【作者】 赵秋洁；

【导师】 李延青；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 背景溃疡性结肠炎(UC)是一种累及直肠和结肠粘膜及粘膜下层的慢性非特异性炎症性疾病。患者临床症状表现为腹痛、腹泻、粘液脓血便等,病情轻重不一,多为慢性病程,表现为发作期与缓解期交替。UC的发病机制尚未明确。已有的研究证实了机体的固有免疫及适应性免疫的异常、遗传易感因素以及环境因素共同参与了该病的发生。乳汁球微粒表皮生长因子8(MFG-E8)是一种分泌型的糖蛋白,可在多种细胞中表达,包括巨噬细胞、乳腺上皮细胞、表皮角质细胞等。MFG-E8参与了许多重要的细胞事件。MFG-E8在巨噬细胞介导的凋亡细胞清除中起了重要作用,同时在乳腺发育以及精子与卵细胞的结合过程中也起了重要作用。MFG-E8在肠粘膜损伤后修复以及肠道炎症调节中都起了重要的作用。已有的研究证实了MFG-E8存在于小鼠肠粘膜固有层的巨噬细胞中。在盲肠结扎穿刺诱导的小鼠败血症模型中,肠道中的MFG-E8含量明显下降。体内实验证实了外源性的重组MFG-E8能够促进其肠上皮细胞沿着隐窝-绒毛轴移行,从而促进肠粘膜损伤后的修复。在右旋葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎模型急性期,炎症累及的结肠段中MFG-E8的表达量与正常的结肠段相比明显降低。预先给予小鼠外源性的重组MFG-E8,能明显减轻DSS诱导的结肠炎症,这些表明了MFG-E8在肠道炎症中起了调节作用。最近的一项研究证实了在DSS诱导的小鼠结肠炎恢复期,给予小鼠重组MFG-E8能加快肠粘膜损伤后的修复,从另一方面有力地证实了MFG-E8在肠道炎症治疗中的积极作用。尽管许多动物实验已经证实了MFG-E8在肠道炎症及损伤修复中的重要作用,迄今为止尚未有关于人类肠道中MFG-E8研究的报道。同时UC患者中MFG-E8表达情况的研究也是缺失的。鉴于MFG-E8在肠道炎症及肠粘膜损伤修复中的重要作用,我们推测UC患者肠粘膜中MFG-E8的含量可能存在异常。所以我们本次研究的目的在于揭示MFG-E8在正常人及UC患者肠粘膜中的表达情况,同时分析UC患者肠粘膜中MFG-E8含量与患者疾病严重程度以及肠粘膜炎症分级的关系。目的1.研究MFG-E8在正常人及UC患者肠粘膜中的表达情况。2.分析UC患者肠粘膜中MFG-E8含量与肠粘膜炎症程度以及患者疾病活动指数的相关性。方法1.研究对象及标本采集本研究入选了26例UC患者及26例对照者。26例UC患者为监测病情而来山东大学齐鲁医院消化内镜中心进行结肠镜检查,其诊断依据我国已有的临床、内镜及病理诊断标准。对照者选用来我院行结肠镜检查的内镜下肠粘膜无异常者,这些对照者中包括息肉切除术后复查者、肠道功能性疾病如肠易激综合征的患者,也有部分为查体而行结肠镜检查。本研究方案经山东大学齐鲁医院医学伦理委员会批准实施,所有的受试对象均被提前告知实验过程,并亲自签署了知情同意书。对于UC患者,肠粘膜标本取自内镜下炎症明显的部位。正常对照者标本取自内镜下正常的肠粘膜。所有的标本均取自直肠或者乙状结肠。每例研究对象从同部位取2块临近的标本,1块用于行组织学分析,另外1块用于RNA或总蛋白提取。2.肠粘膜标本炎症程度分级及UC患者的疾病活动指数计算肠粘膜标本的炎症程度按照Mart’s标准进行分级,根据HE染色结果显示的中性粒细胞浸润程度划分为轻度、中度或重度级别。每位UC患者在进行肠镜检查时均计算Mayo疾病活动指数(DAI,0-12分范围)。该指数的计算基于患者每日的排便次数、便血情况、内镜下表现以及临床医师对患者的整体评价。3.RNA提取、半定量逆转录-聚合酶链反应(RT-PCR)及实时荧光定量PCR.总共有25例肠粘膜活检标本用来提取RNA,包括12例来自对照者的正常肠粘膜,13例来自UC患者的病变肠粘膜。RNA的提取应用Trizol试剂,采用氯仿萃取及异丙醇沉淀的方法获得RNA。反转录应用莫罗尼氏鼠白血病毒(M-MLV)来源的逆转录酶,引物为寡聚脱氧胸腺嘧啶18(oligo-dT18)。普通半定量PCR应用ExTaq DNA聚合酶,反应在Mastercyler热循环仪上进行。实时荧光定量PCR应用SYBR Green试剂,在罗氏Lightcycler2.0机器上进行PCR反应。管家基因人beta-actin作为内参,实时定量PCR的结果采用2-△△CT分析方法。4.蛋白质提取及western boltting总共有27例肠粘膜活检标本用来提取总蛋白,13例为来自对照者的正常肠粘膜,14例为来自UC患者的病变肠粘膜。总蛋白采用RIPA裂解法,蛋白浓度测定采用BCA法。变性后的总蛋白通过不同浓度的聚丙烯酰胺凝胶电泳分离,转移到聚偏二氟乙烯(PVDF)膜上,室温下应用5%脱脂奶粉封闭后依次孵育一抗以及辣根过氧化物酶(HRP)标记的二抗。用ECL化学发光法来检测条带。5.组织学分析每一例研究对象都有1例肠粘膜活检标本用于组织学分析。用于组织学分析的标本首先在10%中性福尔马林缓冲液中固定48h,再行脱水浸蜡。石蜡包埋后,切成3μm厚度的切片。常规组织病理学分析采用伊红-苏木素染色法。MFG-E8免疫染色应用Envision二步法,一抗为小鼠来源的抗人MFG-E8单克隆抗体。结果1.研究对象的基本情况UC患者(26例)与正常对照组(26例)在年龄、性别构成方面相匹配。UC患者中有3例溃疡性直肠炎、14例左半结肠炎以及7例全结肠炎患者。2. MFG-E8在UC患者肠粘膜中的表达情况普通半定量PCR结果证实了在对照者及UC患者的肠粘膜中均能检测到MFG-E8mRNA。实习荧光定量PCR进一步证实了肠粘膜中MFG-E8mRNA水平在UC患者中明显低于正常对照者(P<0.01)。Western blotting结果证实了在UC患者(13例)肠粘膜中MFG-E8蛋白水平明显低于对照者正常肠粘膜(14例)(P<0.01)。3.MFG-E8表达量与UC肠粘膜炎症分级和疾病活动指数的相关性分析在肠粘膜中度至重度炎症的UC患者中MFG-E8蛋白含量下降较之轻度炎症患者更为明显,有明显统计学差异(P<0.01)。Spearman相关性分析证实了MFG-E8蛋白水平与UC患者疾病活动指数呈负相关(r=-0.7713,P<0.01)。4.免疫组化免疫组化结果显示了MFG-E8在人肠粘膜中主要存在于肠上皮细胞中。在UC患者的肠上皮中,较之对照者正常肠粘膜上皮MFG-E8染色明显变弱。肠粘膜间质中未见明显阳性MFG-E8染色。结论1.MFG-E8存在于人肠粘膜之中,由人肠上皮细胞表达。2.溃疡性结肠炎患者炎症累及的肠粘膜组织中MFG-E8mRNA及蛋白表达水平与正常对照者肠粘膜相比明显降低。3.溃疡性结肠炎患者肠粘膜组织中MFG-E8含量与粘膜炎症分级和患者的疾病活动指数均呈负相关。背景MFG-E8是一种多功能的分泌型糖蛋白,最早发现其存在于人乳汁球微粒的表面。MFG-E8可在巨噬细胞、树突状细胞、表皮角质细胞及乳腺上皮细胞等多种细胞中表达。MFG-E8在巨噬细胞清除凋亡细胞、精子和卵细胞结合以及乳腺的发育等多种复杂的细胞事件中起了重要作用。MFG-E8在肠粘膜损伤后修复及肠道炎症调节中也起了重要作用。在盲肠结扎穿刺诱导的小鼠败血症模型中,应用外源重组的MFG-E8能促进小鼠肠上皮细胞沿隐窝-绒毛轴爬行,从而促进损伤后肠粘膜的修复。在DSS诱导的小鼠实验性结肠炎的急性期,炎症累及的结肠粘膜中MFG-E8的表达量明显下调。预先给予小鼠外源性重组的MFG-E8可以减轻DSS所导致的结肠炎症。体外实验中,重组的MFG-E8能抑制LPS所诱导的小鼠腹腔巨噬细胞炎性因子TNF-alpha. IL-1beta和IFN-gamma的分泌。MFG-E8能抑制NF-kappa B的活化,从而进一步下调下游炎性介质的产生。这些研究都充分肯定了MFG-E8在肠粘膜损伤修复以及肠道炎症调节中的作用。我们之前的研究从转录水平及蛋白水平上证实了溃疡性结肠炎患者病变结肠粘膜中MFG-E8表达量较之对照组的正常结肠粘膜明显减少。同时,在UC患者中肠粘膜MFG-E8的表达量与粘膜炎症分级以及患者的Mayo疾病活动指数均成负相关。应用免疫组织化学染色技术,我们首次证实了MFG-E8存在于人肠粘膜上皮细胞中。鉴于在正常人肠上皮细胞中MFG-E8有丰富的表达,同时UC患者的肠上皮细胞中MFG-E8表达量明显减少,我们推测MFG-E8可能在肠上皮细胞中参与了重要的生理功能并且在溃疡性结肠炎的发病机制起了重要作用。肠上皮细胞(IEC)及其之间的紧密连接构成的肠粘膜屏障,将机体内环境与肠腔内的细菌等有害物质隔离开,是人体的一道重要生理防护屏障。正常肠粘膜屏障功能的维持依赖于完整的肠粘膜、肠道的内正常菌群、肠道内分泌物和蠕动以及肠道免疫功能等,其中最基础的是完整的肠粘膜上皮屏障。UC患者中肠上皮细胞凋亡率增加,这使得正常肠上皮细胞的生理周期缩短,加快肠上皮细胞的脱落,从而引起肠粘膜上皮屏障功能的损害。肠上皮细胞能够产生大量的细胞因子及趋化因子,这些因子一方面可以直接作用于肠粘膜上皮,引起肠上皮屏障的损伤；另一方面这些炎性因子也可作用于固有层中的单核细胞及中性粒细胞等,促进其分泌更多的炎性因子。溃疡性结肠炎为慢性病程,肠粘膜损伤与修复过程持续存在。许多促修复因子参与了肠粘膜损伤修复的过程中,溃疡性结肠炎患者肠粘膜中多种修复因子存在异常表达。在这些因子中肠三叶肽及转化生长因子-β1的作用尤为突出。根据我们早期的研究成果及已有的报道,我们推测UC患者的肠上皮细胞中MFG-E8含量下降可能参与了该病的发病机制,患者肠上皮细胞高凋亡率、高炎性反应性以及损伤修复减慢可能与其中MFG-E8含量不足有关。目的1.体外培养人结直肠癌来源的肠上皮细胞株HT-29和Caco-2,应用慢病毒包装的MFG-E8短发夹RNA (ShRNA)载体进行RNA干预,获得MFG-E8沉默的肠上皮细胞克隆。2.检测MFG-E8基因沉默的肠上皮细胞中凋亡以及凋亡相关分子的改变。同时应用重组人MFG-E8蛋白作用于肠上皮细胞,检测凋亡相关分子的改变。3.检测MFG-E8基因沉默的肠上皮细胞对TNF-alpha及Flagellin刺激的炎性反应性变化,并给予肠上皮细胞重组人MFG-E8提前处理,观察处理后的炎性反应性变化。4.检测MFG-E8基因沉默的肠上皮细胞损伤后修复能力、重组人MFG-E8对肠上皮细胞损伤后修复的影响以及相关促修复因子的变化。方法1.细胞培养人结直肠癌来源的肠上皮细胞株HT-29及Caco-2培养于添加了10%胎牛血清(V/V)的高糖型DMEM中,培养条件为37℃、5%CO2和95%湿度。2.慢病毒转导以及MFG-E8沉默的肠上皮细胞克隆的获得应用慢病毒包装的MFG-E8靶向的ShRNA载体来进行MFG-E8沉默,载体同时携带有嘌呤霉素抗性基因。应用coGFP对照病毒来摸索MOI值。两种细胞转导的慢病毒MOI值均为100。细胞融合率达30%-50%时加入聚凝胺及病毒液的混合物,12小时后换液。72小时后荧光显微镜下观察根据荧光情况来监测转导效率并筛选出每种细胞合适的MOI值。加入100ug/ml嘌呤霉素加压培养,每2-3天更换含有新鲜嘌呤霉素的培养基。加压培养约3周后可见明显的细胞克隆形成。挑选出单个克隆,消化传代后继续培养扩增,从而筛选出稳定转导的细胞克隆。3.细胞RNA提取及实习荧光定量PCR细胞RNA提取应用Trizol,反转录应用M-MLV逆转录酶,实时荧光定量PCR中应用SYBR Green试剂,反应在罗氏Lightcycler2.0仪器上进行。人beta-actin基因为内参,结果采用2-△△CT分析方法。4.细胞总蛋白提取及western blotting使用RIPA裂解液,裂解产物经14000rpm4℃离心10分钟,吸取上清液,即为总蛋白。蛋白浓度测定应用BCA法。蛋白中加入上样缓冲液后煮沸变性,经不同浓度的聚丙烯酰胺凝胶电泳分离后,电转至聚偏二氟乙烯(PVDF)膜上。5%脱脂奶粉室温封闭2小时后,将膜依次孵育一抗二抗。次日用ECL化学发光法检测蛋白条带。5.细胞凋亡检测细胞凋亡检测应用流式细胞术以及Annexin Ⅴ-FITC凋亡检测试剂盒。细胞融合率达60-70%时,将漂浮细胞及贴壁细胞均收集起来,PBS洗涤2次后,重悬于binding buffer中。随后加入AnnexinⅤ-FITC及碘化丙啶,室温避光孵育10分钟后,在BD FACSCalibur流式细胞仪上检测Annexin V-FITC结合情况。凋亡形态学检测应用Hoechst33342染色,随后在荧光显微镜下观察细胞核形态。6.细胞伤痕愈合试验细胞选取合适的密度种板,以次日能长满为宜。采用经典的细胞划痕实验,用无菌枪头在长满细胞的平板上划三条直线,PBS冲洗3次冲掉漂浮的细胞,加入完全培养基继续培养24小时。划痕后0小时及24小时后于倒置显微镜下观察拍照,并测量划痕处的宽度,计算出24小时内细胞的迁移距离。结果1.肠上皮细胞中MFG-E8的表达以及MFG-E8沉默的验证普通PCR及western blotting证实了MFG-E8mRNA及蛋白在HT-29及Caco-2两种肠上皮细胞株中均有表达。实时荧光定量PCR证实了与转导LV-C的细胞相比,转导了LV-M的HT-29及Caco-2细胞中,MFG-E8mRNA表达量明显抑制。Western blotting结果进一步证实了转导LV-M后两种细胞中MFG-E8蛋白水平明显受到抑制。2.凋亡及凋亡相关分子的检测Annexin V-FITC染色后的流式细胞术证实了MFG-E8沉默导致了肠上皮细胞中凋亡率增加。Hoechst33342染色结果证实了MFG-E8沉默后的IEC中核固缩、核碎裂等的凋亡细胞比率明显增加。同时实习荧光定量PCR及western blotting发现MFG-E8沉默后的肠上皮细胞中存在促凋亡因子BAX及活化型Caspase-3表达水平的明显上调,而内源性抑制凋亡因子BCL-2含量明显下调。为进一步证实MFG-E8的抗凋亡作用,我们将HT-29及Caco-2细胞与重组人MFG-E8孵育,发现MFG-E8在100ng/ml时即可显著上调凋亡抑制因子BCL-2的表达,同时伴有促凋亡因子BAX以及活化型caspase-3的下降。3.肠上皮细胞炎症反应性检测应用肿瘤坏死因子(TNF)-alpha及鞭毛蛋白(Flagellin)刺激能显著诱导肠上皮细胞IL-8mRNA的产生。MFG-E8沉默后,IEC中基础IL-8mRNA表达量及TNF-alpha、Flagellin诱导的IL-8mRNA表达量并无明显差异。预先给予HT-29及Caco-2细胞重组人MFG-E8处理后再给予TNF-α及flagellin刺激,所诱导的IL-8mRNA表达量与未予重组人MFG-E8处理组比较无统计学差异。4.细胞伤痕愈合实验及肠粘膜促修复因子的检测细胞划痕24小时后,MFG-E8沉默的HT-29细胞的迁移距离明显小于转导对照病毒的HT-29细胞。同时,MFG-E8沉默可抑制IEC中肠粘膜促修复因子肠三叶肽3(TFF3)mRNA的表达,但是对另一修复相关的重要因子转化生长因子(TGF)-β1mRNA水平并无影响。结论1.MFG-E8在IEC中起了抑制凋亡的作用,IEC中MFG-E8的沉默伴随着凋亡相关分子BCL-2和BAX的改变。2.MFG-E8能促进IEC损伤后的修复,IEC中MFG-E8的沉默伴随着肠粘膜修复因子TFF3的表达的下降。3.MFG-E8不影响IEC中基础IL-8mRNA表达水平以及TNF-alpha和Flagellin刺激所诱导的IL-8mRNA表达水平。更多还原

【Abstract】 BackgroundUlcerative colitis (UC) is a chronic and relapsing inflammatory disorder involving the mucosal layer of the colon and rectum. The patients complain about abdominal pain, diarrhea, bloody stool, etc. Most patients have active and remission phases alternatively. However the underlying molecular basis of the disease has not been completely elucidated.Milk fat globule-epidermal growth factor8(MFG-E8), also named lactadherin, BA46or SED1, is a secreted glycoprotein present in several cell types, including macrophages, mammary epithelial cells, and epidermal keratinocytes. It participates in engulfing apoptotic cells. MFG-E8-null mice show severe autoimmune disease like human systemic lupus erythematosis because of accumulation of uncleared apoptotic cells. MFG-E8also participates in other cell events including the promotion of mammary gland branching and facilitation of sperm-egg binding.Several studies of animal models demonstrated that MFG-E8play an important role in intestinal homeostasis. In mice with sepsis, MFG-E8promoted enterocyte migration along the crypt-villus axis and accelerated intestinal mucosal healing. As well, MFG-E8expression was impaired in inflamed mouse colon during the acute phase of dextran sodium sulfate (DSS)-induced colitis, but pre-treatment with recombinant MFG-E8had a protective role against inflammation. Moreover, a recent study reported that recombinant MFG-E8administered during the recovery phase of DSS-induced colitis in mouse could attenuate inflammation and enhance epithelial repair, which suggests a therapeutic role for MFG-E8in DSS-induced colitis. But we lack reports of MFG-E8activity in humans with UC.Objectives1. To evaluate MFG-E8expression in normal human colon and inflamed colon in UC patients.2. To analyze the correlation of MFG-E8levels with colonic mucosal inflammation gradings and patients’disease activity indecies in UC patients.Methods1. Colonic biopsy samplingWe enrolled26patients with UC who underwent colonoscopy for disease surveillance. The diagnosis was based on well-established clinical, endoscopy and histopat-hological criteria in China. Control subjects were26volunteers matched by age and sex who underwent colonoscopy for physical examination, surveillance of polyp recurrence or functional abdominal pain. Our study was approved by the Clinical Ethical Committee of Qilu Hospital of Shandong University. All subjects had given their written informed consent before biopsy sampling.During colonoscopies, intestinal mucosal biopsies were obtained from the macroscopically inflamed area for UC patients or from normal areas for healthy controls in the sigmoid colon or rectum. For each subject, we obtained2adjacent biopsies, one for histology and anotherr for RNA or protein analysis. For histology, biopsies were immediately fixed in10%neutral buffered formaldehyde and those for RNA or protein analysis were snap-frozen in liquid nitrogen and stored at-80℃Biopsies from all of the subjects underwent routine histology, and all biopsies from healthy controls were confirmed as histologically normal.2. Inflammation grading and calculation of disease activity index Mucosal inflammation severity was classified as mild, moderate or severe by neutrophil infiltration according to Matt’s grade. The Mayo disease activity index (0-12scale) calculated for each UC patient at the time of colonoscopy was based on frequency of bowel movements, rectal bleeding, endoscopy findings and the physician’s overall assessment.3. RNA isolation, semi-quantitative RT-PCR and real-time quantitative PCRA total of25biopsy specimens (12from UC patients and13from controls) were used for RNA analysis. Total RNA was isolated by the Trizol reagent method. RNA was reverse-transcribed using MMLV-derived reverse transcriptase and oligodT primer. Semi-quantitative RT-PCR was performed with ExTaq DNA polymerase in a Mastercycler thermal cycler. For real-time PCR, cDNA was amplified with SYBR Green reagent in a Roche fluorescence thermo-cycler. Human beta-actin was used as an internal control. Targeted gene mRNA levels were normalized to those of beta-actin by the2-ΔΔCT method.4. Protein extraction and western blottingTotal protein was extracted from27biopsy samples (14from UC patients and13from controls) in RIPA buffer. Protein was quantified by the use of a BCA protein quantification kit. Protein was separated by SDS-PAGEs and transferred to PVDF membrane (0.22μm pore). The membrane was incubated with primary antibodies and HRP secondary antibodies sequentially. Densitometry of protein bands was quantified by the use of Quantity One4.6.2.5. Immunohistological analysisParaffin-embedded tissues were cut into3-μm-thick sections and underwent routine deparaffinizing and rehydrating. HE staining was used for routine histological analysis. For MFG-E8immunostaining, the Envision-two-step method was used.Results1. Demographic features of participantsNo statistical differences of age or sex ratio between26ulcerative patients and26control subjects were observed. Among26patients, there are3with ulcerative proclitis,14with left-sided colitis and7with ulcerative pancolitis.2. Colonic MFG-E8expression in ulcerative colitisSemi-quantitative RT-PCR proved the existence of MFG-E8mRNA in colonic biopsies from ulcerative colitis patients and controls. Real-time quantitative PCR revealed that colonic MFG-E8mRNA expression was significantly lowered in patients than controls (P<0.01). Western blotting results indicated that MFG-E8protein was dramatically decreased in ulcerative colitis patients than controls (P<0.01).3. Correlation analysis of colonic MFG-E8expression with mucosal inflammation and disease activity index in ulcerative colitisColonic MFG-E8expression in ulcerative colitis, was much lower in biopsies with moderate or severe inflammation than those with mild inflammation (P<0.01). Spearman correlation analysis revealed that MFG-E8expression was inversely correlated with disease activity index in ulcerative colitis (r=-0.7713, P<0.01).4. ImmunohistochemistryWe used immunohistochemistry to investigate the cellular origin of MFG-E8in intestinal tissues. The intestinal epithelium of healthy controls showed strong positive staining for MFG-E8. MFG-E8was present in both of the surface epithelium and crypts of normal intestinal tissues. However, the intestinal epithelium of UC patients showed less staining for MFG-E8.Conclusions1. MFG-E8is present in human colonic mucosa and the intestinal epithelial cell is its mam origin.2. Colonic MFG-E8mRNA and protein expression is decreased in ulcerative colitis patients compared with controls.3. Colonic MFG-E8expression in UC patients is inversely correlated with mucosa inflammation gradings and the Mayo disease activity indecies. BackgroundUlcerative colitis is characterized by elevated production of pro-inflammatory mediators, repeated intestinal injury and cell restitution and increased apoptosis of intestinal epithelial cells (IECs), which leads to impaired mucosal barrier function. However the underlying molecular basis has not been completely elucidated.IECs are a pivotal physiological barrier between the environment and the host. They play a key role in gut innate immunity and contribute to inflammatory progression in inflammatory bowel disease by secreting various chemokines and cytokines. IEC apoptosis has a major role in intestinal epithelial homeostasis and pathogenic mechanisms. Increased IEC apoptosis has been observed in acute inflammatory sites of patients with IBD, which led to impaired intestinal barrier function and contributed to disease development. The intestinal epithelium is exposed to various stimuli, and IEC injury is constant and inevitable. Especially in patients with IBD, repeated intestinal epithelial injury is typical. During intestinal epithelium injury, IECs rapidly depolarize and migrate to cover the denuded area, independent of proliferation. This process is called restitution, and rapid restitution is required to maintain the integrity of intestinal barrier.MFG-E8expression was impaired in inflamed mouse colons during the acute phase of dextran sodium sulfate (DSS)-induced colitis, but pre-treatment with recombinant MFG-E8had a protective role against inflammation. Our previous study has proved that MFG-E8was present in human intestinal epithelium and colonic MFG-E8was dramatically decreased in ulcerative colitis. Additionally, MFG-E8 expression in ulcerative colitis is inversely correlated with mucosal inflammation grading and disease activity index.Overall, we presumed that decreased MFG-E8expression in IECs may be related to increased apoptosis, abnormal inflammatory responsiveness and impaired wound healing in UC patients. Objectives1. To acquire MFG-E8knockdown intestinal epithelial cells (IECs) in vitro to represent IECs in ulcerative colitis.2. To detect apoptosis and apoptosis-related proteins expression in IECs after MFG-E8knockdown.3. To detect wound healing and intestinal restitution-related factors in MFG-E8knockdown IECs.4. To detect the inflammatory responses of IECs after MFG-E8knockdown.Methods1. Cell culture and lentivirus mediated MFG-E8knockdownHuman colorectal cancer-derived IEC lines Caco-2and HT-29were cultured in Dulbecco’s modified Eagle’s medium supplemented with10%(v/v) fetal calf serum. Lentiviral particles encoding short hairpin RNA (shRNA) sequences targeting human MFG-E8mRNA and control lentiviral particles encoding a scrambled shRNA sequence were from Santa Cruz Biotechnology. Lentiviruses all carried a puromycin-resistant gene. When cells were at50%to60%confluence, lentiviral vectors were added with10μg/ml polybrene. Puromycin was added to get stable-transduced clones. MFG-E8-knockdown levels were validated by real-time quantitative PCR and western blot analysis.2. Cellular RNA isolation and real-time quantitative PCRCellular RNA was isolated with Trizol reagent. The following were similar with procedures in Part Ⅰ.3. Cellular protein extraction and western blottingCells were lysised in RIPA buffer. Lysis was centrifuged by14000rpm at4℃for 10min. Supernatants were collected. Protein concentration was quantified with BCA protein assay kit. Total protein was separated by SDS-PAGEs and transferred to PVDF membrane later. The membrane was then incubated with different antibodies. An enhanced chemiluminiscent substrate was used to detect the protein bands.4. Apoptosis detectionApoptosis was detected by flowcytometry with an Annexin-V/FITC kit. Briefly, floating and adherent cells were washed with phosphate buffered saline. After cells were resuspended in binding buffer, FITC-conjugated annexin V and propidium iodide were added to incubate for10min in the dark. Annexin-V binding was determined in a BD FACSCalibur flow cytometer (Becton-Dickinson). Apoptotic nucleus changes were viewed under a fluorescence microscope after incubation with Hoechst33342staining solution.5. Wound healing assayHT-29cells transduced with LV-C or LV-M were seeded into6-well plates and allowed to reach confluence overnight, then wounds were made by scratching cell monolayers with a sterile pipette tip. Detached cells were rinsed off3times with PBS. Serum-deprived culture medium (DMED with0.1%FCS) was added for further culture. Cells were photographed0and24hr after wounding. Distances covered by migrated cells were quantified.Results1. MFG-E8in IEC lines and validation of MFG-E8knockdownMFG-E8mRNA and protein were present in the IEC lines HT-29and Caco-2. After transduction with LV-M, MFG-E8mRNA levels were decreased by93.2%and92.7%in HT-29and Caco-2cells. As well, MFG-E8protein levels were decreased by78%and73%in HT-29and Caco-2cells, respectively.2. MFG-E8has no effect on IEC inflammatory responsesThe basal IL-8mRNA expression levels in HT-29and Caco-2cells with MFG-E8-knockdown was similar to that with LV-C transduction. After stimulation with100ng/ml TNF-alpha or flagellin for12hr, IL-8mRNA levels were both upregulated in MFG-E8-knockdown cells and LV-C-transduced cells. And IL-8mRNA expression induced by TNF-alpha or flagellin was not altered in MFG-E8-knockdown IECs. To further investigate the role of MFG-E8in flagellin induced inflammatory response in IECs, we used recombinant human MFG-E8to treat Caco-2cells (Since Caco-2is more responsive to flagellin than HT-29) before flagellin addition. And pre-incubation with rhMFG-E8from lOng/ml to500ng/ml for12hr didn’t alter IL-8mRNA levels induced by flagellin in Caco-2cells.3. Apoptosis induction in MFG-E8knockdown IECsAnnexin V positive apoptotic cell proportions were significantly higher in MFG-E8knockdown IECs. The pro-apoptotic protein BAX and cleaved caspase-3expression were upregulated after MFG-E8knockdown. The anti-apoptotic protein BCL-2level was downregulated after MFG-E8knockdown.4. MFG-E8knockdown impaired wound healing and TFF3expression in IECsMFG-E8knockdown greatly attenuated wound closure of scratched HT-29cell monolayers24hr after wounding. In addition, expression of TFF3, a pivotal factor in intestinal restitution, was decreased after MFG-E8knockdown.Conclusions1. MFG-E8knockdown can promote basal apoptosis in IEC lines accompanied by the induction of cleaved caspase-3and BAX and the suppression of BCL-2.2. MFG-E8knockdown can impair wound healing in IEC lines as well as the mRNA level of TFF3.3. MFG-E8has no effect on inflammatory responses in IECs induced by TNF-alpha or flagellin.更多还原