Cajal样间质细胞参与胆管梗阻致急性胆囊炎胆囊动力变化的机制研究
发布时间：2018-07-13 17:48
【摘要】：背景和目的：急性非结石性胆囊炎(acute acalculous cholecystitis,AAC)的临床表现无特异性、诊断困难,具有起病急、进展快的特点,病死率高达30%。其发病机制包括胆汁淤积、缺血-再灌注损伤、内源性凝血因子的激活以及细菌感染等因素,而胆囊平滑肌收缩功能减弱是AAC的重要标志。胆总管结扎(commonbile duct ligation, CBDL)术是目前公认的构建AAC的有效动物模型,其病理特征与临床AAC患者的病理特征非常相符。Cajal间质细胞(interstitial cells ofCajal,ICC)是胃肠道慢波活动的起搏细胞,对胃肠道平滑肌的节律运动具有重要调控作用,ICC胞内自发的周期性钙(Ca2+)震荡是产生ICC起搏电流的重要机制。近年来,人们在动物及人体胆囊都发现了与胃肠道类似的ICC,并观察到胆囊ICC内也存在节律性Ca2+振荡,提示胆囊ICC也可能参与节律性电活动的产生和传播,介导胆囊自发性节律的产生和调节平滑肌收缩活动。研究表明,AAC发生早期,胆囊ICC超微结构出现变化。中性粒细胞是机体早期炎症反应的重要炎症细胞、处于炎症的核心位置,中性粒细胞与胆囊ICC结构损伤的关系尚不明确。因此,本研究将采用CBDL术构建AAC动物模型,观察胆囊ICC与中性粒细胞的关系,并探讨ICC参与AAC胆囊平滑肌动力减退的可能机制。 方法：首先,本研究采用健康成年豚鼠,通过CBDL方法构建AAC动物模型；观察CBDL24h、48h、72h各时间点,豚鼠胆囊胆汁的颜色、体积变化,并检测各时间点体内血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、胆红素(Bilirubin)等肝功能指标；通过胆囊HE染色及炎症评分(inflammation score)判断胆囊的炎症情况；采用RM6240多道生理信号采集系统记录CBDL前及CBDL后各时间点,胆囊平滑肌分别对乙酰胆碱(Ach,10-5mol/l)、八肽胆囊收缩素(CCK-8,10-6mol/l)、氯化钾(KCl,60mmol/l)的收缩反应性；电镜观察CBDL前及CBDL后各时间点ICC超微结构变化及其与中性粒细胞的联系。 然后,选取CBDL后电镜下中性粒细胞与胆囊ICC关系密切的时间点,即CBDL-48h组作为重点研究对象,活体内预先注射抗中性粒细胞抗体(anti-polymorphonuclear antibody, anti-PMN)以减少体内中性粒细胞数量及其组织浸润程度；动物分为正常组(normal-2)、假手术48h(sham-2)组、胆总管结扎48h(CBDL-2)组以及anti-PMN+旦总管结扎48h(anti-PMN)组,各组豚鼠12只；观察各组动物胆囊胆汁的颜色及体积变化,同时检测各组血清ALT、AST及Bilirubin水平；HE染色及炎症评分评价各组胆囊的炎症程度；测定循环血液中中性粒细胞数量以及髓氧过化物酶(MPO)活性以判断中性粒细胞的组织浸润情况；张力换能器记录各组胆囊平滑肌分别对Ach(10-5mol/l)、CCK-8(10-6mo1/l)、KCl(60mmol/l)的收缩反应性；电镜观察各组胆囊ICC超微结构。 最后,选择出生10-15d的豚鼠,无菌条件下取出胆囊,钝性分离胆囊平滑肌肌条,采用组织块培养法进行胆囊ICC的离体培养,倒置显微镜下观察胆囊ICC的形态；采用ICC特异性的酪氨酸激酶受体c-kit蛋白进行免疫荧光染色,以鉴定胆囊ICC；为下一步记录胆囊ICC的自发性电活动奠定基础。 结果：CBDL术后的豚鼠活动度明显减少；随着CBDL时间延长,胆囊胆汁颜色由正常浅黄色变成深黄色,最后变成墨绿色；CBDL各组胆汁体积、血清肝功能指标ALT、AST及Bilirubin分别较对应时间组normal组、sham组显著升高,差异有统计学意义(P0.05)；HE染色后,CBDL各组胆囊可见炎性细胞浸润、组织水肿、血管扩张、充血,CBDL组炎症评分与对应时间组normal、sham相比较显著增加,差异有统计学意义(P0.05)；CBDL后,各组胆囊平滑肌对Ach、CCK-8、KCI的收缩反应性分别较对应时间组normal、sham显著降低,差异有统计学意义(P0.05)；电镜显示,随着梗阻时间延长,胆囊ICC超微结构损伤加重；CBDL48h后,可见中性粒细胞胞胞浆内充满大量颗粒,与受损ICC胞体及ICC突起之间紧密接触。 CBDL-2组、anti-PMN组的胆汁体积以及血清AST、Bilirubin水平分别较normal-2及sham-2组显著增加,差异有统计学意义(P0.05),而CBDL-2组与anti-PMN组之间差异无统计学意义(P0.05)；HE染色、炎症评分以及MPO活性检测显示,CBDL-2组.anti-PMN组的炎症程度和MPO活性分别较normal-2、sham-2显著增加,差异有统计学意义(P0.05),其中anti-PMN组与CBDL-2相比较,炎症程度及MPO活性明显降低,差异有统计学意义(P0.05)；肌条张力实验显示,CBDL-2组、anti-PMN组对Ach、CCK-8、KC1的收缩反应性分别较normal-2及sham-2组显著降低,差异有统计学意义(P0.05),与CBDL-2组相比,anti-PMN组对Ach、CCK-8、KCl的收缩反应性均显著提高,差异有统计学意义(P0.05)[Ach.(0.70±0.13vs.0.29±0.03)；CCK-8：(0.26±0.06vs.0.12±0.01)；KCl(0.90±0.18vs.0.34±0.08)]。电镜结果显示,anti-PMN组胆囊ICC肿胀及内质网扩张程度较CBDL-2组减轻,ICC突起不但没有减少、消失,反而有增多的趋势。与CBDL-2组相比,anti-PMN组ICC与ICC之间、ICC与平滑肌之间的缝隙连接明显增加。 采用组织块原代培养3-4d后,ICC胞体呈三角形或椭圆形,有1-2个突起,ICC可单个散在生长,也可以聚集成簇状生长；ICC可与ICC或平滑肌细胞相连接,但未见网络状结构；c-kit免疫荧光表明,3-4d后培养的大多数细胞表面c-kit蛋白表达呈阳性。 结论：在AAC模型豚鼠中,anti-PMN可以降低中性粒细胞在炎性胆囊的浸润密度,减轻胆囊组织的炎症反应；同时,anti-PMN可以缓解胆囊ICC超微结构受损程度,提高胆囊平滑肌对的Ach、CCK-8、KCl的收缩反应；浸润于胆囊组织的中性粒细胞可能先作用于胆囊ICC,继而累及胆囊平滑肌,从而出现胆囊平滑肌收缩功能减退；采用组织块原代培养法可以成功培养出胆囊ICC。
[Abstract]:BACKGROUND & OBJECTIVE : The clinical manifestation of acute acalculous cholecystitis ( AAC ) is not specific , it is difficult to diagnose , it has the characteristics of rapid onset and rapid progress .
Methods : First , healthy adult guinea pigs were used to construct AAC animal model by CBDL method .
The changes of color and volume of gallbladder bile in guinea pig were observed at 24 h , 48 h and 72 h , and the indexes of ALT , AST and bilirubin were detected in every time point .
The inflammation of gallbladder was determined by HE staining and inflammation score .
After CBDL and CBDL were recorded by RM6240 multi - channel physiological signal acquisition system , the contraction reactivity of acetylcholine ( 10 - 5 mol / l ) , CCK - 8 ( 10 - 6 mol / l ) , potassium chloride ( KCl , 60 mmol / l ) was observed in the gallbladder smooth muscle .
The ultrastructural changes of ICC and their association with neutrophils were observed by electron microscope .
Then , CBDL - 48h group was selected as the focal point in CBDL - 48h group , and anti - neutrophil antibody ( anti - PMN ) was injected into the living body to reduce the number of neutrophils in vivo and the degree of tissue infiltration .
The animals were divided into normal group ( normal - 2 ) , sham operation 48h ( sham - 2 ) group , bile duct ligation 48h ( CBDL - 2 ) group and anti - PMN + day manifold ligation 48h ( anti - PMN ) group .
The color and volume of gallbladder bile in each group were observed , and the levels of ALT , AST and AST in each group were detected .
HE staining and inflammatory score were used to evaluate the degree of inflammation in each group .
determining the neutrophil count and myeloperoxidase ( MPO ) activity in circulating blood to determine the tissue infiltration of neutrophils ;
The contractile responses of each group of gallbladder smooth muscle were recorded by the tension transducer ( 10 - 5 mol / l ) , CCK - 8 ( 10 - 6 mol / l ) and KCl ( 60 mmol / l ) .
The ultrastructure of ICC was observed by electron microscope .
Finally , the guinea pigs born 10 - 15d were selected , the gallbladder was taken out under aseptic conditions , the smooth muscle strips of the gallbladder were isolated blunt , the isolated culture of the gallbladder ICC was carried out by using the tissue block culture method , and the morphology of the gallbladder ICC was observed under an inverted microscope ;
Immunofluorescence staining was performed using the ICC - specific tyrosine kinase receptor c - kit protein to identify the gallbladder ICC ;
To lay the foundation for the next step of recording spontaneous electrical activity of the gallbladder ICC .
Results : The activity of guinea pigs was significantly decreased after CBDL operation .
With the prolongation of CBDL time , the color of gallbladder bile changed from normal light yellow to dark yellow , finally became dark green ;
In CBDL group , the volume of bile and serum liver function index were significantly higher than that in normal group and sham group ( P0.05 ) .
After HE staining , the inflammatory cell infiltration , tissue edema , blood vessel dilatation , congestion and CBDL were significantly increased in CBDL group compared with normal and sham phase in CBDL group ( P0.05 ) .
After CBDL , the contraction reactivity of the gallbladder smooth muscle of each group was significantly lower than that in the corresponding time group , and the difference was significant ( P0.05 ) .
Electron microscope showed that the ultrastructure of gallbladder was aggravated with the prolongation of obstruction time .
After 48 hours of CBDL48 , a large amount of particles were found in the cytoplasm of neutrophils , which was in close contact with the damaged ICC cells and the ICC protrusions .
Compared with normal - 2 and sham - 2 groups , CBDL - 2 and anti - PMN increased significantly ( P0.05 ) , but there was no significant difference between CBDL - 2 and anti - PMN groups ( P0.05 ) .
Compared with CBDL - 2 , the inflammatory degree and MPO activity of the anti - PMN group were significantly lower than those of CBDL - 2 ( P0.05 ) .
Compared with CBDL - 2 group , the contraction reactivity of the anti - PMN group was significantly higher than that in normal - 2 and sham - 2 groups ( P0.05 ) .
CCK-8锛，
本文编号：2120247
[Abstract]:BACKGROUND & OBJECTIVE : The clinical manifestation of acute acalculous cholecystitis ( AAC ) is not specific , it is difficult to diagnose , it has the characteristics of rapid onset and rapid progress .
Methods : First , healthy adult guinea pigs were used to construct AAC animal model by CBDL method .
The changes of color and volume of gallbladder bile in guinea pig were observed at 24 h , 48 h and 72 h , and the indexes of ALT , AST and bilirubin were detected in every time point .
The inflammation of gallbladder was determined by HE staining and inflammation score .
After CBDL and CBDL were recorded by RM6240 multi - channel physiological signal acquisition system , the contraction reactivity of acetylcholine ( 10 - 5 mol / l ) , CCK - 8 ( 10 - 6 mol / l ) , potassium chloride ( KCl , 60 mmol / l ) was observed in the gallbladder smooth muscle .
The ultrastructural changes of ICC and their association with neutrophils were observed by electron microscope .
Then , CBDL - 48h group was selected as the focal point in CBDL - 48h group , and anti - neutrophil antibody ( anti - PMN ) was injected into the living body to reduce the number of neutrophils in vivo and the degree of tissue infiltration .
The animals were divided into normal group ( normal - 2 ) , sham operation 48h ( sham - 2 ) group , bile duct ligation 48h ( CBDL - 2 ) group and anti - PMN + day manifold ligation 48h ( anti - PMN ) group .
The color and volume of gallbladder bile in each group were observed , and the levels of ALT , AST and AST in each group were detected .
HE staining and inflammatory score were used to evaluate the degree of inflammation in each group .
determining the neutrophil count and myeloperoxidase ( MPO ) activity in circulating blood to determine the tissue infiltration of neutrophils ;
The contractile responses of each group of gallbladder smooth muscle were recorded by the tension transducer ( 10 - 5 mol / l ) , CCK - 8 ( 10 - 6 mol / l ) and KCl ( 60 mmol / l ) .
The ultrastructure of ICC was observed by electron microscope .
Finally , the guinea pigs born 10 - 15d were selected , the gallbladder was taken out under aseptic conditions , the smooth muscle strips of the gallbladder were isolated blunt , the isolated culture of the gallbladder ICC was carried out by using the tissue block culture method , and the morphology of the gallbladder ICC was observed under an inverted microscope ;
Immunofluorescence staining was performed using the ICC - specific tyrosine kinase receptor c - kit protein to identify the gallbladder ICC ;
To lay the foundation for the next step of recording spontaneous electrical activity of the gallbladder ICC .
Results : The activity of guinea pigs was significantly decreased after CBDL operation .
With the prolongation of CBDL time , the color of gallbladder bile changed from normal light yellow to dark yellow , finally became dark green ;
In CBDL group , the volume of bile and serum liver function index were significantly higher than that in normal group and sham group ( P0.05 ) .
After HE staining , the inflammatory cell infiltration , tissue edema , blood vessel dilatation , congestion and CBDL were significantly increased in CBDL group compared with normal and sham phase in CBDL group ( P0.05 ) .
After CBDL , the contraction reactivity of the gallbladder smooth muscle of each group was significantly lower than that in the corresponding time group , and the difference was significant ( P0.05 ) .
Electron microscope showed that the ultrastructure of gallbladder was aggravated with the prolongation of obstruction time .
After 48 hours of CBDL48 , a large amount of particles were found in the cytoplasm of neutrophils , which was in close contact with the damaged ICC cells and the ICC protrusions .
Compared with normal - 2 and sham - 2 groups , CBDL - 2 and anti - PMN increased significantly ( P0.05 ) , but there was no significant difference between CBDL - 2 and anti - PMN groups ( P0.05 ) .
Compared with CBDL - 2 , the inflammatory degree and MPO activity of the anti - PMN group were significantly lower than those of CBDL - 2 ( P0.05 ) .
Compared with CBDL - 2 group , the contraction reactivity of the anti - PMN group was significantly higher than that in normal - 2 and sham - 2 groups ( P0.05 ) .
CCK-8锛，
本文编号：2120247
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