【作者】 唐乐辉；

【导师】 任国胜；

【作者基本信息】 重庆医科大学 ， 外科学， 2012， 博士

【摘要】 研究背景作为居女性恶性肿瘤死亡原因之首的乳腺癌，确切证据表明，90％以上的患者最终死于肿瘤转移或复发，乳腺癌的复发和转移是影响其疗效的主要因素。探讨导致肿瘤侵袭和转移的致病因素因此显得非常重要。因为乳腺癌是复杂的病理学本质的异质性疾病，所以预测肿瘤远端转移比率的发生还是非常困难的。就因于此，寻找乳腺癌增殖、迁移运动和转移有关的新的预后因素相关的分子生物学标记物在应对乳腺癌的复发和转移有积极意义。诸多的研究证实，炎症与癌症具有密切相关性，而持续性的炎症能诱导癌症的产生。细胞因子和化学因素在增加血管发生、转移和特异性免疫下调中发挥决定性作用。长期慢性炎症刺激能够引起肿瘤组织释放许多诱发肿瘤本身生长的因子,这样形成了一个炎性微环境,促进了肿瘤的发生与发展。并且一旦炎症刺激肿瘤细胞释放多种免疫因子后,我们很难再次诱导肿瘤细胞凋亡。可见，炎症因素能够诱导肿瘤细胞抵抗凋亡。Toll样受体(Toll like receptors,TLRs)的深入研究是近年在先天免疫研究方面的卓越发展。一般情况下，TLRs能够被激动剂激活，分别通过两个下游途径即MyD88依赖性和非依赖性即TRIF途径启动下游信号转导，形成系列反应。有研究提示TLRs的表达与乳腺癌之间呈现较强烈的关系，在乳腺癌中部分TLRs表达与雌激素受体呈负相关，说明TLRs的表达水平在临床对乳腺癌侵袭和转移的诊断中可能发挥重要作用，但TLRs介导的入侵的病理生理学机制还不明确。研究目的本研究在收集如病人的年龄、淋巴转移率、肿瘤大小、TNM病理分期等临床数据后，并通过基因、蛋白、形态学的检测，观察TLR受体下游相关信号的表达分布、变化特点，以及TLRs信号因子MyD88和TRIF表达特点与肿瘤特性之间的相关性。同时，试图从体外细胞研究中，了解MyD88和TRIF对乳腺癌生物学特征的影响及分子机制进行探讨。方法1.临床资料经患者知情同意，随机收集乳腺癌改良根治手术且临床资料完整的患者标本，经病理确诊。所有患者术前均未接受放、化疗。均为女性，平均年龄52（38～72）岁，其中绝经前20例，绝经后15例。病理类型以乳腺浸润性导管癌为主，仅1例为粘液腺癌及1例髓样癌。依照国际乳腺癌TNM分期分为：Ⅰ期1例，Ⅱ期26例，Ⅲ期8例。组织学分类：早期浸润性导管癌1例，浸润性非特殊性癌32例，浸润性特殊性癌2例。2．免疫组织化学染色乳腺癌肿瘤组织及其癌旁正常组织切片，进行常规脱蜡、梯度脱水、抗原热修复后,3%过氧化氢以消除内源性过氧化物酶;滴加山羊血清封闭。甩干血清直接滴入兔抗MyD88和TRIF抗体，4℃孵育过夜。PBS清洗，滴加山羊抗兔二抗。37℃，40min，PBS清洗，加生物素卵白素复合物。显色、脱水、透明、封片。每张切片随机选取3-5个高倍视野(×400)采图，图像分析。3.免疫组化结果判定细胞阳性染色表现在细胞核或细胞质，颜色为棕黄色颗粒定为阳性信号。阳性结果判断：①阳性细胞的百分比：＜1%=0；1%～10%=1；＞10%，≤30%=2；＞30%，≤60%=3；>60%=4。②阳性细胞的染色强弱：阴性=0；淡黄色=1；黄色=2；棕黄色=3。①+②即为标本的免疫组化评分：0～2分（阴性），3～7分+（阳性，+++即强阳性）。4.提取总RNA以及RT-PCR检测按照Trizol试剂说明书和逆转录试剂盒说明书介绍的步骤提取组织总RNA，以及进行后继的反转录过程基因合成。产物用1.4%（含gold view）琼脂糖凝胶电泳和染色。使用紫外光凝胶照像，经Image软件进行灰度测定，实验重复3次。把MyD88和TRIF的灰度值与内参GAPDH灰度值的比值进行统计、分析。5. Western blot表达检测取出收集置-80℃保存备用的临床标本，使用蛋白裂解试剂进行裂解，然后进行蛋白浓度定量确定待测样品的浓度。在胶凝固后，加入上样组织，120V电泳。用PVDF膜进行转膜、加入兔抗MyD88和TRIF一抗，4℃过夜、二抗杂交后显色，拍照。6.细胞培养与转染选用人乳腺癌MCF7细胞，使用含10%新鲜胎牛血清的DMEM培养基，37℃和5%C02的条件培养。后采用MyD88和TRIF siRNA慢病毒转染肿瘤细胞，转染后观察细胞的生长状态并对转染后的细胞进行MyD88和TRIF基因和蛋白表达检测。7. MTT法检测细胞增殖分别将MyD88和TRIF慢病毒转染组后的细胞和对照组细胞悬液接种在96孔培养板里面。接着培养，根据要求在每孔加入MTT溶液，继续培养，然后加入DMSO，采用490nm波长测定，以等体积培养基作对照，根据结果绘制细胞生长曲线。8.细胞侵袭能力检测在transwell小室和下室中加入培养基，上室加入不包含血清的DMEM培养基稀释的Matrigel，再将转染后24小时的细胞接种到上室。37℃，5%CO2孵箱，继续培养一定时间,用枪吸尽培养基，再清洗，甲醇固定后进行苏木素染色，采图。9.统计学分析各组资料利用SPSS18.0进行统计分析。P<0.05有统计学意义。结果1. MyD88和TRIF在乳腺癌性组织（1.5445±0.1833）和乳腺癌性周围正常组织中的基因（1.1058±0.1167）表达显示，二者之间差异有统计学意义（P<0.01）。MyD88和TRIF蛋白在乳腺癌性组织（1.0042±0.0523）和乳腺癌性周围正常组织中的蛋白（0.7191±0.1102）表达显示，二者之间差异有统计学意义（P<0.01）。2.免疫组化法检测结果发现，MyD88和TRIF蛋白在35例乳腺癌组织全部阳性表达,表达阳性率为100%。MyD88和TRIF蛋白在乳腺癌组织则呈现明显强烈高表达，并且不同细胞类型差异性表达。ⅡA期乳腺癌组织癌细胞包括浆细胞均表达MyD88和TRIF蛋白；ⅡB期乳腺癌组织与炎性细胞强烈高表达MyD88和TRIF蛋白;ⅢA期乳腺癌组织中癌细胞及炎性细胞强烈高表达MyD88和TRIF蛋白。在乳腺癌周围正常组织中，MyD88和TRIF在乳腺癌周围正常组织中也有部分表达，部分细胞呈弱阳性表达，表达主要集中于炎性细胞、肌上皮及梭形的纤维母细胞等细胞中，导管上皮未见表达。3.乳腺癌患者中，45岁前后MyD88和TRIF的表达水平有差异，但无统计学意义（P>0.05），MyD88和TRIF的阳性表达与临床肿瘤大小、受侵淋巴细胞数目、临床分期呈正相关（P<0.05），包括临床Ⅲ期乳腺癌患者乳腺癌组织中MyD88和TRIF的表达明显高于Ⅰ、Ⅱ期（P<0.05）。4. MyD88和TRIF慢病毒siRNA基因与空白对照转染MCF-7细胞后,三者之间对其形态结构并没有明显的影响，对乳腺癌细胞的转染效率可以达到75%。基因表达结果显示,与空白质粒慢病毒转染组以及对照肿瘤细胞组（232.35士19.17和232.07士18.29）相比，MyD88和TRIF慢病毒siRNA基因转染的细胞组（77.38士11.57和128.48士13.51），有显著统计学意义（P<0.01）。蛋白印迹结果显示，与空白质粒慢病毒转染组以及对照组（0.986711士0.017和0.952425934士0.029）相比，MyD88和TRIF慢病毒siRNA基因转染的细胞组（0.651553士0.057和0.678224211士0.047），有显著统计学意义（P<0.01）。5.从转染前后的生长曲线中我们可以看出，与对照组细胞（吸光值0.16075士0.022）相比，TRIF和MyD88慢病毒siRNA基因转染两组转染0小时内（0.181士0.035；0.183士0.024），三者之间差别不大。在转染后24小时，三组之间有明显的差异，三组（0.3165士0.032；0.251士0.033；0.25475士0.061）之间但是并没有明显的统计学意义（P>0.05）。但是在后继的培养中，48小时，三组之间有明显的差异（0.5625士0.081；0.312士0.053；0.30275士0.042），具有统计学意义（P<0.05），说明转染siRNA干扰TRIF和MyD88基因后，与对照组相比较，人乳腺癌MCF7细胞生长能力明显下降。6.从干预影响结果来看，在未干预肿瘤细胞对照组细胞表现随着时间的推移，MCF7细胞不断向划痕的区域进行浸润与扩散。在MyD88和TRIF siRNA基因干预后0小时内，两者并没有明显区别。但是随着时间的推移，在12小时后，两者之间出现明显的差异。可以看出未干预组细胞表现明显的浸润与扩散，划痕间隙明显缩小，侵袭能力明显减弱，而MyD88和TRIF基因干预组并没有看出明显的浸润与扩散，划痕间隙明显缩扩散。说明MyD88和TRIF基因阻断后对于乳腺癌肿瘤的细胞有明显的迁移能力抑制或减轻作用。7.在该实验中发现，与未干预肿瘤细胞对照组细胞穿膜数（163.5士8.5）相比，MyD88和TRIF siRNA基因干预细胞穿膜数（32.9士4.1和30.9士7.8）有明显下降，二者之间差异有明显统计学意义（P<0.01）。说明MyD88和TRIF阻断后基因对于乳腺癌肿瘤的细胞有明显的抑制转移的能力。结论1.MyD88和TRIF蛋白在乳腺癌组织中均呈现高表达，其表达与乳腺癌肿瘤组织病理特征如临床肿瘤大小、受侵淋巴细胞数目、临床分期呈正相关。2.MyD88和TRIF蛋白在人乳腺癌MCF-7细胞中广泛表达。通过RNA干扰技术，沉默MyD88和TRIF基因，发现可以调控MyD88和TRIF蛋白的低表达，有着明显的显著抑制MCF-7细胞的侵袭和转移能力，在乳腺癌的发生、发展中起到重要的作用。更多还原

【Abstract】 BackgoundThe incidence of breast cancer has a sustainable and rapid growth inthe past20years in China. The annual growth rate is about3%to4%,much higher than the world average of0.5%. Breast cancer is still one ofthe important reasons in the world cause of death for women today. Clearevidence of more than90percent of breast cancer patients eventually diedof tumor metastasis or recurrence of breast cancer recurrence andmetastasis is the main factor affecting its efficacy. The reason for researchand looking for leads to tumor invasion and metastasis of tumor treatmentis imperative. It is very difficult to predict the occurrence of tumor distantmetastasis rate.Because of this, looking for new prognostic factors ofbreast cancer proliferation, migration movements and the transfer thatrelated to the molecular biological markers of positive significance, whichhelp to take more effective and timely treatment.Many studies have confirmed that inflammation and cancer areclosely related and persistent inflammation can induce the generation ofcancer. Cytokines and chemical factors in the increased vascular play a decisive role in the transfer and the downward adjustment of specificimmune stimulation of chronic inflammation leads to tumor releasefactors that directly promote its own growth, which constitutes aninflammatory microenvironment conducive to tumor the occurrence anddevelopment. Inflammatory stimuli, tumor cells can release a variety ofimmune factors, and after that, it is difficult to re-stimulate the tumor cellsor induce their apoptosis. In other words, the inflammation induced tumorcell resistance to apoptosis.The discovery of the Toll-like receptors （Toll like receptors, TLRs） isthe most important advances in the innate immune research in recent years.Usually TLRs is activated by agonist, must be start by the pathwaydownstream signal transduction of MyD88-dependent and-independentTRIF that the formation of the cascade reaction. Another study alsosuggested showing a strong relationship of TLRs with breast cancer.Breast cancer or any other cancer, the pathophysiological mechanisms ofTLRs-mediated invasion is not clear. TLRs expression levels that relativeto normal breast epithelial cells in breast cancer is increased. Somereacher found that the estrogen receptor negative correlation with TLRs inbreast cancer. The level of expression of TLRs plays an important role inthe clinical diagnosis of invasion and metastasis of breast cancer. Newideas can provide anti-tumor through the induction of high expression. Research purposesThis research is collecting clinical data, including patient age, tumorsize, lymph node metastasis, the TNM pathological type data, and by theslice, the detection of genes, proteins, and morphology, observed TLRreceptor expression of downstream signal changes, as well as theexpression pattern of change, understanding the relationship betweenclinical data and the innate immune downstream signals. Further studies toexplore the molecular mechanisms of MyD88and TRIF expression withbreast cancer biological characteristics,and explore if it is the the newtarget of molecular biology markers predictive of human breast cancermetastasis and molecular therapy points.Methods:1. Clinical dataInformed consent of patients, randomly collected from January2010to September201135cases of breast cancer, modified radical surgery andclinical data of patients with complete specimens, and confirmed bypathology. All patients had not received radiotherapy and chemotherapy.Were female, average age52（38⁷2） years of age, premenopausal20cases,15cases of post-menopausal.2. Immunohistochemical stainingSlice conventional dewaxing and hydration, sliced into citrate bufferantigens hot fixes, natural cooling to room temperature, dropping3%hydrogen peroxide, at room temperature incubated; dropping goat serum closed for20min. adding rabbit anti-MyD88and TRIF antibody4°Cincubation overnight. PBS washed dropping goat anti-rabbit secondaryantibody.37℃for40min, after the PBS wash, horseradish peroxidase（HRP） labeled biotin-avidin complex. Color, dehydration, transparent, andwere mounted after microscopic examination. Each slice was randomlyselected three high-power field （×400） image analysis.3Immunohistochemical results to determinePositive staining brown granules positive signal in the nucleus orcytoplasm. Immunohistochemical interpretation of positive results,according to the following two aspects:（1） the percentage of positive cells:<1%=0;1%¹0%=1;>10%≤30%=2;>30%≤60%3;>60%=4.②positive cells, strong or weak staining: negative=0; light yellow=1;yellow=2; brown=3.①+②is the specimen Immunohistochemicalscore:0to2-（negative）,3to7+（positive,++strongly positive）.4total RNA extraction and RT-PCRExtracted total RNA using Trizol kit （days root Corporation）according to the instructions. Reverse transcription reagent instructions.The PCR products were1.5%agarose gel electrophoresis, gold viewdyeing. The use of UV gel camera, gray determination by the Imagesoftware, the experiment was repeated three times. Last valve the grayvalue of GAPDH ratio between of MyD88and TRIF. 5. Western blot expression detectionRemove the collection of clinical specimens of the set stored at-80℃, the use of proteolytic cleavage reagent for cracking, then theprotein concentration to quantitatively determine the concentration of thesample. Glue, add on samples energized electrophoresis. Transfermembrane PVDF membrane by adding rabbit anti-MyD88and TRIF ananti-4°C overnight, after two anti-hybrid color cameras.6. Cell culture and transfectionDMEM medium containing10%fresh fetal calf serum, cultured at37°C,5%C02under the conditions of human breast cancer cell line wastransfected to dye siRNA MyD88and TRIF adenovirus, specifictransfection step by liposomes LipofectamineTM reagent instructions.Untransfected cells and transfected with empty vector cells served ascontrols.7. MTT assay cell proliferationAfter transfection of a single cell suspension was inoculated in96-Lculture plates, each well containing an expansion of cells cultured for24hours, each hole by adding MTT solution to continue to train for fourhours, adding of DMSO, were holes in the490nm wavelength absorptionvalues, control the volume of medium without cells.8. Cell invasion ability to detectRoom added to the media in transwell chambers, the upper chamberto join the pre-cooling of the Matrigel, serum-free DMEM, dilution, hours after transfection, cells were seeded into the upper chamber.37°C,5%CO₂, incubator, cultured for hours after the exhaustion of the medium, thePBS washing, fixed methanol at room temperature with a cotton swab towipe the microporous membrane surface of cells, hematoxylin, dried atroom temperature overnight. Remove the microporous membrane, andplacing the slides on the microscope.9statistical analysesEach set of data using the SPSS18.0for statistical analysis. P <0.05was considered statistically significant.Results1. Of MyD88and TRIF in the nature of breast cancer tissue （1.5445±0.1833） and in the surrounding normal tissue of the breast cancerresistance gene （1.1058±0.1167） expression showed that the differencesbetween the two was statistically significant （P <0.01）. MyD88and TRIFprotein expression in the nature of breast cancer tissue （1.0042±0.0523）and the surrounding normal tissue of the breast cancer resistance gene（0.7191±0.1102）, the difference between the two was statisticallysignificant （P <0.01）.2immunohistochemical assay results of MyD88and TRIF protein allpositive in35cases of breast cancer tissue expression, expression was100%. In the surrounding normal tissue in breast cancer, MyD88andTRIF in the surrounding normal tissue of breast cancer, some expression, some cells were weakly positive expression mainly in cells of theinflammatory cells, myoepithelial and spindle-shaped fibroblasts ductalepithelium did not express. of MyD88and TRIF protein showed stronghigh expression in breast cancer tissues and different cell types aredifferentially expressed. A stage II breast cancer tissue of cancer cells,including plasma cell expression of MyD88and TRIF protein; II B breastcancer organizations were strongly associated with inflammatory cellsstrongly express MyD88and TRIF protein; III A breast cancer tissue ofcancer cells and inflammatory cells strongly express high MyD88andTRIF protein.3. Breast cancer patients45years of age before and after the MyD88and TRIF expression levels are different, but no statistical significance（P>0.05） of MyD88and TRIF were correlated with clinical tumor size,invasion of the number of lymphocytes, clinical stage were positivelycorrelated （P <0.05）, including clinical stage Ⅲpatients with breastcancer breast cancer tissue of MyD88expression was significantly higherⅠ and Phase Ⅱ （P <0.05）.4MyD88and TRIF lentivirus siRNA gene and control transfectedMCF-7cells, among its morphology and had no significant effect,transfection efficiency can reach75%. Gene expression results showedthat:Slow virus with blank plasmid transfection group and the normalcontrol group （232.35±19.17and232.07±18.29） compared to cells of MyD88and TRIF lentivirus siRNA gene-transfected group （77.38士11.57and128.48士13.51）, a statistically significant significance （P<0.01）. Western blot results showed that transfection group and the normalcontrol group （0.986711±0.017and0.952425934±0.029） comparedwith blank plasmid lentiviral of MyD88and TRIF lentivirus siRNAgene-transfected cells （0.651553士0.057and0.678224211士0.047）There are statistically significant （P <0.01）.5.From the growth curve before and after transfection can be seen,the two groups compared with control cells （0.16075士0.022）, TRIF andMyD88lentivirus siRNA gene transfection transfection （0.181士0.035;0.183±0.024）, little difference between the three. After transfection24hours between the three groups, significant differences between thethree groups （0.3165±0.032;0.251士0.033;0.25475士0.061） but notstatistically significant （P>0.05）. But in the subsequent culture,48hoursbetween the three groups significant differences （0.5625士0.081;0.312士0.053;0.30275士0.042）, statistically significant （P <0.05）, the siRNAtransfected TRIF and MyD88gene, compared with the control group,human breast cancer MCF7cell growth was significantly decreased.6. From the results of the impact of interventions, in tumor cells withoutintervention control group, cell performance over time, MCF7cellscontinue to scratch the area for infiltration and proliferation.0hours afterthe intervention of siRNA of MyD88and TRIF genes, the two and there is no significant difference. However, there are different significantdifferences after12hours between the two groups. It can be seen notinterfere with the group of cells showed obvious infiltration and diffusion,the scratch gap has significantly narrowed the invasion capacity wassignificantly decreased, MyD88and TRIF genes in the intervention groupand did not see the obvious infiltration and proliferation, the scratches gapsignificantly shrink the spread. The MyD88and TRIF blocked the abilityto inhibit the transfer of genes for breast tumor cells.7. We apply the experimental methods of in vitro tanswell small room ofMyD88and TRIF genes in breast cancer cell line MCF-7metastaticability. Found in the experiment, compared to control cells and tumor cellsdid not interfere with the number of transmembrane （163.5±8.5） ofsiRNA of MyD88and TRIF genes intervene in cell-penetrating number（32.9±4.1;30.9士7.8） have decreased significantly, both The differencebetween the statistically significant （P <0.01）. Our results find that theMyD88and TRIF blocked the ability to inhibit the transfer of genes forbreast tumor cells.Conclusion1. MyD88and TRIF protein is highly expressed in breast cancer, itsexpression and pathological features of the tumor tissue is closelyrelatieve with the clinical feature of breast cancer such as clinical tumorsize, the number of invaded lymphocytes, clinical stage was positively correlated,etc..2. MyD88and TRIF protein is widely expressed in human breastcancer MCF-7cells, play an important role in the incidence of breastcancer development. Found that low expression regulation of MyD88andTRIF protein by RNA interference that silence MyD88and TRIF genes,significant inhibit of MCF-7cell invasion and metastasis.更多还原