發(fā)布時(shí)間：2018-04-10 10:53

  本文選題：重癥急性胰腺炎 + 腎損傷��；參考：《武漢大學(xué)》2014年博士論文

【摘要】：第一部分：糖原合酶激酶-3p在重癥急性胰腺炎大鼠腎損傷中的表達(dá)及作用研究 目的：探討重癥急性胰腺炎(SAP)大鼠模型中腎臟的組織形態(tài)、超微結(jié)構(gòu)和功能變化,研究GSK-3β在SAP大鼠腎臟組織的變化規(guī)律及其可能作用。 方法：采用SPF級(jí)雄性Wistar大鼠,隨機(jī)分為5組(N=12)。假手術(shù)組(SO組),重癥急性胰腺炎組(SAP3h、6h、12h、24h四個(gè)亞組),通過膽胰管逆行注射5%�；悄懰徕c溶液制備大鼠SAP模型。分別測(cè)定各組大鼠血清淀粉酶(AMY)、脂肪酶(LIPA)、肌酐(Cr)和尿素氮(BUN)水平,應(yīng)用HE染色法觀察各組大鼠胰腺和腎臟組織病理學(xué)改變,透射電鏡觀察大鼠腎臟細(xì)胞超微結(jié)構(gòu)變化,采用Western-blot法檢測(cè)腎臟組織中GSK-3β及其磷酸化形式p-GSK-3p ser9在各組中的表達(dá)變化。 結(jié)果：SAP大鼠血清AMY.LIPA.胰腺病理評(píng)分隨著時(shí)間進(jìn)展水平逐漸升高,而Cr、BUN及腎臟病理評(píng)分在SAP3h、6h、12h呈現(xiàn)逐漸增高的趨勢(shì),而SAP12h、24h組基本呈現(xiàn)一個(gè)水平表達(dá)狀態(tài),差異無統(tǒng)計(jì)學(xué)意義(P0.05),SAP各組大鼠腎臟GSK-3β蛋白表達(dá)較SO組表達(dá)明顯增高(P0.05),但SAP各組之間比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)；β-GSK-3β ser9在SO組表達(dá)不高,而在SAP3h時(shí)組,表達(dá)較SO組增高,但從SAP6h組開始,隨著病程的進(jìn)展,p-GSK-3β ser9的表達(dá)卻呈現(xiàn)出逐漸減弱的趨勢(shì)(P0.05)。結(jié)論：隨SAP病程進(jìn)展,大鼠胰腺、腎臟病理損傷呈進(jìn)行性加重,腎功能檢測(cè)指標(biāo)Cr、BUN亦出現(xiàn)相應(yīng)變化。大鼠腎臟組織中GSK-3β并未隨SAP的病程進(jìn)展出現(xiàn)明顯表達(dá)變化,但其磷酸化形式的p-GSK-3β ser9表達(dá)卻呈現(xiàn)出先增強(qiáng),后逐漸減弱的趨勢(shì),表明GSK-3β的磷酸化調(diào)控可能在SAP并發(fā)腎損傷的發(fā)病過程中發(fā)揮重要作用。第二部分：不同類型GSK-3p抑制劑對(duì)重癥急性胰腺炎大鼠腎損傷的作用比較及量效關(guān)系探討 目的：觀察TDZD-8干預(yù)大鼠SAP腎損傷的量效關(guān)系,并將三種常用GSK-3β抑制劑TDZD-8、氯化鋰(LiCL)、SB216763對(duì)該模型的作用效果進(jìn)行對(duì)比,以探討針對(duì)SAP并發(fā)腎損傷大鼠模型最有效的GSK-3β抑制劑類別及其有效、安全的最佳劑量。方法：96只SPF級(jí)雄性Vistar大鼠,隨機(jī)分為8組(N=12)：假手術(shù)組(SO組)、重癥急性胰腺炎組(SAP組)、TDZD-8預(yù)處理組0.25、0.5、1、2mg/kg(TD組,分別標(biāo)記為TD1、TD2、TD3、TD4組),LiCL預(yù)處理組(L組)和SB216763預(yù)處理組(SB組),胰膽管逆行注射5%�；悄懰徕c制作SAP模型。SO組、SAP組均于術(shù)前30min經(jīng)股靜脈注射溶劑10%DMSO (O.lml/100g), TDZD-8各劑量預(yù)處理組SAP造模前30min經(jīng)股靜脈注射等量10%DMSO溶解的不同劑量的TDZD-8；L組和SB組分別經(jīng)股靜脈注射等體積10%DMSO溶解的LiCL(60mg/kg)和SB216763(1mg/kg)。術(shù)后12h剖殺各組大鼠,測(cè)定各組大鼠死亡率、腹水量、血清AMY、Cr、BUN和ALT水平,并觀察胰腺、腎臟組織病理學(xué)變化。 結(jié)果：SO、SAP、TD3、L和SB組大鼠死亡率分別為0%、33.3%、0%、8.3%和16.6%；SAP組腹水量、AMY、Cr、BUN、ALT值以及胰腺、腎臟病理評(píng)分均較SO組顯著升高(P0.05)；TD1組幾乎對(duì)SAP無緩解作用,TD2、TD3、TD4、L組和SB組均能不同程度地減少SAP大鼠的腹水量,降低血清AMY、Cr、BUN值,并顯著降低胰腺組織病理評(píng)分,差異有統(tǒng)計(jì)學(xué)意義P0.05)；TD2、TD3均能不同程度地減少ALT值(P0.05),而TD4組ALT值較高,與SAP組相似；TD2與TD3組比較,對(duì)各個(gè)指標(biāo)均有效果,但效果不如TD3組作用顯著,兩者比較各項(xiàng)指標(biāo)均有顯著差異(P0.05)。TD組中最佳劑量組TD3組在腹水量、ALT值之間與L組和SB組比較,無顯著性差異(P0.05)；而TD3組的AMY、Cr、BUN值以及胰腺、腎臟病理評(píng)分均較L組和SB降低更顯著,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；L組Cr、BUN值和胰腺、腎臟病理學(xué)評(píng)分與SB組比較,均較低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)； 結(jié)論：通過對(duì)TDZD-8、LiCL和SB216763對(duì)大鼠SAP腎損傷模型的作用比較可知TDZD-8是針對(duì)該模型最有效的GSK-3β抑制劑。對(duì)于�；悄懰徕c誘導(dǎo)的SAP并發(fā)腎損傷大鼠模型,靜脈給予TDZD-81mg/kg預(yù)處理對(duì)該模型是安全有效的最佳劑量。第三部分：GSK-3p抑制劑TDZD-8對(duì)大鼠重癥急性胰腺炎腎損傷的保護(hù)作用機(jī)制探討目的：觀察抑制GSK-3β活性對(duì)SAP大鼠腎臟組織病理和超微結(jié)構(gòu)的影響,檢測(cè)腎臟組織NF-κB激活及其依賴性基因的蛋白表達(dá)變化,探討GSK-3β抑制劑TDZD-8對(duì)SAP腎損傷保護(hù)作用的機(jī)制。方法：SPF級(jí)雄性Wistar大鼠,隨機(jī)分為4組(N=20)。SO組(sham+vehicle組)、SAP組(SAP+vehicle組)、TDZD-8治療組(SAP+TDZD-8組)和TDZD-8藥物對(duì)照組(sham+TDZD-8組),以12h為觀察點(diǎn)。SO組、SAP組均于術(shù)前30min經(jīng)股靜脈注射TDZD-8溶劑10%DMSO(0.1ml/100g),TDZD-8治療組在SAP造模前30min經(jīng)大鼠股靜脈注射GSK-3β抑制劑TDZD-8,TDZD-8藥物對(duì)照組在操作前30min經(jīng)大鼠股靜脈注射與TDZD-8治療組等體積的GSK-3β抑制劑TDZD-8,余操作同SO組。術(shù)后12h剖殺各組大鼠,測(cè)定各組大鼠血清AMY、Cr、BUN水平并觀察胰腺、腎臟組織病理學(xué)變化,電鏡觀察腎臟細(xì)胞超微形態(tài)結(jié)構(gòu)變化,比色法檢測(cè)腎臟組織髓過氧化物酶(MPO)活性,ELISA法檢測(cè)IL-1β、IL-6在各組大鼠血清中含量,免疫組化法檢測(cè)NF-κB p65在大鼠腎臟中的定位表達(dá),Western-blot法檢測(cè)腎臟組織GSK-3β、p-GSK-3β ser9、NF-κB p65、TNF-α、iNOS、ICAM-1和IL-10的表達(dá)水平。 結(jié)果：與SAP組比較,TDZD-8可顯著降低SAP大鼠血清中AMY、LIPA、Cr、BUN含量,減輕胰腺、腎臟病理損傷,并減輕大鼠腎臟細(xì)胞超微結(jié)構(gòu)損傷(P0.05)。TDZD-8治療組腎臟組織MPO活性、以及血清IL-1p、IL-6活性與SAP組比較,均有明顯下降(P0.05)。免疫組化結(jié)果示：SAP組中NF-κB p65的表達(dá)較SO組增強(qiáng),且主要在細(xì)胞核內(nèi)表達(dá),而TDZD-8治療組則較SAP組表達(dá)明顯減少。Western-blot結(jié)果顯示：SAP組和TDZD-8治療組GSK-3p表達(dá)較SO組和TDZD-8藥物對(duì)照組明顯增強(qiáng)(P0.05),而SAP組和TDZD-8治療組表達(dá)無顯著差異。SAP組腎臟組織中p-GSK-3β ser9的表達(dá)較SO組明顯減弱(P0.05),而TDZD-8治療組大鼠腎臟組織中p-GSK-3β ser9的表達(dá)較SAP組表達(dá)則明顯增多(P0.05)；SAP組腎臟組織中NF-kB p65、TNF-α、ICAM-1、iNOS的表達(dá)較SO組明顯增強(qiáng),IL-10表達(dá)明顯減弱(P0.05),阻斷GSK-3β活性能夠抑制NF-κB p65、TNF-α、ICAM-1、iNOS的表達(dá),提高IL-10的表達(dá)(P0.05)。各項(xiàng)指標(biāo)檢測(cè)結(jié)果顯示TDZD-8藥物對(duì)照組均與SO組無顯著差異(P0.05)。 結(jié)論：GSK-3β抑制劑TDZD-8可有效導(dǎo)致SAP大鼠腎臟GSK-3β磷酸化而使其活性受到抑制,從而抑制腎臟NF-κB炎癥通路激活以及中性粒細(xì)胞的募集,進(jìn)一步抑制其下游炎癥介質(zhì)(TNF-α、IL-1β、IL-6、ICAM-1、iNOS)的釋放,提高保護(hù)因子IL-10的釋放,從而減輕腎臟炎癥及病理損傷,緩解SAP病情進(jìn)展。
[Abstract]:Part one: expression and role of glycogen synthase kinase -3p in renal injury of severe acute pancreatitis in rats
Objective: To investigate the changes of renal morphology, ultrastructure and function in rats with severe acute pancreatitis (SAP), and to explore the possible role of GSK-3 beta in renal tissue of SAP rats.
Methods: male Wistar SPF rats were randomly divided into 5 groups (N=12). Sham operation group (SO group), severe acute pancreatitis group (SAP3h, 6h, 12h, 24h four groups), SAP rat model by retrograde injection of 5% sodium taurocholate solution prepared by serum bile duct. The rats were measured by amylase, lipase (AMY) (LIPA), creatinine (Cr) and urea nitrogen (BUN) level, observe changes of pancreas and kidney tissue of rats with HE pathological staining, observe the ultrastructure changes of rat kidney cells by transmission electron microscope, the expression of GSK-3 in renal tissue and form beta phosphate Western-blot method was used to detect p-GSK-3p ser9 in each group.
Results: SAP rats serum AMY.LIPA. pancreatic pathological score as time progresses levels increased gradually, while Cr, BUN and renal pathological score in SAP3h, 6h, 12h increased markedly, while SAP12h, 24h group showed a level of expression, the difference was not statistically significant (P0.05), compared with the SO group increased significantly. Beta protein GSK-3 in rat kidney SAP expression in each group (P0.05), but there was no significant difference between the groups of SAP (P0.05); beta -GSK-3 beta ser9 expression is higher in the SO group, in SAP3h group, the expression was higher than the SO group, but in SAP6h group, along with the progress of the disease, the expression of p-GSK-3 beta ser9 has gradually weakened (P0.05). Conclusion: with the progression of SAP, rat pancreas, kidney pathological damage is progressive, Cr detection index of renal function, BUN also changed. The renal tissues of rats in GSK-3 beta with SAP progression did not appear Clear expression, but the p-GSK-3 beta ser9 phosphorylated forms of expression are enhanced first, after gradually weakened, showed that phosphorylation of GSK-3 beta may play an important role in the pathogenesis of SAP with renal injury. The second part: comparative study on renal injury in rats with severe acute pancreatitis and the effect of the amount of effect of different types of GSK-3p inhibitors
Objective: To observe the dose effect relationship of TDZD-8 intervention in SAP rats with kidney injury, and three kinds of commonly used GSK-3 beta TDZD-8 inhibitors, lithium chloride (LiCL), SB216763 compares the effect of the model, to explore for SAP patients with renal injury in rats model of GSK-3 beta inhibitors most effective and effective optimal dose not. Safety. Methods: 96 male Vistar SPF rats were randomly divided into 8 groups (N=12): sham operation group (SO group), severe acute pancreatitis group (SAP group), TDZD-8 pretreatment group (group TD, 0.25,0.5,1,2mg/kg were labeled as TD1, TD2, TD3, TD4 group), LiCL treatment group (L group) and SB216763 pretreatment group (SB group), pancreatic duct retrograde injection of 5% sodium taurocholate SAP model.SO group, SAP group were on preoperative 30min by intravenous injection solvent 10%DMSO (O.lml/100g), different doses of TDZD-8 preconditioning group SAP before modeling 30min by intravenous injection of the same amount of 10%DMSO the dissolution of different agents The amount of TDZD-8; group L and group SB were injected with equal volume 10%DMSO dissolved LiCL (60mg/kg) and SB216763 (1mg/kg) respectively via femoral vein. After operation, 12h was used to kill rats in each group. The mortality, ascites volume, serum AMY, Cr, level of 12h and level of rats in each group were measured, and pathological changes in pancreas and kidney were observed.
緇撴灉錛歋O,SAP,TD3,L鍜孲B緇勫ぇ榧犳浜＄巼鍒嗗埆涓，

本文編號(hào)：1730896