Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice
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摘要:
目的 探讨肝细胞生长因子（HGF）修饰的人脂肪间充质干细胞（ADSC）外泌体对糖尿病小鼠全层皮肤缺损创面的作用及其机制。 方法 采用实验研究方法。取2021年2—5月于空军军医大学第一附属医院整形科行腹部手术的3名健康女性（10~25岁）废弃脂肪组织，采用胶原酶消化法获取原代ADSC，培养7 d，用倒置相差显微镜观察细胞形态。取第3代ADSC，转染HGF慢病毒，培养5 d，采用成像系统观察细胞荧光情况并计算转染率。取第3~6代ADSC和第3~6代转染HGF的ADSC，均分别采用密度梯度离心法提取其外泌体，即为ADSC外泌体和HGF-ADSC外泌体。采用透射电子显微镜观察外泌体微观形态，采用流式细胞仪检测外泌体的CD9、CD63、CD81阳性表达。取24只6周龄雄性昆明小鼠，制作糖尿病模型后，在其背部制作全层皮肤缺损创面。按照随机数字表法将小鼠分为磷酸盐缓冲液（PBS）组、单纯HGF组、单纯ADSC外泌体组和HGF-ADSC外泌体组，每组6只，分别作相应处理。伤后3、7、10、14 d，观察创面愈合情况并计算其愈合率，使用多普勒血流仪检测创基血流强度并计算伤后10 d创基相对血流强度百分比。伤后10 d，采用免疫荧光法检测创面中Ki67阳性细胞数，采用免疫组织化学法检测创面中CD31阳性染色且成管样结构的新生血管数，采用蛋白质印迹法检测创面中内皮型一氧化氮合酶（eNOS）、磷脂酰肌醇3激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（Akt）、磷酸化Akt（p-Akt）的蛋白表达并计算p-PI3K与PI3K比值及p-Akt与Akt比值。伤后14 d，采用苏木精-伊红染色法、Masson染色法分别检测创面皮肤组织缺损长度及胶原新生情况，并计算胶原容积分数（CVF）。样本数均为3。对数据行重复测量方差分析、单因素方差分析及Tukey检验。 结果 原代ADSC培养7 d，细胞呈梭形，生长密集后呈旋涡状排列。培养5 d后，第3代转染HGF的ADSC携带绿色荧光，转染率为85%。ADSC外泌体及HGF-ADSC外泌体微观形态相似，均呈囊泡状结构，平均粒径分别为103、98 nm，均呈CD9、CD63、CD81阳性。伤后3 d，4组小鼠创面的创周均红肿且有少量渗液；伤后7 d，HGF-ADSC外泌体组创面逐渐缩小，其余3组创面缩小不明显；伤后10 d，4组创面均缩小、结痂；伤后14 d，HGF-ADSC外泌体组创面基本愈合，其余3组均有残余创面。伤后3 d，4组创面愈合率接近（P>0.05）；伤后7、10 d，HGF-ADSC外泌体组创面愈合率均分别明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为13.11、13.11、11.89，12.85、11.28、7.74，P<0.01）；伤后14 d，HGF-ADSC外泌体组创面愈合率明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为15.50、11.64、6.36，P值均<0.01）。伤后3 d，4组小鼠创基均无明显血运；伤后7 d，HGF-ADSC外泌体组创基开始有微血管形成，其余3组创基仅创缘充血；伤后10 d，除PBS组创基仍无明显血运外，其余3组均有微血管形成；伤后14 d，HGF-ADSC外泌体组创基血流信号强于其余3组且分布均匀。伤后10 d，HGF-ADSC外泌体组创基相对血流强度百分比明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为23.73、19.32、9.48，P值均<0.01）；HGF-ADSC外泌体组创面中Ki67阳性细胞数及新生血管数均明显多于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为19.58、18.20、11.04，20.68、13.79、8.12，P<0.01）。伤后10 d，HGF-ADSC外泌体组创面中eNOS蛋白表达水平明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为53.23、42.54、26.54，P值均<0.01）；HGF-ADSC外泌体组创面中p-PI3K与PI3K的比值及p-Akt与Akt的比值均分别明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为16.11、11.78、6.08，65.54、31.63、37.86，P<0.01）。伤后14 d，HGF-ADSC外泌体组创面皮肤中组织缺损长度明显短于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为20.51、18.50、11.99，P值均<0.01）；HGF-ADSC外泌体组创面中CVF明显高于PBS组、单纯HGF组及单纯ADSC外泌体组（q值分别为31.31、28.52、12.35，P值均<0.01）。 结论 人HGF-ADSC外泌体可增加创基组织新生血管形成，从而显著促进糖尿病小鼠全层皮肤缺损创面愈合，其机制可能与HGF-ADSC外泌体通过激活PI3K/Akt信号通路从而促进创面中eNOS的表达增高有关。 Abstract:Objective To investigate the effects and mechanism of exosomes from hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on full-thickness skin defect wounds in diabetic mice. Methods The experimental study method was adopted. Discarded adipose tissue of 3 healthy females (10-25 years old) who underwent abdominal surgery in the Department of Plastic Surgery of First Affiliated Hospital of Air Force Medical University from February to May 2021 was collected, and primary ADSCs were obtained by collagenase digestion method and cultured for 7 days. Cell morphology was observed by inverted phase contrast microscope. The ADSCs of third passage were transfected with HGF lentivirus and cultured for 5 days, and then the fluorescence of cells was observed by imaging system and the transfection rate was calculated. The exosomes of ADSCs of the third to sixth passages and the HGF transfected ADSCs of the third to sixth passages were extracted by density gradient centrifugation, respectively, and named, ADSC exosomes and HGF-ADSC exosomes. The microscopic morphology of exosomes was observed by transmission electron microscopy, and the positive expressions of CD9, CD63, and CD81 of exosomes were detected by flow cytometry, respectively. Twenty-four 6-week-old male Kunming mice were selected to make the diabetic models, and full-thickness skin defect wounds were made on the backs of mice. According to the random number table method, the mice were divided into phosphate buffer solution (PBS) group, HGF alone group, ADSC exosome alone group, and HGF-ADSC exosome group, with 6 mice in each group, and treated accordingly. On post injury day (PID) 3, 7, 10, and 14, the wounds were observed and the wound healing rate was calculated; the blood flow intensity of wound base was detected by Doppler flowmeter and the ratio of relative blood flow intensity on PID 10 was calculated. On PID 10, the number of Ki67 positive cells in wounds was detected by immunofluorescence method, and the number of new-vascularity of CD31 positive staining and tubular neovascularization in the wounds was detected by immunohistochemistry method; the protein expressions of protein endothelial nitric oxide synthase (eNOS), phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt) and phosphorylated Akt (p-Akt) in wounds were detected by Western blotting, and the ratios of p-PI3K to PI3K and p-Akt to Akt were calculated. On PID 14, the defect length and collagen regeneration of wound skin tissue were detected by hematoxylin and eosin staining and Masson staining, respectively, and the collagen volume fraction (CVF) was calculated. The number of samples is 3 in all cases. Data were statistically analyzed with repeated measurement analysis of variance, one-way analysis of variance, and Tukey test. Results After 7 days of culture, the primary ADSCs were spindle shaped and arranged in vortex shape after dense growth. After 5 days of culture, HGF transfected ADSCs of the third passage carried green fluorescence, and the transfection rate was 85%. The ADSC exosomes and HGF-ADSC exosomes were similar in microscopic morphology, showing vesicular structures with an average particle size of 103 nm and 98 nm respectively, and both were CD9, CD63, and CD81 positive. On PID 3, the wounds of mice in the 4 groups were all red and swollen, with a small amount of exudate. On PID 7, the wounds of HGF-ADSC exosome group were gradually reduced, while the wounds of the other three groups were not significantly reduced. On PID 10, the wounds in the 4 groups were all reduced and scabbed. On PID 14, the wounds in HGF-ADSC exosome group were basically healed, while the residual wounds were found in the other three groups. On PID 3, the healing rates of wounds in the four groups were similar (P>0.05); On PID 7 and 10, the wound healing rates in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 13.11, 13.11, 11.89, 12.85, 11.28, and 7.74, respectively, all P<0.01); on PID 14, the wound healing rate in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 15.50, 11.64, and 6.36, respectively, all P<0.01). On PID 3, there was no obvious blood supply in wound base of mice in the 4 groups. On PID 7, microvessels began to form in the wound base of HGF-ADSC exosome group, while the wound base of the other three groups was only congested at the wound edge. On PID 10, microvessel formation in wound base was observed in the other 3 groups except in PBS group, which had no obvious blood supply. On PID 14, the blood flow intensity of wound base in HGF-ADSC exosome group was stronger than that in the other 3 groups, and the distribution was uniform. On PID 10, the ratio of wound base relative blood flow intensity in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 23.73, 19.32, and 9.48, respectively, all P<0.01); The numbers of Ki67-positive cells and new-vascularity of wounds in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 19.58, 18.20, 11.04, 20.68, 13.79, and 8.12, respectively, P<0.01). On PID 10, the protein expression level of eNOS of wounds in HGF-ADSC exosome group was higher than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 53.23, 42.54, and 26.54, respectively, all P<0.01); the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt of wounds in HGF-ADSC exosome group were significantly higher than those in PBS group, HGF alone group, and ADSC exosome alone group, respectively (with q values of 16.11, 11.78, 6.08, 65.54, 31.63, and 37.86, respectively, P<0.01). On PID 14, the length of skin tissue defect in the wounds of HGF-ADSC exosome group was shorter than that in PBS group, HGF alone group, and ADSC exosome alone group (with q values of 20.51, 18.50, and 11.99, respectively, all P<0.01); the CVF of wounds in HGF-ADSC exosome group was significantly higher than that in PBS group, HGF alone group and ADSC exosome alone group (with q values of 31.31, 28.52, and 12.35, respectively, all P<0.01). Conclusions Human HGF-ADSC exosomes can significantly promote wound healing in diabetic mice by increasing neovascularization in wound tissue, and the mechanism may be related to the increased expression of eNOS in wounds by activating PI3K/Akt signaling pathway. -
参考文献
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1 原代培养7 d的人脂肪间充质干细胞（ADSC）形态及转染肝细胞生长因子的第5代ADSC的鉴定。1A.ADSC呈梭状生长，胞核呈球形、核质分界清晰 倒置相差显微镜×20；1B.大部分转染细胞携带绿色荧光 异硫氰酸荧光素×40
2 人脂肪间充质干细胞（ADSC）外泌体微观形态和肝细胞生长因子（HGF）修饰的ADSC外泌体（简称HGF-ADSC外泌体）微观形态及其粒径。2A.ADSC外泌体呈囊泡状结构 透射电子显微镜×40 000；2B.HGF-ADSC外泌体外观与图2A相似 透射电子显微镜×40 000；2C.HGF-ADSC外泌体粒径分布在60~160 nm之间
注：图2C横坐标为经过lg处理后的数据
3 4组糖尿病小鼠伤后各时间点全层皮肤缺损创面愈合情况。3A、3B、3C、3D.分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组伤后10 d创面，4组创面均结痂；3E、3F、3G、3H.分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组伤后14 d创面，均分别较图3A、3B、3C、3D明显缩小，其中图3H中创面基本愈合、新生上皮致密
注：PBS为磷酸盐缓冲液，HGF为肝细胞生长因子，ADSC为脂肪间充质干细胞；各图中的外环起固定小鼠创周皮肤作用
4 激光多普勒血流仪检测4组糖尿病小鼠伤后各时间点全层皮肤缺损创面中血流强度变化情况。4A、4B、4C、4D.分别为磷酸盐缓冲液（PBS）组伤后3、7、10、14 d创基及创周血运情况，创基血运均较差；4E、4F、4G、4H.分别为单纯肝细胞生长因子（HGF）组伤后3、7、10、14 d创基及创周血运情况，除伤后7 d创周血流强度增强外，其余时间点的创基血运均不佳；4I、4J、4K、4L.分别为单纯脂肪间充质干细胞（ADSC）外泌体组伤后3、7、10、14 d创基及创周血运情况，均分别较图4E、4F、4G、4H与图4A、4B、4C、4D血流信号强；4M、4N、4O、4P.分别为HGF-ADSC外泌体组伤后3、7、10、14 d创基及创周血运情况，伤后7 d该组创基血流强度明显高于同时间点其余3组，伤后10 d创基血流强度最强，伤后14 d创基血运反而下降
注：颜色越接近红色表示血流强度越高，颜色越接近蓝色表示血流强度越低
5 4组糖尿病小鼠全层皮肤缺损创面免疫荧光染色及免疫组织化学染色情况。5A、5B、5C、5D.分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组创面组织伤后10 d的Ki67染色情况，图5D中阳性细胞数多于其余3图 花青素3-4′，6- 二脒基-2-苯基吲哚×100；图5E、5F、5G、5H分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组创基组织CD31免疫组织化学染色情况，其中图5H阳性染色且成管样结构的新生血管数量多于其余3图 辣根过氧化物酶-苏木精×100
注：PBS为磷酸盐缓冲液，HGF为肝细胞生长因子，ADSC为脂肪间充质干细胞；Ki67阳性细胞染色为红色，CD31阳性细胞染色为棕色
6 4组糖尿病小鼠伤后10 d全层皮肤缺损创面中eNOS的表达及PI3K与Akt的磷酸化水平
注：eNOS为内皮型一氧化氮合酶，Akt为蛋白激酶B，p-Akt为磷酸化AKT，PI3K为磷脂酰肌醇3-激酶，p-PI3K为磷酸化PI3K，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指磷酸盐缓冲液（PBS）组、单纯肝细胞生长因子（HGF）组、单纯脂肪间充质干细胞（ADSC）外泌体组、HGF-ADSC外泌体组
7 4组糖尿病小鼠伤后14 d全层皮肤缺损创面上皮化情况及胶原新生情况。7A、7B、7C、7D.分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组创面，图7D的创面缺损较长度明显短于其余3图 苏木精-伊红×20；7E、7F、7G、7H.分别为PBS组、单纯HGF组、单纯ADSC外泌体组及HGF-ADSC外泌体组创面，图7H较其余3图的蓝染面积更大，胶原排列更疏松有序 Masson×100
注：PBS为磷酸盐缓冲液，HGF为肝细胞生长因子，ADSC为脂肪间充质干细胞；图7A、7B、7C、7D中黑色线段为创面缺损长度；图7E、7F、7G、7H中胶原阳性染色为蓝色
表1 4组糖尿病小鼠伤后各时间点全层皮肤缺损创面愈合率比较（%，
组别 鼠数（只） 3 d 7 d 10 d 14 d HGF-ADSC外泌体组 3 10.7±0.6 27.0±1.1 76.1±0.7 89.7±0.4 PBS组 3 10.3±0.8 18.5±1.7 54.4±3.0 70.5±3.0 单纯HGF组 3 10.1±0.3 18.5±1.0 57.0±1.8 75.3±2.1 单纯ADSC外泌体组 3 10.8±0.6 19.3±0.3 63.0±2.0 81.8±2.2 F值 0.93 40.67 65.59 45.21 P1值 0.472 <0.001 <0.001 <0.001 q1值 0.90 13.11 12.85 15.50 P2值 0.708 <0.001 <0.001 <0.001 q2值 1.31 13.11 11.28 11.64 P3值 0.458 <0.001 <0.001 <0.001 q3值 0.07 11.89 7.74 6.36 P4值 0.100 <0.001 <0.001 0.008 注：HGF为肝细胞生长因子，ADSC为脂肪间充质干细胞，PBS为磷酸盐缓冲液；处理因素主效应，F=73.43，P<0.001；时间因素主效应，F=8 679.77，P<0.001；两者交互作用，F=38.18，P<0.001；F值、P1值为4组间各时间点总体比较所得；q1值、P2值，q2值、P3值，q3值、P4值分别为HGF-ADSC外泌体组与PBS组、单纯HGF组、单纯ADSC外泌体组各时间点比较所得 
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