本文關(guān)鍵詞： 刀豆球蛋白A 肝損傷 P物質(zhì) 神經(jīng)激肽1-受體拮抗劑 神經(jīng)源性炎癥 枯否細(xì)胞 細(xì)胞因子 腫瘤壞死因子-α 白細(xì)胞介素-6　出處：《山東大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分小鼠免疫性肝炎模型的建立及NK-1受體拮抗劑L-703,606對小鼠免疫性肝損傷的影響 目的 采用經(jīng)小鼠的尾靜脈注射刀豆蛋白A (Concanavalin A, Con A),制造小鼠免疫性肝損傷模型,通過檢測肝組織中P物質(zhì)(substance P, SP)的含量變化,證實(shí)SP介導(dǎo)的神經(jīng)源性炎癥在肝炎的發(fā)生和發(fā)展中的作用；同時(shí)從生化、病理學(xué)、細(xì)胞因子等方面分析比較,SP受體即神經(jīng)激肽-1受體(Neurokinin-1receptor, NK-1R)的阻滯劑L-703,606對小鼠免疫性肝炎肝損傷的干預(yù)作用,進(jìn)一步證實(shí)神經(jīng)內(nèi)分泌系統(tǒng)對肝炎的調(diào)控機(jī)制,為各種肝炎的發(fā)病機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法 將60只雌性小鼠隨機(jī)平分為生理鹽水對照組、刀豆球蛋白A (Con A)免疫性肝炎模型組和NK-1R阻滯劑L-703,606干預(yù)組,每組20只。生理鹽水對照組給予尾靜脈注射生理鹽水0.2ml；刀豆球蛋白A免疫性肝炎模型組給予尾靜脈注射20mg/kg Con A; NK-1R阻滯劑L-703,606干預(yù)組在尾靜脈注射10mg/kgL-703,606的同時(shí)給與等量Con A注射。造模后6小時(shí),用水合氯醛麻醉小鼠,處死所有小鼠并收集血液和肝臟標(biāo)本,應(yīng)用酶聯(lián)免疫吸附(ELISA)法測定對照組和模型組肝臟組織中P物質(zhì)的含量；應(yīng)用酶聯(lián)免疫吸附(ELISA)法分別測定各組小鼠肝組織中的腫瘤壞死因子-α (TNF-α)和白介素-6(IL-6)的蛋白含量及各組小鼠血清中轉(zhuǎn)氨酶ALT、AST水平,HE染色觀察肝臟病理變化。 結(jié)果 模型組小鼠肝組織中SP含量顯著高于對照組(387.23±29.36pg/mL VS138.52±13.23pg/mL),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；模型組和干預(yù)組血清AST、ALT水平均明顯升高(P0.01),但與模型組相比,干預(yù)組ALT和AST明顯下降(402.22±16.42U/L VS782.37±21.51U/L；581.45±17.51U/L VS1004.25±18.24U/L),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);ConA模型組TNF-α和IL-6蛋白含量水平均明顯高于生理鹽水對照組(49.15±9.33pg/ml VS12.21±7.15pg/ml；281.17±13.12pg/ml VS131.25±10.34pg/ml),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。同ConA模型組小鼠比較,L-703,606干預(yù)組小鼠TNF-α和IL-6蛋白含量水平下降明顯(P0.05)(20.34±10.14pg/ml VS49.15±9.33pg/ml；152.53±11.16pg/ml VS281.17±13.12pg/ml)。模型組小鼠肝組織病理可見肝細(xì)胞索破壞,肝竇稍增寬,內(nèi)有大量瘀血,肝細(xì)胞彌漫性腫脹,核固縮、溶解甚至消失,點(diǎn)、灶狀及片狀壞死多見,匯管區(qū)及中央靜脈肝竇內(nèi)可見中性粒細(xì)胞和淋巴細(xì)胞浸潤；而干預(yù)組的肝組織病理改變明顯減輕,肝細(xì)胞腫脹壞死及淋巴細(xì)胞浸潤也較模型組明顯改善。 結(jié)論 本實(shí)驗(yàn)制造的模型,組織病理顯示模型組肝臟點(diǎn)、灶狀及片狀壞死多見,匯管區(qū)及中央靜脈肝竇內(nèi)可見中性粒細(xì)胞和淋巴細(xì)胞浸潤,與急性肝損害較為符合。模型組小鼠肝組織SP含量、TNF-α和IL-6蛋白含量水平顯著高于對照組,說明SP介導(dǎo)的神經(jīng)源性炎癥在肝炎的發(fā)生中起到重要的作用。應(yīng)用NK-1受體阻滯劑L-703,606進(jìn)行干預(yù),可使干預(yù)組小鼠血清AST、ALT水平下降,TNF-α和IL-6蛋白含量水平下降,并伴有肝臟組織病理的減輕,證明阻斷SP與其受體的結(jié)合對肝臟炎癥具有一定的抑制和治療作用。 第二部分P物質(zhì)和NK-1受體拮抗劑L-703,606對Kupffer細(xì)胞產(chǎn)生細(xì)胞因子的影響 目的 探討P物質(zhì)對小鼠枯否細(xì)胞(Kupffer Cell,KC)分泌功能的影響。觀察小鼠枯否細(xì)胞在P物質(zhì)作用下炎癥細(xì)胞因子水平的變化,進(jìn)一步探討SP受體(NK-1R)阻滯劑L-703,606的作用及其作用機(jī)制,為臨床應(yīng)用提供新的啟示。 方法 采用Percoll密度梯度離心法分離得到高純度的小鼠枯否細(xì)胞,在過夜培養(yǎng)后沖洗兩遍,以5×105/ml的細(xì)胞濃度,接種于24孔培養(yǎng)板。采用免疫熒光技術(shù)檢測NK-1R在枯否細(xì)胞中的定位。分離并培養(yǎng)枯否細(xì)胞48小時(shí)后,分別設(shè)立不同濃度SP組,確立其對細(xì)胞無毒性的劑量范圍。將枯否細(xì)胞分為三組,為正常培養(yǎng)液+內(nèi)毒素100ng/ml組(B組),正常培養(yǎng)液+內(nèi)毒素100ng/ml+SPluM組(C組),正常培養(yǎng)液+內(nèi)毒100ng/ml+SPluM+L-703,60610uM組(D組),刺激原代培養(yǎng)的枯否細(xì)胞3小時(shí)。應(yīng)用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測小鼠枯否細(xì)胞上清液中TNF-α和IL-6水平。應(yīng)用實(shí)時(shí)逆轉(zhuǎn)錄多聚酶鏈?zhǔn)椒磻?yīng)((reverse transcriptase—polymeras chain reaction,RT-PCR)檢測小鼠枯否細(xì)胞中NK-lRmRNA的表達(dá)。 結(jié)果 離體正常小鼠的枯否細(xì)胞NK-1R免疫熒光主要集中在細(xì)胞質(zhì)中。與對照組比較,P物質(zhì)能增加枯否細(xì)胞中NK-lRmRNA的表達(dá)增加2.5倍；與對照培養(yǎng)基比較應(yīng)用NK-1R拮抗劑能使NK-1R mRNA表達(dá)的顯著減少。經(jīng)過P物質(zhì)刺激后,枯否細(xì)胞上清液中TNF-α及IL-6分泌水平均明顯高于對照組(P0.01)(92.15±8.56pg/ml VS31.02±7.26pg/ml;194.12±11.09pg/ml VS71.13±9.36pg/ml)而NK-1R阻滯劑即L-703,606能減弱P物質(zhì)對枯否細(xì)胞分泌功能的刺激作用,即應(yīng)用NK-1R阻滯劑即L-703,606后,枯否細(xì)胞上清液中TNF-α及IL-6分泌水平均明顯下降(P0.01)(28.38±5.04pg/ml VS92.15±8.56pg/ml;68.16±9.51pg/ml VS194.12±11.09pg/ml)。 結(jié)論 小鼠枯否細(xì)胞中表達(dá)NK-1R,體外實(shí)驗(yàn)證實(shí)P物質(zhì)能增加枯否細(xì)胞中NK-lRmRNA的表達(dá),應(yīng)用NK-1R拮抗劑能減少NK-lRmRNA的表達(dá)。P物質(zhì)對枯否細(xì)胞的分泌功能有激活作用,能使TNF-α及IL-6分泌量增加,而應(yīng)用NK-1R阻滯劑L-703,606能減弱P物質(zhì)對枯否細(xì)胞分泌功能的刺激。
[Abstract]:Establishment of immune hepatitis model and the effects of NK - 1 receptor antagonist L - 703,606 on immune liver injury in mice Purpose A model of immune liver injury in mice was established by using Concanavalin A ( Con A ) . The effects of SP - mediated neurovascular inflammation in the pathogenesis and development of hepatitis were confirmed by detecting the changes of substance P and SP in liver tissues . method Sixty female mice were randomly divided into physiological saline control group , Con A immune hepatitis model group and NK - 1R blocker group L - 703,606 . The contents of TNF - 偽 and IL - 6 in liver tissues were determined by enzyme - linked immunosorbent assay ( ELISA ) . Results Compared with the model group , the levels of ALT and AST in the intervention group were significantly higher than those in the normal saline control group ( 49.15 鹵 9.33pg / ml VS782.37 鹵 21.51U / L ; 585.45 鹵 17.51U / L VS4.25 鹵 10.34pg / ml ) . The pathological changes of liver tissues in the model group were found to be slightly widened , there were a large number of blood stasis , diffuse swelling , nuclear fixation , dissolution and even disappearance , point , focal and lamellar necrosis . The pathological changes of liver tissues in the intervention group were obviously reduced , and the cell swelling and necrosis and lymphocyte infiltration were obviously improved compared with the model group . Conclusion The levels of serum AST , ALT , TNF - 偽 and IL - 6 in the model group were significantly higher than those in the control group . The levels of serum AST , ALT , TNF - 偽 and IL - 6 in the model group were decreased , and the level of TNF - 偽 and IL - 6 protein decreased . Effects of the second part of P - substance and NK - 1 receptor antagonist L - 703,606 on the production of cytokines Purpose To investigate the effect of substance P on the secretion function of ffer cells ( KC ) in mice . To observe the changes of inflammatory cytokine levels in mouse cuffer cells under the action of substance P , further study the role and mechanism of SP receptor ( NK - 1R ) blocking agents L - 703,606 and provide new revelations for clinical application . method The levels of NK - 1R mRNA were determined by enzyme - linked immunosorbent assay ( ELISA ) , and the expression of NK - lRmRNA was detected by reverse transcriptase polymerase chain reaction ( RT - PCR ) . Results Compared with the control group , the levels of TNF - 偽 and IL - 6 in the supernatant of cuffer cells decreased significantly ( P0.01 ) ( 28.38 鹵 5.04pg / ml VS92.15 鹵 8.56pg / ml ; 68.16 鹵 9.51pg / ml VS191.12 鹵 11.09pg / ml ) . Conclusion The NK - 1R mRNA expression in cuffer cells of mice showed that P - substance could increase the expression of NK - lRmRNA in cuffer cells . NK - 1R antagonist could reduce the expression of NK - lRmRNA . The secretion of TNF - 偽 and IL - 6 could be increased , and NK - 1R blocking agents L - 703,606 could attenuate the secretion of NK - 1R mRNA .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R965

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