李紅昌 雷若慶 許志偉 王慶剛 柴春宇 鄧漾 吳旭波 武鈞 陳勝 韓天權 湯耀卿 張圣道
·論著·
急性胰腺炎大鼠血和組織中降鈣素原水平的變化
李紅昌 雷若慶 許志偉 王慶剛 柴春宇 鄧漾 吳旭波 武鈞 陳勝 韓天權 湯耀卿 張圣道
目的觀察急性胰腺炎(AP)大鼠血和組織中降鈣素原(procalcitonin,PCT)水平的變化,并探討其意義。方法將102只雄性Wistar大鼠按數(shù)字表法隨機分為正常對照組(6只)、脂多糖(LPS)組(24只)、急性水腫性胰腺炎(AEP)組(24只)、急性壞死性胰腺炎(ANP)組(24只)和ANP+LPS組(24只)。皮下注射雨蛙素制備AEP模型,逆行膽胰管注射?;悄懰徕c制備ANP模型。術后3、6、18、24 h分批處死動物,胰腺組織常規(guī)病理檢查并評分;取血檢測PCT水平;取肝、肺、脾、胰腺、小腸及大腸組織檢測PCT含量。結果AEP和ANP模型制備成功。術后6 h時,對照組、LPS組、AEP組、ANP組和ANP+LPS組血PCT水平分別為(0.0144±0.0082)ng/ml、(0.1722±0.0449)ng/ml、(0.4751±0.0572)ng/ml、(0.7070±0.1040)ng/ml和(1.1960±0.8644)ng/ml,各組間相差顯著(P<0.05)。正常大鼠肝、肺、脾、胰腺、小腸及大腸組織均檢測到PCT。與之相比,ANP組肝和胰腺PCT含量無明顯變化;肺、脾及大腸PCT含量顯著降低[分別為(5.63±0.62)ng/ml對(6.85±0.46)ng/ml,(4.73±1.27)ng/ml對(6.88±0.31)ng/ml,(1.08±0.52)ng/ml對(4.12±1.02)ng/ml,P值均<0.01],而小腸組織明顯升高[(2.51±0.90)ng/ml對(0.98±0.20)ng/ml,P<0.01]。結論血清PCT水平與AP的嚴重程度及感染相關,AP時各組織PCT含量的不同變化可能與器官功能的改變有一定的關系。
胰腺炎; 降鈣素原; 壞死; 組織水平
急性胰腺炎(AP)的發(fā)病機制是一個復雜的、多因素參與的病理生理過程,至今尚未完全闡明,但炎癥細胞因子參與AP的發(fā)生和發(fā)展已是共識。降鈣素原(procalcitonin,PCT)是一種對感染的炎性反應具有放大效應的繼發(fā)介質[1],其在AP中的應用已受到國內(nèi)外學者的廣泛關注。本文觀察AP大鼠血清和組織PCT的變化,并探討其意義。
一、動物分組
102只健康雄性Wistar大鼠,體重200~250 g,SPF級,由上海交通大學醫(yī)學院附屬瑞金醫(yī)院實驗動物中心提供。按數(shù)字表法隨機分為正常對照組(6只)、脂多糖(LPS)組(24只)、急性水腫性胰腺炎(AEP)組(24只)、急性壞死性胰腺炎(ANP)組(24只)和ANP+LPS組(24只)。連續(xù)4次皮下注射雨蛙素20 μg/kg體重(1次/h)的方法制備AEP模型;胰膽管逆行推注5%?;悄懰徕c1 ml/kg體重的方法制備ANP模型;LPS組向胰膽管推注LPS 2 mg/kg體重;ANP+LPS組在ANP建模后即刻加注LPS 2 mg/kg體重。術后3、6、18、24 h分批處死動物,取血,并留取肝臟、肺臟、脾臟、胰腺、小腸及大腸標本。
二、檢測項目及方法
1.血清PCT:采用酶聯(lián)免疫吸附試驗(ELISA)法,試劑盒購自美國R&D公司。
2.組織PCT:各組織按0.1 g組織加300 μl生理鹽水的比例制作組織勻漿,離心后取上清液,測定方法同血清PCT。
3.胰腺組織病理變化:行常規(guī)病理檢查,并按Schmidt等[2]的標準進行評分。
三、統(tǒng)計學方法
一、一般情況
AEP組大鼠造模后活動減少,無其他異常,10 h左右基本恢復正?;顒樱籄NP組大鼠精神萎靡,蜷曲,活動少,反應差;ANP+LPS組大鼠,精神極度萎靡,嗜睡,反應極差;LPS組大鼠無明顯異常表現(xiàn)。
二、胰腺病理改變
正常組和LPS組大鼠胰腺大體及鏡下無明顯變化。AEP組在造模后6 h胰腺明顯水腫,鏡下見腺泡、間質水腫,少量炎細胞浸潤;18 h后基本恢復正常。ANP組腹腔內(nèi)有淡黃色至深褐色腹水,胃腸擴張明顯,胰腺重度腫脹,有點、片狀出血,腸系膜、網(wǎng)膜、胰腺表面等處可見皂化斑;鏡下見胰腺小葉結構模糊,間質充血、出血,胰腺組織壞死,大量炎細胞浸潤。ANP+LPS組與ANP組變化相似。AEP組、ANP組及ANP+LPS組的胰腺病理評分分別為4.83±0.88、11.08±0.97和13.92±1.24。
三、血清PCT水平變化
正常大鼠血清PCT水平極低;LPS組、AEP組、ANP組和ANP+LPS組在制模后3 h血清PCT水平較正常組顯著升高(P<0.01),18 h達峰值,24 h時仍維持在較高水平,各組間差異顯著(P<0.05,表1)。
四、組織PCT含量的變化
正常大鼠肝、肺、脾、胰腺、小腸及大腸中PCT含量分別為(4.34±0.36)ng/ml、(6.85±0.46)ng/ml、(6.88±0.31)ng/ml、(1.11±0.23)ng/ml、(0.98±0.20)ng/ml、(4.12±1.02)ng/ml;ANP組大鼠6 h時各組織PCT含量分別為(4.44±0.72)ng/ml、(5.63±0.62)ng/ml、(4.73±1.27)ng/ml、(1.33±0.35)ng/ml、(2.51±0.90)ng/ml、(1.08±0.52)ng/ml。與正常組比較,ANP組PCT水平在肝和胰腺中無明顯變化,在肺、脾及大腸中顯著減少(P<0.01),而在小腸中明顯升高(P<0.01)。
表1 各組大鼠血清PCT水平的變化
注:與正常組比較,aP<0.01,與LPS組比較,bP<0.05;與AEP組比較,cP<0.05;與ANP組比較,dP<0.05
PCT是由116個氨基酸組成的蛋白質,是降鈣素的前體物質。PCT可能是一種繼發(fā)介質,對感染的炎性反應具有放大效應,在調(diào)控細胞因子網(wǎng)絡中發(fā)揮重要作用。文獻報道[3-4],血PCT在AEP、無菌性ANP和感染性ANP中均顯著升高,且感染性胰腺壞死時PCT的升高較無菌壞死時更顯著,因此檢測血PCT水平是判斷AP發(fā)生、病情嚴重程度、胰腺壞死感染的一個十分有價值的指標。本實驗結果顯示,血清PCT水平在成模后3 h明顯升高,18 h達峰值,且ANP組的升高較AEP組更顯著,提示血清PCT水平可以較早地評估AP的嚴重程度。ANP的感染主要來源于腸道,血內(nèi)毒素水平的升高提示腸源性感染的存在。因為內(nèi)毒素是刺激LPS升高的一個重要因素,故本實驗增設一個LPS對照組。結果顯示,單純給予LPS可使血PCT水平升高,但其程度有限,而在ANP基礎上給予LPS,則血PCT水平升高極為明顯,說明PCT也可以較早地反映內(nèi)毒素的水平。
PCT的組織來源還不是很清楚。正常生理狀況下血PCT主要來源于甲狀腺C細胞,也可由其他內(nèi)分泌細胞內(nèi)的前降鈣素原水解產(chǎn)生。正常人不同的組織均存在PCT,以肝、胰腺、腎和睪丸中含量最高。在病理狀態(tài)下,肝、肺、腎、腎上腺、腦和胰腺等多種組織器官都可能是PCT的生成場所。本實驗結果顯示,正常大鼠的肝、肺、脾、胰腺、小腸及大腸組織均可檢測到PCT,而在ANP大鼠,肝和胰腺的PCT含量無明顯變化,但肺、脾及大腸組織的PCT含量顯著減少,小腸的PCT含量明顯升高。Ammori等[5]曾報道過血PCT與AP腸屏障損傷的關系,提示血PCT水平與腸屏障損害的關聯(lián)性較其與全身炎癥還要好。由此我們推想,ANP大鼠早期血清、小腸PCT的變化與腸功能的改變存在一定的關系。
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2010-02-10)
(本文編輯:呂芳萍)
Levelsofprocalcitonininbloodandtissueofacutepancreatitisrats
LIHong-chang,LEIRuo-qing,XUZhi-wei,WANGQing-gang,CHAIChun-yu,DENGYang,WUXu-bo,WUJun,CHENSheng,HANTian-quan,TANGYao-qing,ZHANGSheng-dao.
DepartmentofSurgery,RuijinHospital,MedicineSchool,ShanghaiJiaotongUniversity,Shanghai200025,China
Correspondingauthor:LEIRuo-qing,Email:ruoqinglei@yahoo.com.cn
ObjectiveTo investigate the variation of procalcitonin(PCT) in blood and tissue level of acute pancreatitis rats and probe its significant.MethodsOne hundred and two male Wistar rats were randomly divided into control group (n=6), lipopolysaccharide group (LPS,n=24), acute edematous pancreatitis (AEP) group (n=24), acute necrotizing pancreatitis (ANP) group (n=24), ANP+LPS group (n=24). Subcutaneous injection of cerulein was used for AEP induction, while ANP model was induced by retrograde injection of sodium taurocholate into the biliary and pancreatic duct. The rats were sacrificed at 3, 6, 18 and 24 hours after model induction. Pancreatic tissue was harvested and the pathological scores were assessed. Levels of PCT in serum, liver, lung, spleen, pancreas, small intestine, large intestine tissue was harvested and tissue levels of PCT were determined.ResultsAEP and ANP models were established successfully. At 6 h, the serum levels of PCT in control group, LPS group, AEP group, ANP group and ANP+LPS group were (0.0144±0.0082) ng/ml, (0.1722±0.0449) ng/ml,(0.4751±0.0572) ng/ml, (0.7070±0.1040) ng/ml and (1.1960±0.8644) ng/ml, respectively; and the difference was statistically significant (P<0.05). PCT could be detected in liver, lung, spleen, pancreas, small intestine and large intestine tissue of normal rats. PCT levels in liver and pancreas of ANP group were not statistically different, but the PCT levels in lung, spleen, and large intestine tissue significantly decreased, and the corresponding values were (5.63±0.62) ng/ml vs. (6.85±0.46) mg/ml, (4.73±1.27) mg/ml vs.(6.88±0.37) ng/ml, (1.08±0.52) ng/ml vs. (4.12±1.02) ng/ml (P<0.01). However, the PCT levels in small intestine significantly increased, which were (2.51±0.90) ng/mlvs(0.98±0.12) ng/ml (P<0.01).ConclusionsSerum PCT level was associated with the severity of AP and infection; the changes of PCT levels in different tissues may be related with the changes of organ′s function.
Pancreatitis; Procalcitonin; Necrosis; Tissue level
10.3760/cma.j.issn.1674-1935.2010.03.013
200025 上海,上海交通大學醫(yī)學院附屬瑞金醫(yī)院膽胰外科
雷若慶,Email:ruoqinglei@yahoo.com.cn