發(fā)布時(shí)間：2018-08-19 10:37

【摘要】：目的：腹瀉是潰瘍性結(jié)腸炎常見(jiàn)的臨床癥狀,其產(chǎn)生主要與水鹽(包括Cl-、 HCO3-和Na+)分泌和吸收不平衡相關(guān)。SLC26A3是位于腸上皮細(xì)胞頂端膜的離子轉(zhuǎn)運(yùn)蛋白,參與調(diào)控腸道Cl-、HCO3-和水的吸收與分泌。已有體外研究證實(shí)溶血磷脂酸(LPA)可提高SLC26A3的基因表達(dá)和離子轉(zhuǎn)運(yùn)功能,并可能成為治療炎癥性腹瀉的候選藥物,但是關(guān)于LPA在體內(nèi)是否能提高SLC26A3表達(dá)并改善炎癥性腹瀉癥狀尚無(wú)研究報(bào)道,本課題首先對(duì)此進(jìn)行觀察。既往研究提示,LPA可以通過(guò)提高SLC26A3的啟動(dòng)子活性而上調(diào)SLC26A3的細(xì)胞膜表達(dá)和離子轉(zhuǎn)運(yùn)功能,但對(duì)LPA是否有助于提高SLC26A3細(xì)胞膜表達(dá)的穩(wěn)定性和持續(xù)性卻未見(jiàn)研究報(bào)道。鑒于SLC26A3的糖基化在腸道環(huán)境中對(duì)維持SLC26A3細(xì)胞膜表達(dá)的穩(wěn)定性具有重要意義和PDZ結(jié)構(gòu)蛋白NHERF4對(duì)SLC26A3黏膜表達(dá)持續(xù)性的影響,課題利用Caco2細(xì)胞觀察LPA對(duì)SLC26A3糖基化水平的影響和NHERF4對(duì)LPA促SLC26A3蛋白細(xì)胞膜表達(dá)的影響,以期能進(jìn)一步了解LPA調(diào)控SLC26A3細(xì)胞膜表達(dá)的相關(guān)機(jī)制。 方法：利用4%葡聚糖硫酸鈉(DSS)構(gòu)建C57BL/6J小鼠急性結(jié)腸炎動(dòng)物模型,設(shè)立正常對(duì)照組,觀察小鼠的體質(zhì)量、腸道出血情況、糞便含水量、結(jié)腸大體組織炎癥損害和鏡下病理組織學(xué)表現(xiàn),參考Butzner JD標(biāo)準(zhǔn)進(jìn)行結(jié)腸損傷組織學(xué)大體評(píng)分,參考Sutherland LR標(biāo)準(zhǔn)進(jìn)行結(jié)腸炎疾病活動(dòng)指數(shù)(DAI)評(píng)分,運(yùn)用實(shí)時(shí)定量熒光聚合酶鏈反應(yīng)和蛋白免疫印跡實(shí)驗(yàn)分析炎癥結(jié)腸的SLC26A3mRNA水平和蛋白表達(dá),評(píng)估該動(dòng)物模型應(yīng)用于觀察分析LPA對(duì)炎癥性腹瀉和離子轉(zhuǎn)運(yùn)體SLC26A3黏膜表達(dá)的影響的可行性。然后,對(duì)DSS誘導(dǎo)的急性結(jié)腸炎小鼠給予LPA灌腸治療,設(shè)立正常小鼠組、模型組、磷酸鹽緩沖溶液(PBS)灌腸處理組做為對(duì)照,觀察LPA對(duì)結(jié)腸炎小鼠炎癥性腹瀉相關(guān)指標(biāo)的影響,如體質(zhì)量的下降、DAI評(píng)分、稀便的程度和黏膜炎癥損傷程度,并觀察分析LPA對(duì)中遠(yuǎn)段炎癥結(jié)腸的SLC26A3mRNA水平和蛋白表達(dá)的影響,以評(píng)估LPA做為治療炎癥性腹瀉候選藥物的可能性。利用Caco2細(xì)胞,將LPA與Caco2細(xì)胞共孵育,以共孵育時(shí)間0小時(shí)、6小時(shí)、12小時(shí)、18小時(shí)為觀察時(shí)間點(diǎn),觀察LPA對(duì)SLC26A3的基因表達(dá)、總蛋白表達(dá)、糖基化蛋白表達(dá)的影響。利用胰蛋白酶對(duì)SLC26A3蛋白的降解作用,設(shè)立LPA處理Caco2細(xì)胞組和LPA空白Caco2細(xì)胞組,以細(xì)胞和胰酶共孵育時(shí)間Omin、5min、10min、30min為觀察時(shí)間點(diǎn),觀察分析LPA對(duì)SLC26A3細(xì)胞膜表面表達(dá)穩(wěn)定性的影響。構(gòu)建NHERF4質(zhì)粒,轉(zhuǎn)染Caco2細(xì)胞,設(shè)立轉(zhuǎn)染NHERF4質(zhì)粒的Caco2組(pIRES2-ZsGreenl-NHERF4-Caco2組,即NHERF4-Caco2組)、轉(zhuǎn)染空質(zhì)粒的Caco2組(pIRES2-ZsGreen1-NP-Caco2組,即NP-Caco2組)、LPA處理的轉(zhuǎn)染NHERF4質(zhì)粒的Caco2組(LPA+pIRES2-ZsGreenl-NHERF4-Caco2組,即LPA+NHERF4-Caco2組)、LPA處理的轉(zhuǎn)染空質(zhì)粒的Caco2組(LPA+pIRES2-ZsGreen1-NP-Caco2組,即LPA+NP-Caco2組)、LPA處理的Caco2組(即LPA+Caco2組)和LPA空白Caco2組(即Caco2組),以LPA與Caco2細(xì)胞共孵育12小時(shí)為觀察時(shí)間點(diǎn),分析Caco2細(xì)胞轉(zhuǎn)染NHERF4質(zhì)粒后,LPA對(duì)SLC26A3的細(xì)胞表達(dá)的影響是否發(fā)生改變,并觀察分析LPA對(duì)NHERF4蛋白表達(dá)的影響,初步評(píng)價(jià)LPA、SLC26A3、NHERF4之間的相互作用關(guān)系。 結(jié)果：1.在構(gòu)建DSS誘導(dǎo)的C57BL/6J小鼠急性結(jié)腸炎動(dòng)物模型實(shí)驗(yàn)中,模型組小鼠表現(xiàn)出糞便含水量增加、血便、炎癥活動(dòng)指數(shù)DAI值迅速上升、體質(zhì)量明顯下降。實(shí)驗(yàn)結(jié)束時(shí),與對(duì)照組相比,模型組的結(jié)腸大體組織學(xué)炎癥評(píng)分升高(p0.05),結(jié)腸明顯縮短(正常組vs.模型組：7.53±0.3cm vs.4.8±0.8cm,p0.05),炎癥結(jié)腸黏膜中SLC26A3mRNA表達(dá)明顯下降(正常組vs.模型組：14.03±1.4vs.1.0,p=0.00),蛋白表達(dá)明顯下降。2.在觀察LPA對(duì)結(jié)腸炎小鼠炎癥性腹瀉和對(duì)炎癥結(jié)腸段SLC26A3表達(dá)影響的實(shí)驗(yàn)中,所有飲用DSS的小鼠均表現(xiàn)出體質(zhì)量下降,PBS處理組(實(shí)驗(yàn)前vs.實(shí)驗(yàn)后：17.02±0.19gvs.14.46±0.97g,p=0.001)和模型對(duì)照組(實(shí)驗(yàn)前vs.實(shí)驗(yàn)后：16.90±0.49g vs.13.5±0.90g,p=0.001)體質(zhì)量明顯下降,LPA處理組的體質(zhì)量下降趨勢(shì)明顯減緩(實(shí)驗(yàn)前vs.實(shí)驗(yàn)后：17.36±0.67g vs.16.05±0.92g,p=0.06)。LPA處理組的糞便含水量上升幅度(18.89±8.67%)較PBS處理組(29.48±6.71%)和模型對(duì)照組(28.97±6.95%)明顯減緩(p=0.049,p=0.041)。LPA處理組SLC26A3mRNA(2.27±0.4)較模型對(duì)照組(1.0)明顯上升(p=0.03),模型對(duì)照組和PBS處理組(1.41±0.45)SLC26A3mRNA差異無(wú)顯著性(p=0.09)。LPA處理組、模型對(duì)照組、PBS處理組的SLC26A3蛋白表達(dá)水平較正常對(duì)照組均降低,但LPA處理組SLC26A3蛋白表達(dá)較模型對(duì)照組和PBS處理組升高。3.在觀察LPA對(duì)SLC26A3糖基化水平影響的實(shí)驗(yàn)中,LPA孵育12小時(shí)的Caco2細(xì)胞與LPA空白Caco2細(xì)胞相比,SLC26A3基因表達(dá)量增加1.67±0.03倍,蛋白表達(dá)量亦增加(相對(duì)β-actin的表達(dá)量,0.92±0.10vs.0.46±0.05,p0.05),SLC26A3的細(xì)胞膜表達(dá)與細(xì)胞漿表達(dá)比增加(膜表達(dá)/漿表達(dá)：2.17±0.17vs.1.72±0.12,p=0.023),提示LPA在提高Caco2細(xì)胞SLC26A3總蛋白表達(dá)的同時(shí),也有助于SLC26A3的細(xì)胞膜定位和表達(dá)。在觀察LPA對(duì)SLC26A3蛋白抵抗胰酶降解的影響的實(shí)驗(yàn)中,LPA空白Caco2細(xì)胞的細(xì)胞膜SLC26A3蛋白在與胰酶共孵育10min后明顯減少,而LPA孵育12小時(shí)的Caco2細(xì)胞的細(xì)胞膜SLC26A3蛋白在與胰酶共孵育后含量未出現(xiàn)明顯減少,提示LPA可提高細(xì)胞膜SLC26A3蛋白抵抗胰酶降解的能力。4.在觀察LPA與NHERF4在SLC26A3表達(dá)中的相互作用的實(shí)驗(yàn)中,與LPA+NP-Caco2組相比,LPA+NHERF4-Caco2組SLC26A3蛋白表達(dá)量明顯減少(相對(duì)β-actin的表達(dá)量,0.27±0.042vs.0.56±0.022,p=0.003),提示NHERF4可弱化LPA的促SLC26A3蛋白表達(dá)作用。與NHERF4-Caco2組相比LPA+NHERF4-Caco2組NHERF4的蛋白表達(dá)量無(wú)明顯改變,提示LPA對(duì)NHERF4的蛋白表達(dá)無(wú)明顯影響。 結(jié)論：LPA可提高炎癥結(jié)腸黏膜的SLC26A3表達(dá),并減輕DSS誘導(dǎo)的結(jié)腸炎性腹瀉的嚴(yán)重程度,提示其可做為治療結(jié)腸炎相關(guān)性腹瀉的候選藥物。LPA在Caco2細(xì)胞可提高SLC26A3的蛋白表達(dá)和糖基化水平并增強(qiáng)SLC26A3抗胰蛋白酶降解的能力。NHERF4可弱化LPA促進(jìn)Caco2細(xì)胞SLC26A3蛋白表達(dá)的作用,但LPA對(duì)NHERF4的表達(dá)影響不顯著。提示LPA可提高SLC26A3細(xì)胞膜表達(dá)的穩(wěn)定性,但對(duì)SLC26A3細(xì)胞膜表達(dá)持續(xù)性的影響尚需進(jìn)一步實(shí)驗(yàn)探討。
[Abstract]:OBJECTIVE: Diarrhea is a common clinical symptom of ulcerative colitis. Its production is mainly related to the imbalance of secretion and absorption of water and salt (including Cl-, HCO3 - and Na +). SLC26A3 is an ion transporter located at the apical membrane of intestinal epithelial cells. It is involved in regulating the absorption and secretion of Cl-, HCO3 - and water in the intestine. Increasing gene expression and ion transport of SLC26A3 may be a candidate drug for the treatment of inflammatory diarrhea. However, there is no report on whether LPA can increase the expression of SLC26A3 and improve the symptoms of inflammatory diarrhea in vivo. This study first observed this effect. Previous studies suggest that LPA can increase the promoter activity of SLC26A3. Sexually up-regulated cell membrane expression and ion transport of SLC26A3, but no studies have been reported on whether LPA contributes to the stability and persistence of cell membrane expression of SLC26A3. In view of the important significance of glycosylation of SLC26A3 in intestinal environment for maintaining the stability of cell membrane expression of SLC26A3 and the role of PDZ structural protein NHERF4 in the adhesion of SLC26A3 The aim of this study was to investigate the effect of LPA on the glycosylation level of SLC26A3 and the effect of NHERF4 on the membrane expression of SLC26A3 protein promoted by LPA in Caco2 cells.
Methods: Acute colitis model of C57BL/6J mice was established by using 4% dextran sodium sulfate (DSS). Normal control group was set up to observe the body mass, intestinal bleeding, fecal water content, inflammation damage of colon gross tissues and microscopic histopathological manifestations. Butzner JD standard was used for histological gross score of colon injury. Sutherland LR was used to assess the activity index (DAI) of colitis. Real-time quantitative fluorescence polymerase chain reaction and Western blot were used to analyze the expression of SLC26A3 mRNA and protein in inflammatory colon. The animal model was used to observe and analyze the effect of LPA on inflammatory diarrhea and the expression of SLC26A3, an ion transporter. Feasibility. Then, the mice with acute colitis induced by DSS were given LPA enema treatment. Normal mice, model group, phosphate buffer solution (PBS) enema treatment group were set up as control group. The effects of LPA on inflammatory diarrhea-related indexes such as body mass loss, DAI score, degree of stool dilution and degree of mucosal inflammation injury were observed. The effects of LPA on the expression of SLC26A3 mRNA and protein in the middle and distal inflammatory colon were observed and analyzed to evaluate the possibility of LPA as a candidate drug for the treatment of inflammatory diarrhea. The effects of trypsin on the degradation of SLC26A3 protein were studied. LPA-treated Caco2 cells and LPA-treated blank Caco2 cells were established. Cell and trypsin co-incubation time of Omin, 5 min, 10 min and 30 min were used to observe the effect of LPA on the stability of membrane expression of SLC26A3 cells. NHERF4 plasmid was constructed, Caco2 cells were transfected with NHERF4 plasmid, Caco2 cells were transfected with NHERF4 plasmid, Caco2 group (pIRES2-ZsGreenl-NHERF4-Caco2 group, namely NHERF4-Caco2 group), Caco2 group transfected emptemptplasmid (pIRES2-ZsGreen1-NP-NP-Caco2 group, NP-Caco2 group, NP-Caco2 group, LPA-LPA-treated with LPA transfected NHERF4 plasmid, Caco2 group (A+pES2-ZES2-ZsGreenl-ZsGreenl-NHERF4-Caco4-Caco2 group), LPA+LPA+LPA+pES2-Z4-Caco 2 group, L Cell expression of SLC26A3 was analyzed by incubation of LPA with Caco 2 (LPA+pIRES2-ZsGreen 1-NP-Caco 2 group), LPA with Caco 2 (LPA+Caco 2 group) and LPA with Caco 2 (Caco 2 group) for 12 hours. The effect of LPA on the expression of NHERF4 protein was observed and analyzed. The interaction among LPA, SLC26A3 and NHERF4 was preliminarily evaluated.
Results: 1. In the animal model of acute colitis induced by DSS in C57BL/6J mice, the water content of feces, blood stool, DAI value of inflammatory activity index increased rapidly, and body weight decreased significantly. At the end of the experiment, compared with the control group, the colon gross histological inflammation score of the model group increased (p0.05), colon bright. The expression of SLC26A3 mRNA in inflammatory colonic mucosa was significantly decreased (vs. normal vs. model group: 14.03 + 1.4 vs. 1.0, P = 0.00), and the expression of SLC26A3 protein was significantly decreased (vs. model group: 7.53 + 0.3 cm vs. 4.8 cm, P 0.05). The mice who drank DSS showed a significant decrease in body mass. The body mass of the PBS treatment group (before and after the experiment vs. 17.02 + 0.19 g vs. 14.46 + 0.97 g, P = 0.001) and the model control group (before and after the experiment vs. 16.90 + 0.49 g vs. 13.5 + 0.90 g, P = 0.001) decreased significantly, and the body mass of the LPA treatment group decreased significantly (before and after the experiment vs. 17.36). The increase of fecal water content in LPA treatment group (18.89+8.67%) was significantly slower than that in PBS treatment group (29.48+6.71%) and model control group (28.97+6.95%) (p=0.049, p=0.041). SLC26A3 mRNA (2.27+0.4) in LPA treatment group was significantly higher than that in model control group (1.0), model control group and PBS treatment group (1.41+0.45). There was no significant difference in SLC26A3 mRNA (p = 0.09). The expression of SLC26A3 protein in LPA treatment group, model control group and PBS treatment group was lower than that in normal control group, but the expression of SLC26A3 protein in LPA treatment group was higher than that in model control group and PBS treatment group. Compared with LPA blank Caco2 cells, SLC26A3 gene expression increased by 1.67+0.03 times and protein expression increased (relative expression of beta-actin, 0.92+0.10 vs.0.46+0.05, p0.05). The ratio of membrane expression to cytoplasmic expression of SLC26A3 increased (membrane expression/plasma expression: 2.17+0.17 vs.1.72+0.12, p=0.023), suggesting that LPA increased the expression of SLC26A3 in Caco2 cells. The expression of SLC26A3 protein was also helpful for the localization and expression of SLC26A3. In the experiment of observing the effect of LPA on the resistance of SLC26A3 protein to trypsin degradation, the expression of SLC26A3 protein in the cell membrane of LPA blank Caco2 cells decreased significantly after 10 minutes incubation with trypsin, while that in the cell membrane of Caco2 cells incubated with LPA for 12 hours decreased significantly after incubation with trypsin. There was no significant decrease in the content of SLC26A3 protein after co-incubation, suggesting that LPA could enhance the ability of SLC26A3 protein to resist trypsin degradation. 4. In the experiment of observing the interaction between LPA and NHERF4 in the expression of SLC26A3, the expression of SLC26A3 protein in LPA+NHERF4-Caco2 group was significantly lower than that in LPA+NP-Caco2 group (relative expression of beta-actin, 0.27 +0.042 vs. 0). The expression of NHERF4 protein in LPA+NHERF4-Caco2 group had no significant change compared with NHERF4-Caco2 group, suggesting that LPA had no significant effect on the expression of NHERF4 protein.
CONCLUSION: LPA can increase the expression of SLC26A3 in inflammatory colonic mucosa and reduce the severity of DSS-induced inflammatory diarrhea, suggesting that LPA can be used as a candidate drug for the treatment of colitis-associated diarrhea. LPA in Caco2 cells can increase the protein expression and glycosylation level of SLC26A3 and enhance the anti-trypsin degradation ability of SLC26A3. It is suggested that LPA can improve the stability of SLC26A3 cell membrane expression, but the effect of LPA on the persistence of SLC26A3 cell membrane expression needs further study.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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本文編號(hào)：2191413