【作者】 王春艳；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的:单核-巨噬细胞的活化在诱导炎症反应中起关键作用。内毒素的主要活性成分脂多糖(Lipopolysaccharide,LPS)或其它致炎因素激活巨噬细胞释放大量炎症介质,包括TNF-α、IL-1β和IL-6 等,引起机体自身组织损伤,导致过度的全身性炎症反应,最终导致多器官功能障碍以至死亡。CD14 是巨噬细胞识别LPS 的关键受体,LPS 与LPS结合蛋白(Lipopolysaccharide binding protein, LBP)形成复合物后,与CD14 结合激活巨噬细胞,因而CD14 是LPS激活细胞发挥效应的始动环节。CD14 包括膜结合型CD14(membrane bound CD14, mCD14)和可溶性CD14(solubleCD14, sCD14)两种形式。八肽胆囊收缩素(cholecystokinin octapeptide, CCK-8)具有一定的抗炎及抗休克作用,可明显减轻内毒素休克(endotoxin shock, ES)大鼠肺脏、肝脏、脾脏、肾脏间质水肿及白细胞浸润等炎性病理损伤。但有关CCK-8 的抗炎作用及其机制尚未完全阐明。蛋白激酶C(Protein Kinase C,PKC)作为重要的信息分子广泛参与机体免疫调节和炎症反应的信号转导。许多研究证实PKC 是参与LPS 诱导CD14表达的重要信号介质。最近本室研究表明,在肺间质巨噬细胞(pulmonaryinterstitial macrophages,PIMs),CCK-8 可明显抑制LPS 诱更多还原

【Abstract】 Objective: It is well known that the activation ofmonocytes/macrophages plays a key role in inducing theinflammatory response. Macrophages stimulated by LPS （maincomponent of endotoxin） or other inflammatory factors canproduce and release a lot of various proinflammatory cytokinesincluding TNF-α, IL-1β, IL-6 and so on. Overproduction of thecytokines can result in systemic inflammatory responsesyndrome （SIRS） and multiple organ dysfunction syndrome（MODS）, which might lead to death. CD14 is a key receptorrecognizing LPS in macrophages. LPS can activate macrophagethrough interaction with CD14 after its binding tolipopolysaccharide binding protein （LBP）. So, CD14 is a initialdot of LPS activating cells. CD14 exists in membrane-bound（mCD14） form or soluble form （sCD14）.Cholecystokinin octapeptide （CCK-8） had anti-shock andanti-inflammation effects, and could alleviate the inflammatorylesions of the lung, liver and spleen tissues in endotoxin shock（ES） rat significantly. However, the mechanism of alleviating theinflammatory response by CCK-8 is not clear. It is well knownthat protein kinase C （PKC） acts as an important messagemolecular, which involved in immune regulation and signaltransduction in inflammatory response. Previous studies showedthat PKC was an important signal mediator participating in theCD14 expression induced by LPS.Recently, our laboratory reported that CCK-8 inhibitedCD14 mRNA and mCD14 protein expression and sCD14 releasein pulmonary interstitial macrophages （PIMs） induced by LPS,which causing PIMs low sensitization to endotoxin（lipopolysaccharide）. This effect may be one of the mechanismsfor CCK-8 against ES. However, the signal transductionmechanism of CCK-8 regulating the CD14 expression inducedby LPS is not clear. To elucidate the anti-inflammatory andanti-shock mechanism of CCK-8, we used murine macrophageline RAW264.7 to investigate the effect of PKC on the signaltransduction pathway of CCK-8 regulating CD14 expression, andto analyze the signal transduction mechanism of CD14expression mediated by CCK-8.Methods: The murine macrophage line RAW264.7 werecultured and passaged to logarithm growth phase. 1×10⁷ cells perbottle were treated with serum-free RPMI-1640 or other reagents.The groups as follows: （1） control group: serum-free RPMI-1640as vehide. （2） LPS group: cells were subject to 1mg/L LPS. （3）LPS+GF109203X group: pretreatment with 10^-9, 10^-7 or 10^-5mol/L GF109203X 30min before LPS was used. （4） PMA group:cells were treated with PMA 10^-7 mol/L. （5） PMA+CCK group:pretreatment with PMA 10-7 mol/L 30min before 10^-10, 10^-8 or10^-6 mol/L CCK-8 administration. The RAW264.7 cells had beenincubated with above treatment for 24h. The mCD14 expressionon the membrane of the RAW264.7 cells was assayed byflowcytometry. The sCD14 protein level in the supernatant wasmeasured by Western blot, and the CD14 mRNA expression inthe RAW264.7 cells was analyzed by RT-PCR. Data werepresented as x ±s and analyzed with ANOVA and LSD usingSPSS statistical program. A level of P<0.05 was consideredstatistically significant.Results: （1）Non-stimulated RAW264.7 cells expressed alittle of mCD14. At 24h after LPS stimulation, mCD14expression was markedly increased compared with the contralgroup, and the percentage of fluorescent cells was24.76%±4.81% （P<0.01）. While the RAW264.7 cells wereincubated with both LPS and 10^-9, 10-7 or 10^-5 mol/L GF109203Xfor 24h, the percentage of fluorescent cells was reduced to16.22%±4.76% （P>0.05）, 11.62%±3.00% （P<0.05） and7.35%±1.23% （P<0.01） respectively. There was no difference inmCD14 expression between 10-9 mol/L GF109203X-pretreatedgroup and LPS-treated group （P>0.05）. At 24h after PMA 10^-7mol/L incubation, mCD14 expression was up-regulatedsignificantly in comparison with the contral group, and thepercentage of fluorescent cells was 32.09%±6.01% （P<0.01）.While the RAW264.7 cells were incubated with both PMA andCCK 10^-10, 10^-8 or 10^-6 mol/L for 24h, the percentage offluorescent cells was reduced to 26.38%±3.29% （P>0.05）,15.84%±4.71% （P<0.05） and 12.66%±2.62% （P<0.01） in adose-dependent manner. No significant change in mCD14expression was observed between CCK 10^-10 mol/L-pretreatedgroup and PMA-treated group （P>0.05）. （2）We detected a littleof sCD14αin the supernatant of the control group. At 24h afterLPS stimulation, sCD14αprotein in supernatant was notablyincreased compared with the contral group （P<0.01）.Co-incubation with LPS and GF109203X (10^-7 or 10-5 mol/L)clearly reduced the sCD14αlevel （P<0.05）, and the inhibitorypercentage was 25% and 50%. 10^-9 mol/L GF109203X-pretreatedgroup also decreased the sCD14αlevel, but the result was nodifference from that of the contral group （P>0.05）. At 24h afterPMA 10^-7 mol/L incubation, sCD14αprotein in supernatant wasmarkedly increased in comparison with the contral group（P<0.01）. CCK 10^-10, 10^-8 and 10^-6 mol/L reduced clearly thesCD14αlevel induced by PMA in a dose-dependent manner（P<0.01）, and the inhibitory percentage was 37%, 45% and 76%respectively. （3） Non-stimulated RAW264.7 cells expressed alittle of CD14 mRNA. Incubation with 1mg/L LPS for 24hresulted in an obvious elevation in CD14 mRNA expression（P<0.01）. 10^-7 and 10-5 GF109203X-pretreatment could marklydecrease the CD14 mRNA level induced by LPS （P<0.05）, andthe inhibitory percentage was 54% and 70% respectively. 10^-9mol/L GF109203X-pretreatment also inhibited the CD14 mRNAexpression, but the result was no difference from that of the LPSgroup （P>0.05）. At 24h after PMA 10^-7 mol/L incubation, amarked increase was observed in CD14 mRNA expression in更多还原