本文選題：腸三葉因子　切入點(diǎn)：表皮生長(zhǎng)因子　出處：《第三軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 背景: 腸三葉因子(intestinal trefoil factor,ITF)是由腸道杯狀細(xì)胞特異分泌的具有特殊空間結(jié)構(gòu)的小分子多肽,ITF不僅能促進(jìn)細(xì)胞增殖和移行,還由于其空間結(jié)構(gòu)中有和粘蛋白的寡聚糖或多糖側(cè)鏈結(jié)合的位點(diǎn),使之形成穩(wěn)定的凝膠復(fù)合物,穩(wěn)定腸粘液層。此外,ITF具有蛋白酶水解抗性和酸堿穩(wěn)定性,可減輕多種致傷因子造成的消化道損害,這些特性決定了ITF在腸道的自我保護(hù)機(jī)制中占重要地位。目前對(duì)ITF的研究已很深入,但有關(guān)ITF受體的研究相對(duì)滯后,ITF是否具有獨(dú)特的受體存在爭(zhēng)議,一些學(xué)者認(rèn)為ITF沒(méi)有特異受體,它依賴表皮生長(zhǎng)因子受體(EGFR)發(fā)揮生物學(xué)效應(yīng);另一些學(xué)者則持不同觀點(diǎn),他們發(fā)現(xiàn)了一類能和ITF特異結(jié)合的蛋白,將其稱之為ITF結(jié)合蛋白,并對(duì)蛋白的基本特性進(jìn)行了初步分析,但這些蛋白是否就是ITF受體還缺乏具有說(shuō)服力的直接證據(jù)。 目的: 在成功獲取重組表達(dá)ITF的基礎(chǔ)上,通過(guò)配體受體動(dòng)力學(xué)研究和受體定位研究,探索在腸上皮細(xì)胞上是否存在ITF特異受體,以及受體在細(xì)胞上的分布特點(diǎn)。 方法: 1.ITF在酵母中的表達(dá)和純化:將成功轉(zhuǎn)化的含ITF成熟肽序列的酵母X-33菌株進(jìn)行Zeocin篩選,將陽(yáng)性轉(zhuǎn)化子接種于YPD液體培養(yǎng)基中搖瓶表達(dá),表達(dá)產(chǎn)物上清經(jīng)硫酸銨分級(jí)沉淀、Ni-NTA親和層析、超濾離心三步純化以獲取重組表達(dá)的ITF。Tricine SDS-PAGE及Western bloting鑒定重組蛋白。 2.腸三葉因子受體放射性配基結(jié)合實(shí)驗(yàn)(Radioligand binding assay,RLBA):摸索ITF受體放射性配基結(jié)合分析的最佳反應(yīng)溫度、時(shí)間、細(xì)胞濃度和非標(biāo)記ITF的濃度。在此條件下在IEC-6細(xì)胞上進(jìn)行ITF受體放射性配基結(jié)合實(shí)驗(yàn),得到親和常數(shù)(K_D)及最大結(jié)合容量(B_(max))。在相同的條件下對(duì)HT-29、5E6L、HaCat細(xì)胞上ITF受體進(jìn)行分析,比較不同細(xì)胞上ITF受體的差異。通過(guò)大量EGF、ITF及EGFR阻斷肽對(duì)ITF受體和EGF受體競(jìng)爭(zhēng)抑制,證實(shí)ITF特異受體的存在。 3.腸三葉因子受體在腸上皮細(xì)胞上的定位:將傳代培養(yǎng)的IEC-6細(xì)胞用0.5%的胰酶消化后,細(xì)胞濃度調(diào)為約2.5×10~5個(gè)/ml,加入放有8×8mm蓋玻片的24孔板于37℃,5%CO_2培養(yǎng)箱孵育,待IEC-6腸上皮細(xì)胞貼壁,細(xì)胞爬片用PBS清洗3遍后與B-藻紅蛋白(B-PE)標(biāo)記的ITF(BPE-ITF)在4℃下分別共同孵育0.5,1.0,2.0,4.0h,孵育結(jié)束后PBS清洗3遍×3min,細(xì)胞片置于載玻片上并用90%緩沖甘油封片,激光共聚焦顯微鏡下觀察ITF受體在IEC-6細(xì)胞上的分布規(guī)律。并使用過(guò)量的EGF、ITF和EGFR阻斷肽進(jìn)行競(jìng)爭(zhēng)抑制實(shí)驗(yàn),觀察熒光的變化情況。 結(jié)果: 1.酵母表達(dá)獲得ITF,產(chǎn)物經(jīng)Tricine SDS-PAGE、Western blot鑒定具有良好的抗原性和特異性,三步純化后重組蛋白的純度超過(guò)95%,表達(dá)量達(dá)到約50mg/L。 2.放射性配基結(jié)合實(shí)驗(yàn)最佳反應(yīng)條件為4℃,孵育30min,細(xì)胞濃度為5.0×10~5個(gè)/ml,非特異結(jié)合配基濃度為標(biāo)記配基的10000倍。在此條件下,對(duì)IEC-6、HT-29、5E6L和HaCat細(xì)胞上ITF受體進(jìn)行放射性結(jié)合分析,并通過(guò)Scatchard線性回歸分析得到IEC-6細(xì)胞最大結(jié)合容量B_(max)=62.713×10~6結(jié)合位點(diǎn)/細(xì)胞,親和常數(shù)K_D=4.3478×10~(-10)mol/L;HT-29細(xì)胞最大結(jié)合容量B_(max)=61.1583×10~6結(jié)合位點(diǎn)/細(xì)胞,親和常數(shù)KD=0.8849×10~(-10)mol/L;5E6L細(xì)胞最大結(jié)合容量B_(max)=184.89×10~6結(jié)合位點(diǎn)/細(xì)胞,親和常數(shù)KD=3.448×10~(-10)mol/L。根據(jù)所得到的K_D值和B_(max)分析,ITF與IEC-6、HT-29、5E6L細(xì)胞結(jié)合呈高親和力,但受體密度有所差異。表皮細(xì)胞HaCat與ITF結(jié)合呈低親和力。EGF對(duì)ITF受體及ITF對(duì)EGF受體均無(wú)競(jìng)爭(zhēng)抑制作用,EGFR阻斷肽對(duì)ITF與IEC-6細(xì)胞的結(jié)合并未受影響。 3.激光共聚焦觀察發(fā)現(xiàn),熒光標(biāo)記的ITF首先聚集在細(xì)胞膜上,隨時(shí)間和濃度的增加,熒光標(biāo)記的ITF逐漸向胞漿與胞核轉(zhuǎn)移。過(guò)量的ITF可明顯抑制熒光標(biāo)記的ITF與細(xì)胞結(jié)合,熒光強(qiáng)度明顯降低。EGF和EGFR阻斷肽對(duì)ITF在細(xì)胞膜上的熒光強(qiáng)度和向胞漿、胞核的轉(zhuǎn)移沒(méi)有影響。 結(jié)論: 1.重組表達(dá)的ITF產(chǎn)量約50mg/L,純度＞95%,符合受體研究中對(duì)配體的要求。 2.確定了放射性配基結(jié)合實(shí)驗(yàn)最佳反應(yīng)條件,并證明腸上皮細(xì)胞IEC-6、結(jié)腸癌細(xì)胞HT-29、小腸上皮細(xì)胞5E6L上存在能與ITF結(jié)合的受體,受體的親和力和受體密度存在一定差異,表皮細(xì)胞HaCat上沒(méi)有與ITF結(jié)合的受體。 3.EGF和ITF與各自的受體結(jié)合,無(wú)相互競(jìng)爭(zhēng)抑制作用,證明ITF不是通過(guò)EGF受體發(fā)揮生物學(xué)效應(yīng)。 4.ITF受體存在于IEC-6細(xì)胞膜上,ITF與其受體結(jié)合后呈現(xiàn)向胞漿和胞核轉(zhuǎn)移的特性。
[Abstract]:Background:
Intestinal trefoil factor (intestinal trefoil, factor, ITF) is a small molecule with special structure of polypeptide secreted by intestinal goblet cell specific, ITF can not only promote cell proliferation and migration, but also because of its spatial structure and mucin oligosaccharides or polysaccharides of side chain binding sites, so as to form a composite gel a stable, stable intestinal mucus layer. In addition, ITF with protease hydrolysis resistance and acid-base stability, can reduce the injury caused by a variety of factors of digestive tract damage, these characteristics determine that ITF occupies an important position in the self - protection mechanism of intestine. At present the study on ITF has been very thorough, but the Research on ITF receptor the relative lag, whether ITF has a unique receptor is controversial, some scholars believe that there is no specific ITF receptor, it is dependent on the epidermal growth factor receptor (EGFR) exert biological effects; others have different opinions, they A class of proteins that can specifically bind to ITF have been discovered. They are called ITF binding proteins. The basic characteristics of proteins have been preliminarily analyzed. But whether these proteins are ITF receptors still lack convincing direct evidence.
Objective:
Based on successful acquisition of recombinant expression of ITF, we investigated the existence of ITF specific receptors on intestinal epithelial cells and the distribution of receptors on cells by ligand receptor kinetic studies and receptor localization studies.
Method:
Expression and purification of 1.ITF in yeast: successfully transformed with the mature peptide sequence of ITF strain X-33 was screened by Zeocin, the positive transformants were inoculated in YPD liquid culture medium in shake flask expression, expression supernatant by ammonium sulfate precipitation, Ni-NTA affinity chromatography, ultrafiltration from three steps of purification to obtain recombinant expression ITF.Tricine SDS-PAGE and Western bloting identification of recombinant protein.
Intestinal trefoil factor 2. receptor radioligand binding assay (Radioligand binding, assay, RLBA): exploring ITF receptor radioligand binding analysis of the optimum reaction temperature, time, concentration of cell concentration and non labeled ITF. Under these conditions, ITF receptor radioactive ligand binding experiments were performed on IEC-6 cells by affinity constants (. The maximum binding capacity (K_D) and B_ (max)). In the same conditions of HT-29,5E6L, analyzes on the HaCat cell receptor ITF, ITF receptor in different cell differences. Through a large number of EGF, ITF and EGFR blocking peptide inhibition and EGF receptor competition on ITF, confirmed that the ITF specific receptor.
3. localization of intestinal trefoil factor receptor in intestinal epithelial cells: IEC-6 cells with 0.5% trypsin digestion, the cell concentration to about 2.5 * 10~5 /ml, adding 8 * 8mm were placed in 24 holes plate at 37 DEG C, box were incubated for 5%CO_2, IEC-6 for intestinal epithelial cell paste the wall, cell body cleaning 3 times after B- and phycoerythrin (B-PE) labeled with PBS ITF (BPE-ITF) at 4 C were incubated for 0.5,1.0,2.0,4.0h, after incubation of PBS cleaning 3 times * 3min, slides and 90% glycerol buffer mounting piece is placed on the distribution of load cell, confocal laser under the microscope of ITF receptor in IEC-6 cells. And the excessive use of EGF, ITF and EGFR were blocking peptide competitive inhibition experiment, observe the fluorescence changes.
Result:
1. ITF was obtained from yeast expression. The product was identified by Tricine SDS-PAGE and Western blot. It had good antigenicity and specificity. After three steps, the purity of recombinant protein was over 95%, and its expression reached about 50mg/L..
2. radioligand binding experiments the optimum reaction conditions of 4 DEG C, incubated for 30min, cell concentration of 5 * 10~5 /ml, non-specific binding ligand concentrations 10000 times labeled ligands. Under this condition, the IEC-6, HT-29,5E6L and HaCat on ITF cell receptor binding analysis and analysis of radioactive, IEC-6 cells the maximum binding capacity of B_ by Scatchard linear regression (max) =62.713 * 10~6 binding sites per cell, the affinity constant of K_D=4.3478 * 10~ (-10) mol/L; HT-29 cell maximum binding capacity of B_ (max) =61.1583 * 10~6 binding sites per cell, the affinity constant of KD= 0.8849 * 10~ (-10) mol/L; 5E6L (maximum binding capacity of B_ cells max) =184.89 * 10~6 binding sites per cell, the affinity constant of KD=3.448 * 10~ (-10) mol/L. according to the values of K_D and B_ (max) analysis, ITF and IEC-6, HT-29,5E6L cells with a high affinity receptor, but the density difference. Epidermal cells of HaCat combined with ITF The low affinity.EGF has no competitive inhibition effect on ITF receptor and ITF to EGF receptor, and the binding of EGFR blocking peptide to ITF and IEC-6 cells is not affected.
3. laser scanning confocal microscope, fluorescence labeled ITF first gather in the cell membrane, increase the time and concentration of fluorescent labeled ITF to cytosolic and nuclear transfer. The combination of ITF and ITF can inhibit the cell excessive fluorescent marker, the fluorescence intensity decreased.EGF and EGFR blocking peptide on fluorescence intensity ITF on the cell membrane and the cytoplasm to the nucleus, did not affect the transfer.
Conclusion:
1. the yield of recombinant ITF was about 50mg/L, and the purity was more than 95%, which was in line with the requirement for ligand in the receptor study.
2. determine the combination of experimental optimum reaction conditions of radioactive ligand, and that intestinal epithelial cell IEC-6, colon cancer cell HT-29, are capable of binding ITF receptor 5E6L in intestinal epithelial cells, there are some differences in receptor affinity and receptor density, epidermal cells HaCat no binding to ITF receptor.
3.EGF and ITF are combined with their respective receptors, and there is no mutual competitive inhibition, which proves that ITF does not play biological effects through the EGF receptor.
The 4.ITF receptor exists on the IEC-6 cell membrane, and the ITF and its receptors bind to the cytoplasm and nucleus.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

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8 解放軍307醫(yī)院腫瘤研究室 朱運(yùn)峰邋鮑云華;尋找靶向藥物療效的預(yù)測(cè)方法[N];健康報(bào);2007年

9 胡德榮;上海瑞金醫(yī)院研制抗P-選擇素功能域單抗[N];中國(guó)醫(yī)藥報(bào);2007年

10 紫箕;盤(pán)尼圖姆：安進(jìn)稱雄的重要籌碼[N];醫(yī)藥經(jīng)濟(jì)報(bào);2006年

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10 任于果;大鼠胚腦皮層源性神經(jīng)干細(xì)胞體外培養(yǎng)及增殖分化特點(diǎn)[D];安徽醫(yī)科大學(xué);2005年

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