神经肽P物质在小鼠免疫性肝炎中的作用及对枯否细胞分泌功能影响的研究

发布时间：2018-02-26 17:30

本文关键词： 刀豆球蛋白A 肝损伤 P物质神经激肽1-受体拮抗剂神经源性炎症枯否细胞细胞因子肿瘤坏死因子-α 白细胞介素-6　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分小鼠免疫性肝炎模型的建立及NK-1受体拮抗剂L-703,606对小鼠免疫性肝损伤的影响目的采用经小鼠的尾静脉注射刀豆蛋白A (Concanavalin A, Con A),制造小鼠免疫性肝损伤模型,通过检测肝组织中P物质(substance P, SP)的含量变化,证实SP介导的神经源性炎症在肝炎的发生和发展中的作用；同时从生化、病理学、细胞因子等方面分析比较,SP受体即神经激肽-1受体(Neurokinin-1receptor, NK-1R)的阻滞剂L-703,606对小鼠免疫性肝炎肝损伤的干预作用,进一步证实神经内分泌系统对肝炎的调控机制,为各种肝炎的发病机制提供实验依据。方法将60只雌性小鼠随机平分为生理盐水对照组、刀豆球蛋白A (Con A)免疫性肝炎模型组和NK-1R阻滞剂L-703,606干预组,每组20只。生理盐水对照组给予尾静脉注射生理盐水0.2ml；刀豆球蛋白A免疫性肝炎模型组给予尾静脉注射20mg/kg Con A; NK-1R阻滞剂L-703,606干预组在尾静脉注射10mg/kgL-703,606的同时给与等量Con A注射。造模后6小时,用水合氯醛麻醉小鼠,处死所有小鼠并收集血液和肝脏标本,应用酶联免疫吸附(ELISA)法测定对照组和模型组肝脏组织中P物质的含量；应用酶联免疫吸附(ELISA)法分别测定各组小鼠肝组织中的肿瘤坏死因子-α (TNF-α)和白介素-6(IL-6)的蛋白含量及各组小鼠血清中转氨酶ALT、AST水平,HE染色观察肝脏病理变化。结果模型组小鼠肝组织中SP含量显著高于对照组(387.23±29.36pg/mL VS138.52±13.23pg/mL),差异具有统计学意义(P0.05)；模型组和干预组血清AST、ALT水平均明显升高(P0.01),但与模型组相比,干预组ALT和AST明显下降(402.22±16.42U/L VS782.37±21.51U/L；581.45±17.51U/L VS1004.25±18.24U/L),差异具有统计学意义(P0.05);ConA模型组TNF-α和IL-6蛋白含量水平均明显高于生理盐水对照组(49.15±9.33pg/ml VS12.21±7.15pg/ml；281.17±13.12pg/ml VS131.25±10.34pg/ml),差异有统计学意义(P0.05)。同ConA模型组小鼠比较,L-703,606干预组小鼠TNF-α和IL-6蛋白含量水平下降明显(P0.05)(20.34±10.14pg/ml VS49.15±9.33pg/ml；152.53±11.16pg/ml VS281.17±13.12pg/ml)。模型组小鼠肝组织病理可见肝细胞索破坏,肝窦稍增宽,内有大量瘀血,肝细胞弥漫性肿胀,核固缩、溶解甚至消失,点、灶状及片状坏死多见,汇管区及中央静脉肝窦内可见中性粒细胞和淋巴细胞浸润；而干预组的肝组织病理改变明显减轻,肝细胞肿胀坏死及淋巴细胞浸润也较模型组明显改善。结论本实验制造的模型,组织病理显示模型组肝脏点、灶状及片状坏死多见,汇管区及中央静脉肝窦内可见中性粒细胞和淋巴细胞浸润,与急性肝损害较为符合。模型组小鼠肝组织SP含量、TNF-α和IL-6蛋白含量水平显著高于对照组,说明SP介导的神经源性炎症在肝炎的发生中起到重要的作用。应用NK-1受体阻滞剂L-703,606进行干预,可使干预组小鼠血清AST、ALT水平下降,TNF-α和IL-6蛋白含量水平下降,并伴有肝脏组织病理的减轻,证明阻断SP与其受体的结合对肝脏炎症具有一定的抑制和治疗作用。第二部分P物质和NK-1受体拮抗剂L-703,606对Kupffer细胞产生细胞因子的影响目的探讨P物质对小鼠枯否细胞(Kupffer Cell,KC)分泌功能的影响。观察小鼠枯否细胞在P物质作用下炎症细胞因子水平的变化,进一步探讨SP受体(NK-1R)阻滞剂L-703,606的作用及其作用机制,为临床应用提供新的启示。方法采用Percoll密度梯度离心法分离得到高纯度的小鼠枯否细胞,在过夜培养后冲洗两遍,以5×105/ml的细胞浓度,接种于24孔培养板。采用免疫荧光技术检测NK-1R在枯否细胞中的定位。分离并培养枯否细胞48小时后,分别设立不同浓度SP组,确立其对细胞无毒性的剂量范围。将枯否细胞分为三组,为正常培养液+内毒素100ng/ml组(B组),正常培养液+内毒素100ng/ml+SPluM组(C组),正常培养液+内毒100ng/ml+SPluM+L-703,60610uM组(D组),刺激原代培养的枯否细胞3小时。应用酶联免疫吸附实验(ELISA)检测小鼠枯否细胞上清液中TNF-α和IL-6水平。应用实时逆转录多聚酶链式反应((reverse transcriptase—polymeras chain reaction,RT-PCR)检测小鼠枯否细胞中NK-lRmRNA的表达。结果离体正常小鼠的枯否细胞NK-1R免疫荧光主要集中在细胞质中。与对照组比较,P物质能增加枯否细胞中NK-lRmRNA的表达增加2.5倍；与对照培养基比较应用NK-1R拮抗剂能使NK-1R mRNA表达的显著减少。经过P物质刺激后,枯否细胞上清液中TNF-α及IL-6分泌水平均明显高于对照组(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阻滞剂即L-703,606能减弱P物质对枯否细胞分泌功能的刺激作用,即应用NK-1R阻滞剂即L-703,606后,枯否细胞上清液中TNF-α及IL-6分泌水平均明显下降(P0.01)(28.38±5.04pg/ml VS92.15±8.56pg/ml;68.16±9.51pg/ml VS194.12±11.09pg/ml)。结论小鼠枯否细胞中表达NK-1R,体外实验证实P物质能增加枯否细胞中NK-lRmRNA的表达,应用NK-1R拮抗剂能减少NK-lRmRNA的表达。P物质对枯否细胞的分泌功能有激活作用,能使TNF-α及IL-6分泌量增加,而应用NK-1R阻滞剂L-703,606能减弱P物质对枯否细胞分泌功能的刺激。
[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 - 伪 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 卤 9.33pg / ml VS782.37 卤 21.51U / L ; 585.45 卤 17.51U / L VS4.25 卤 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 - 伪 and IL - 6 in the model group were significantly higher than those in the control group . The levels of serum AST , ALT , TNF - 伪 and IL - 6 in the model group were decreased , and the level of TNF - 伪 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 - 伪 and IL - 6 in the supernatant of cuffer cells decreased significantly ( P0.01 ) ( 28.38 卤 5.04pg / ml VS92.15 卤 8.56pg / ml ; 68.16 卤 9.51pg / ml VS191.12 卤 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 - 伪 and IL - 6 could be increased , and NK - 1R blocking agents L - 703,606 could attenuate the secretion of NK - 1R mRNA .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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