本文關(guān)鍵詞： 鹽酸戊乙奎醚 內(nèi)毒素 腸損傷 缺氧誘導(dǎo)因子-1α　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：新生兒腸損傷是兒科常見的疾病之一，其發(fā)生的主要原因有早產(chǎn)、窒息引起的缺血缺氧以及感染等。其中嚴(yán)重感染常可導(dǎo)致危重患兒胃腸功能衰竭。腸道在全身炎癥反應(yīng)綜合征（SIRS）和多器官功能障礙綜合征(MODS)的發(fā)生發(fā)展過程中扮演著極其重要的角色。腸道感染或缺血缺氧會導(dǎo)致腸黏膜屏障功能受損，引起細(xì)菌及內(nèi)毒素的移位，引發(fā)敗血癥和膿毒性休克，從而進(jìn)一步加重腸組織的損傷，甚者可伴隨遠(yuǎn)隔器官受累。脂多糖（LPS）作為典型的內(nèi)毒素,可誘發(fā)內(nèi)毒素血癥，導(dǎo)致腸黏膜損害。國內(nèi)外諸多研究發(fā)現(xiàn)缺氧誘導(dǎo)因子-1α（HIF-1α）在內(nèi)毒素性腸損傷的病理改變過程中具有重要作用。有研究指出腸道缺血缺氧可以誘導(dǎo)腸黏膜HIF-1α持續(xù)高表達(dá)，這一結(jié)果至少在部分程度上是由于腸道細(xì)菌的移位及其產(chǎn)物（如LPS）的增加所造成的。HIF-1α能夠通過激活其下游的相關(guān)基因轉(zhuǎn)錄并促進(jìn)炎癥因子的大量釋放和引起細(xì)胞凋亡等，從而加重內(nèi)毒素血癥或缺血/再灌注時的組織損傷。鹽酸戊乙奎醚（PHC）是我國自主研制的抗膽堿藥。研究表明，鹽酸戊乙奎醚因能改善微循環(huán)、降低毛細(xì)血管壁通透性和減少溶酶體釋放而具有多器官保護(hù)作用。目前鹽酸戊乙奎醚對內(nèi)毒素性肺損傷的保護(hù)作用得到了廣泛地證實和認(rèn)可，我們通過大量的臨床觀察發(fā)現(xiàn)鹽酸戊乙奎醚在解除腸痙攣、減輕腸道水腫方面同樣具有良好的效果�；谝陨媳尘�，本課題旨在研究內(nèi)毒素致新生大鼠腸損傷時鹽酸戊乙奎醚預(yù)先給藥對腸組織HIF-1α表達(dá)的影響，并對其腸保護(hù)作用機(jī)制進(jìn)行初步探討。 目的 探討鹽酸戊乙奎醚預(yù)先給藥對內(nèi)毒素致新生大鼠腸損傷時腸組織HIF-1α和其他相關(guān)細(xì)胞因子表達(dá)的影響及其發(fā)揮腸保護(hù)作用的可能機(jī)制，為臨床防治新生兒腸損傷提供一條新的思路。 材料與方法 1.研究對象 69只SPF級健康新生7日齡SD大鼠，由鄭州大學(xué)河南省實驗動物中心提供，雌雄不限，體重18±2g。 2.實驗方法 2.1實驗動物分組 本實驗分為兩部分。第一部分：將30只幼鼠隨機(jī)分為3組，每組10只。分別為生理鹽水對照組（C組）、內(nèi)毒素致腸損傷模型組（L組）和鹽酸戊乙奎醚干預(yù)組（P組）。本部分實驗主要進(jìn)行腸組織濕/干重比的測定、腸組織HE染色后光鏡下形態(tài)學(xué)改變的觀察以及相關(guān)細(xì)胞因子的檢測。第二部分：同樣將另39只健康新生7日齡SD大鼠隨機(jī)分為C組、L組和P組，每組13只。本部分實驗用于觀察造模后各組幼鼠行為活動的改變并計算每組24h生存率。 2.2動物模型的制備 內(nèi)毒素致腸損傷模型組腹腔注射內(nèi)毒素（細(xì)菌脂多糖LPS，E.Coli055：B5，Sigma公司，美國）5mg/kg，配制濃度為0.5mg/ml；鹽酸戊乙奎醚干預(yù)組在注射內(nèi)毒素前30min，先腹腔注射鹽酸戊乙奎醚（成都力思特制藥股份有限公司，批號：120603）2mg/kg，配制濃度為0.05mg/ml；生理鹽水對照組腹腔注射生理鹽水10ml/kg。第一部分實驗于腹腔注射內(nèi)毒素或生理鹽水后6h麻醉并解剖幼鼠。兩部分實驗均在造模完成后將幼鼠放回鼠籠與母鼠共同喂養(yǎng)。 2.3腸組織的標(biāo)本采集 在密閉玻璃容器中吸入七氟烷快速麻醉幼鼠后，將其固定于操作臺，解剖，用1ml注射器迅速進(jìn)行心臟采血，靜置2小時，4℃下4000r/min離心15分鐘，取上清液，-20℃冰箱中保存待檢。距回盲部3cm向上取回腸組織7cm，4℃生理鹽水沖洗腸管3遍，濾紙吸干腸組織表面水分后，將7cm回腸組織分為4段：上段1cm用于光鏡下形態(tài)學(xué)觀察，中上段2cm用于測量腸組織濕重和干重，中下段2cm用于制備腸組織勻漿測定相關(guān)細(xì)胞因子的表達(dá)，下段2cm用于RT-PCR檢測腸組織HIF-1α mRNA的表達(dá)。 2.4檢測指標(biāo) 2.4.1計算腸組織濕/干重比（W/D） 電子稱重計稱量中上段2cm回腸組織并記錄濕重，隨后放入70℃恒溫烘干箱烘干48h至重量不再發(fā)生變化，測量干重，計算濕/干重比。 2.4.2腸組織形態(tài)學(xué)觀察 將上段1cm回腸組織放入4%多聚甲醛中保存待檢。石蠟包埋后制作5μm病理切片，，行HE染色。400倍光鏡下觀察回腸組織黏膜上皮細(xì)胞形態(tài)學(xué)的改變。 2.4.3ELISA法測定腸組織中TNF-α、IL-6、HIF-1α、Gln、二胺氧化酶（DAO）及血清DAO的含量 中下段2cm回腸組織稱重后制備組織勻漿，4℃下3000r/min離心15分鐘，取上清液，置于-20℃冰箱中保存待檢。TNF-α、IL-6、HIF-1α、Gln和DAO的檢測嚴(yán)格按照ELISA試劑盒（RD公司，美國）說明書進(jìn)行。 2.4.4RT-PCR法測定腸組織HIF-1α mRNA的表達(dá) 將下段2cm回腸組織經(jīng)DEPC水沖凈后濾紙吸干，經(jīng)液氮迅速冷卻后放入凍存管中，置于-80℃冰箱保存待檢。經(jīng)腸組織總RNA的提取、逆轉(zhuǎn)錄、DNA擴(kuò)增和瓊脂糖凝膠電泳等步驟檢測幼鼠腸組織HIF-1α mRNA的表達(dá)情況。 3．統(tǒng)計學(xué)處理 采用SPSS17.0統(tǒng)計軟件包進(jìn)行統(tǒng)計學(xué)分析，計量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，多組間比較采用單因素方差分析，兩兩比較采用最小顯著差異法（LSD-t），以α=0.05為檢驗水準(zhǔn)，即P0.05時，組間差異有統(tǒng)計學(xué)意義。 結(jié)果 1.肉眼觀察，腹腔注射LPS后，1小時內(nèi)L組與P組幼鼠開始出現(xiàn)渾身抖動，呼吸急促。2~3小時左右出現(xiàn)周身發(fā)涼，精神萎靡，活動度下降，并且進(jìn)食減少。6小時左右出現(xiàn)明顯的倦怠乏力，幾乎不活動也不進(jìn)食，甚者口唇青紫。P組幼鼠癥狀相對較輕，C組幼鼠活動、進(jìn)食等情況正常。C組、L組及P組的24h生存率分別為100%、69.2%和92.3%，幼鼠死亡主要發(fā)生在注射LPS后12~24小時期間。 2.與C組比較，L組和P組腸組織濕/干重比均顯著升高（P0.05）；與L組比較，P組的這一比值顯著降低（P0.05）。差異均有統(tǒng)計學(xué)意義。 3.HE染色光鏡結(jié)果顯示：C組回腸絨毛結(jié)構(gòu)完整，上皮細(xì)胞排列整齊，形態(tài)正常；L組可見絨毛細(xì)胞水腫，上皮細(xì)胞排列紊亂；P組絨毛水腫較L組明顯減輕，上皮細(xì)胞排列較整齊。 4.與C組比較，L組和P組腸組織TNF-α、IL-6、HIF-1α的含量均升高（P0.05），而Gln的含量降低（P0.05）；與L組比較，P組腸組織Gln的含量較高（P0.05），而余各指標(biāo)的含量均較低（P0.05）。差異均有統(tǒng)計學(xué)意義。 5.與C組比較，L組和P組腸組織DAO含量均顯著降低，而血清DAO含量升高（P0.05）；與L組比較，P組腸組織DAO含量較高，而血清DAO含量較低（P0.05）。差異均有統(tǒng)計學(xué)意義。 6.與C組比較，L組和P組腸組織HIF-1α mRNA的表達(dá)量均升高（P0.05）；與L組相比，P組腸組織HIF-1α mRNA的表達(dá)量較低（P0.05）。差異均有統(tǒng)計學(xué)意義。 結(jié)論 鹽酸戊乙奎醚預(yù)先給藥能夠減輕內(nèi)毒素血癥時的腸道水腫，減輕腸組織炎癥反應(yīng)，降低腸黏膜屏障的通透性。其腸保護(hù)作用機(jī)制可能與其下調(diào)HIF-1α的表達(dá)有關(guān)。
[Abstract]:Neonatal intestinal injury is one of the most common diseases in children, the main reasons for its occurrence are premature, caused by ischemia and hypoxia and asphyxia. The infection of severe infection often leads to failure in critically ill children. Gastrointestinal gut in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) plays a very important the role in the pathogenesis of intestinal infection or ischemia. Hypoxia can lead to intestinal mucosal barrier dysfunction, caused by the translocation of bacteria and endotoxin, sepsis and septic shock, so as to further increase the intestinal tissue damage, even accompanied with remote organ involvement. Lipopolysaccharide (LPS) as a typical endotoxin induced endotoxemia that lead to intestinal mucosal damage. Many studies found that hypoxia inducible factor -1 alpha (HIF-1 alpha), intestinal injury pathological change process plays an important role on. That intestinal anoxia can induce intestinal mucosal HIF-1 a high expression, this result is at least partly due to the shift of intestinal bacteria and their products (such as LPS) caused by an increase in.HIF-1 alpha through transcriptional activation of its downstream genes and promote the inflammatory factor release and induce cell apoptosis. In order to increase the tissue damage during reperfusion endotoxemia or ischemia. Penehyclidine hydrochloride (PHC) is China's self-developed anticholinergic drugs. The study shows that penehyclidine hydrochloride can improve microcirculation, reduce capillary wall permeability and reduce the release of lysosomal function has multiple organ protection. Current protection effect of penehyclidine hydrochloride on endotoxin induced acute lung injury has been widely recognized and confirmed, we found that penehyclidine hydrochloride in relieving intestinal spasm through a number of clinical observation, reduce intestinal edema. Based on the above background, the aim of this study is to investigate the effect of penehyclidine hydrochloride on the expression of HIF-1 alpha in intestinal tissue after intestinal injury induced by lipopolysaccharide in neonatal rats, and to explore the mechanism of intestinal protection.
objective
Objective to investigate the effect of penehyclidine hydrochloride on the expression of HIF-1 and other related cytokines in intestinal tissue of neonatal rats with endotoxin induced intestinal injury, and to explore the possible mechanisms of intestinal protection, so as to provide a new idea for the prevention and treatment of neonatal intestinal injury.
Materials and methods
1. research objects
69 SPF grade healthy newborn 7 day old SD rats were provided by the experimental animal center of Henan province of Zhengzhou University. The male and female were not limited, and the weight was 18 + 2g.
2. experimental method
2.1 group of experimental animals
This experiment is divided into two parts. The first part: 30 rats were randomly divided into 3 groups, 10 rats in each group respectively. Normal saline control group (group C), endotoxin induced intestinal injury model group (L group) and penehyclidine hydrochloride group (group P). The main part of the experiment of intestine tissue wet / dry weight ratio determination, detection of intestinal tissue HE staining was used to observe the morphological changes under light microscopy and related cytokines. The second part: the same to the other 39 healthy SD rats of 7 days old were randomly divided into C group, L group and P group, 13 rats in each group. This part of the experiment for observation in different group activities after the model change and calculate each 24h survival rate.
The preparation of 2.2 animal models
Endotoxin induced intestinal injury model by intraperitoneal injection of endotoxin (lipopolysaccharide LPS, E.Coli055:B5, Sigma, 5mg/kg, USA) with a concentration of 0.5mg/ml; penehyclidine hydrochloride in the intervention group before the first injection of endotoxin 30min, intraperitoneal injection of penehyclidine hydrochloride (Chengdu Lisite pharmaceutical Limited by Share Ltd, batch number: 120603) 2mg/kg concentration was prepared 0.05mg/ml; saline control group received intraperitoneal injection of saline 10ml/kg. the first part of the experiment on intraperitoneal injection of endotoxin or saline 6h after anesthesia and the anatomy of rats. The two part experiments were made in rats after the rats were returned to the cage and the mother feeding.
Sample collection of 2.3 intestinal tissues
In a closed glass container seven inhalation halothane anesthesia in rats after rapid, which is fixed on the operating table, anatomy, with 1ml syringe rapid heart blood, standing 2 hours, at 4 4000r/min centrifuge for 15 minutes, the supernatant was detected, kept in the refrigerator. From -20 DEG 3cm to ileocecus intestinal tissue on the back 7cm, 4 C saline intestine 3 times, intestinal tissue surface water filter paper blot, 7cm ileum is divided into 4 sections: the upper 1cm for morphological observation under light microscope, in the upper 2cm used to measure intestinal tissue wet weight and dry weight, the lower section of 2cm for the preparation of expression of intestinal tissue homogenate were determined by cell the lower section of the 2cm factor, used to detect the expression of intestinal RT-PCR HIF-1 alpha mRNA.
2.4 detection index
2.4.1 calculation of the wet / dry weight ratio of intestinal tissue (W/D)
The electronic weigher weighed the upper and middle 2cm ileum tissue and recorded the wet weight. Then it was put into the 70 degree temperature drying oven and dried to 48h until the weight was no longer changed. The dry weight was measured and the wet / dry weight ratio was calculated.
Observation of 2.4.2 intestinal histomorphology
The upper 1cm ileum tissue was placed in 4% paraformaldehyde and kept for inspection. A 5 m pathological section was made after paraffin embedding, and HE staining was used to observe the morphological changes of ileum tissue epithelial cells after.400 staining.
Determination of TNF- alpha, IL-6, HIF-1 alpha, Gln, two amine oxidase (DAO) and the content of serum DAO in intestinal tissue by 2.4.3ELISA
The middle and lower 2cm ileum tissues were weighed and then homogenized, then centrifuged for 15 minutes at 4 degrees, and the supernatant was taken in the refrigerator at -20 degrees. The detection of.TNF-, IL-6, HIF-1 alpha, Gln and DAO was performed strictly in accordance with the instructions of ELISA Kit (RD company, USA). The contents of 3000r/min, IL-6 and HIF-1 were determined.
Determination of the expression of HIF-1 alpha mRNA in intestinal tissue by 2.4.4RT-PCR
The lower section of ileal tissue in 2cm by DEPC water rinse, dry with filter paper, rapid cooling by liquid nitrogen in freezing tube, placed in the refrigerator to be detected. C -80 extraction, the total RNA of intestinal tissue for reverse transcription to detect HIF-1 expression of alpha mRNA in intestinal tissue DNA amplification and agarose gel electrophoresis and other steps.
3. statistical treatment
The data were analysed by SPSS17.0 statistical software, measurement data to mean + standard deviation (x + s) said that the comparison among multiple groups using ANOVA, 22 compared with the least significant difference method (LSD-t), to test the level of a =0.05, namely P0.05 group, there was significant difference between two groups.
Result
1. eye, 1 hours after intraperitoneal injection of LPS, L group and P group rats began to appear all over the body was shaking, chills, shortness of breath apathetic about.2~3 hours, decreased activity, and eating less lassitude was.6 hours, almost no activity is not eating, even purple lips.P rats were relatively mild symptoms, C of rats in group activities, eating normal.C group, L group and P group 24h survival rates were 100%, 69.2% and 92.3%, neonatal death occurred mainly in LPS after injection of 12~24 hour period.
2. compared with group C, the wet / dry weight ratio of L group and P group increased significantly (P0.05). Compared with L group, the ratio of P group decreased significantly (P0.05). The difference was statistically significant.
3.HE staining and light microscopy showed that the villi of the C group were intact, the epithelial cells arranged neatly, and the morphology was normal. L group showed villous cell edema and epithelial cell disorder. The villus edema in group P was significantly lower than that in L group, and epithelial cells arranged orderly.
4. compared with group C, the contents of TNF-, IL-6, HIF-1 and alpha in intestinal tissue of group L and P increased (P0.05), while Gln content decreased (P0.05). Compared with L group, the content of P0.05 in intestinal tissue of the P group was higher than that of the L group, while the contents of all other indexes were all low.
5. compared with group C, DAO content in intestinal tissue of L group and P group was significantly decreased, while serum DAO level increased (P0.05). Compared with L group, DAO content in intestinal tissue of P group was higher, while serum DAO level was lower (P0.05). The difference was statistically significant.
6. compared with group C, the expression level of HIF-1 alpha and mRNA in intestinal tissue of L group and P group increased (P0.05). Compared with L group, the expression of HIF-1 mRNA mRNA in P group was low (P0.05). The difference was statistically significant.
conclusion
Penehyclidine hydrochloride can reduce the intestinal edema, reduce the inflammatory reaction and reduce the permeability of intestinal barrier in endotoxemia. The mechanism of intestinal protection may be related to its downregulation of the expression of HIF-1.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R726.1

【共引文獻(xiàn)】

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