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中华胸部外科电子杂志 ›› 2024, Vol. 11 ›› Issue (03) : 180 -187. doi: 10.3877/cma.j.issn.2095-8773.2024.03.06

论著

aFGF修饰自体成纤维细胞治疗食管吻合口瘘的实验研究
徐立1, 阎岩2,()   
  1. 1. 200433 上海,同济大学附属上海市肺科医院
    2. 310007 杭州,中国人民解放军联勤保障部队第九○三医院
  • 收稿日期:2024-06-22 修回日期:2024-07-13 接受日期:2024-08-08 出版日期:2024-08-28
  • 通信作者: 阎岩
  • 基金资助:
    浙江省医药卫生科技计划项目面上项目计划(2020KY774)

aFGF-modified autologous fibroblasts therapy improves the outcome of esophageal anastomotic leakage

Li Xu1, Yan Yan2,()   

  1. 1. Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Shanghai Tongji University, Shanghai 200433, China
    2. Department of Cardiothoracic Surgery, No. 903 Hospital of Chinese People’s Liberation Army, Hangzhou 310007, China
  • Received:2024-06-22 Revised:2024-07-13 Accepted:2024-08-08 Published:2024-08-28
  • Corresponding author: Yan Yan
引用本文:

徐立, 阎岩. aFGF修饰自体成纤维细胞治疗食管吻合口瘘的实验研究[J]. 中华胸部外科电子杂志, 2024, 11(03): 180-187.

Li Xu, Yan Yan. aFGF-modified autologous fibroblasts therapy improves the outcome of esophageal anastomotic leakage[J]. Chinese Journal of Thoracic Surgery(Electronic Edition), 2024, 11(03): 180-187.

目的

验证酸性成纤维细胞生长因子(aFGF)基因修饰的自体成纤维细胞(FB),联合纤维蛋白胶在促进食管吻合口瘘愈合过程中的作用,探究其作为治疗食管癌术后吻合口瘘新方法的可行性。

方法

通过兔颈段食管切开缝合置管法构建食管吻合口模型,同时获取皮肤组织,采用酶消化方法原代培养自体皮肤FB。采用aFGF过表达腺病毒转染FB,并通过蛋白免疫印迹和酶联免疫吸附实验检测修饰细胞中aFGF的表达和分泌情况。将20只实验兔随机分为aFGF修饰FB联合纤维蛋白胶治疗组(aFGF+FBs组)和单纯纤维蛋白胶治疗组(对照组)。通过大体标本和颈部食管增强计算机断层扫描(CT)观察吻合口瘘的愈合情况。采用感染率和生存曲线比较aFGF+FBs组和对照组的治疗效果。

结果

本实验成功构建了兔颈段食管吻合口瘘模型。修饰后的自体皮肤FB能够显著表达和分泌aFGF。与对照组相比,aFGF+FBs组的感染率低(20% vs. 70%),生存率获得了显著提升(P<0.05)。

结论

aFGF修饰的自体FB联合纤维蛋白胶能够显著降低食管吻合口瘘的感染率,促进瘘口愈合,降低死亡率。因此,本方法为防治食管吻合口瘘这一临床亟待解决的难题提供了有希望的替代方案。

Objective

To verify the role of autologous fibroblasts modified with acidic fibroblast growth factor (aFGF) gene, combined with fibrin glue, in promoting the treatment of esophageal anastomotic leakage through a new animal model, and to explore the feasibility of this approach as a new method for treating anastomotic leakage after esophageal cancer surgery.

Methods

The esophageal anastomotic model was established by cutting and suturing the cervical esophagus of rabbits, and the skin tissue was obtained. The primary culture of autologous skin fibroblasts was performed by enzymatic digestion method. The aFGF overexpression adenovirus was transfected into fibroblasts, and the expression and secretion of aFGF in the modified cells were detected by Western blot and enzyme-linked immunosorbent assay. Twenty rabbits were randomly divided into two groups: aFGF modified fibroblasts combined with fibrin glue treatment group (aFGF + FBS group) and fibrin glue treatment group (control group) . The healing of anastomotic fistula was observed by gross specimens and cervical esophageal enhanced CT. The infection rate and survival curve were used to compare the therapeutic effect of aFGF + FBs group and control group.

Results

A rabbit model of cervical esophageal anastomotic leakage was successfully established. The modified autologous skin fibroblasts can significantly express and secrete aFGF. Compared with the control group, the infection rate of aFGF + FBs group was lower (20% vs. 70%) , and the survival rate was significantly improved (P<0.05) .

Conclusions

The application of autologous fibroblasts modified with aFGF, in conjunction with fibrin glue, can significantly reduce the infection rate of esophageal anastomotic leaks, facilitate the healing of the leaks, and decrease mortality. Consequently, this method offers a promising alternative for addressing the clinical challenge of esophageal anastomotic leaks, which urgently requires resolution.

图1 皮肤FB的分离培养和鉴定。A:兔自体FB的原代培养;B:蛋白质印迹法检测vimentin蛋白表达量;C:荧光显微镜下观察vimentin蛋白的表达和分布。Control为阴性对照。#:P>0.05。PC:阳性对照;FB:成纤维细胞
图2 腺病毒转染的成纤维细胞能够有效表达和分泌aFGF。A:光镜和相同视野的荧光显微镜观察修饰的细胞,可见大部分细胞均表达绿色荧光蛋白;B:病毒载体转染细胞后的时间梯度蛋白质印迹法的定量结果;C:细胞培养液的aFGF-酶联免疫吸附实验结果。**:P<0.01。aFGF:酸性成纤维细胞生长因子
图3 兔颈部吻合口瘘模型的构建和治疗
图4 颈部吻合口瘘的组织标本和CT检查。A:aFGF+FBs组瘘口周围大体标本;B:对照组瘘口周围大体标本;C:对照组颈段食管CT影像,白色箭头指示瘘口周围的高信号区;D:治疗组颈段食管CT影像。aFGF:酸性成纤维细胞生长因子;FB:成纤维细胞;CT:计算机断层扫描
图5 aFGF+FBs联合纤维蛋白胶及单纯纤维蛋白胶治疗食管吻合口瘘的感染率(A)及生存率(B)比较。aFGF:酸性成纤维细胞生长因子;FB:成纤维细胞
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