Cajal樣間質(zhì)細(xì)胞參與膽管梗阻致急性膽囊炎膽囊動(dòng)力變化的機(jī)制研究
發(fā)布時(shí)間：2018-07-13 17:48
【摘要】：背景和目的：急性非結(jié)石性膽囊炎(acute acalculous cholecystitis,AAC)的臨床表現(xiàn)無(wú)特異性、診斷困難,具有起病急、進(jìn)展快的特點(diǎn),病死率高達(dá)30%。其發(fā)病機(jī)制包括膽汁淤積、缺血-再灌注損傷、內(nèi)源性凝血因子的激活以及細(xì)菌感染等因素,而膽囊平滑肌收縮功能減弱是AAC的重要標(biāo)志。膽總管結(jié)扎(commonbile duct ligation, CBDL)術(shù)是目前公認(rèn)的構(gòu)建AAC的有效動(dòng)物模型,其病理特征與臨床AAC患者的病理特征非常相符。Cajal間質(zhì)細(xì)胞(interstitial cells ofCajal,ICC)是胃腸道慢波活動(dòng)的起搏細(xì)胞,對(duì)胃腸道平滑肌的節(jié)律運(yùn)動(dòng)具有重要調(diào)控作用,ICC胞內(nèi)自發(fā)的周期性鈣(Ca2+)震蕩是產(chǎn)生ICC起搏電流的重要機(jī)制。近年來(lái),人們?cè)趧?dòng)物及人體膽囊都發(fā)現(xiàn)了與胃腸道類似的ICC,并觀察到膽囊ICC內(nèi)也存在節(jié)律性Ca2+振蕩,提示膽囊ICC也可能參與節(jié)律性電活動(dòng)的產(chǎn)生和傳播,介導(dǎo)膽囊自發(fā)性節(jié)律的產(chǎn)生和調(diào)節(jié)平滑肌收縮活動(dòng)。研究表明,AAC發(fā)生早期,膽囊ICC超微結(jié)構(gòu)出現(xiàn)變化。中性粒細(xì)胞是機(jī)體早期炎癥反應(yīng)的重要炎癥細(xì)胞、處于炎癥的核心位置,中性粒細(xì)胞與膽囊ICC結(jié)構(gòu)損傷的關(guān)系尚不明確。因此,本研究將采用CBDL術(shù)構(gòu)建AAC動(dòng)物模型,觀察膽囊ICC與中性粒細(xì)胞的關(guān)系,并探討ICC參與AAC膽囊平滑肌動(dòng)力減退的可能機(jī)制。 方法：首先,本研究采用健康成年豚鼠,通過CBDL方法構(gòu)建AAC動(dòng)物模型；觀察CBDL24h、48h、72h各時(shí)間點(diǎn),豚鼠膽囊膽汁的顏色、體積變化,并檢測(cè)各時(shí)間點(diǎn)體內(nèi)血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、膽紅素(Bilirubin)等肝功能指標(biāo)；通過膽囊HE染色及炎癥評(píng)分(inflammation score)判斷膽囊的炎癥情況；采用RM6240多道生理信號(hào)采集系統(tǒng)記錄CBDL前及CBDL后各時(shí)間點(diǎn),膽囊平滑肌分別對(duì)乙酰膽堿(Ach,10-5mol/l)、八肽膽囊收縮素(CCK-8,10-6mol/l)、氯化鉀(KCl,60mmol/l)的收縮反應(yīng)性；電鏡觀察CBDL前及CBDL后各時(shí)間點(diǎn)ICC超微結(jié)構(gòu)變化及其與中性粒細(xì)胞的聯(lián)系。 然后,選取CBDL后電鏡下中性粒細(xì)胞與膽囊ICC關(guān)系密切的時(shí)間點(diǎn),即CBDL-48h組作為重點(diǎn)研究對(duì)象,活體內(nèi)預(yù)先注射抗中性粒細(xì)胞抗體(anti-polymorphonuclear antibody, anti-PMN)以減少體內(nèi)中性粒細(xì)胞數(shù)量及其組織浸潤(rùn)程度；動(dòng)物分為正常組(normal-2)、假手術(shù)48h(sham-2)組、膽總管結(jié)扎48h(CBDL-2)組以及anti-PMN+旦總管結(jié)扎48h(anti-PMN)組,各組豚鼠12只；觀察各組動(dòng)物膽囊膽汁的顏色及體積變化,同時(shí)檢測(cè)各組血清ALT、AST及Bilirubin水平；HE染色及炎癥評(píng)分評(píng)價(jià)各組膽囊的炎癥程度；測(cè)定循環(huán)血液中中性粒細(xì)胞數(shù)量以及髓氧過化物酶(MPO)活性以判斷中性粒細(xì)胞的組織浸潤(rùn)情況；張力換能器記錄各組膽囊平滑肌分別對(duì)Ach(10-5mol/l)、CCK-8(10-6mo1/l)、KCl(60mmol/l)的收縮反應(yīng)性；電鏡觀察各組膽囊ICC超微結(jié)構(gòu)。 最后,選擇出生10-15d的豚鼠,無(wú)菌條件下取出膽囊,鈍性分離膽囊平滑肌肌條,采用組織塊培養(yǎng)法進(jìn)行膽囊ICC的離體培養(yǎng),倒置顯微鏡下觀察膽囊ICC的形態(tài)；采用ICC特異性的酪氨酸激酶受體c-kit蛋白進(jìn)行免疫熒光染色,以鑒定膽囊ICC；為下一步記錄膽囊ICC的自發(fā)性電活動(dòng)奠定基礎(chǔ)。 結(jié)果：CBDL術(shù)后的豚鼠活動(dòng)度明顯減少；隨著CBDL時(shí)間延長(zhǎng),膽囊膽汁顏色由正常淺黃色變成深黃色,最后變成墨綠色；CBDL各組膽汁體積、血清肝功能指標(biāo)ALT、AST及Bilirubin分別較對(duì)應(yīng)時(shí)間組normal組、sham組顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；HE染色后,CBDL各組膽囊可見炎性細(xì)胞浸潤(rùn)、組織水腫、血管擴(kuò)張、充血,CBDL組炎癥評(píng)分與對(duì)應(yīng)時(shí)間組normal、sham相比較顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；CBDL后,各組膽囊平滑肌對(duì)Ach、CCK-8、KCI的收縮反應(yīng)性分別較對(duì)應(yīng)時(shí)間組normal、sham顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；電鏡顯示,隨著梗阻時(shí)間延長(zhǎng),膽囊ICC超微結(jié)構(gòu)損傷加重；CBDL48h后,可見中性粒細(xì)胞胞胞漿內(nèi)充滿大量顆粒,與受損ICC胞體及ICC突起之間緊密接觸。 CBDL-2組、anti-PMN組的膽汁體積以及血清AST、Bilirubin水平分別較normal-2及sham-2組顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而CBDL-2組與anti-PMN組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；HE染色、炎癥評(píng)分以及MPO活性檢測(cè)顯示,CBDL-2組.anti-PMN組的炎癥程度和MPO活性分別較normal-2、sham-2顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),其中anti-PMN組與CBDL-2相比較,炎癥程度及MPO活性明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；肌條張力實(shí)驗(yàn)顯示,CBDL-2組、anti-PMN組對(duì)Ach、CCK-8、KC1的收縮反應(yīng)性分別較normal-2及sham-2組顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與CBDL-2組相比,anti-PMN組對(duì)Ach、CCK-8、KCl的收縮反應(yīng)性均顯著提高,差異有統(tǒng)計(jì)學(xué)意義(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)]。電鏡結(jié)果顯示,anti-PMN組膽囊ICC腫脹及內(nèi)質(zhì)網(wǎng)擴(kuò)張程度較CBDL-2組減輕,ICC突起不但沒有減少、消失,反而有增多的趨勢(shì)。與CBDL-2組相比,anti-PMN組ICC與ICC之間、ICC與平滑肌之間的縫隙連接明顯增加。 采用組織塊原代培養(yǎng)3-4d后,ICC胞體呈三角形或橢圓形,有1-2個(gè)突起,ICC可單個(gè)散在生長(zhǎng),也可以聚集成簇狀生長(zhǎng)；ICC可與ICC或平滑肌細(xì)胞相連接,但未見網(wǎng)絡(luò)狀結(jié)構(gòu)；c-kit免疫熒光表明,3-4d后培養(yǎng)的大多數(shù)細(xì)胞表面c-kit蛋白表達(dá)呈陽(yáng)性。 結(jié)論：在AAC模型豚鼠中,anti-PMN可以降低中性粒細(xì)胞在炎性膽囊的浸潤(rùn)密度,減輕膽囊組織的炎癥反應(yīng)；同時(shí),anti-PMN可以緩解膽囊ICC超微結(jié)構(gòu)受損程度,提高膽囊平滑肌對(duì)的Ach、CCK-8、KCl的收縮反應(yīng)；浸潤(rùn)于膽囊組織的中性粒細(xì)胞可能先作用于膽囊ICC,繼而累及膽囊平滑肌,從而出現(xiàn)膽囊平滑肌收縮功能減退；采用組織塊原代培養(yǎng)法可以成功培養(yǎng)出膽囊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錛，
本文編號(hào)：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錛，
本文編號(hào)：2120247
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