人脂肪间充质干细胞多层膜片对促进裸鼠皮肤缺损愈合的效果观察

doi:10.3877/cma.j.issn.1673-9450.2024.04.012

目的

探讨人脂肪间充质干细胞(ADSCs)膜片对BALB/c-nu裸鼠全层皮肤缺损的治疗效果。

方法

体外分离培养人ADSCs，制备成3层的人ADSCs膜片。选择健康无特殊病原体级雄性BALB/c-nu裸鼠36只，建立背部直径为12 mm的圆形全层皮肤缺损创面。将裸鼠随机分为2组，对照组单纯使用羊脱细胞真皮基质(ADM)覆盖创面；ADSCs膜片组在3层ADSCs膜片移植后使用羊ADM覆盖创面。术后7 d进行组织学评价，测量创面肉芽组织厚度。术后7、12、17 d，比较两组创面愈合率和创面组织表皮生长因子(EGF)的表达。各组各时间点样本数均为6。对数据行t检验。

结果

从人脂肪组织分离培养的细胞呈梭形，漩涡状排列，流式细胞术检测和分化实验证实为ADSCs。经过成膜培养，ADSCs形成3层膜片。术后7 d，ADSCs膜片组创面肉芽组织厚度为(456.35±57.05)μm，明显大于对照组的(295.08±38.86)μm(t＝5.721，P<0.001)。术后7、12、17 d，ADSCs膜片组创面愈合率[(45.07±1.58)%、(83.29±1.54)%、(98.49±0.85)%]均高于对照组[(29.99±0.90)%、(78.18±1.02)%、(92.38±0.54)%]，差异均有统计学意义(t＝20.294、6.777、14.733，P<0.001)。术后7、12、17 d，ADSCs膜片组创面组织EGF表达[(90.32±2.36)pg/ml、(107.84±1.82)pg/ml、(111.93±2.25)pg/ml]均高于对照组[(81.14±2.15)pg/ml、(102.46±2.18)pg/ml、(106.14±2.10)pg/ml]，差异均有统计学意义(t＝7.012、4.621、4.630，P<0.001)。

结论

人ADSCs膜片能显著加速裸鼠创面愈合，其机制可能与EGF表达增加从而促进肉芽组织生长有关。

关键词: 脂肪间充质干细胞, 细胞膜片技术, 创面愈合

Objective

To investigate the therapeutic effect of human adipose-derived stem cells (ADSCs) sheet on full-thickness skin defect in BALB/c-nu nude mice. The sample number was 6 in each group at each time point. Data were statistically analyed with t test.

Methods

Human ADSCs were isolated and cultured in vitro. Three layers of human ADSCs sheet were prepared. A 12 mm-diameter, round, full-thickness cutaneous wound was made on the back of 36 nude mice. The nude mice were divided into two groups, sheep acelluar dermal matrix (ADM) was used alone to cover the wound in control group, and ADSCs sheet combined with sheep ADM were used in ADSCs sheet group. On 7 days after surgery, the thickness of granulation tissue was measured. On 7, 12 and 17 days after surgery, the wound healing rate and epidermal growth factor (EGF) expression in wound tissue were compared between the two groups.

Results

The cells isolated and cultured from adipose tissue were spindle shaped, arranged in whirlpool. Flow cytometry and differentiation experiments confirmed that the cells were ADSCs. After sheet formation culture, ADSCs formed membrane. On 7 days after surgery, the thickness of wound granulation tissue in ADSCs sheet group was (456.35±57.05)μm, which was significantly larger than (295.08±38.86)μm in control group(t=5.721, P<0.001). On 7, 12 and 17 days after surgery, the wound healing rates in ADSCs sheet group [(45.07±1.58)%, (83.29±1.54)%, (98.49±0.85)%] were significantly higher than those in control group [(29.99±0.90)%, (78.18±1.02)%, (92.38±0.54)%], the differences were statistically significant (t=20.294, 6.777, 14.733; P<0.001). On 7, 12 and 17 days after surgery, EGF expressions in wound tissue in ADSCs sheet group [(90.32±2.36)pg/ml, (107.84±1.82)pg/ml, (111.93±2.25)pg/ml] were significantly higher than those in control group [(81.14±2.15)pg/ml, (102.46±2.18)pg/ml, (106.14±2.10)pg/ml], with statistically significant difference(t=7.012, 4.621, 4.630; P<0.001).

Conclusion

Human ADSCs sheet could accelerate wound healing in nude mice, its mechanism may be related to the increase of EGF expression, which promotes the growth of granulation tissue.

Key words: Adipose-derived stem cells, Cell sheet technology, Wound healing

图1 ADSCs的形态。A示ADSCs原代细胞(×50)；B示ADSCs P₃代细胞(×50)

图2 ADSCs生长曲线

图3 ADSCs表面标记物表达

图4 ADSCs成脂、成骨诱导实验。A示成脂诱导实验细胞，B示成脂诱导对照细胞，油红O染色(×200)；C示成骨诱导实验细胞，D示成骨诱导对照细胞，茜素红染色(×100)

图5 ADSCs膜片。A示培养第8天大体外观；B示培养第8天显微镜下所见(×50)；C示培养第21天大体外观；D示培养第21天显微镜下所见(×50)；E示培养第21天苏木精-伊红染色(×400)

图6 2组术后7 d创面肉芽组织生长情况(苏木精-伊红染色，×40)。A示对照组创面肉芽组织生长情况；B示ADSCs膜片组创面肉芽组织生长情况

图7 2组术后各时间点创面情况。A-C示对照组术后7、12、17 d创面情况；D-F示ADSCs膜片组术后7、12、17 d创面情况

表1 两组术后7、12、17 d创面愈合率比较(%,

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	45.07±1.58	83.29±1.54	98.49±0.85
对照组	6	29.99±0.90	78.18±1.02	92.38±0.54
t值		20.294	6.777	14.733
P值		<0.001	<0.001	<0.001

表1 两组术后7、12、17 d创面愈合率比较(%,

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	45.07±1.58	83.29±1.54	98.49±0.85
对照组	6	29.99±0.90	78.18±1.02	92.38±0.54
t值		20.294	6.777	14.733
P值		<0.001	<0.001	<0.001

表2 两组术后7、12、17 d创面组织EGF表达量(pg/ml，

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	90.32±2.36	107.84±1.82	111.93±2.25
对照组	6	81.14±2.15	102.46±2.18	106.14±2.10
t值		7.012	4.621	4.630
P值		<0.001	<0.001	<0.001

表2 两组术后7、12、17 d创面组织EGF表达量(pg/ml，

±s)

组别	样本数	术后7 d	术后12 d	术后17 d
ADSCs膜片组	6	90.32±2.36	107.84±1.82	111.93±2.25
对照组	6	81.14±2.15	102.46±2.18	106.14±2.10
t值		7.012	4.621	4.630
P值		<0.001	<0.001	<0.001

[1]	Kondo M，Kameishi S，Grainger DW，et al. Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells[J]. Emerg Top Life Sci，2020，4(6)：677-689.
[2]	Nourian Dehkordi A, Mirahmadi Babaheydari F, Chehelgerdi M，et al. Skin tissue engineering: wound healing based on stem-cell-based therapeutic strategies[J]. Stem Cell Res Ther，2019，10(1)：111.
[3]	Miana VV，González EAP. Adipose tissue stem cells in regenerative medicine[J]. Ecancermedicalscience，2018，12：822.
[4]	Mazini L，Rochette L，Amine M，et al. Regenerative capacity of adipose derived stem cells (ADSCs)，comparison with mesenchymal stem Cells (MSCs)[J]. Int J Mol Sci，2019，20(10)：2523.
[5]	Bou-Ghannam S, Kim K, Grainger DW，et al. 3D cell sheet structure augments mesenchymal stem cell cytokine production[J]. Sci Rep，2021，11(1)：8170.
[6]	曹胜军，王凌峰，巴特，等. 异体小鼠脂肪源性间充质干细胞-微孔化羊脱细胞真皮基质对小鼠全层皮肤缺损创面愈合的影响及相关机制[J].中华烧伤杂志，2018，34(12)：901-906.
[7]	Koto Y, Iwata T, Morikawa S，et al. Allogeneic transplantation of an adipose-derived stem cell sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity[J]. Diabetes，2015，64(8)：2723-2734.
[8]	Laloze J, Fiévet L, Desmoulière A. Adipose-derived mesenchymal stromal cells in regenerative medicine: state of play，current clinical trials and future prospects[J]. Adv Wound Care (New Rochelle)，2021，10(1)：24-48.
[9]	Lechanteur C，Briquet A，Giet O，et al. Clinical-scale expansion of mesenchymal stromal cells：a large banking experience[J]. J Transl Med，2016，14(1)：145.
[10]	Alstrup T，Eijken M，Brunbjerg ME，et al. Measured levels of human adipose tissue-derived stem cells in adipose tissue is strongly dependent on harvesting method and stem cell isolation technique[J]. Plast Reconstr Surg，2020，145(1)：142-150.
[11]	Sukho P，Cohen A，Hesselink JW，et al. Adipose tissue-derived stem cell sheet application for tissue healing in vivo: a systematic review[J]. Tissue Eng Part B Rev，2018，24(1)：37-52.
[12]	Li M, Ma J, Gao Y, et al. Cell sheet technology: a promising strategy in regenerative medicine[J]. Cytotherapy，2019，21(1)：3-16.
[13]	杨霄彦，郝春光，李俊亮，等．微孔化羊ADM复合hUCMSC促进创面修复的机制研究[J].中华损伤与修复杂志(电子版)，2021，16(2)：97-103.

[1]	李亚龙, 王星童, 申传安. 异体富血小板血浆在创面修复中的临床应用进展[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 541-545.
[2]	李煜, 王鹏, 陆翮, 冯蓉琴, 韩军涛. 采用低频脉冲电刺激治疗深Ⅱ度烧伤创面的临床观察[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 474-478.
[3]	周清洁, 蒋萍萍, 梁云, 李琰. 脂质水胶体技术在创面愈合中的应用进展[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(04): 360-363.
[4]	刘高雨, 罗鹏, 史春梦. 成纤维细胞重编程与创面修复的研究进展[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(02): 176-179.
[5]	冯蓉琴, 王鹏, 李煜, 陆翮, 白晓智, 韩军涛. 抗菌肽在糖尿病创面愈合中作用的研究进展[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(01): 78-82.
[6]	赵雅玫, 谢斌, 陈艳, 吴健. 抗生素骨水泥联合负压封闭引流对糖尿病足溃疡临床疗效的荟萃分析[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(05): 427-433.
[7]	何雪锋, 赵世新, 李珮珊, 刘恒登, 谢举临. 卡奴卡叶提取物通过增强真皮成纤维细胞功能促进大鼠创面修复的效果观察[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(05): 405-412.
[8]	汪国建, 谭雨龙, 龙爽, 吕晓凡, 赵娜, 冉新泽, 王军平, 王涛. 高温高湿环境暴露对重度放创复合伤小鼠损伤恢复的影响[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(04): 285-292.
[9]	程飚. 浓缩血小板制品在创面修复中应用与思考[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(03): 276-276.
[10]	何泽亮, 李锦, 张程亮, 随振阳, 安亮恩, 刘玲玲, 姚媛媛, 张聚磊, 仇树林, 李晓东. 采用超声清创联合负压吸引疗法治疗深度烧伤溶痂创面的临床观察[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(02): 123-127.
[11]	尚强强, 王凌峰, 巴特, 曹胜军, 周彪, 李全, 侯智慧, 闫增强, 陈强. 外泌型汗腺参与创面愈合的研究进展[J/OL]. 中华损伤与修复杂志(电子版), 2022, 17(05): 450-453.
[12]	郑刚, 谢志, 黄涛, 谷才之. 自体邮票植皮术联合冲洗治疗在烧伤患者中的疗效观察[J/OL]. 中华损伤与修复杂志(电子版), 2022, 17(02): 141-144.
[13]	陈旭渊, 罗仕云, 李文忠, 李毅. 腺源性肛瘘经手术治疗后创面愈合困难的危险因素分析[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(01): 82-85.
[14]	王大伟, 陆雅斐, 皇甫少华, 陈玉婷, 陈澳, 江滨. 间充质干细胞通过调控免疫机制促进创面愈合的研究进展[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(06): 361-366.
[15]	郭庆, 李冠琳, 刘慧, 魏炜, 于洋, 张纯. 脂肪间充质干细胞治疗糖尿病及其慢性并发症的研究进展[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(01): 58-62.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Observation on the efficacy of human adipose-derived stem cells multilayer sheet promoting wound healing in nude mice

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Observation on the efficacy of human adipose-derived stem cells multilayer sheet promoting wound healing in nude mice