【作者】 于莉娜；

【导师】 丁彦青；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 研究背景和目的Tiam1是目前最受重视的Db1家族的鸟嘌呤核酸转换因子（guaninenucleotide exchange factor,GEFs）之一,GEFs主要功能是调节Rho家族活性。Rho家族包括Rho、Rac和Cdc42,是Ras超家族的成员,它们均参与细胞骨架形成,在调节细胞骨架重组、细胞周期进程、基因转录、细胞迁移和粘附等细胞生命活动中发挥重要作用。早期研究表明Tiam1是诱导人类T细胞淋巴瘤侵袭和转移的基因。Tiam1在体内外均可特异性地激活Rho GTPase家族中的Rac1。新近研究发现Tiam1还可以直接与胞浆和胞膜上的蛋白质相互作用,将它们耦合到Tiam1-Rac信号通路上,从而影响Rac信号通路的特异性。Tiam1基因最初在小鼠T淋巴瘤细胞高侵袭变异株中分离鉴定。随后,在T细胞淋巴瘤、B细胞淋巴瘤、胰腺癌、乳腺癌、膀胱癌和肺癌等肿瘤细胞中证实Tiam1具有明显的促进肿瘤进展和转移的作用,是多种肿瘤的促癌基因。目前,有关Tiam1的研究多基于体外细胞水平。由于体外和体内基因功能研究尚存在一定差异,体外基因功能研究往往不能够完全模拟基因在体作用形式和机制,因此,基因功能研究到一定阶段,大多会引入转基因（Transgene）或基因打靶（Gene targeting）等技术。建立表达或缺失特异目的基因的遗传工程小鼠（Genetically modified mice,GMMs）,整体动态观察目的基因功能,是研究人类疾病发病机制、解答特定人群对某种疾病的易感性、识别药靶、新药筛选和疗效判定等最为有效的手段。转基因技术就是将某种特异性外源基因导入到另一种不具备这种性状的生物内,并能使该生物表现插入基因特性的技术,是将外源基因导入动物体内的重要方法,以此方法得到的能正常繁衍的动物称为转基因动物（Transgenic animals）。Malliri曾应用在体RNA干扰技术建立了Tiam1沉默小鼠进行小鼠皮肤癌研究,结果显示Tiam1沉默小鼠皮肤癌的发生率明显降低,并且肿瘤进展缓慢,认为Tiam1在Ras诱导皮肤癌发生的启动和进展阶段发挥关键作用,这一效应与Tiaml表达量有显著相关性。目前尚没有Tiam1转基因动物模型。由于在正常组织中,Tiam1基因除了在正常脑、睾丸和皮肤中高表达外,在其他组织低表达或不表达,因此建立Tiam1转基因小鼠,能够为整体水平研究Tiam1基因功能和作用机制提供理想动物模型。建立转基因小鼠方法主要有四种,应用比较多的方法是受精卵原核显微注射技术和慢病毒感染技术。原核显微注射法为目前最经典的和最成熟制备转基因小鼠的方法,被广泛采用,但是显微注射制备转基因动物的效率很低。慢病毒是逆转录病毒的一种,可以感染分裂和非分裂细胞,并稳定整合到细胞的基因组内,因此,它是一个极好的基因导入工具。本研究所用的慢病毒载体是一个自我失活载体,携带一个GFP报告基因,即增强了载体的安全性,又提高了携带基因的检出率。本研究拟建立pCDF1-Tiam1-copGFP慢病毒表达载体,并转染内源性不表达Tiam1的结直肠癌HT29细胞株,细胞水平探讨Tiam1的生物学功能,同时验证所构建慢病毒载体的正确性;通过慢病毒卵周隙显微注射的转基因方法制备Tiam1转基因小鼠,并观察其阳性表型,检测增殖标志物Ki-67,探讨Tiam1在活体内整体水平的生物学功能,并为Tiam1研究提供动物模型。方法1.pCDF1-Tiam1-copGFP慢病毒表达载体的构建利用载体克隆技术构建pCDF1-Tiam1-copGFP慢病毒表达载体,应用酶切及测序方法进行鉴定,将慢病毒载体质粒与辅助质粒共转染293FT病毒包装细胞,收获Tiam1慢病毒上清。2.Tiam1表达对结直肠癌细胞生物学特性的影响利用慢病毒感染技术,将Tiam1导入内源性不表达Tiam1基因的人结直肠癌HT29细胞株中,应用RT-PCR、免疫组化及Western blot方法鉴定转染后Tiam1基因在细胞中的表达情况,获得稳定表达Tiam1的细胞株HT29/Tiam1;观察Tiam1基因转染前后,细胞形态以及增殖、运动、侵袭能力的改变。3.慢病毒载体法制备Tiam1转基因小鼠按标准程序准备受精卵供体母鼠、种公鼠、结扎公鼠和假孕母鼠;将超速离心浓缩后的慢病毒注射入ICR小鼠受精卵的卵周隙中,将注射后状态良好的受精卵移植进ICR假孕母鼠输卵管内,并将假孕母鼠送回笼内并观察,直至恢复知觉,仔鼠一般19.5～20.0d后出生。4.Tiam1转基因首建鼠的筛选与鉴定应用PCR、PCR产物测序等方法鉴定转基因小鼠目的基因的整合情况,以获得Tiam1转基因首建鼠。5.Tiam1转基因首建鼠繁殖传代及转基因遗传和表达稳定性检测将PCR阳性的首建鼠与野生型ICR鼠交配以传代,获得F₁后,用体视荧光显微镜检测GFP在F₁脏器中的表达及分布;利用免疫组化法检测GFP、Tiam1表达来进一步验证上述结果,对其表达稳定性做出判断,进而间接判断转基因是否稳定遗传。6.Tiam1转基因阳性表型的检测对Tiam1转基因小鼠各脏器组织进行包埋、制片,进行病理形态学观察,应用免疫组化检测和分析转基因小鼠的增殖标志物Ki-67表达情况。7.统计学方法用SPSS 13.0软件进行数据分析,细胞周期、平板克隆形成实验、迁移实验和侵袭实验采用两样本t检验,体外增殖实验和皮下成瘤实验采用重复测量方差分析。结果1.所构建慢病毒表达载体pCDF1-Tiam1-copGFP,经EcorI酶切鉴定,可见6771bp载体和3800bpTiam1片段;目的片段测序结果与NCBI网站上公布的Tiam1序列（NM₀03253）进行Blast比对一致;将慢病毒载体质粒与辅助质粒共转染293FT病毒包装细胞,48h后倒置荧光显微镜下可见绿色荧光,预示病毒包装成功。2.用收集的病毒上清感染内源性不表达Tiam1的结直肠癌细胞株HT29,48h后倒置荧光显微镜下可观察到绿色荧光,通过有限稀释法获得表达Tiam1的稳定克隆HT29/Tiam1。HE染色可见,HT29/Tiam1较HT29/mock细胞排列更为弥散,腺管样结构减少,细胞异型性更为明显;细胞骨架染色及细胞扫描电镜可见,HT29/Tiam1部分细胞由圆形、卵圆形向梭形转变,细胞表面的伪足增多且更加宽厚,并出现纤长的伪足。流式细胞术分析细胞周期,发现HT29/Tiam1及HT29/mock细胞的S期细胞平均比例分别为43.0%和31.4%,差异有统计学意义（t=19.546,P=0.000）。MTT法观察表达Tiam1基因后细胞的增殖情况,HT29/mock细胞相比,HT29/Tiam1细胞的增殖能力增强,有统计学差异（F=177.125,P=0.000）。平板克隆形成实验显示HT29/Tiam1细胞的活力显著下降,差异有统计学意义（t=3.222,P=0.032）。接种HT29/Tiam1细胞的裸鼠皮下肿瘤生长快于接种HT29/mock细胞的裸鼠,有统计学差异（F=53.040,P=0.002）。这些结果均说明Tiam1表达后能够促进细胞体外增殖。运动小室实验证明,HT29/Tiam1和HT29/mock细胞的运动能力是HT29/mock细胞的1.5倍,差异具有统计学意义（t=4.832,P=0.001）,说明Tiam1与细胞的运动迁移能力有关。体外侵袭实验证明,肿瘤细胞侵袭实验24h后,HT29/Tiam1和HT29/mock细胞均穿透基底膜向下侵袭,在聚碳酸酯膜上可见变形的肿瘤细胞,HT29/Tiam1细胞侵袭能力是HT29/mock细胞的1.4倍,差异具有显著性（t=3.779,P=0.005）,说明Tiam1基因的表达增强了结直肠癌细胞的侵袭能力。3.慢病毒注射入628枚单细胞受精卵的卵周隙,存活胚胎556枚,共移植入33只假孕鼠,其中21只回种假孕鼠怀孕,受孕率为64%（21/33）,共获仔鼠76只,产仔率为14%（76/556）。PCR和PCR产物测序证明获得Tiam1阳性首建鼠5只,首建鼠Tiam1整合率为7%;4.体视荧光显微镜和冰冻切片的荧光观察发现,F₁代的Tiam1转基因小鼠的结直肠、胃、肺、肾和睾丸五个脏器可见绿色荧光;免疫组化显示GFP表达与Tiam1转基因定位完全一致。提示外源基因不仅可以从一代向下一代稳定传递,且能够稳定表达。5.F₁代转基因小鼠和对照小鼠的各脏器组织标本的形态学观察比较显示,Tiam1过表达的五个脏器（结直肠、胃、肺、肾和睾丸）表现出显著的增殖性改变:结直肠和胃腺上皮细胞增生,腺体排列密集、粘膜层增厚;肺支气管上皮由假复层纤毛柱状上皮转变为复层纤毛柱状上皮,细胞密集;肾小管上皮细胞增生,细胞排列紧密;睾丸初级精母细胞显著增生,充满曲细精管管腔,成熟精子数量减少。增殖标志物Ki-67表达定位、强度与增殖性形态学改变一致。结论1.成功构建慢病毒表达载体pCDF1-Tiam1-copGFP,并获得携带Tiam1和GFP融合基因的重组慢病毒,为Tiam1基因相关功能研究提供了优质的稳定转染载体;2.通过慢病毒感染人结直肠癌HT29细胞株,在细胞水平证明Tiam1是细胞增殖、运动和侵袭的促进基因;3.运用慢病毒载体法成功建立Tiam1转基因小鼠,并稳定传代,为Tiam1体内研究提供理想的可视化动物模型。4.Tiam1转基因鼠的多个脏器（结直肠、胃、肺、肾和睾丸）Tiam1显著过表达,相应脏器细胞增殖活性显著增强,其中结直肠增殖性改变尤为明显,与体外细胞研究结果一致,表明Tiam1过表达可能与细胞增殖有关,是促进细胞增殖的基因之一。本研究的创新之处1.首次成功建立可稳定传代的Tiam1转基因小鼠,实现Tiam1基因的非损伤和可视化体内示踪;2.首次利用Tiam1转基因小鼠,在活体状态下整体水平证明Tiam1能够促进小鼠多脏器细胞增殖,表明Tiam1过表达可能与细胞增殖有关,为细胞分裂增殖机制的研究提供了重要靶点。更多还原

【Abstract】 BACKGROUND & OBJECTIVETiam1 is one of guanine nucleotide exchange factors（GEFs）,which activate GTPases by promoting the exchange of the GDP-bound forms from inactive to active. Although Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro,it specifically activates Rac in vivo.Recent evidence suggests that Tiam1 could also influence Rac GTPases signaling specificity by promoting their activation.Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins,which couples Tiam1-Rac activity to specific signaling pathways.Tiam1 was originally identified as the invasion-and metastasis-inducing gene in T lymphoma cells.The role of Tiam1 in cellular migration,invasion and metastasis may not be limited to T lymphoma.It was reported that Tiam1 plays an important role in promoting the tumor progression in a variety of cancers such as breast cancer,lung cancer and Ras-induced skin tumors.However,the possible role of Tiam1 in the metastasis of colorectal cancer is still not very clear.The transgenic animal is the one that carries a foreign gene which has been deliberately inserted into its genome.The transgenic animal could express exogenous genes and make it an indispensable tool for modern biologists.Transgenic mice are currently generated by pronuclear injection;another approach is the use of retroviruses as gene delivery vehicles.Retroviruses are able to stably integrate into the genome of cells.Lentiviruses are a class of retroviruses which can cause host organisms chronic illnesses.Among retroviruses,lentiviruses have the distinguished property of being able to infect both split-phase cells and non-split-phase cells.The lentiviral vector used in these experiments is a self-inactivating vector,which carry an internal promoter driving the GFP reporter gene.The lentiviral vector we used not only promoted the safety of the vector,but also increased the detection ratio of the exogenous gene it carried.After established the pCDF1-Tiam1-copGFP lentivirus vector,we successfully transfected the HT29 colorectal cancer cell line which do not express Tiam1,also we investigated Tiam1 biological functions in vitro.Meanwhile,by means of lentivirus-mediated perivitelline space microinjection of Tiam1 gene into zygotes,we generated the Tiam1 transgenic mice.Based on the Tiam1 transgenic mice model, this study will further investigate the functions of Tiam1 gene in vivo.METHODS1.Construction of lentiviral vector pCDF1-Tiam1-copGFPLentiviral vector pCDF1-Tiam1-copGFP was constructed by gene cloning.The recombinant plasmid of pCDF1-Tiam1-copGFP was identified by restriction endonuclease analysis and DNA sequencing.Recombinant lentivirus was harvested from 293FT cells cotransfected with the pPACKF1 Packaging Plasmid Mix.2.Effect of Tiam1 gene on the biological behaviors of human CRCTiam1/C1199HA cDNA was transfected into HT29.The expression level of Tiam1 gene was determined by RT-PCR,immunohistochemistry and Western blot. The biological behaviors of transfected HT29 were investigated by flow cytometry, MTT assay and plate colony formation assay in vitro.Cell migration and invasion were assessed by Transwell chamber and Boydon chamber. 3.Generation of Tiam1 transgenic mice by lentivirus-mediated gene transferLentiviruses harboring Tiam1 were subzonally injected into single-cell fertilized eggs of ICR mice,and subsequently embryos were transplanted into the pseudopregnant mice to attain F₀ mice.4.Screening Tiam1 transgenic mice（Founder,F₀）PCR,sequence analysis and Southern Blot were used to explore the Tiam1 gene combination in transgenic mice.5.Mouse propagation and transgene transmissionF₁ generation was produced after Tiam1 transgenic mice mated with wild-type ICR mouse.Detect GFP expression（F₁）under fluorescent stereo microscope. Immunohistochemistry analysis were used to explore the expressions of Tiam1 and GFP in F₁ mice,confirming that Tiam1 and GFP transgene actually integrated into genome of transgenic mice and could be transmitted to the next generation.6.Detection of positive phenotype of Tiam1 transgenic micePathological morphology of transgenic mice was detected.Corresponded cell proliferation activities were analysised by immunohistochemistry.7.Statistical analysisStatistical analysis was done using SPSS 13.0.Cell cycle,plate colony formation efficiency assay,cell migration and invasion assay were analyzed by two sample t-test. In vitro proliferation assay and subcutaneous tumor growth assay were analyzed by repeated measure square analyzeRESULTS1.Recombinant pCDF1-Tiam1-copGFP lentivirus vector was constructed and identified by restriction endonuclease analysis and DNA sequencing.6771bp vector segment and 3800bpTiam1 were got after EcoRI digested.After 48 hours of transferring pCDF1-Tiam1-copGFP and packaging plasmid into 293FT cell,the green fluorescence could be observed in most of the transferring cells.2.Single clone HT29 cells infected with recombinant lentivirus were selected and observed in inversion fluorescence microscope after 48 hours.HT29/Tiam1 cells showed a significantly enhanced proliferation features compared with the HT29/mock cells as determined by in vitro MTT assay.In addition,HT29/Tiam1 cells had a significant promotion in their ability to form colonies in plate compared with HT29/mock cells.Next,the effect of Tiam1 on tumor growth was assessed by subcutaneous injection of HT29/Tiam1 and HT29/mock cells for thirty days in vivo. Compared with HT29/mock cells,the expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 15,up to 2.3-fold increase in mice of tumor volume at day 30 after cell injection.The results of migration and invasion assays showed that the HT29/Tiam1 cells posses a remarkable increasement in migration and invasiveness compared with HT29/mock cells in vitro.3.Transgenic mice were generated by subzonal injection of lentivirus harboring Tiam1 gene into single-cell fertilized eggs of ICR mice.We transplanted 588 virus-injected eggs into 33 pseudopregnant mice and attained 76 F0 mice（potential transgenic founders）.Founder mice were selected from 76 potential transgenic founders（F0）by PCR assay,sequence analysis and Southern Blot.A total of 5 PCR-positive founders were attained,all of them transmitted Tiam1 gene to their offspring.The ratio of founders to born mice was 7%.4.Green fluorescence was detected in tissue samples.Those tissues including colon, stomach,lung,kidney and testis,which isolated from the Tiam1 transgene-positive mice but not from wild-type mice.Immunohistochemistry showed that the localization of GFP is consistent with that of Tiam1.Tiam1 can stablely transfer from one generation to the next generation. 5.Comparing to the control mice,significantly over-expressed Tiam1 was detected in multiple organs（colon,stomach,lung,kidney and testis）of Tiam1 transgenic mice. Correspondingly,cell proliferation activity was significantly enhanced in those organs,especially in the colon.CONCLUSION1.Successfully constructed lentiviral expression vector pCDF1-Tiam1-copGFP;2.Tiam1 gene plays an important role in proliferation,metastasis and invasion of CRC in vitro;3.Tiam1 transgenic mice were established by lentivirus-mediated gene transfer. Tiam1 gene could be transmitted from founders to subsequent generation;4.Significantly over-expressed Tiam1 was detected in multiple organs（colon, stomach,lung,kidney and testis）of Tiam1 transgenic mice.Correspondingly,cell proliferation activity was significantly enhanced in those organs,especially in the colon.The results of these experiments in vivo are consistent with that in vitro, which we have mentioned before.更多还原