肠道不同层次对合缝合与支架法吻合的动物实验研究
摘要
目的：研究大鼠肠道壁的不同层次之间互相对合缝合与捆绑式支架法吻合是否安全可行,其肠道吻合口愈合结果有无差异；采用捆绑式支架法肠吻合有无优势。
方法：以SD大鼠为研究对象,随机分为5组,每组24只大鼠,观察4个不同的时间点(术后3天、7天、14天、30天),每个时间点6只大鼠,采用不同层次对合缝合(浆膜层与浆膜层对合缝合SM vs SM组、粘膜层与粘膜层对合缝合MMvs MM组、浆膜层与粘膜层对合缝合SM vs MM组)、相同层次对合缝合(单纯缝合吻合Control组)以及捆绑式支架法吻合(Stent组)的方式,行大鼠小肠端端吻合术,对比各组吻合方式所需的缝合或吻合时间,术后一般状况,术后腹腔粘连评分,术后各个观测点吻合口的大体愈合情况,包含吻合口肠段的造影结果,吻合口爆破压,羟脯氨酸含量,吻合口HE染色、Masson染色以及TGF-β1、b-FGF、 α-SMA的免疫组化染色,TGF-β1、b-FGF.α-SMA的mRNA的表达量的RT-PCR测定等,通过统计学数据分析结果,得出结论。
结果：总共完成120只大鼠的实验,其中两只大鼠在实验过程中死亡：一只手术日晚被同笼大鼠咬死,另一只术中麻醉过量致死,其余大鼠术后均正常存活。各组大鼠的平均吻合时间测定结果显示：SM vs SM组、MM vs MM组和SM vs MM组均比Control组所需吻合时间长,而Control组比Stent组吻合所需时间长,结果具有显著统计学意义(P<0.01),可见支架法吻合方便快速,比传统的各种手工缝合方法均简单,易于掌握并推广。各组大鼠的平均腹腔粘连评分结果显示：MM vs MM组和Stent组的腹腔粘连评分高于Control组,结果具有统计学意义(P<0.05),说明粘膜层与粘膜层对合缝合以及支架法吻合相对更容易导致腹腔粘连,但短期内不导致梗阻,而浆膜层与浆膜层对合缝合则腹腔粘连较轻,提示浆膜层外翻的吻合原则可以减少腹腔内产生粘连的几率。各组大鼠的吻合口肠段造影结果显示：在术后30天各组吻合口通畅,均未见明显狭窄及梗阻,也未见肠瘘发生,说明肠道的不同层次对合缝合以及支架法吻合对大鼠肠道的愈合是安全可行的,不会导致明显的肠吻合口瘘以及肠道狭窄和梗阻。吻合口爆破压结果显示：在术后7天和14天,SM vs SM组、MM vs MM组和SM vs MM组的平均爆破压均比Control组的要低,结果具有统计学意义(P<0.05)；在术后3天和术后30天,各组吻合口的爆破压与Control组相比无明显差异(P-0.05),说明在力学愈合上,在术后早期,各种缝合或者吻合方式对大鼠肠道的力学性能无明显影响,在术后中期开始相同层次对合缝合法和支架法吻合的方式比其他三种缝合方式在力学性能上更牢固,但是在后期,各种缝合或者吻合方式均能达到一致的力学性能,对大鼠肠道的愈合结果不产生影响。吻合口羟脯氨酸含量测定结果显示：每组的平均羟脯氨酸含量随时间的增长逐渐增加；而在各个时间点上各组间吻合口羟脯氨酸含量与Control组相比无统计学差异(／>0,05),说明各组吻合口胶原蛋白的形成过程基本一致,均随着时间的增长而增加,均能正常愈合,且各种缝合方法及支架法吻合方式对胶原蛋白量的生成影响不大。吻合口组织切片HE染色和Masson染色结果显示：通过对组织切片评分量化分析,得出Stent组及Control组相比其余几组炎症反应轻,愈合开始早,但在术后30天时,各组肠吻合口在组织学上基本愈合一致；而吻合口组织的TGF-β1、b-FGF和α-SMA免疫组化染色显示TGF-β1和b-FGF的变化较为一致,随着时间的增长均出现逐渐下降的趋势,而α-SMA呈现先增长后下降的趋势,各个组间的差异无统计学意义(／>0,05)；RT-PCR对吻合口TGF-β1、b-FGF和α-SMA的mRNA含量测定结果与免疫组化结果基本一致；且免疫组化结果和RT-PCR的结果与文献报道一致,说明在TGF-β1.b-FGF和α-SMA的表达上,本研究的几种肠道缝合方法以及捆绑式支架吻合法对大鼠肠道的愈合无显著性差异。
结论：1、肠壁不同层次对合缝合以及支架法吻合对大鼠肠道的愈合是安全可行的。2、从力学愈合上来看,肠道壁相同层次对合缝合和不同层次对合缝合对肠道的愈合在早期无明显影响,中期相同层次对合缝合具有更好的力学性能,而后期则各种方式均具有相同的力学性能。从组织学愈合上来看,肠道壁相同层次对合缝合能够更早开始组织修复过程,但是在后期几乎无吻合口愈合的差异,各种缝合方法及支架法吻合愈合结果一致。3、肠道支架法吻合在肠道愈合过程中在力学愈合上和组织愈合上与按相同层次对合缝合的肠道缝合方法基本一致,并且具有吻合方式简单、快速的优点,无需缝合,易于推广。
Purpose This experiment verifies whether there are differences between different-layer suture and the same-layer suture anastomosis of small intestine and whether there are advantages of stent anastomosis compared with traditional hand-sewn method.
Methods One hundred and twenty male Sprague-Dawley rats (8-week-old,270Π30g) were randomly divided into five groups(n=6):Control group(use simple interrupted suture, the same-layer anastomosis), SM vs SM group(use interrupted inverting suture, serous membrane to serous membrane anastomosis), MM vs MM group(use interrupted everting suture, mucous membrane to mucous membrane anastomosis), SM vs MM group(use intussusception-like suture, serous membrane to mucous membrane anastomosis), and Stent group (use stent anastomosis). Time to perform the anastomosis, mortality and complications, abdominal adhesion scores, bursting pressures, hydroxyproline contents were determined at postoperative days3d,7d,14d,30d. Intestinal contrast imaging under X-rays was carried out at postoperative days30in vitro. Anastomotic tissues were histologically analyzed under the hematoxylin-eosin(HE) staining, Masson staining, the immunohistochemical staining of anti-TGF-β1, anti-b-FGF and anti-α-SMA. mRNA expressions of TGF-β1, b-FGF and α-SMA were evaluated as well via real-time reverse transcription polymerase chain reactions(RT-PCR).
Results Two rats died during the experiments, one was ate off by others the next day, and another died of over dose of anesthetic drug in operation. All of the other rats had no complications during the experiments. Anastomotic time of Control group, SM vs SM group, MM vs MM group, SM vs MM group and stent group were3.96±1.803min,6.75±1.113min,6.29±1.042min,6.54±1.103min,1.75±0.794min, respectively, stent anastomosis is an easy and convienient method as a result it needs least time to perform, which has significant differences statistically(P<0.01). Abdominal adhesion scores of Control group, SM vs SM group, MM vs MM group, SM vs MM group and stent group are1.00±0.905,0.83±0.702,1.87±0.815,1.13±0.741,1.46±0.509, respectively, method of serous membrane to serous membrane suture anastomosis causes least abdominal adhesion, mucous membrane to mucous membrane suture anastomosis and stent anastomosis seem to cause more abdominal adhesion than other groups, the results are significant different statistically(P<0.01). Bursting pressure show that there were no difference among5groups in the early(POD3d) and last stage(POD30d)(P>0.01), but Control group and stent group seemed stronger than other group in the middle stage(POD7d and14d)(P<0.01). Determination of hydroxyproline contents show hydroxyproline contents go up gradually over time but there were no significant differences compared with Control group at the same period(P>0.01). Intestinal contrast imaging under X-rays show none of a group had leakage or stenosis, every group has the same results on this respect. Intestine tissue histological studies show that Control group and stent group had less inflammation and began healing earlier than other groups, but each group reached the same end at POD30d, there are no differences statistically. The immunohistochemical staining of anti-TGF-β1, anti-b-FGF and anti-α-SMA in each group show that TGF-β1and b-FGF decreased gradually over time while α-SMA increased at beginning then decreased after that, and there were no significant differences compared with Control group on each time point(P>0.01); RT-PCR assay of mRNA expressions of TGF-β1, b-FGF and α-SMA show almost the same results as the immunohistochemical staining; results of expressions of TGF-β1, b-FGF and a-SMA were more or less the same as reported in literatures.
Conclusion Different-layer suture anastomosis makes an equal healing ending to the same-layer suture which begins the healing process earlier. Stent anastomosis is a safe and feasible method of intestine anastomosis which makes the anstomosis easier and faster.
方法：以SD大鼠为研究对象,随机分为5组,每组24只大鼠,观察4个不同的时间点(术后3天、7天、14天、30天),每个时间点6只大鼠,采用不同层次对合缝合(浆膜层与浆膜层对合缝合SM vs SM组、粘膜层与粘膜层对合缝合MMvs MM组、浆膜层与粘膜层对合缝合SM vs MM组)、相同层次对合缝合(单纯缝合吻合Control组)以及捆绑式支架法吻合(Stent组)的方式,行大鼠小肠端端吻合术,对比各组吻合方式所需的缝合或吻合时间,术后一般状况,术后腹腔粘连评分,术后各个观测点吻合口的大体愈合情况,包含吻合口肠段的造影结果,吻合口爆破压,羟脯氨酸含量,吻合口HE染色、Masson染色以及TGF-β1、b-FGF、 α-SMA的免疫组化染色,TGF-β1、b-FGF.α-SMA的mRNA的表达量的RT-PCR测定等,通过统计学数据分析结果,得出结论。
结果：总共完成120只大鼠的实验,其中两只大鼠在实验过程中死亡：一只手术日晚被同笼大鼠咬死,另一只术中麻醉过量致死,其余大鼠术后均正常存活。各组大鼠的平均吻合时间测定结果显示：SM vs SM组、MM vs MM组和SM vs MM组均比Control组所需吻合时间长,而Control组比Stent组吻合所需时间长,结果具有显著统计学意义(P<0.01),可见支架法吻合方便快速,比传统的各种手工缝合方法均简单,易于掌握并推广。各组大鼠的平均腹腔粘连评分结果显示：MM vs MM组和Stent组的腹腔粘连评分高于Control组,结果具有统计学意义(P<0.05),说明粘膜层与粘膜层对合缝合以及支架法吻合相对更容易导致腹腔粘连,但短期内不导致梗阻,而浆膜层与浆膜层对合缝合则腹腔粘连较轻,提示浆膜层外翻的吻合原则可以减少腹腔内产生粘连的几率。各组大鼠的吻合口肠段造影结果显示：在术后30天各组吻合口通畅,均未见明显狭窄及梗阻,也未见肠瘘发生,说明肠道的不同层次对合缝合以及支架法吻合对大鼠肠道的愈合是安全可行的,不会导致明显的肠吻合口瘘以及肠道狭窄和梗阻。吻合口爆破压结果显示：在术后7天和14天,SM vs SM组、MM vs MM组和SM vs MM组的平均爆破压均比Control组的要低,结果具有统计学意义(P<0.05)；在术后3天和术后30天,各组吻合口的爆破压与Control组相比无明显差异(P-0.05),说明在力学愈合上,在术后早期,各种缝合或者吻合方式对大鼠肠道的力学性能无明显影响,在术后中期开始相同层次对合缝合法和支架法吻合的方式比其他三种缝合方式在力学性能上更牢固,但是在后期,各种缝合或者吻合方式均能达到一致的力学性能,对大鼠肠道的愈合结果不产生影响。吻合口羟脯氨酸含量测定结果显示：每组的平均羟脯氨酸含量随时间的增长逐渐增加；而在各个时间点上各组间吻合口羟脯氨酸含量与Control组相比无统计学差异(／>0,05),说明各组吻合口胶原蛋白的形成过程基本一致,均随着时间的增长而增加,均能正常愈合,且各种缝合方法及支架法吻合方式对胶原蛋白量的生成影响不大。吻合口组织切片HE染色和Masson染色结果显示：通过对组织切片评分量化分析,得出Stent组及Control组相比其余几组炎症反应轻,愈合开始早,但在术后30天时,各组肠吻合口在组织学上基本愈合一致；而吻合口组织的TGF-β1、b-FGF和α-SMA免疫组化染色显示TGF-β1和b-FGF的变化较为一致,随着时间的增长均出现逐渐下降的趋势,而α-SMA呈现先增长后下降的趋势,各个组间的差异无统计学意义(／>0,05)；RT-PCR对吻合口TGF-β1、b-FGF和α-SMA的mRNA含量测定结果与免疫组化结果基本一致；且免疫组化结果和RT-PCR的结果与文献报道一致,说明在TGF-β1.b-FGF和α-SMA的表达上,本研究的几种肠道缝合方法以及捆绑式支架吻合法对大鼠肠道的愈合无显著性差异。
结论：1、肠壁不同层次对合缝合以及支架法吻合对大鼠肠道的愈合是安全可行的。2、从力学愈合上来看,肠道壁相同层次对合缝合和不同层次对合缝合对肠道的愈合在早期无明显影响,中期相同层次对合缝合具有更好的力学性能,而后期则各种方式均具有相同的力学性能。从组织学愈合上来看,肠道壁相同层次对合缝合能够更早开始组织修复过程,但是在后期几乎无吻合口愈合的差异,各种缝合方法及支架法吻合愈合结果一致。3、肠道支架法吻合在肠道愈合过程中在力学愈合上和组织愈合上与按相同层次对合缝合的肠道缝合方法基本一致,并且具有吻合方式简单、快速的优点,无需缝合,易于推广。
Purpose This experiment verifies whether there are differences between different-layer suture and the same-layer suture anastomosis of small intestine and whether there are advantages of stent anastomosis compared with traditional hand-sewn method.
Methods One hundred and twenty male Sprague-Dawley rats (8-week-old,270Π30g) were randomly divided into five groups(n=6):Control group(use simple interrupted suture, the same-layer anastomosis), SM vs SM group(use interrupted inverting suture, serous membrane to serous membrane anastomosis), MM vs MM group(use interrupted everting suture, mucous membrane to mucous membrane anastomosis), SM vs MM group(use intussusception-like suture, serous membrane to mucous membrane anastomosis), and Stent group (use stent anastomosis). Time to perform the anastomosis, mortality and complications, abdominal adhesion scores, bursting pressures, hydroxyproline contents were determined at postoperative days3d,7d,14d,30d. Intestinal contrast imaging under X-rays was carried out at postoperative days30in vitro. Anastomotic tissues were histologically analyzed under the hematoxylin-eosin(HE) staining, Masson staining, the immunohistochemical staining of anti-TGF-β1, anti-b-FGF and anti-α-SMA. mRNA expressions of TGF-β1, b-FGF and α-SMA were evaluated as well via real-time reverse transcription polymerase chain reactions(RT-PCR).
Results Two rats died during the experiments, one was ate off by others the next day, and another died of over dose of anesthetic drug in operation. All of the other rats had no complications during the experiments. Anastomotic time of Control group, SM vs SM group, MM vs MM group, SM vs MM group and stent group were3.96±1.803min,6.75±1.113min,6.29±1.042min,6.54±1.103min,1.75±0.794min, respectively, stent anastomosis is an easy and convienient method as a result it needs least time to perform, which has significant differences statistically(P<0.01). Abdominal adhesion scores of Control group, SM vs SM group, MM vs MM group, SM vs MM group and stent group are1.00±0.905,0.83±0.702,1.87±0.815,1.13±0.741,1.46±0.509, respectively, method of serous membrane to serous membrane suture anastomosis causes least abdominal adhesion, mucous membrane to mucous membrane suture anastomosis and stent anastomosis seem to cause more abdominal adhesion than other groups, the results are significant different statistically(P<0.01). Bursting pressure show that there were no difference among5groups in the early(POD3d) and last stage(POD30d)(P>0.01), but Control group and stent group seemed stronger than other group in the middle stage(POD7d and14d)(P<0.01). Determination of hydroxyproline contents show hydroxyproline contents go up gradually over time but there were no significant differences compared with Control group at the same period(P>0.01). Intestinal contrast imaging under X-rays show none of a group had leakage or stenosis, every group has the same results on this respect. Intestine tissue histological studies show that Control group and stent group had less inflammation and began healing earlier than other groups, but each group reached the same end at POD30d, there are no differences statistically. The immunohistochemical staining of anti-TGF-β1, anti-b-FGF and anti-α-SMA in each group show that TGF-β1and b-FGF decreased gradually over time while α-SMA increased at beginning then decreased after that, and there were no significant differences compared with Control group on each time point(P>0.01); RT-PCR assay of mRNA expressions of TGF-β1, b-FGF and α-SMA show almost the same results as the immunohistochemical staining; results of expressions of TGF-β1, b-FGF and a-SMA were more or less the same as reported in literatures.
Conclusion Different-layer suture anastomosis makes an equal healing ending to the same-layer suture which begins the healing process earlier. Stent anastomosis is a safe and feasible method of intestine anastomosis which makes the anstomosis easier and faster.
引文
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