山东大学耳鼻喉眼学报 ›› 2025, Vol. 39 ›› Issue (3): 141-147.doi: 10.6040/j.issn.1673-3770.0.2023.433

• 论著 • 上一篇    

TNF-α诱导的人脐带间充质干细胞培养基对小鼠角膜缘干细胞缺乏的治疗作用

于浩南1,钟莹莹2,王新萌1,张敏3,姜清敏2,李娜2,李艳2   

  1. 临床医学院)潍坊医学院, 山东 潍坊 261031;
    2.潍坊医学院附属医院 眼科中心, 山东 潍坊 261041;
    3.暨南大学番禺校区第一临床医学院, 广东 广州 511436
  • 发布日期:2025-06-04
  • 通讯作者: 李艳. E-mail:liyanmails@126.com

Therapeutic effects of human umbilical cord mesenchymal stem cells induced by TNF-α on limbal stem cell deficiency in mice

YU Haonan1, ZHONG Yingying2, WANG Xinmeng1, ZHANG Min3, JIANG Qingmin2, LI Nan2, LI Yan2   

  1. 1. Affiliated Hospital of Weifang Medical University , School of Clinical Medicine , Weifang Medical University, Weifang 261031, Shandong, China2. The Eye Center, Affiliated Hospital of Weifang Medical University, Weifang 261031, Shandong, China3. The First Clinical Medical College, Jinan University, Guangzhou Panyu District Campus, Guangzhou 511436, Guangdong, China
  • Published:2025-06-04

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摘要： 目的 探讨局部应用肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)诱导的人脐带间充质干细胞条件培养基(conditioned medium for human umbilical cord mesenchymal stem cells, hUCMSC-CM)对小鼠角膜缘干细胞缺乏(limbal stem cell deficiency, LSCD)的修复作用。 方法 应用组织块贴壁培养技术培育原代人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)。用TNF-α诱导第4代hUCMSCs得到TNF-α诱导的人脐带间充质干细胞条件培养基(conditioned medium for human umbilical cord mesenchymal stem cells induced by TNF-α, hUCMSC-CMT)。48只健康6周龄C57雄性小鼠,随机分为对照组(N组)和LSCD实验组,LSCD实验组模型采取角膜缘碱烧伤方式建立,依据造模后结膜下注射的成分不同,分为PBS注射阴性对照组(A组)、hUCMSC-CM注射治疗组(B组)及hUCMSC-CMT注射治疗组(C组),结膜下注射为每天1次,连续7 d。于LSCD造模后第3、7天裂隙灯显微镜下观察角膜上皮缺损程度并评分,于LSCD造模后第7、21天裂隙灯显微镜下观察角膜新生血管情况并评分。于LSCD造模后第7、21天应用免疫组织化学染色法检测α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)和眼表黏蛋白5AC(mucin 5AC, Muc-5AC)的表达情况。 结果 造模后3 d,A组小鼠见角膜上皮大片缺失;7 d见角膜缘血管扩张充血,角膜上皮缺损面积大于2/3角膜;21天见角膜缘较多新生血管长入,部分可及2/3角膜直径。B、C各时间点在减少角膜新生血管和修复角膜上皮缺损方面都有明显改善,且C组效果强于B组。免疫组织化学染色观察发现,对照组角膜基质中均无α-SMA表达,角膜上皮层未见Muc-5AC表达。造模后7、21 d,各实验组α-SMA表达均为阳性,B组和C组均低于A组。三个实验组第7天的Muc-5AC平均光密度值组间异无统计学意义,而第21天组间两两比较均具有统计学意义。 结论 结膜下注射hUCMSC-CMT或hUCMSC-CM可以增强角膜缘干细胞功能,促进角膜上皮修复,保护眼表组织;且hUCMSC-CMT对小鼠角膜缘干细胞缺乏的修复和治疗作用强于hUCMSC-CM。

关键词: 肿瘤坏死因子α, 间充质干细胞, 角膜缘干细胞缺乏, 条件培养基

Abstract: Objective To investigate the repairing effect of local application of tumor necrosis factor alpha(TNF-α)-induced human umbilical cord mesenchymal stem cell conditioned medium(hUCMSC-CM)on limbal stem cell deficiency(LSCD)in mice. Methods Primary human umbilical cord mesenchymal stem cells were isolated and cultured in vitro by the adherent tissue block culture method, the fourth generation hUCMSCs were induced with TNF-α, and the TNF-α-induced human umbilical cord mesenchymal stem cell conditioned medium(hUCMSC-CMT)was collected. Forty-eight healthy 6-week-old C57 male mice were randomly divided into the control group(group N)and LSCD experimental group, and the LSCD experimental group model was established by limbal alkali burn and divided into negative PBS injection control group(group A), hUCMSC-CM injection treatment group(group B), and the injection treatment group hUCMSC-CMT(group C)according to the different components of subconjunctival injection after modeling, and the subconjunctival injection was once a day for 7 consecutive days. On the 3rd and 7th days after LSCD modeling, the degree of corneal epithelial defect was observed and scored by slit-lamp microscopy, and corneal neovascularization was observed and scored by slit-lamp microscopy on the 7th and 21st days after LSCD modeling. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin(α-SMA)and ocular surface mucin 5AC(Muc-5AC). Results The mice in group A showed large areas of corneal epithelial defect on day 3 after modeling; obvious congested limbal blood vessels were observed on day 7 after modeling. The corneal epithelial defect developed in more than 2/3 of the cornea. On the 21st day after modeling, more neovascularization in the corneal limbus was observed, and some could reach 2/3 of the corneal diameter. According to the statistical results, both group B and group C had significant improvements in reducing corneal neovascularization and repairing corneal epithelial defect, and the curative effect was stronger in group C than in group B. Immunohistochemical staining showed that there was no expression of α-SMA in the corneal stroma and no expression of Muc-5AC in the corneal epithelial layer of the normal group. At 7 and 21 days after modeling, the cornea of each experimental group showed positive expression of α-SMA. The expression of α-SMA was significantly lower in groups B and C than in group A. The average optical density values of Muc-5AC in three experimental groups on the 7th day after modeling were not significantly different. Conclusion Subconjunctival injection of hUCMSC-CMT or hUCMSC-CM could enhance the function of limbal stem cells, promote corneal epithelial repair, and protect ocular surface tissues. In addition, hUCMSC-CMT had stronger repairing and therapeutic effects on limbal stem cell deficiency in mice than hUCMSC-CM.

Key words: Tumor necrosis factor alpha, Human umbilical cord mesenchymal stem cells, Limbal stem cell deficiency, Conditioned medium

中图分类号: 

  • R772.2
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