.5±1.1）个相近（P>0.05）；GAS6/BMSC组小鼠创面组织中F4/80和MPO双阳性细胞数为（8.2±1.2）个,显著多于BMSC组（P<0.05）。 结论 糖尿病小鼠全层皮肤缺损创面中存在巨噬细胞的胞葬功能障碍,而GAS6/BMSC可通过恢复巨噬细胞的胞葬作用促进创面愈合。 Abstract:Objective To investigate the influence and mechanism of bone marrow mesenchymal stem cells (BMSCs) overexpressing growth arrest specific 6, i.e. GAS6/BMSCs on full-thickness skin defect wounds in diabetic mice. Methods This study was an experimental study. Twelve 8-week-old male C57BL/6J mice were divided into a control wound group with only full-thickness skin defects and a diabetic wound group with diabetic full-thickness skin defects according to the random number table method, with 6 mice in each group. The wound healing rates were calculated at 3, 7, 14, and 21 days after injury. At 21 days after injury, wound tissue specimens were collected for hematoxylin-eosin staining to observe the histopathological conditions; Masson staining was performed to detect collagen deposition; immunohistochemical staining was performed to detect the number of proliferating cell nuclear antigen (PCNA)-positive cells and CD31-positive cells, representing cell proliferation and capillary density, respectively; immunofluorescence staining was performed to detect the number of F4/80 and myeloperoxidase (MPO) double-positive cells, indicating efferocytosis. Two 4-week-old male C57BL/6J mice were used to extract BMSCs, and GAS6/BMSCs were constructed through adenovirus transfection and successfully identified. Eighteen 8-week-old male C57BL/6J mice were used to create diabetic full-thickness skin defect wound models and divided into phosphate buffered solution (PBS) group, BMSC group, and GAS6/BMSC group (with 6 mice in each group) according to the random number table method. Immediately after injury, PBS, BMSC single-cell suspension, and GAS6/BMSC single-cell suspension were injected locally into the wounds of the three groups of mice, respectively. The wound healing rates were calculated, and the cell proliferation, capillary density, and efferocytosis were detected at the same time points as the previous experiments. Results At 3, 7, 14, and 21 days after injury, the wound healing rates of mice in diabetic wound group were significantly lower than those in control wound group (with t values of 7.99, 8.62, 9.80, and 5.85, respectively, P<0.05). Compared with those in control wound group, the wound tissue of mice in diabetic wound group showed the infiltration of a large number of inflammatory cells and reduced collagen deposition at 21 days after injury. At 21 days after injury, the number of PCNA-positive cells and CD31-positive cells in the wound tissue of mice in diabetic wound group were significantly less than that in control wound group (with t values of 6.61 and 5.38, respectively, P<0.05). At 21 days after injury, the number of F4/80 and MPO double-positive cells in the wound tissue of mice in diabetic wound group was 3.3±0.8, which was significantly less than 12.7±1.8 in control wound group (t=11.00, P<0.05). At 14 and 21 days after injury, the wound healing rates of mice in BMSC group were significantly higher than those in PBS group (P<0.05); at 3, 7, 14, and 21 days after injury, the wound healing rates of mice in GAS6/BMSC group were significantly higher than those in BMSC group (P<0.05). At 21 days after injury, the number of PCNA-positive cells in the wound tissue of mice in BMSC group was significantly higher than that in PBS group (P<0.05), and the number of PCNA-positive cells and CD31-positive cells in the wound tissue of mice in GAS6/BMSC group were significantly higher than that in BMSC group (P<0.05). At 21 days after injury, the number of F4/80 and MPO double-positive cells in the wound tissue of mice in BMSC group was 4.2±1.2, which was similar to 3.5±1.1 in PBS group (P>0.05); the number of F4/80 and MPO double-positive cells in the wound tissue of mice in GAS6/BMSC group was 8.2±1.2, which was significantly more than that in BMSC group (P<0.05). Conclusions Dysfunctional efferocytosis of macrophage exists in the full-thickness skin defect wounds of diabetic mice, while GAS6/BMSC can promote wound healing by restoring the efferocytosis of macrophages. -
Key words:
- Diabetes mellitus, type 2 /
- Ulcer /
- Mesenchymal stem cells /
- Macrophages /
- Efferocytosis /
- Growth arrest specific 6 /
- Wound repair
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参考文献
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图 1 2组小鼠全层皮肤缺损创面伤后各时间点面积情况。1A、1B.分别为对照创面组与糖尿病创面组伤后0 d（即刻）创面情况,创面面积一致；1C、1D.分别为对照创面组与糖尿病创面组伤后7 d创面情况,图1C创面面积显著小于图1D；1E、1F.分别为对照创面组与糖尿病创面组伤后14 d创面情况,图1E创面面积显著小于图1F；1G、1H.分别为对照创面组与糖尿病创面组伤后21 d创面情况,图1G创面面积显著小于图1H
注：糖尿病创面组小鼠制成糖尿病模型后致伤,对照创面组小鼠单纯致伤
图 2 2组小鼠全层皮肤缺损创面伤后21 d组织中细胞增殖情况、毛细血管密度和胞葬情况。2A、2B.分别为糖尿病创面组与对照创面组PCNA阳性细胞（棕褐色）情况,图2A中阳性细胞数显著少于图2B 二氨基联苯胺-苏木精×200；2C、2D.分别为糖尿病创面组与对照创面组CD31阳性细胞（棕褐色）情况,图2C中阳性细胞数显著少于图2D 二氨基联苯胺-苏木精×200；2E、2F.分别为糖尿病创面组与对照创面组F4/80和MPO双阳性细胞（黄色）情况,图2E中双阳性细胞数显著少于图2F Alexa Fluor 488-Cy3-4',6-二脒基-2-苯基吲哚×200
注：糖尿病创面组小鼠制成糖尿病模型后致伤,对照创面组小鼠单纯致伤；分别以增殖细胞核抗原（PCNA）阳性细胞数、CD31阳性细胞数、F4/80（阳性为红色）和髓过氧化物酶（MPO,阳性为绿色）双阳性细胞数表示细胞增殖情况、毛细血管密度、胞葬情况
图 3 过表达GAS6的小鼠BMSC的鉴定。3A.培养48 h,GAS6/BMSC形态为长梭形 倒置荧光显微镜（明场通道）×200；3B.蛋白质印迹法检测显示GAS6/BMSC中GAS6蛋白表达水平显著高于作为对照的BMSC；3C.成脂诱导15 d后,GAS6/BMSC内可见脂滴（红色） 油红O×200；3D.成骨诱导15 d后,GAS6/BMSC内可见钙盐结节（红色） 茜素红S×200
注：GAS6为生长停滞特异性蛋白6,BMSC为骨髓间充质干细胞,GAS6/BMSC为过表达GAS6的BMSC
图 4 3组糖尿病小鼠全层皮肤缺损创面伤后各时间点面积情况。4A、4B、4C.分别为PBS组、BMSC组与GAS6/BMSC组伤后0 d（即刻）创面情况,创面面积一致；4D、4E、4F.分别为PBS组、BMSC组与GAS6/BMSC组伤后7 d创面情况,图4D与图4E创面面积相近,图4F创面面积显著小于图4E；4G、4H、4I.分别为PBS组、BMSC组与GAS6/BMSC组伤后14 d创面情况,图4H创面面积显著小于图4G,图4I创面面积显著小于图4H；4J、4K、4L.分别为PBS组、BMSC组与GAS6/BMSC组伤后21 d的创面情况,图4K创面面积显著小于图4J,图4L创面面积显著小于图4K
注：磷酸盐缓冲液（PBS）组、骨髓间充质干细胞（BMSC）组与过表达生长停滞特异性蛋白6的BMSC（GAS6/BMSC）组小鼠创面局部分别注射PBS、BMSC单细胞悬液、GAS6/BMSC单细胞悬液
图 5 3组糖尿病小鼠全层皮肤缺损创面伤后21 d组织中细胞增殖情况、毛细血管密度和胞葬情况。5A、5B、5C.分别为PBS组、BMSC组与GAS6/BMSC组PCNA阳性细胞（棕褐色）情况,图5B中阳性细胞数显著多于图5A,图5C中阳性细胞数显著多于图5B；5D、5E、5F.分别为PBS组、BMSC组与GAS6/BMSC组CD31阳性细胞（棕褐色）情况,图5E与图5D中阳性细胞数相近,图5F中阳性细胞数显著多于图5E；5G、5H、5I.分别为PBS组、BMSC组与GAS6/BMSC组F4/80和MPO双阳性细胞（黄色）情况,图5G与图5H中双阳性细胞数相近,图5I中双阳性细胞数显著多于图5H
注：磷酸盐缓冲液（PBS）组、骨髓间充质干细胞（BMSC）组与过表达生长停滞特异性蛋白6的BMSC（GAS6/BMSC）组小鼠创面局部分别注射PBS、BMSC单细胞悬液、GAS6/BMSC单细胞悬液；分别以增殖细胞核抗原（PCNA）阳性细胞数、CD31阳性细胞数、F4/80（阳性为红色）和髓过氧化物酶（MPO,阳性为绿色）双阳性细胞数表示细胞增殖情况、毛细血管密度、胞葬情况
Table 1. 2组小鼠全层皮肤缺损创面伤后各时间点愈合率比较（%,
组别 样本数 3 d 7 d 14 d 21 d 对照创面组 6 31.7±4.8 48.3±5.5 86.8±3.4 92.0±4.1 糖尿病创面组 6 10.8±4.2 24.3±4.0 65.8±4.0 79.0±3.6 t值 7.99 8.62 9.80 5.85 P值 <0.001 <0.001 <0.001 <0.001 注：糖尿病创面组小鼠制成糖尿病模型后致伤,对照创面组小鼠单纯致伤；处理因素主效应,F=263.80,P<0.001；时间因素主效应,F=1 090.00,P<0.001；两者交互作用,F=19.47,P<0.001 
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Table 2. 3组糖尿病小鼠全层皮肤缺损创面伤后各时间点愈合率比较（%,
组别 样本数 3 d 7 d 14 d 21 d PBS组 6 12.8±5.8 27.2±4.1 63.0±3.4 74.5±4.3 BMSC组 6 12.3±3.6 29.3±5.9 68.3±2.2 80.2±3.2 GAS6/BMSC组 6 31.7±4.4 49.7±6.4 84.0±2.9 92.0±2.6 F值 33.38 30.18 86.95 40.69 P值 <0.001 <0.001 <0.001 <0.001 P1值 0.980 0.780 0.015 0.030 P2值 <0.001 <0.001 <0.001 <0.001 注：磷酸盐缓冲液（PBS）组、骨髓间充质干细胞（BMSC）组与过表达生长停滞特异性蛋白6的BMSC（GAS6/BMSC）组小鼠创面局部分别注射PBS、BMSC单细胞悬液、GAS6/BMSC单细胞悬液；处理因素主效应,F=197.30,P<0.001；时间因素主效应,F=1 399.00,P<0.001；两者交互作用,F=10.41,P<0.001；F值、P值为各时间点组间总体比较所得,P1值为BMSC组与PBS组各时间点比较所得,P2值为GAS6/BMSC组与BMSC组各时间点比较所得
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