丝线线结在小鼠腹壁不同解剖层次诱导局部免疫应答的比较研究

Hypersensitivities induced by silk knot at differential levels in murine abdominal wall

目的小儿腹腔镜监视下经皮腹膜外内环结扎术后有时会出现线结反应。本研究旨在探索腹壁不同层次打结与其诱导迟发型超敏反应过程中抗原呈递细胞(APC)和CD4+ Th细胞浸润的相关性。方法在同一Balb/C小鼠腹壁自左向右分别纵向剪开3个皮肤切口(0.5 cm长),分别经切口处用无菌平镊提起全腹壁,采用4-0带针慕斯线进针贯穿腹壁至壁层腹膜内,经切口直视下经原针孔采用钩针将带针丝线折返钩出,经原线道出针,分别打结于皮下、肌层内和腹膜外间隙。术后14 d取材打结部位腹壁标本,连续冰冻切片进行组织学观察和分析。结果HE染色显示各组线准确位于腹壁不同解剖层次内(皮下、肌内和腹膜外间隙组)。APC膜表面表达抗原肽-MHC Ⅰ/Ⅱ类分子复合物的免疫组织化学显示皮下组线结周围APC浸润最明显,腹膜外间隙组线结周围浸润最轻微。半定量检测结果显示:皮下组(2.23±0.31)与肌层组(1.67±0.12)比较,差异有统计学意义(P=0.043 9);肌层组与腹膜外间隙组(0.89±0.06)比较,差异亦有统计学意义(P=0.002 0)。三组线结周围CD4+ Th细胞免疫组织化学检测显示皮下组线结周围CD4+分子表达最明显,腹膜外间隙组线结周围表达最轻微。半定量检测结果显示:皮下组(1.79±0.17)与肌层组(1.42±0.20)比较,差异有统计学意义(P=0.009 5);肌层组与腹膜外间隙组(0.51±0.15)比较,差异亦有统计学意义(P=0.002 8)。结论丝线位于不同层次腹壁打结与其诱导的迟发型超敏反应过程中APC和CD4+ Th细胞浸润具有相关性;线结位于深层腹膜外间隙引起超敏反应轻微,皮下浅层线结引起超敏反应最明显。提示对于腹腔镜监视下经皮腹膜外内环结扎术,将线结打在腹膜外间隙有利于降低线结诱导的超敏反应和超敏反应相关并发症的发生率。

Objective Silk knot induced inflammatory response occurs after percutaneous extracorporeal knotting for laparoscopic inguinal inner ring closure in children. The present study was intended to explore the correlation between antigen-presenting cell (APC) and CD4+ Th cell infiltrations during knot-induced delayed hepersensitivity and differential knot locations in murine abdominal wall. Methods Three 0.5 cm-long vertical and parallel incisions in abdominal wall were created in the same Balb/C mice, followed by initialized stitch in the same path using MERSILK non-absorbable suture. The knots were located in subcutaneous, intramuscular and extra peritoneal space in abdominal wall. The knot segments in each group were harvested at Day 14 post-operation. Hematoxylin & eosin (H&E) and imunohistochemical (IHC) stains were employed for examining the location of knots in each group and determining the infiltration of APC/CD4+ Th cells. Results H&E staining indicated that knot was created accurately in subcutaneous, intramuscular and extra peritoneal space in abdominal wall. IHC stain indicated a less infiltration of APC and CD4+ Th cells in extra peritoneal space in abdominal wall. On the contrary, more could be noted in subcutaneous layer (0.89±0.06). Quantitative evaluation revealed that significant differences in APC infiltration existed between subcutaneous and intramuscular layer groups [2.23±0.31 vs. 1.67±0.12, P=0.043 9] and between intramuscular and extra peritoneal space groups (P=0.002 0). Regarding CD4+ Th cell infiltration, quantitative evaluations revealed that significant difference between subcutaneous and intramuscular layer groups [1.79±0.17 vs. 1.42±0.20, P=0.009 5] and between intramuscular layer and extra peritoneal space groups [0.51±0.15, P=0.002 8]. Conclusions Correlation exists between APC and CD4+ Th cell infiltrations during knot-induced delayed type hepersensitivity and differential knot locations in murine abdominal wall. Knot in extra peritoneal space induces milder hypersensitivity in abdominal wall than other subcutaneous and intramuscular layers. Therefore knot in extra peritoneal space is more prone to lower knot-induced hypersensitivity and inflammation. Knotting in extra peritoneal space contributes to fewer complications after laparoscopic inguinal inner ring closure in children.