【作者】 袁庆丰；

【导师】 姒健敏；

【作者基本信息】 浙江大学 ， 消化内科学， 2007， 博士

【摘要】 表皮生长因子（epidermal growth factor,缩写EGF）,是一强有力的细胞分裂因子,主要由颌下腺管细胞分泌。EGF对上皮细胞有促增殖作用,外源性EGF在临床中的应用开始于创伤治疗方面:如体表创伤、溃疡,角膜损伤等。由于人体大多数的EGF存在于消化道,远远高于循环中的EGF浓度,因此,EGF对消化道黏膜有效的保护作用使外源性EGF用于消化道疾病治疗成为了一个研究热点。研究同时也发现EGF和EGF受体（EGFR）拮抗剂可以影响肿瘤的发生、发展和消退,因此在临床中EGF的应用由于它潜在的安全性问题而受到了制约。由于EGF与EGFR激活及其信号转导通路十分复杂,病理生理功能多样,而Bcl-2、p53基因蛋白表达异常与肿瘤发生、发展有关,故迫切需要进一步研究它们在基础研究和临床实践中的地位。本实验首先检测不同浓度EGF在不同时间段对人胚胎羊膜细胞FL的促增殖作用,进一步检测不同浓度EGF与激活Ras/Raf/MEK/ERK信号通路的时效关系以及对Bcl-2、p53蛋白表达的影响,为整体动物实验提供EGF应用的理论依据和实验数据。目前在临床中已经被采用的EGF外用材料主要来源于基因工程合成或小鼠颌下腺提取,价格十分昂贵,而且来源非常有限,这是制约EGF应用于临床的另一个重要原因。如果不能解决这个问题,即使研究阐明了EGF治疗的信号转导机制并证明其在临床实践中的积极意义,也无法赋予这项研究的现实的理论意义和应用价值。这是使得基础研究的成果具有应用可行性的最基本条件。联系我国目前的实际情况,如能简便,价廉地从家畜腺体提取到有生物学活性的EGF将是一件利国利民的好事。而同时我国每年又有大量的牲畜颌下腺被废弃,因此原材料来源丰富且成本低廉,并且作为重要的经济动物,羊类制品为人类接受程度高,这些都为EGF广阔的应用前景提供了最基本的保障。本论文的内容分以下两个部分:第一部分:外源性EGF促FL细胞增殖作用的信号转导机制及其安全性评价研究目的探讨EGF促FL细胞增殖时激活Ras/Raf/MEK/ERK信号转导通路时间-剂量-效应关系及对Bcl-2、p53蛋白表达的影响。材料与方法1.MTT法检测细胞增殖:细胞以2×10⁸/L的密度接种于96孔板上,每孔100μ1。待细胞贴璧生长后换含不同浓度rhEGF（0.1、1、10、30、60μg/L）的无血清培养基继续培养,对照孔以生理盐水代替rhEGF。观察细胞生长形态学变化并分别在第1、2、3、4、5 d取样;或换含rhEGF（10μg/L）+不同浓度PD98059（25、50、75μmol/L）的MEM培养基100μ1,继续培养48 h。取样后每孔加入MTT 20μ1,孵育4 h后弃上清,每孔加150μ1 DMSO,振荡10 min,于酶标仪490 nm波长下测定各孔的吸光度A值。细胞增殖率=（实验组A值-对照组A值）/对照组A值×100%。2.Western蛋白印迹法检测Ras/Raf/MEK/ERK通路及Bcl-2、p53蛋白水平:待细胞贴璧生长后换用rhEGF终浓度为1、10、60μg/L无血清的MEM培养基继续培养:测定MAPK通路抑制剂PD98059的影响则分别加入终浓度为25,50,75μmol/L的PD98059,预处理1 h。实验开始后根据需要在不同时段用胰酶消化,收集细胞并加细胞裂解液,取上清液,Bradford法测蛋白浓度。每孔加50μg的蛋白样品电泳,电泳结束后转PVDF膜,将转有蛋白的膜置于5%BSA封闭,经一抗和HRP标记的二抗处理后,在暗室,取ECL发光试剂A、B液各1 ml,混合后覆盖于膜上1.5 min,最后用X光胶片进行曝光、显影及定影。结果1.FL细胞经培养48小时后,生理盐水对照组基本以多边形居多,而加入rhEGF后细胞以长条纺锤形居多。2.第1-5天中,1-60μg/L浓度组促细胞增殖率明显增高（P＜0.01）。rhEGF浓度10μg/L第3天时达到最高（42.4%,P＜0.01）,第4、5天后1-60μg/L浓度组促细胞增殖率开始下降,但仍高于对照组（P＜0.01）。3.各浓度PD98059可明显抑制EGF促FL细胞增殖作用（P＜0.01）,且随着剂量的加大其抑制作用愈明显。4.不同浓度rhEGF刺激30 min后p-Raf、p-ERK1/2蛋白诱导表达灰度分析结果示rhEGF 1-60μg/L浓度组p-Raf、p-ERK1/2蛋白较对照组明显升高（P＜0.05）,尤以10μg/L浓度组的作用最强。而非磷酸化ERK 1/2的表达无明显变化。5.rhEGF浓度10μg/L刺激FL细胞后测定连续时间段（0 min,5 min,30 min,1h,2 h）p-Raf、p-ERK1/2蛋白诱导表达结果示刺激第5 min-2 h各时间段p-Raf、p-ERK1/2活性表达呈峰型曲线,其中第5 min时p-Raf、p-ERK1/2活性最高（P＜0.01）,随后各时间段p-Raf、p-ERK1/2活性开始下降。2 h后p-Raf表达的水平即明显下降到刺激前的水平,与对照组无明显差别（P＞0.05）:而2 h后p-ERK1/2表达的水平甚至低于刺激前的水平（P＜0.05）。6.与对照组比较,灰度分析结果示各浓度组PD98059（25,50,75μmol/L）可明显抑制rhEGF促p-ERK1/2蛋白诱导表达（P＜0.01）。且PD98059抑制p-ERK1/2蛋白的表达有剂量依赖性,随着PD98059剂量的加大,抑制p-ERK1/2蛋白表达的作用增强。7.与对照组比较,受不同浓度rhEGF刺激1 h后Bcl-2蛋白表达灰度分析结果示1-60μg/L浓度组Bcl-2蛋白表达较对照组明显增高（P＜0.05）;rhEGF 1-60μg/L浓度组p53蛋白明显下降（P＜0.01）。其中rhEGF 1μg/L浓度组的作用最弱,10μg/L浓度组的最强,两组之间比较有显著差异（P＜0.01）。8.以rhEGF 10μg/L的刺激浓度观测0 min,30 min,1 h,2 h,4 h五个时间段的Bcl-2蛋白诱导表达结果示30 min-4 h时间段的表达呈峰型曲线,以1h时间段的表达最高（P＜0.01）,随后开始下降。与rhEGF 10μg/L的浓度刺激后p-ERK1/2蛋白的表达水平比较,各时间段Bcl-2与p-ERK1/2的峰型变化曲线基本一致。结论EGF促FL细胞增殖、激活Ras/Raf/MEK/ERK信号转导通路及对Bcl-2、p53蛋白的表达有明显的剂量和时间依赖性作用。EGF浓度10μg/L是最强的刺激浓度,FL细胞对此浓度EGF的刺激具有自适应控制作用。1μg/L浓度的EGF可能是既发挥生理效应又较为安全的用量。第二部分:羊颌下腺表皮生长因子提取、纯化及生物活性鉴定的研究目的从山羊颌下腺分离、纯化具有生物学活性的EGF。方法1.羊EGF的提取、纯化:首先进行EGF的粗分离,称取山羊颌下腺（新鲜或冷冻）,解冻后剪去结缔组织,用剪刀剪成小块,用搅拌机搅碎,然后放入匀浆器匀浆,按比例加入冰醋酸（HAC）,操作在4℃冰浴中进行。50 000×g,离心30 min,抽取上清液。进行DEAE Sphacel阴离子交换柱分离,收集洗脱液,UV-280nm检测图分析吸光度,选出的管数做ELISA实验。并以0.2 M醋酸铵和EGF标准品做为阴性、阳性对照,以检测EGF活性成分。不加硫酸（在652nm处的读数）及加酸后（在450nm处读数）的结果分析。读数高、和EGF标准品读数接近为有EGF活性组份的可能,将这些组份合并后进行透析脱去盐分（透析袋分子量为3 500）,透析过夜。将透析好的组份分别用甘油（丙三醇）或聚乙二醇（分子量20 000）进行浓缩以除去水分。透析浓缩后的组份再次进行ELISA实验。将含有EGF成分的浓缩液过分子筛色谱（Sephadex G50）柱层析,根据层析图所示,选取对应的收集管再次进行ELISA实验,步骤同前,结果吸光度值高的几管与层析图中的一个峰所对应,因此该峰就是含有EGF的活性峰。2.对提取、纯化的羊EGF进行生物活性检测2.1 MTT法测定不同浓度羊EGF对人胚胎羊膜细胞FL的促增殖作用。2.2流式细胞术检测羊EGF促细胞增殖时细胞周期变化。3.纯化的山羊颌下腺EGF行SDS-PAGE电泳观测其可见的分子量条带位置。结果1.48小时后贴璧生长的FL细胞逐渐以多边形居多,而加入EGF后细胞以长条纺锤形居多。2.MTT法促增殖实验:结果显示不同浓度羊颌下腺EGF组（稀释倍数:1:100、1:500、1:1000）、rhEGF（10μg/L）组对FL细胞的促增殖率分别为20.90%、39.43%、24.90%和40.60%。与对照组比较,羊颌下腺分离的EGF可明显促进FL细胞的生长（P＜0.01）,以稀释1:500倍的羊EGF作用效果最强,其促FL细胞增殖率与人重组EGF比较无明显差异（P＞0.05）。3.流式细胞术:根据细胞实验中确立的最佳羊EGF稀释浓度观测流式细胞术中羊EGF对细胞周期的影响,与对照组相比较,结果显示作用48 h后,G₀/G₁期细胞百分比由71.1±1.9减少至60.3±2.2（P＜0.01）,S期细胞百分比由18.8±1.4增高至25.8±1.1（P＜0.01）,纯化的山羊颌下腺EGF显著刺激细胞由G₀/G₁期进入S期,使分布在S期的细胞数增加。3.SDS-聚丙烯酰胺凝胶电泳:电泳结果显示在分子量6 kD处,可见明显的条带。结论从山羊颌下腺分离得到的蛋白质经鉴定为EGF,该EGF能明显促进FL细胞生长、显著提高细胞的S期细胞分数,为具有生物学活性的EGF。羊颌下腺可能成为外源性EGF的药源。更多还原

【Abstract】 Epidermal growth factor（EGF）,a powerful cell division factor,was originally secretion from salivary gland cells.EGF has promotion effect on epithelial cells. Preliminary studies in humans suggest that exogenous EGF enhances healing of skin and cornea wounds or ulcers.The concentration of EGF is higher in gastrointestinal tract than in blood circulation.Recently,the study of exogenous EGF in treating the benign diseases of gastrointestinal tract becomes a hotspot in this area owing to the protection effect of EGF on alimentary canal.Some researches also find that EGF and the antagonist of EGFR can contribute to the occurrence,development or decrescence of the gastrointestinal tumors,therefore the application of EGF on clinic is cautious.The signal transduction mechanism of EGF on the proliferation and the activation of EGFR are very complex.Abnormal expression of Bcl-2,p53 concemed with occurrence and development of tumor,so lots of researches are need to illustrate the mechanism of its clinical effects.We first analysis the proliferation of FL cells stimulated by EGF of various concentration and time,then detect the effect of EGF on Ras/Raf/MEK/ERK signaling transduction pathway（s）and the change of Bcl-2 and p53 protein,provide theory and experimental data for the application of EGF on entirety animal experiment.Now,the main source of EGF products applied on clinical come from gene recombinant or isolation from the submaxillary glands of mices.But their disadvantages,such as high investment,shortness of source etc,are hardly to be overcomed,so it is difficult to utilize EGF on clinic extensively.If we can’t solve this problem,even our study has illuminated the signal transduction mechanism and demonstrated the effect of EGF in treating the disease,it will also be valueless.If we can extract inexpensive bioactive EGF from the submaxillary glands of goats expediently,it will benefit the nation and the people overwhelmingly.Being a fundamental economic animal,native material of goats was abundance and the cost is cheap,all of these provide a spacious perspective.There are two parts of this thesis.PartⅠ:The signal transduction mechanism of exogenous EGF on the proliferation of human amnion cells and the study of its safetyObjectionTo explore the effect of EGF on Ras/Raf/MEK/ERK signal transduction pathway（s） and the change of Bcl-2 and p53 protein when FL cells were promoted by rhEGFMaterial and methods1.The proliferation of FL cells was evaluated by MTT assay:FL cells were cultured in MEM containing 10%FBS,incubated in 37℃,5%CO₂ for 48 hours,colleted the cells,wash with PBS solution,dilluted to 2×10⁸ cell /L number,then added 100 ul samples to every reaction.When cells stuck wall,cultured them in serum-free medium containing rhEGF of various concentration（0.1,1,10,30, 60μg/L）for 1,2,3,4,5 days,or containing rhEGF（10μg/L）and PD98059 of various concentration（25,50,75μmol/L）for 48 hours.In control group,rhEGF was substituted with saline solution.Then added MTT（5 mg/ml）20ul and cultured for another 4 hours, added DMSO 150ul to stop the reaction.Read the absorbance value on OD490 nm,the cell growth acceleration rate was calculated as follows acceleration rate=(Ai-Ao/（Ao）×100% Ao,Ai:absorbance value of control group and experiment group respectively.2.Western blot was used to detect Ras/Raf/MEK/ERK signaling transduction pathway（s）and the expression of Bcl-2 and p53 protein:When cells stuck wall,cultured in serum-free medium containing rhEGF of various concentration（1,10,60μg/L）.In control group,rhEGF was substituted with saline solution.37℃,5%CO₂ incubated for 5 min - 4 h,colleted the cells,added cell disruption lysate,collected supernate and detected the concentration of protein,added 50μg samples to every reaction.After electrophoresis,transfer protein to PVDF membrane,blocked with 5%BSA in 4℃for 2 hours.Added first antibody,enzyme labeled second antibody reaction for 2 hours or overnight,washed with PBST for 3 times, added ECL reagent for 1.5 min,then exposed to theⅩfilm.3.To explore the effect of MAPK pathway inhibitor PD98059 on the proliferation of FL cell and the activation of ERK1/2:FL cells were cultured with PD98059 of various concentration（25,50,75μmol/L） for 1 h,then added 10μg/L rhEGF,cultured for 30 min,Western blot was used to detect the change of ERK1/2 protein.Results1.Incubated in 37℃,5%CO₂ for 48 hours,Morphology of FL cells cultured in MEM medium with rhEGF became fusiform mostly.2.At 1st day to 5th day,rhEGF of various concentration（1,10,30,60μg/L）can significantly accelerate the growth of FL cells and there was no significant difference when rhEGF concentration was 0.1μg/L and.The proliferation rate of FL cells reached the maximum on third day when concentration of EGF were 10μg/L（42.4%,P＜0.01） and 60μg/L（36.1%,P＜0.01）respectively.At 4th day to 5th day,the proliferation rate of FL cells began to decline.3.Stimulated by rhEGF of various concentration for 30 min,Western blotting showed that the activity of p-Raf increased significantly when rhEGF ranged in 1μg/L～60μg/L（P＜0.05）.Furthermore,we detect the level of p-Raf when FL cells were treated with 10μg/L rhEGF of different time（0 min,5 min,30 min,1 h,2 h respectively）.Result showed the expression of p-Raf protein was highest at 5 min（P＜0.01）,then began to decline.2 h after cells were treated with rhEGF,the level of p-Raf had no significant difference when compared with control group（P＞0.05）.4.Stimulated by rhEGF of various concentration for 30 min,Western blot showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 1μg/L～60μg/L（P＜0.01）.Further more,we detect the level of p-ERK1/2 when FL cells were treated with 10μg/L rhEGF of different time（0 min,5 min,30 min,1 h,2 h respectively）.Result showed the expression of p-ERK1/2 protein was highest at 5 min（P ＜0.01）,then began to decline.2 h after cells were treated with rhEGF,the level of p-ERK1/2 was lower when compared with control group（P＜0.05）.5.Compared with control group,Western blot showed PD98059 of various concentration（25,50,75μmol/L）can inhibit the expressive of p-ERK1/2.The effect of PD98059 has dose dependent.6.Stimulated by rhEGF of various concentration for 1 hour,Western blot showed that the activity of Bcl-2 protein increased significantly when rhEGF ranged in 1μg/L～60μg/L（P＜0.05）,but p53 protein decreased significantly.The effect reach maximal when rhEGF in 10μg/L but 1μg/L rhEGF was minimum,there was significant difference between two groups（P＜0.01）.7.We detect the level of Bcl-2 when FL cells were treated with 10μg/L rhEGF of different time.Result showed the expression of Bcl-2 protein was highest at 1 h（P＜0.01）,then began to descend.It is similar with the change of p-ERK1/2 when compared with control group.ConclusionsrhEGF stimulated FL cell proliferation,Ras/Raf/MEK/ERK signaling transduction pathway in a time or dose dependent manner.EGF of 10μg/L concentration had the best effect.FL cells stimulated by rhEGF had self-adapting control action.EGF activate Bcl-2 and p53 also in a time or dose dependent manner.It was saft when rhEGF concentration was in 1μg/L.PartⅡ:The isolation,purification and identification of EGF from goats’ submaxillary glandsObjectionIsolate,purify and identify EGF from goats’ submaxillary glandsMaterial and methods1.Isolation and purification EGF from the goats’ submaxillary glands:We took several fresh or freezed goats’ submaxillary glands,cut off connective tissue and smashed it with blender,then put it into homogenizer.Added HAC（4℃）,centrifigated 50 000G for 30 mins,collected the supernatant.Pass it through DEAE Sephadacel anion exchange column,collected the eluent,and detected the protein flow peak at wave length 280 nm.ELISA test was done to detect active component of EGF,designed 0.2 M ammonium acetate as negative control,EGF standard preparation as positive control. Read the number on OD652 nm or on OD490 nm（added 50 ul 2 M sulphuric acid to stop the reaction）.Component whose reading are close to EGF standard preparation were considered active constituent.Undertook dialyze to take off saline matter from active constituent,molecular weight of bag filter was 3500,then condense with glycerin or PEG（molecular weight:20 000）to remove water.All the proceed was on ice（4℃）. ELISA test was done to detect active component of EGF of concentrated solution,then took the concentrated solution pass through Sephadex G50 column.Detected the protein flow peak at wave length 280nm.Collected the eluent,and ELISA test was done to detect active component of EGF.Consequence,the absorbance of one protein flow peak was higher than others.Therefore,this protein flow peak was the peak of EGF identified by the ELISA.2.Identification the bioactivity of goats’ EGF2.1.FL cells were stimulated by goats’ EGF of various concentration.The proliferation of FL cells were evaluated by MTT assay.2.2.FL cells were stimulated by goats’ EGF of various concentration.The cell cycle was detected by flow cytometry.2.3.SDS-PAGE electrophoresis was used to detect goats’EGF band.Results1.Incubated in 37℃,5%CO₂ for 48 hours,morphology of FL cells cultured in MEM medium with rhEGF became fusiform mostly.2.MTT methods:MTT methods showed the cell growth acceleration rate of goat EGF group（diluted 1∶100,1∶500,1∶1000 respectively）and rhEGF group were 20.9%,39.4%,24.9%and 40.6%respectively.So the goats’ EGF can significantly accelerate the growth of FL cells（P＜0.01）.The effect of goats’ EGF diluted to 1∶500 was best and there was no significant difference with the rhEGF（P＞0.05）.3.Flow cytometry:compare with control group,goats’ EGF also increased the S-phase cells fraction obviously,G₀/G₁ phase cells fraction decreased from 71.1±1.9 to 60.3±2.2（P＜0.01）,S-phase cells fraction increased from 18.8±1.4 to 25.8±1.1（P＜0.01）.4.SDS-PAGE electrophoresis showed a band in 6 KD.ConclusionsThe protein isolated from goats’ submaxillary glands was identified as EGF,it accelerates the growth of FL cells,changes the morphology of the cultured cells and increases the S-phase cells fraction obviously.Therefore it is bioactive EGF.Goats’ submaxillary glands can be the medicinal herbs resource of EGF.更多还原