肝细胞生长因子修饰人脂肪间充质干细胞对糖尿病大鼠全层皮肤缺损创面愈合的影响及其机制

张月; 韩飞; 何亭; 计鹏; 张智; 陶克

doi:10.3760/cma.j.cn501120-20200626-00329

肝细胞生长因子修饰人脂肪间充质干细胞对糖尿病大鼠全层皮肤缺损创面愈合的影响及其机制

doi: 10.3760/cma.j.cn501120-20200626-00329

空军军医大学第一附属医院全军烧伤中心，烧伤与皮肤外科，西安　710032

基金项目:

国家自然科学基金面上项目 81871561

国家自然科学基金青年科学基金 82002039

详细信息

通讯作者:
陶克，Email：taoke918@fmmu.edu.cn

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出版历程
- 收稿日期: 2020-06-26

Effects and mechanism of hepatocyte growth factor-modified human adipose mesenchymal stem cells on wound healing of full-thickness skin defects in diabetic rats

Department of Burns and Cutaneous Surgery, Burn Center of PLA, the First Affiliated Hospital of Air Force Medical University, Xi'an 710032, China

Funds:

General Program of National Natural Science Foundation of China 81871561

Youth Science Foundation of National Natural Science Foundation of China 82002039

More Information

Corresponding author: Tao Ke, Email: taoke918@fmmu.edu.cn

摘要

摘要: 目的探讨肝细胞生长因子（HGF）修饰人脂肪间充质干细胞（ADSC）对糖尿病大鼠全层皮肤缺损创面愈合的影响及其机制。方法采用实验研究方法。取2019年12月于空军军医大学第一附属医院整形科行腹部抽脂术的1名35岁健康女性术后弃用腹部脂肪组织，采用胶原酶消化法获取呈长梭形的原代ADSC，其第3代细胞经流式细胞仪鉴定阳性表达ADSC表面标志物CD29、CD90，阴性表达CD34、CD45。取第3代ADSC进行后续实验。采用慢病毒介导的HGF转染ADSC 4 h（获得HGF修饰ADSC）后，常规培养24 h，倒置相差显微镜下观察细胞形态并计算转染率。取81只4周龄雄性SD大鼠，通过高糖高脂饮食联合链脲佐菌素注射诱导为糖尿病大鼠模型后，在每只大鼠背部制作1.5 cm×1.5 cm全层皮肤缺损创面。采用随机数字表法，将致伤大鼠分为磷酸盐缓冲液（PBS）组、单纯ADSC组、HGF修饰ADSC组，每组27只，均分别于伤后1、3、7 d在创周注射相同体积的相应物质。采用随机数字表法选取各组9只大鼠，于伤后0（即刻）、3、7、10、14 d（注射日注射后）测量创面面积并计算伤后3、7、10、14 d创面愈合率。伤后3、7 d行当日注射后分别处死各组剩余大鼠中的9只，取创周皮肤组织，采用实时荧光定量反转录PCR法检测伤后3 d肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-10及伤后7 d Ⅰ、Ⅲ型胶原的mRNA表达，采用酶联免疫吸附测定法检测伤后7 d血管内皮生长因子（VEGF）的表达水平，采用蛋白质印迹法检测伤后3 d核因子κB-p65蛋白表达、伤后7 d蛋白激酶B（Akt）的磷酸化水平。对数据行重复测量方差分析、单因素方差分析、LSD-t检验及Bonferroni校正。结果培养24 h，转染HGF的ADSC形态良好，与未转染ADSC无差异，转染率达90%。伤后3、7、10、14 d，HGF修饰ADSC组大鼠创面愈合率分别为（31.5±1.0）%、（75.2±2.0）%、（92.2±1.3）%、（99.1±1.8）%，显著高于PBS组的（21.4±1.3）%、（61.4±1.5）%、（80.1±2.1）%、（92.4±1.8）%和单纯ADSC组的（25.1±2.1）%、（67.2±1.3）%、（89.3±1.4）%、（95.1±2.1）%（t=1.452、0.393、0.436、0.211，4.982、3.011、4.211、7.503，P<0.05或P<0.01）。伤后3 d，与PBS组和单纯ADSC组比较，HGF修饰ADSC组大鼠创周皮肤组织中TNF-α、IL-1β的mRNA表达及核因子κB-p65的蛋白表达均显著降低（t=7.281、17.700、9.447，6.231、13.083、7.783，P<0.01），IL-10的mRNA表达显著升高（t= - 6.644、 - 6.381，P<0.01）。伤后7 d，与PBS组和单纯ADSC组比较，HGF修饰ADSC组大鼠创周皮肤组织中Ⅰ、Ⅲ型胶原的mRNA表达及VEGF表达水平、Akt磷酸化水平均明显升高（t= - 5.126、 - 4.347、 - 5.058、 - 3.367， - 10.694、 - 19.876、 - 4.890、 - 6.819，P<0.05或P<0.01）。结论 HGF修饰的人ADSC可显著促进糖尿病大鼠全层皮肤缺损创面愈合，其机制可能与抑制TNF-α、IL-1β表达，促进IL-10、Ⅰ型胶原、Ⅲ型胶原及VEGF的表达相关，且可能与抑制核因子κB信号通路、促进Akt信号通路有关。
- 糖尿病 /
- 伤口愈合 /
- 肝细胞生长因子 /
- 脂肪间充质干细胞 /
- 慢性创面 /
- 炎症反应
Abstract: Objective To investigate the effects and mechanism of hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on the wound healing of full-thickness skin defects in diabetic rats. Methods The experimental research method was adopted. The discarded abdominal adipose tissue was collected from a 35-year-old healthy female who underwent abdominal liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University in December 2019. The long spindle-shaped primary ADSCs were obtained by collagenase digestion, and the third passage of cells were identified by flow cytometry to positively express ADSCs surface markers CD29 and CD90 and negatively express CD34 and CD45. The third passage of ADSCs were used for the subsequent experiments. ADSCs were transfected with lentivirus-mediated HGF for 4 h (obtaining HGF modified ADSCs) and then routinely cultured for 24 h. The cell morphology was observed under an inverted phase contrast microscope, and the transfection rate was calculated. Eighty-one male Sprague-Dawley rats aged 4 weeks were induced into diabetic rat model by high glucose and high fat diet combined with streptozotocin injection. A full-thickness skin defect wound of 1.5 cm×1.5 cm was made on the back of each rat. The injured rats were divided into phosphate buffer solution (PBS) group, ADSCs alone group, and HGF-modified ADSCs group according to the random number table, with 27 rats in each group. The rats were injected with the same volume of corresponding substances around the wound on post injury day (PID) 1, 3, and 7, respectively. Nine rats in each group were selected according to the random number table, the wound area of whom was measured on PID 0 (immediately), 3, 7, 10, and 14 (after injection on injection day), and the wound healing rates on PID 3, 7, 10, and 14 were calculated. Nine remaining rats in each group were sacrificed after injection on PID 3 and 7, respectively, and the skin tissue around the wound were collected. The mRNA expressions of inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 on PID 3 and collagen type Ⅰ and Ⅲ on PID 7 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The expression level of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay on PID 7. The protein expression of nuclear factor κb-p65 on PID 3 and phosphorylation level of protein kinase B (Akt) on PID 7 were detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results After 24 h of culture, the HGF-transfected human ADSCs showed good morphology, which was not different with the non-transfected ADSCs, and the transfection rate reached 90%. On PID 3, 7, 10, and 14, the wound healing rates of rats in HGF-modified ADSCs group were (31.5±1.0)%, (75.2±2.0)%, (92.2±1.3)%, and (99.1±1.8)%, respectively, being significantly higher than (21.4±1.3)%, (61.4±1.5)%, (80.1±2.1)%, and (92.4±1.8)% in PBS group and (25.1±2.1)%, (67.2±1.3)%, (89.3±1.4)%, and (95.1±2.1)% in ADSCs alone group (t=1.452, 0.393, 0.436, 0.211, 4.982, 3.011, 4.211, 7.503, P<0.05 or P<0.01). On PID 3, compared with those in PBS group and ADSCs alone group, the mRNA expressions of TNF-α and IL-1β and protein expression of nuclear factor κb-p65 in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly decreased (t=7.281, 17.700, 9.447, 6.231, 13.083, 7.783, P<0.01), and the mRNA expression of IL-10 in the skin tissue around the wound of rats in HGF-modified ADSCs group was significantly increased (t=-6.644, -6.381, P<0.01). On PID 7, compared with those in PBS group and ADSCs alone group, the mRNA expressions of collagen type Ⅰ and Ⅲ, the expression level of VEGF, and the phosphorylation level of Akt in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly increased (t=-5.126, -4.347, -5.058, -3.367, -10.694, -19.876, -4.890, -6.819, P<0.05 or P<0.01). Conclusions HGF-modified human ADSCs can significantly promote the wound healing of full-thickness skin defects in diabetic rats. The mechanism may be related to the inhibition of TNF-α and IL-1β expression, the promotion of IL-10, collagen type Ⅰ and Ⅲ, and VEGF expression, which could be related to the inhibition of nuclear factor κB signaling pathway, and the promotion of Akt signaling pathway.
- Diabetes mellitus /
- Wound healing /
- Hepatocyte growth factor /
- Adipose mesenchymal stem cells /
- Chronic wounds /
- Inflammatory response
所有作者均声明不存在利益冲突

促进伤口愈合一直以来都是外科营养的关注点之一。加速康复外科理念中特别提到，围手术期的营养支持能减少患者的创伤应激反应、促进伤口愈合、减少并发症、缩短住院时间。悉尼大学Concord医院ANZAC研究所烧伤研究与重建外科Yiwei Wang教授团队近期在《Burns & Trauma》发文《Low-protein diet accelerates wound healing in mice post-acute injury》，本文在小鼠模型上利用几何框架法（一种营养素建模方法，能分析多种营养素的广谱交互作用）来探究促进皮肤伤口愈合的最佳宏量营养素配比。在雄性C57BL/6小鼠背侧构建2个等大的全层皮肤缺损模型，随机分配小鼠喂养12种不同宏量营养素配比的高能饮食（能量密度17 kJ/g）。结论提出重要观点，低蛋白饮食结合碳水化合物和脂肪的均衡摄入能促进小鼠皮肤切除后的伤口愈合。为了解释这一结果，研究者进一步评估了小鼠伤口愈合过程中的细胞因子表达、能量消耗、身体组成以及肌肉和脂肪的储存情况，观察到胶原沉积增强、早期但短时间的炎症反应、棕色和白色脂肪组织中解偶联蛋白1依赖的产热作用、以及瘦体重的自噬可能是低蛋白饮食促进伤口愈合的原因。这一结论打破了目前临床提倡运用高蛋白配方进行营养支持的理念。营养支持的时机、剂量和途径一直是外科临床营养所讨论和关注的热点，该结论的提出对于临床实践来说具有重要的意义。这项研究探讨了不同营养素配比对伤口愈合的作用机制，其亮点在于，同时对三大宏量营养配比进行建模，打破以往就单一营养成分进行研究的限制。未来的研究将集中于解释宏量营养素摄入的机制研究及人群验证，特别是可变量的蛋白质摄入和血糖控制的影响，如何改变内源性蛋白质储备的分解，以及对可用于愈合组织的氨基酸配制的影响。同时，研究的结论为进一步指导临床开展临床试验提供新思路，例如评估低蛋白质、平衡碳水化合物和脂肪摄入在加速康复外科中的有效性等。

翁婷婷，编译自《Burns & Trauma》, 2021,9:tkab010;韩春茂、秦环龙,审校

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1 原代人脂肪间充质干细胞培养24 h呈长梭形倒置相差显微镜×20

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2 大部分人脂肪间充质干细胞成功转染肝细胞生长因子（绿色荧光），细胞呈长梭形倒置相差显微镜×40

注：转染由自带绿色荧光的慢病毒介导

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3 3组糖尿病全层皮肤缺损大鼠伤后各时间点创面愈合情况。3A、3B、3C、3D.分别为磷酸盐缓冲液组伤后3、7、10、14 d创面情况，创面逐渐缩小；3E、3F、3G、3H.分别为单纯脂肪间充质干细胞（ADSC）组伤后3、7、10、14 d创面情况，图3E、3F、3G、3H 中创面分别较图3A、3B、3C、3D有所缩小；3I、3J、3K、3L.分别为肝细胞生长因子修饰ADSC组伤后3、7、10、14 d创面情况，图3J、3K、3L中创面分别较图3F、3G、3H有所缩小，较图3B、3C、3D明显缩小

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4 蛋白质印迹法检测3组糖尿病全层皮肤缺损大鼠创周皮肤组织中伤后3 d核因子κB-p65蛋白表达与伤后7 d Akt磷酸化水平。4A.核因子κB-p65；4B.Akt磷酸化

注：1、2、3分别为磷酸盐缓冲液组、单纯脂肪间充质干细胞（ADSC）组、肝细胞生长因子修饰ADSC组；Akt为蛋白激酶B

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表1 实时荧光定量反转录PCR法检测大鼠创周皮肤组织炎症因子mRNA表达的引物序列及产物大小

指标名称	引物序列（5′→3′）	产物大小（bp）
肿瘤坏死因子α	上游：AGAACTCCAGGCGGTGTCTGTG	225
肿瘤坏死因子α	下游：GTGGCAAATCGGCTGACGGTGT	225
白细胞介素1β	上游：CACTTCACAAGTCGGAGGCT	114
白细胞介素1β	下游：TCTGACAGTGCATCATCGCT	114
白细胞介素10	上游：TCCCAGTCAGCCAGACCCACA	144
白细胞介素10	下游：GGCAACCCAAGTAACCCTTAA	144
3-磷酸甘油醛脱氢酶	上游：GGCACAGTCAAGGCTGAGAATG	143
3-磷酸甘油醛脱氢酶	下游：ATGGTGGTGAAGACGCCAGTA	143

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表2 3组糖尿病全层皮肤缺损大鼠伤后各时间点创面愈合率比较（%，x¯±s）

组别	鼠数（只）	3 d	7 d	10 d	14 d
PBS组	9	21.4±1.3	61.4±1.5	80.1±2.1	92.4±1.8
单纯ADSC组	9	25.1±2.1	67.2±1.3	89.3±1.4	95.1±2.1
HGF修饰ADSC组	9	31.5±1.0	75.2±2.0	92.2±1.3	99.1±1.8
t₁值		1.452	0.393	0.436	0.211
P₁值		0.005	0.028	0.008	0.015
t₂值		4.982	3.011	4.211	7.503
P₂值		0.010	0.032	0.016	0.021
注：PBS为磷酸盐缓冲液，ADSC为脂肪间充质干细胞，HGF为肝细胞生长因子；时间因素主效应，F=1 968.321，P<0.001；处理因素主效应，F=592.785，P<0.001；两者交互作用，F=13.353，P<0.001；t₁值、P₁值，t₂值、P₂值分别为HGF修饰ADSC组与PBS组、单纯ADSC组各时间点比较所得

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表3 3组糖尿病全层皮肤缺损大鼠伤后3 d创周皮肤组织中3种炎症因子mRNA表达比较（x¯±s）

组别	鼠数（只）	TNF-α	IL-1β	IL-10
PBS组	9	1.00±0.16	1.00±0.08	1.00±0.11
单纯ADSC组	9	0.60±0.04	0.47±0.05	1.57±0.23
HGF修饰ADSC组	9	0.35±0.06	0.21±0.03	2.32±0.28
F值		97.003	484.640	85.088
P值		<0.001	<0.001	<0.001
t₁值		7.281	17.700	-6.644
P₁值		<0.001	<0.001	<0.001
t₂值		6.231	13.083	-6.381
P₂值		<0.001	<0.001	<0.001
注：PBS为磷酸盐缓冲液，ADSC为脂肪间充质干细胞，HGF为肝细胞生长因子，TNF-α为肿瘤坏死因子α，IL为白细胞介素；F值、P值为3组间各指标总体比较所得；t₁值、P₁值，t₂值、P₂值分别为HGF修饰ADSC组与PBS组、单纯ADSC组各指标比较所得

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表4 3组糖尿病全层皮肤缺损大鼠伤后7 d创周皮肤组织中2种胶原的mRNA表达比较（x¯±s）

组别	鼠数（只）	Ⅰ型胶原	Ⅲ型胶原
PBS组	9	1.00±0.06	1.00±0.03
单纯ADSC组	9	1.28±0.17	1.31±0.21
HGF修饰ADSC组	9	2.16±0.18	2.88±0.16
F值		158.373	388.256
P值		<0.001	<0.001
t₁值		-5.126	-4.347
P₁值		<0.001	<0.001
t₂值		-10.694	-19.876
P₂值		<0.001	<0.001
注：PBS为磷酸盐缓冲液，ADSC为脂肪间充质干细胞，HGF为肝细胞生长因子；F值、P值为3组间各指标总体比较所得；t₁值、P₁值，t₂值、P₂值分别为HGF修饰ADSC组与PBS组、单纯ADSC组各指标比较所得

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表5 3组糖尿病全层皮肤缺损大鼠创周皮肤组织中伤后3 d核因子κB-p65蛋白表达与伤后7 d Akt磷酸化水平比较（x¯±s）

组别	鼠数（只）	核因子κB-p65	Akt磷酸化
PBS组	9	0.86±0.14	0.23±0.08
单纯ADSC组	9	0.38±0.07	0.38±0.11
HGF修饰ADSC组	9	0.17±0.04	0.74±0.12
F值		132.627	59.502
P值		<0.001	<0.001
t₁值		9.447	-3.367
P₁值		<0.001	0.004
t₂值		7.783	-6.819
P₂值		<0.001	<0.001
注：PBS为磷酸盐缓冲液，ADSC为脂肪间充质干细胞，HGF为肝细胞生长因子，Akt为蛋白激酶B；F值、P值为3组间各指标总体比较所得；t₁值、P₁值，t₂值、P₂值分别为HGF修饰ADSC组与PBS组、单纯ADSC组各指标比较所得

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