本文選題：猴頭爰舌多裙 + 胃黏膜損傷　； 參考：《廣州中醫(yī)藥大學(xué)》2014年碩士論文

【摘要】：目的： 通過觀察猴頭菇多糖對無水乙醇、吲哚美辛、醋酸、水浸拘縛所致大鼠胃黏膜損傷的作用,研究猴頭菇多糖對胃黏膜的保護(hù)作用及其作用機(jī)制,并指導(dǎo)配方中的有效添加劑量；通過觀察猴頭菇多糖對LPS誘導(dǎo)Caco-2細(xì)胞及Caco-2/RAW264.7共培養(yǎng)體系應(yīng)激炎癥反應(yīng)的保護(hù)作用,在細(xì)胞水平研究猴頭菇多糖對腸黏膜屏障的保護(hù)作用。 方法： 1、猴頭菇多糖對胃黏膜保護(hù)作用的實(shí)驗(yàn)研究 采用無水乙醇致大鼠胃黏膜損傷模型觀察猴頭菇多糖對急性胃黏膜損傷的保護(hù)作用,動物隨機(jī)分成空白對照組、模型組對照組、陽性對照組、猴頭菇多糖各劑量組。以猴頭菇多糖的人體最小參考劑量(0.2g/d)的5倍作為大鼠的最小給藥劑量(HEP1),人體最大參考劑量(0.4g/d)的20倍作為最大給藥劑量(HEP4),再在最大、最小劑量間再設(shè)置2個梯度(HEP2、HEP3)。劑量設(shè)置分別為：HEP117mg/kg, HEP268mg/kg, HEP3102mg/kg, HEP4136mg/kg。實(shí)驗(yàn)以無水乙醇模型大鼠的胃黏膜潰瘍指數(shù)為根據(jù)篩選出有效劑量。 在篩選出有效劑量的基礎(chǔ)上,分別采用吲哚美辛、醋酸、水浸拘縛致大鼠胃黏膜損傷模型以進(jìn)一步觀察猴頭菇多糖對胃黏膜的保護(hù)作用并探討其保護(hù)機(jī)理。SD大鼠,雄性,180-220g,隨機(jī)分空白對照組、模型組對照組、陽性對照組、HEP劑量組。灌胃給藥1次/d,共10天,對照組給予等量蒸餾水。 (1)末次給藥1h后,除空白組外,動物灌胃吲哚美辛80mg/kg,空白組給予等體積蒸餾水。7h后處死動物,取出全胃,測量胃黏膜潰瘍面積,酶聯(lián)免疫法測定胃黏膜組織勻漿上清中的前列腺素(PGE2)、表皮生長因子(EGF)含量。 (2)給藥前,以微量注射器平刺入漿膜注射30%醋酸制備大鼠慢性胃黏膜損傷模型,末次給藥后禁食24h處死動物,取出全胃,測量胃黏膜潰瘍面積,酶聯(lián)免疫法測定胃黏膜組織勻漿上清中的堿性成纖維生長因子(bFGF)含量,實(shí)時熒光定量RT-PCR法測定轉(zhuǎn)化生長因子(TGF-α) mRMA表達(dá)的影響。 (3)末次給藥0.5h后固定于金屬網(wǎng)籠內(nèi),直立浸于水溫20±1℃的水槽中6h制備大鼠應(yīng)激性胃黏膜損傷模型,10%水合氯醛麻醉動物,于劍突下正中切開腹前壁,于胃體及胃竇處各作一個長度0.5cm的橫向切口,運(yùn)用激光多普勒灌注監(jiān)視儀測定胃體及胃竇處血流量(GMBF)。以激光多普勒信號表示血流量的相對數(shù)值,以胃體及胃竇處血流量均值表示胃黏膜血流量；處死動物,取出全胃,測量胃黏膜潰瘍面積。 2、猴頭菇多糖對腸黏膜保護(hù)作用的實(shí)驗(yàn)研究： 將Caco-2細(xì)胞以105濃度接種在Transwell轉(zhuǎn)運(yùn)培養(yǎng)槽的微孔濾膜上進(jìn)行培養(yǎng),培養(yǎng)基為DMEM高糖(pH7.4,含10%胎牛血清,2mmol·L-1L-谷氨酰胺,10mmol-L-1Hepes、NEAA、青鏈霉素),于37℃,5%CO2培養(yǎng)箱中培養(yǎng)24h換液,以后每隔48h換液,約4-6d細(xì)胞融合達(dá)90%進(jìn)行傳代。采用形態(tài)學(xué)觀察、跨膜電阻測定、表觀滲透系數(shù)測定和堿性磷酸酶活性檢測對細(xì)胞模型進(jìn)行評估。以脂多糖(LPS)刺激1h引起Caco-2細(xì)胞及Caco-2/RAW264.7共培養(yǎng)體系應(yīng)激炎癥反應(yīng),加入猴頭菇多糖干預(yù)24h,收集下側(cè)轉(zhuǎn)運(yùn)液檢測相關(guān)炎癥因子的分泌情況。 (1)細(xì)胞培養(yǎng)至15d,光鏡下觀察細(xì)胞分布均勻,邊界清晰,以單層致密的方式排列時,即可進(jìn)行實(shí)驗(yàn)。在Transwell板上部加LPS50pg/ml,1h后在上部加入猴頭菇多糖500μg/ml,24h后取下側(cè)培養(yǎng)液酶聯(lián)免疫法測定細(xì)胞因子IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ的含量。 (2)細(xì)胞培養(yǎng)至15d,光鏡下觀察細(xì)胞分布均勻,邊界清晰,以單層致密的方式排列時,在Transwell板下部以105接種小鼠單核巨噬細(xì)胞(RAW264.7),共培養(yǎng)24h。在Transwell板上部加LPS50pg/ml,1h后再加入猴頭菇多糖500μg/ml,24h后取下側(cè)培養(yǎng)液酶聯(lián)免疫法測定細(xì)胞因子IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ的含量。 結(jié)果： 1、猴頭菇多糖對胃黏膜保護(hù)作用的實(shí)驗(yàn)研究 (1)劑量篩選實(shí)驗(yàn)結(jié)果表明,猴頭菇多糖的17mg/kg、68mg/kg和136mg/kg劑量均能對無水乙醇造成的胃黏膜急性損傷有抑制作用。后續(xù)實(shí)驗(yàn)研究選擇17mg/kg、68mg/kg劑量作為猴頭菇多糖的低、高劑量。 (2)猴頭菇多糖對吲哚美辛模型作用的實(shí)驗(yàn)表明,猴頭菇多糖的低、高劑量均能降低模型大鼠胃黏膜潰瘍面積,增加模型大鼠胃黏膜組織中前列腺素(PGE2)、表皮生長因子(EGF)含量的表達(dá)； (3)猴頭菇多糖對醋酸模型作用的實(shí)驗(yàn)表明,猴頭菇多糖低、高劑量均能降低該模型大鼠胃黏膜塤瘍面積,增加模型大鼠胃黏膜組織中堿性成纖維生長因子(bFGF)的含量,并提高其轉(zhuǎn)化生長因子(TGF-α) mRMA的表達(dá)； (4)猴頭菇多糖對水浸拘縛模型作用的實(shí)驗(yàn)表明,猴頭菇多糖低、高劑量均能降低模型大鼠胃黏膜潰瘍面積,升高大鼠胃黏膜血流量(GMBF);2、猴頭菇多糖對腸黏膜保護(hù)作用的實(shí)驗(yàn)研究： (1)LPS刺激下,Caco-2細(xì)胞以及Caco-2/RAW264.7細(xì)胞共培養(yǎng)體系IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ分泌量均顯著增加； (2)猴頭菇多糖可以顯著抑制Caco-2細(xì)胞以及Caco-2/RAW264.7細(xì)胞共培養(yǎng)體系促炎細(xì)胞因子IL-6、IL-8, IL-12的分泌,而促進(jìn)抗炎細(xì)胞因子IL-10的分泌,對以上細(xì)胞模型的應(yīng)激炎癥反應(yīng)發(fā)揮免疫調(diào)節(jié)作用。 結(jié)論： 胃黏膜保護(hù)實(shí)驗(yàn)研究表明猴頭菇多糖對急慢性、藥物誘發(fā)型、應(yīng)激性胃黏膜損傷模型均具有明顯的預(yù)防和改善作用。進(jìn)一步機(jī)制研究發(fā)現(xiàn),猴頭菇多糖可通過增加胃膜黏膜血流量,促進(jìn)黏膜組織中PGE2、EGF、bFGF及TGF-a mRMA的分泌,從而增強(qiáng)胃黏膜自身的防御功能,達(dá)到保護(hù)胃黏膜、抗?jié)兊哪康摹?腸黏膜保護(hù)實(shí)驗(yàn)研究表明猴頭菇多糖通過抑制促炎細(xì)胞因子分泌,促進(jìn)抗炎細(xì)胞因子分泌來緩解模擬腸黏膜屏障環(huán)境的Caco-2細(xì)胞及Caco-2/RAW264.7共培養(yǎng)體系的應(yīng)激炎癥反應(yīng),其保護(hù)作用機(jī)制與其免疫調(diào)節(jié)的生理活性相關(guān)。
[Abstract]:Purpose :

The protective effect and mechanism of polysaccharide on gastric mucosa were studied by observing the effect of the polysaccharide on the gastric mucosa of rats induced by absolute ethanol , indometacin , acetic acid and water , and the effective additive amount in the formulation was instructed .
The protective effect of the polysaccharide on LPS - induced inflammatory reaction of Caco - 2 cells and Caco - 2 / RAW264.7 co - culture system was observed .

Method :

An Experimental Study on the Protective Effect of Polysaccharide on Gastric Mucosa

The rats were randomly divided into two groups : blank control group , model group control group , positive control group and monkey head mushroom polysaccharide . The minimum reference dose ( 0 . 2g / d ) of the human body was taken as the minimum dose ( HEP1 ) of rats , the maximum reference dose ( 0.4 g / d ) was 20 times as the maximum dose ( HEP4 ) , then two gradients ( HEP2 , HEP3 ) were set up between the maximum and minimum doses . The dose setting was HEP117mg / kg , HEP26mg / kg , HEP3102mg / kg and HEP4 136 mg / kg , respectively .

On the basis of screening the effective dose , the rat gastric mucosa injury model was induced by indometacin , acetic acid and water . The protective mechanism of the polysaccharide on gastric mucosa was further investigated . The rats were divided into two groups : SD rats , male rats , 180 - 220g , randomly divided control group , model group control group , positive control group and HEP dose group .

( 1 ) After the last dose of 1h , in addition to the blank group , the animals were treated with indometacin 80 mg / kg and the blank group was given equal volume of distilled water . After 7 hours , the animals were sacrificed , the whole stomach was taken out , the area of gastric mucosa ulcer was measured , and the content of prostaglandin ( PGE2 ) and epidermal growth factor ( EGF ) in the supernatant of gastric mucosa tissue homogenate was measured by ELISA .

( 2 ) To prepare rat chronic gastric mucosa injury model by injecting 30 % acetic acid into plasma membrane by a micro - injector . After the last administration , the rats were sacrificed for 24h , the whole stomach was taken out , the area of gastric mucosa ulcer was measured , the alkaline fibroblast growth factor ( bFGF ) content in the supernatant of gastric mucosa tissue homogenate was measured by enzyme - linked immunosorbent assay , and the influence of the expression of transforming growth factor ( TGF - 偽 ) mRMA was measured by real - time fluorescence quantitative RT - PCR .

( 3 ) After the last dose of 0.5 h , the rats were fixed in a metal mesh cage , and the rats were immersed in a water tank at a temperature of 20 鹵 1 鈩，

本文編號：1818957