微针法注射大鼠表皮干细胞在大鼠全层皮肤缺损创面愈合中的作用

王雪; 程微; 刘小俊; 王顺生; 吴卓凡; 苏建东

doi:10.3760/cma.j.cn501225-20241031-00427

微针法注射大鼠表皮干细胞在大鼠全层皮肤缺损创面愈合中的作用

doi: 10.3760/cma.j.cn501225-20241031-00427

1.
南京医科大学附属苏州医院烧伤整形科,苏州　　215000
2.
苏州大学物理科学与技术学院,江苏省薄膜材料重点实验室,苏州　　215006

基金项目:

苏州市临床重点病种诊疗技术专项项目 LCZX202315

苏州市科技局-医疗器械与新医药项目 SLJ2021018

详细信息

通讯作者:
苏建东,Email：jiandongsu@njmu.edu.cn

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出版历程
- 收稿日期: 2024-10-31

Effects of microneedle injection of rat epidermal stem cells on wound healing of full-thickness skin defects in rats

1.
Department of Burn Plastic Surgery, the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215000, China
2.
School of Physical Science and Technology, Jiangsu Key Laboratory of Thin Film Materials, Soochow University, Suzhou 215006, China

Funds:

Suzhou Special Project for Diagnosis and Treatment of Key Clinical Diseases LCZX202315

Suzhou Science and Technology Bureau-Medical Devices and New Medicine Project SLJ2021018

More Information

Corresponding author: Su Jiandong, Email: jiandongsu@njmu.edu.cn

摘要

摘要: 目的探讨微针法注射大鼠表皮干细胞（ESC）在大鼠全层皮肤缺损创面愈合中的作用。方法该研究为实验研究。采用激光刻蚀技术成功制备空心硅基微针注射装置。从2只2 d龄雌性SD大鼠皮肤中分离培养细胞并采用流式细胞术、免疫荧光法成功鉴定为ESC。取18只8周龄雌性SD大鼠,采用随机数字表法分为对照组、喷洒组和微针组,每组6只。在所有大鼠背部造成1个全层皮肤缺损创面,伤后0 d（即刻）,于对照组、喷洒组大鼠创面分别喷洒0.5 mL磷酸盐缓冲液、0.5 mL大鼠ESC悬液,用空心硅基微针注射装置于微针组大鼠创面注射0.5 mL大鼠ESC悬液。伤后3、7、14 d,观察创面愈合情况并计算创面愈合率；伤后14 d,取大鼠创面组织,行苏木精-伊红染色观察创面上皮化情况,行Masson染色检测创面组织中胶原纤维含量,行免疫组织化学染色检测创面组织中新生血管数量。结果 3组大鼠伤后14 d内创面逐渐愈合。伤后3 d,3组大鼠创面愈合率总体比较,差异无统计学意义（P>0.05）；伤后7、14 d,微针组大鼠创面愈合率分别为（77.0±4.6）%、（95.0±2.1）%,均明显高于喷洒组的（66.5±8.6）%、（88.3±3.1）%和对照组的（44.5±5.7）%、（78.8±6.3）%（P<0.05）；喷洒组大鼠创面愈合率均明显高于对照组（P<0.05）。伤后14 d,微针组大鼠创面上皮化程度较喷洒组和对照组更高,新生皮肤组织中有毛囊生成。伤后14 d,微针组和喷洒组大鼠创面组织中胶原纤维含量均明显高于对照组（P<0.05）,微针组大鼠创面组织中胶原纤维含量明显高于喷洒组（P<0.05）；微针组及喷洒组大鼠创面组织中新生血管数量均明显多于对照组（P<0.05）,微针组大鼠创面组织中新生血管数量明显多于喷洒组（P<0.05）。结论 ESC能够促进创面组织中血管生成和胶原纤维形成,从而加速全层皮肤缺损大鼠创面愈合、改善创面愈合质量。通过空心硅基微针注射装置注射大鼠ESC悬液的方式较常规方式展现出更好的促创面愈合效果,是有效的细胞递送途径。
- 微针疗法 /
- 干细胞 /
- 血管生成 /
- 胶原纤维生成 /
- 创面修复
Abstract: Objective To investigate the effects of microneedle injection of rat epidermal stem cells (ESCs) on wound healing of full-thickness skin defects in rats. Methods This study was an experimental study. The hollow silicon-based microneedle injection device was developed successfully by utilizing laser-etching technology. ESCs were isolated and cultured from the skin of two 2-day-old female Sprague-Dawley rats and identified successfully by using flow cytometry and immunofluorescence method. A total of 18 eight-week-old female Sprague-Dawley rats were divided into control group, spray group, and microneedle group according to the random number table method, with 6 rats in each group. All rats were inflicted with a full-thickness skin defect wound on the dorsum. On day 0 (immediately after injury), the wounds in control group and spray group were sprayed with 0.5 mL phosphate-buffered solution and 0.5 mL rat ESCs suspension, respectively, and the wounds in microneedle group of rats were injected with 0.5 mL rat ESCs suspension through the hollow silicon-based microneedle injection device. On days 3, 7, and 14 after injury, the wound healing was observed and the wound healing rate was calculated. On day 14 after injury, the wound tissue of rats were harvested for hematoxylin and eosin staining to observe the epithelialization of wounds, for Masson staining to detect the collagen fiber content in wound tissue, and for immunohistochemical staining to detect the neovascular number in wound tissue. Results Within 14 days after injury, the wounds in three groups of rats progressively healed. On day 3 after injury, there was no statistically significant difference in the overall comparison of the wound healing rate among the three groups of rats (P>0.05). On days 7 and 14 after injury, the wound healing rates in microneedle group of rats were (77.0±4.6)% and (95.0±2.1)%, respectively, which were significantly higher than (66.5±8.6)% and (88.3±3.1)% in spray group and (44.5±5.7)% and (78.8±6.3)% in control group (P<0.05); the wound healing rates in spray group of rats were significantly higher than those in control group (P<0.05). On day 14 after injury, the degree of wound epithelialization in the microneedle group of rats was higher than that in spray group and control group, and hair follicles developed in the newly regenerated skin tissue. On day 14 after injury, the collagen fiber content in the wound tissue in microneedle group and spray group of rats was significantly higher than that in control group (P<0.05), and the collagen fiber content in the wound tissue in microneedle group of rats was significantly higher than that in spray group (P<0.05); the neovascular number in the wound tissue in microneedle group and spray group of rats was significantly more than that in control group (P<0.05), and the neovascular number in the wound tissue in microneedle group of rats was significantly more than that in spray group (P<0.05). Conclusions ESCs can promote neovascularization and collagen fiber formation in the wound tissue, thereby accelerating the wound healing and improve the quality of wound healing in rats with full-thickness skin defects. Injecting rat ESCs suspension through hollow silicon-based microneedle injection device shows better wound healing effects than using conventional approaches, making it an effective cell delivery method.
- Microneedle acupuncture therapy /
- Stem cells /
- Angiogenesis /
- Collagen fiber formation /
- Wound repair
王雪、程微：实验设计、实验实施、文章撰写；刘小俊、王顺生、吴卓凡：数据整理、统计学分析、文章撰写；苏建东：研究指导、论文审阅、经费支持

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参考文献(40)

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图 1 单个空心硅基微针形态场发射扫描电子显微镜×100。1A.俯视下见微针针尖旁激光打孔通道；1B.侧视下见单个微针呈四棱锥形

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图 2 大鼠原代ESC的形态及免疫荧光法检测第2代ESC的标志物表达。2A.培养7 d后,原代ESC呈铺路石状倒置相差显微镜×200；2B.细胞核、CK19、整合素β₁染色重叠图片,CK19、整合素β₁呈阳性表达 Alexa Fluor 488-花青素3-4',6-二脒基-2-苯基吲哚×300

注：ESC为表皮干细胞,CK19为细胞角蛋白19；细胞核、整合素β₁、CK19阳性染色分别为蓝色、绿色、红色

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图 3 3组全层皮肤缺损大鼠伤后各时间点创面愈合情况。3A、3B、3C.分别为对照组伤后3、7、14 d创面情况；3D、3E、3F.分别为喷洒组伤后3、7、14 d创面情况；3G、3H、3I.分别为微针组伤后3、7、14 d创面情况,图3A、3D、3G创面面积相近,图3H创面面积较图3B、3E小,图3I创面面积较图3C、3F小

注：于对照组大鼠创面喷洒0.5 mL磷酸盐缓冲液,于喷洒组大鼠创面喷洒等量大鼠表皮干细胞（ESC）悬液,通过空心硅基微针注射装置于微针组大鼠创面注射等量大鼠ESC悬液；黄色实线圆圈的面积为原始创面面积

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图 4 3组全层皮肤缺损大鼠伤后14 d创面上皮化情况和创面组织中胶原纤维形成情况。4A、4B、4C.分别为对照组、喷洒组、微针组上皮化情况,图4C上皮化程度、毛囊形成优于图4A、4B 苏木精-伊红×30；4D、4E、4F.分别为对照组、喷洒组、微针组创面组织中胶原纤维形成情况,图4F中胶原纤维明显多于图4D、4E Masson×30

注：于对照组大鼠创面喷洒0.5 mL磷酸盐缓冲液,于喷洒组大鼠创面喷洒等量大鼠表皮干细胞（ESC）悬液,通过空心硅基微针注射装置于微针组大鼠创面注射等量大鼠ESC悬液；红色箭头指示毛囊

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图 5 3组全层皮肤缺损大鼠伤后14 d创面组织中CD31的表达倒置相差显微镜×400。5A、5B、5C.分别为对照组、喷洒组、微针组,图5C中CD31表达明显多于图5A、5B

注：于对照组大鼠创面喷洒0.5 mL磷酸盐缓冲液,于喷洒组大鼠创面喷洒等量大鼠表皮干细胞（ESC）悬液,通过空心硅基微针注射装置于微针组大鼠创面注射等量大鼠ESC悬液；CD31阳性染色为棕色,反映新生血管情况

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Table 1. 3组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	创面数（个）	3 d	7 d	14 d
对照组	6	12.1±4.7	44.5±5.7	78.8±6.3
喷洒组	6	17.7±7.6	66.5±8.6	88.3±3.1
微针组	6	19.8±9.3	77.0±4.6	95.0±2.1
F值		1.70	39.00	22.12
P值		0.220	<0.001	<0.001
P₁值		—	<0.001	0.004
P₂值		—	<0.001	<0.001
P₃值		—	0.040	0.046
注：于对照组大鼠创面喷洒0.5 mL磷酸盐缓冲液,于喷洒组大鼠创面喷洒等量大鼠表皮干细胞（ESC）悬液,通过空心硅基微针注射装置于微针组大鼠创面注射等量大鼠ESC悬液；时间因素主效应,F=877.56,P<0.001；处理因素主效应,F=26.13,P<0.001；两者交互作用,F=9.44,P<0.001；P₁值、P₂值分别为对照组与喷洒组、微针组各时间点比较所得；P₃值为喷洒组与微针组各时间点比较所得；“—”表示无此项

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Table 2. 3组全层皮肤缺损大鼠伤后14 d创面组织中胶原纤维含量和新生血管数量比较（x¯±s）

组别	样本数	胶原纤维含量（%）	新生血管数量（支）
对照组	3	33±6	28±5
喷洒组	3	50±6	45±4
微针组	3	55±4	53±6
F值		44.93	53.52
P值		<0.001	<0.001
P₁值		<0.001	<0.001
P₂值		<0.001	<0.001
P₃值		0.036	0.002
注：于对照组大鼠创面喷洒0.5 mL磷酸盐缓冲液,于喷洒组大鼠创面喷洒等量大鼠表皮干细胞（ESC）悬液,通过空心硅基微针注射装置于微针组大鼠创面注射等量大鼠ESC悬液；P₁值、P₂值分别为对照组与喷洒组、微针组各指标比较所得；P₃值为喷洒组与微针组各指标比较所得