本文選題：重癥急性胰腺炎　切入點(diǎn)：氯化鋰　出處：《武漢大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分：靜脈注射GSK-3β的抑制劑LiCl干預(yù)重癥急性胰腺炎大鼠量效關(guān)系的探討 目的：探討靜脈注射糖原合成酶激酶-3β (Glycogen synthase kinase-3β, GSK-3β)抑制劑氯化鋰(Lithium Chloride, LiCl)干預(yù)重癥急性胰腺炎(severe acute pancreatitis, SAP)大鼠模型的量效關(guān)系,從而為LiCl干預(yù)重癥急性胰腺炎肝臟損傷(acute pancreatitis associated liver injury)選擇適合的用藥劑量提供理論依據(jù)。 方法：雄性、SPF級(jí)、Wistar大鼠50只,體重200-250g,隨機(jī)將大鼠分為5組(N=10)即為：假手術(shù)組(sham operation group, SO組),重癥急性胰腺炎模型組(severe acute pancreatitis, SAP組),LiCl-40mg/kg預(yù)處理組、LiCl-60mg/kg預(yù)處理組以及LiCl-80mg/kg預(yù)處理組(LiCl組)。所有大鼠操作之前均需禁食12小時(shí),但可以自由飲水。10%水合氯醛腹腔注射(0.3ml/100g)麻醉大鼠后,取上腹部正中切口入腹,采用微量泵、經(jīng)膽胰管逆行勻速注射新鮮配制的5%�；悄懰徕c溶液(sodium taurocholate, STC)(0.1ml/100g)制備重癥急性胰腺炎大鼠模型；SO組操作如上說述,但是在膽胰管內(nèi)注射等量生理鹽水替代�；悄懰徕c。LiCl干預(yù)各劑量組均于造模前30min給予股靜脈注射對(duì)應(yīng)濃度的LiCl溶液,劑量分別為40、60、80mg/kg。各組大鼠于模型制作后12h剖殺,棉球吸收法檢測腹水量,下腔靜脈穿刺取血,離心分裝凍存待測,使用大鼠右下肺葉檢測肺組織濕干比,使用大鼠胰頭部組織,固定包埋。切片染色。分別測定各組大鼠腹水量和肺濕干比(反應(yīng)肺臟含水量)、全自動(dòng)生化儀測定血清淀粉酶(amylase, AMY)和肝腎功能指標(biāo)如谷丙轉(zhuǎn)氨酶(alanine aminotransferase, ALT)、肌酐(creatinine, Cr)水平。光鏡下觀察胰腺組織病理學(xué)變化并進(jìn)行病理學(xué)評(píng)分。 結(jié)果：SAP組大鼠的腹水量、血清淀粉酶、谷丙轉(zhuǎn)氨酶、肌酐水平、肺濕干比以及胰腺病理評(píng)分均較SO組顯著升高,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。LiCl-40mg/kg的干預(yù)組上述指標(biāo)(即腹水量、淀粉酶、肺臟組織濕干比以及胰腺病理學(xué)評(píng)分)與SAP組比較無明顯差異(P0.05)。LiCl-60mg/kg的干預(yù)組的全部指標(biāo)(包括腹水量、淀粉酶、谷丙轉(zhuǎn)氨酶、肌酐、肺組織濕干比以及胰腺病理評(píng)分)與SAP組相比較均有明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。LiCl-80mg/kg預(yù)處理組中上述部分指標(biāo)(如腹水量、血清AMY、肺濕干比、胰腺病理評(píng)分)較SAP組顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；但是其ALT水平升高大于SAP組,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)、Cr值與SAP組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：60mg/kgLiCl能有效緩解胰腺炎病情,表現(xiàn)為減少腹水量、降低血清淀粉酶水平和緩解胰腺損傷的病理學(xué)評(píng)分,同時(shí)可緩解肝腎功能的損傷。而80mg/kgLiCl雖能緩解胰腺炎病情,但使用此濃度干預(yù)存在潛在的肝毒性與腎毒性。因此通過綜合分析,我們認(rèn)為60mg/kg的LiCl劑量是干預(yù)重癥急性胰腺炎大鼠模型的相對(duì)最佳有效劑量。 第二部分：GSK-3β抑制劑LiCl對(duì)重癥急性胰腺炎大鼠肝損傷的作用 目的：探討GSK-3β抑制劑LiCl對(duì)重癥急性胰腺炎(severe acute pancreatitis, SAP)大鼠肝損傷的作用。 方法：雄性、SPF級(jí)、Wistar大鼠70只,體重200～250g,隨機(jī)分為4組。假手術(shù)組(SO組)(N=10)；重癥急性胰腺炎模型組(SAP組)(N=40),按照時(shí)間點(diǎn)分為1h,3h,6h,12h四個(gè)亞組,每組大鼠10只；GSK-3β抑制劑LiCl預(yù)處理組(LiCl組)(N=10), LiCl藥物對(duì)照組(Drug-CON組)(N=10)。重癥急性胰腺炎模型組及LiCl預(yù)處理組大鼠需使用逆行膽胰管注射�；悄懰徕c法制作SAP模型。需要使用LiCl的LiCl預(yù)處理組及LiCl藥物對(duì)照組需要在制作相關(guān)模型之前使用靜脈穿刺注射法給予60mg/kg的LiCl溶液。假手術(shù)組、GSK-3β抑制劑LiCl預(yù)處理組、GSK-3β抑制劑LiCl藥物對(duì)照組于均選擇在12h處死大鼠,SAP各亞組大鼠需根據(jù)分組情況,在相應(yīng)的時(shí)間點(diǎn)剖殺。棉球吸收法檢測腹水量,下腔靜脈穿刺取血,離心分裝凍存待測,使用大鼠右下肺葉檢測肺組織濕干比,使用大鼠胰頭部組織,固定包埋。分別記錄和匯總各組大鼠的死亡率、腹水量,全自動(dòng)生化儀血清淀粉酶(AMY)和磷脂酶水平(PLA2),肝功能通過測定谷丙轉(zhuǎn)氨酶(ALT)與谷草轉(zhuǎn)氨酶(AST)來評(píng)價(jià),各組大鼠均行常規(guī)胰腺與肝臟病理學(xué)HE染色檢查,并評(píng)分分級(jí)。 結(jié)果：藥物對(duì)照組與SO組在死亡率、腹水量、AMY、ALT、AST、PLA2以及胰腺和肝臟的病理評(píng)分與分級(jí)無明顯差異(P0.05)。 SAP各組,隨著造模時(shí)間的延長,上述指標(biāo)均有所增加,各組與SO組對(duì)比均升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)比LiCl干預(yù)組與SAP組相應(yīng)時(shí)間點(diǎn)亞組發(fā)現(xiàn),使用LiCl干預(yù)后,上述指標(biāo)均有所改善,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),但這種改變并不能達(dá)到治愈效果,相比于SO組水平依舊有所升高(P0.05)。 結(jié)論：在使用LiCl干預(yù)后,大鼠的胰腺與肝臟酶學(xué)指標(biāo)均有所下降,胰腺組織與肝臟病理學(xué)評(píng)分分級(jí)有所下降,提示LiCl對(duì)于SAP大鼠的胰腺炎以及由胰腺炎導(dǎo)致的肝損傷具有一定的保護(hù)作用。 第三部分：GSK-3β抑制劑LiCl對(duì)重癥急性胰腺炎大鼠肝損傷保護(hù)作用的機(jī)制探討 目的：通過實(shí)驗(yàn)進(jìn)一步探討靜脈給藥對(duì)大鼠重癥急性胰腺炎肝損傷保護(hù)作用的機(jī)制。 方法：雄性、SPF級(jí)、Wistar大鼠60只,體重200-250g,隨機(jī)分為3組。假手術(shù)組(SO組)(N=10)：重癥急性胰腺炎模型組(SAP組)(N=40),根據(jù)造模后的時(shí)間段分為1h,3h,6h,12h四個(gè)亞組,每組大鼠10只；GSK-3β抑制劑LiCl預(yù)處理組(LiCl組)(N=10)。重癥急性胰腺炎模型組及LiCl預(yù)處理組大鼠需使用逆行膽胰管注射�；悄懰徕c法制作SAP模型。需要使用LiCl的LiCl預(yù)處理組需要在制作相關(guān)模型之前使用靜脈穿刺注射法給予60mg/kg的LiCl溶液。假手術(shù)組、GSK-3β抑制劑LiCl預(yù)處理組均于12h處死大鼠,SAP各亞組大鼠需根據(jù)分組情況,在相應(yīng)的時(shí)間點(diǎn)剖殺。取大鼠胰頭部位組織和肝臟右葉部分只固定包埋,切片并行免疫組織化學(xué)染色,檢測GSK-3β和NF-κB。使用胰尾部分與剩余的右葉肝臟,以Western-Blot法檢測NF-κB與ICAM-1水平。 結(jié)果：Western-Blot的結(jié)果提示：SAP各組大鼠肝臟與胰腺組織NF-κB水平隨著造模時(shí)間的延長而升高；各組大鼠NF-κB水平相比于SO組均增高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；使用LiCl干預(yù)組的大鼠NF-κB水平相比于SAP組明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。ICAM-1的變化與NF-κB基本一致,使用LiCl干預(yù)組的ICAM-1表達(dá)與同時(shí)間點(diǎn)的SAP組對(duì)比明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。免疫組化檢測結(jié)果顯示,SAP各組大鼠GSK-3β的水平隨著造模時(shí)間的延長而不斷增加,至12h時(shí)達(dá)到峰值,在給予LiCl干預(yù)后GSK-3β的表達(dá)量下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。NF-ΚB的改變除了進(jìn)一步證實(shí)了Western的結(jié)果之外,還提示在SAP過程中NF-ΚB的定位發(fā)生了改變,出現(xiàn)由胞漿到核內(nèi)的移動(dòng)。 結(jié)論：GSK-3p抑制劑Lic1通過抑制GSK-3β的活化下調(diào)了肝臟組織NF-κB的活化,通過抑制NF-κB的活化又抑制了ICAM-1的表達(dá),從而通過抑制炎癥瀑布效應(yīng)從而減輕急性胰腺炎肝損傷的程度
[Abstract]:Part one: A Study on the dose effect relationship of GSK-3 beta inhibitor LiCl in rats with severe acute pancreatitis
Objective: To investigate the effect of intravenous injection of glycogen synthase kinase -3 beta (Glycogen synthase kinase-3 GSK-3 beta, beta) inhibitor lithium chloride (Lithium Chloride, LiCl) intervention for severe acute pancreatitis (severe acute, pancreatitis, SAP) dose effect relationship of rat model, and intervention of hepatic injury in severe acute pancreatitis (LiCl acute pancreatitis associated liver injury) selection the ideal dose and provide a theoretical basis.
Methods: male, SPF, 50 Wistar rats, weight 200-250g, the rats were randomly divided into 5 groups (N=10) namely: sham operation group (sham operation, group, SO group), severe acute pancreatitis group (severe acute, pancreatitis, SAP group), LiCl-40mg/kg pretreatment group, LiCl-60mg/kg pretreatment group and LiCl-80mg/kg pretreatment group (LiCl group). All rats before operation are required 12 hours of fasting, but free drinking water.10% intraperitoneal injection of chloral hydrate (0.3ml/100g) in anesthetized rats after taking median incision into the abdomen, the micro pump, 5% sodium taurocholate solution after bile duct retrograde infusion freshly prepared (sodium taurocholate, STC) (0.1ml/100g) prepared in a rat model of severe acute pancreatitis; SO group said the operation above, but instead of sodium taurocholate.LiCl intervention groups were given 30min before modeling in intraductal injection of saline Stock solution of LiCl intravenous injection of the corresponding concentration, dose rate of 40,60,80mg/kg. rats in model 12h after slaughter, cotton absorption method was used to detect the amount of ascites, inferior vena cava puncture blood centrifugal freezing test using packaging, detection of lung tissue of rats with right lobe under wet and dry, the head of the pancreas tissue of rats fixed, embedded. Staining of rats were measured. The ascites volume and lung wet dry ratio (reaction lung moisture), serum amylase automatic biochemical analyzer (amylase, AMY) and liver function indexes such as alanine aminotransferase (alanine aminotransferase ALT), creatinine (creatinine, Cr) level of light. Observe the pathologic changes of the pancreas and the pathological score.
Results: serum amylase, ascites volume, the rats in group SAP, alanine aminotransferase, creatinine level, wet / dry ratio of the lung and pancreas pathological score were significantly increased compared with the SO group, the difference was statistically significant (P0.05.LiCl-40mg/kg) of the intervention group the index (i.e. abdominal content, amylase, lung wet to dry ratio and pancreatic pathology no significant difference was found between the scores) and group SAP (P0.05).LiCl-60mg/kg in the intervention group all indexes (including the amount of ascites, amylase, alanine aminotransferase, creatinine, lung wet / dry ratio and pancreas pathological score) compared with the SAP group were significantly decreased, the difference was statistically significant (P0.05) above indicators.LiCl-80mg/kg treatment group (such as abdominal water, serum AMY, lung wet to dry ratio, pancreatic pathological score) decreased significantly compared with SAP group, the difference was statistically significant (P0.05); but the elevated levels of ALT than in the SAP group, the difference was statistically significant (P0.05 There was no significant difference between the Cr value and the SAP group (P0.05).
Conclusion: 60mg/kgLiCl can effectively alleviate pancreatitis and to reduce the amount of ascites, reduce the level of serum amylase and alleviate the pathological score of pancreatic injury, also can relieve liver and kidney function damage. 80mg/kgLiCl can alleviate pancreatitis, but with this concentration of intervention has the potential for hepatotoxicity and renal toxicity. Therefore, through comprehensive analysis, we that LiCl dose 60mg/kg intervention model of rats with severe acute pancreatitis is the best effective dose.
The second part: the effect of GSK-3 beta inhibitor LiCl on liver injury in rats with severe acute pancreatitis
Objective: To investigate the effect of GSK-3 beta inhibitor LiCl on liver injury in severe acute pancreatitis (SAP) rats.
鏂規(guī)硶錛氶泟鎬，

本文編號(hào)：1578467