【作者】 陈辉；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 胃癌是消化道最常见的恶性肿瘤之一,其死亡率居各种恶性肿瘤之首位。近年来,随着分子生物学、免疫学、细胞生物学、病理学等高新技术的迅速发展,国内外对胃癌的基础和防治研究取得了长足进步。但这些研究大部分是在临床基础上对胃癌患者的回顾性研究,在时间和空间上存在许多局限性。如要验证各种可疑的致癌、促癌因素,阐明胃癌发生的发病机制,探讨抗癌药物的作用机制并对其进行筛选,则必须建立胃癌动物模型。人类疾病动物模型在医学发展中起着十分重要的作用。多年来一般采用诱变剂处理的方法来筛选建立肿瘤动物模型,但此方法诱变发生的不确定性、肿瘤产生的随机性、动物无法在整体水平产生生物学效应等缺点,使其应用受到一定的限制。而转基因动物模型则克服了以上缺点,能在动物整体水平观察所转入基因的生物学效应,探讨其在动物体内的组织特异性表达或调控过程,为癌基因作用机理、癌细胞发生、发展过程、基因表达调控等方面研究提供宝贵资料,并为建立人类疾病动物模型开创新的科研途径。转基因动物（transgenic animal）是用基因工程技术将外源基因通过生殖细胞或早期胚胎导入动物胚胎染色体基因组中,使之稳定整合并传递给后一代的动物。转基因技术是生物学领域最新重大进展之一,在医学、分子生物学、分子遗传学、畜牧兽医学等研究领域有无限广阔的前景。目前转基因技术大多采用受精卵细胞的显微基因注射技术或电转移技术,其操作包括外源基因重组载体的构建、受精卵细胞的采集、外源基因的显微注射、注射后受精卵（或胚胎）的移植、转基因小鼠的检测等步骤。国外已建立了人遗传病和癌基因的转基因动物模型,用于肿瘤和遗传学研究,但这方面的研究在我国尚属起步阶段。转基因动物模型研究中广泛采用的SV40（Simian Virus 40,SV40）属乳多空病毒科（papovaviridae）多瘤病毒属（polyomavirus）,在病毒DNA复制前编码2种转化蛋白,即大T抗原（large Tantigen,Tag）和小T抗原（small tantigen,tag）。Tag是一种磷酸化蛋白,在细胞转化中起决定性作用,为细胞转化所必需。tag是非磷酸化蛋白,对细胞转化并非必需,但可起加强作用。SV40基因编码的大T抗原已广泛应用于转基因动物肿瘤模型的建立。如Santarelli等将SV40T基因导入小鼠细胞构建了小鼠乳腺癌动物模型:Brister等将SV40T基因和含金属巯基基因的融合基因质粒导入小鼠生殖系产生的转基因小鼠成年后可发生脑脉络丛乳头状瘤,Hochman建立了眼部肿瘤转基因小鼠模型。胃壁细胞是胃底腺的主要细胞类型之一,能合成和分泌盐酸,从而刺激胃肠道内分泌细胞和胰液的分泌。H⁺/K⁺ATP酶（H⁺/K⁺ATPase）基因在胃壁细胞中特异性表达,和胃酸的合成与分泌有着直接关系。Gordon建立了在胃壁细胞中特异性表达人生长激素（human growth hormone,hGH）、内在因子（intrinsic factor,INF）的转基因动物模型,为研究胃黏膜上皮细胞的发生、演化过程奠定了基础。我们构建胃壁细胞H⁺/K⁺ATPaseβ亚基启动子驱动下的SV40T基因的真核表达载体,并将其导入小鼠受精卵细胞建立转基因动物模型。该模型动物将实现SV40T基因在胃壁细胞中的特异性表达,并诱发小鼠胃壁细胞增生,胃黏膜发生癌前病变、癌变等一系列病理性改变,从而为胃癌发生机理的研究、胃癌的诊断与治疗、胃癌特异性药物的筛选提供十分有用的实验动物模型。方法一、SV40T基因胃壁细胞特异性表达载体的构建1.扩增H⁺/K⁺ATPaseβ亚基启动子:采用酚-氯仿法从小鼠肝细胞中提取基因组DNA,PCR扩增H⁺/K⁺ATPaseβ亚基启动子,PCR产物命名为HK。2.pMT/HK的制备:将PCR产物纯化回收后与pMT18-T载体相连,转化感受态E.coli BH5α细胞,从转化平板上随机挑取8个单菌落,37℃摇菌过夜培养,取菌液提取质粒DNA,XbaⅠ、BamHⅠ双酶切鉴定出阳性克隆,命名为pMT/HK并进行测序鉴定。3.构建pcDNA3.1/HK质粒:将pMT/HK用XbaⅠ、BamHⅠ双酶切,琼脂糖凝胶电泳,切胶回收约1060bp的DNA片段。将回收产物与用XbaⅠ、BamHⅠ双酶切的pcDNA3.1（-）质粒连接,转化感受态DH5α细胞,从转化平板上挑单菌落,提取质粒DNA,限制性内切酶消化鉴定出阳性重组克隆,记作pcDNA3.1/HK。4.构建pcDNA3.1/HKSV质粒:将pLITAg（含SV40T基因片段）质粒用BamHⅠ酶切,琼脂糖凝胶电泳,切胶回收约2700bp的DNA片段,将回收产物与用BamHⅠ酶切的pcDNA3.1/HK连接,转化感受态细胞DH5α,从转化平板上挑取单菌落,37℃摇菌过夜培养,取菌液提取质粒DNA,酶切鉴定重组体,并测序鉴定SV40T基因插入的方向及DNA序列,插入方向正确的重组体记为pcDNA3.1/HKSV。5.构建pcDNA3.1/SV质粒:将pLITAg质粒用BamHⅠ酶切,琼脂糖凝胶电泳,切胶回收约2700bp的DNA片段,将回收产物与用BamHⅠ酶切的pcDNA3.1（-）连接,提取质粒DNA,酶切鉴定重组体,经测序鉴定插入方向正确的重组体记为pcDNA3.1/SV。二、特异性表达SV40T基因转基因小鼠的建立1.制备转基因用DNA:提纯pcDNA3.1/HKSV质粒DNA,用限制性内切酶XbaⅠ、KpnⅠ双酶切。反应产物用0.7%琼脂糖凝胶电泳,凝胶回收试剂盒回收3.7kbH⁺/K⁺ATPaseβ亚基启动子/SV40T基因片断,用于显微基因注射。2.准备结扎雄鼠、供体雌鼠、假孕雌鼠等实验动物,采集受精卵,进行受精卵雄原核的显微基因注射,将注射后形态良好的胚胎移植到假孕雌鼠输卵管中。三、特异性表达SV40T基因转基因小鼠的鉴定与繁殖1.PCR检测转基因阳性首建鼠与F1代仔鼠:剪取出生仔鼠尾尖,提取基因组DNA,PCR扩增鉴定转基因阳性首建鼠与F1代仔鼠。2.Southern blot检测F1代转基因阳性鼠:取PCR检测阳性F1代鼠的肝组织,提取基因组DNA,Southern blot鉴定转基因阳性F1代仔鼠。3.RT-PCR检测SV40T mRNA的表达:Trizol试剂盒提取68^#首建鼠、未转基因B6阴性鼠胃及转基因阳性鼠胃、食管、十二指肠、盲肠、空肠、肝脏、心脏、肺、肾、脾、骨骼肌等组织的RNA,进行逆转录PCR,检测SV40T mRNA的表达。4.免疫组化检测SV40T蛋白表达:取F1代阳性鼠与未转基因B6阴性鼠胃、食管、十二指肠、盲肠、小肠、肝脏、心脏、肺、肾、脾等组织进行石蜡包埋与常规切片,免疫组化检测SV40T抗原表达。四、特异性表达SV40T基因转基因小鼠的生物学特性1.HE染色观察转基因鼠胃粘膜形态学改变:取F1代阳性鼠与未转基因B6阴性鼠胃组织进行常规切片,HE染色后观察转基因鼠胃粘膜厚度与形态学改变。2.透射电镜观测胃粘膜细胞亚显微结构的变化:取未转基因B6鼠与转基因阳性鼠的胃组织,固定、脱水、包埋、聚合、切片、染色后透射电镜观测胃粘膜壁细胞亚显微结构的变化。3.小鼠胃液酸度值的测定:取未转基因B6鼠与阳性转基因鼠各8只,禁食24h后,采用幽门结扎法收集胃内容物,离心分离胃液上清液,检测胃液PH值。4.TUNEL实验检测细胞凋亡:取未转基因B6鼠与转基因阳性鼠的胃组织,常规操作制备石蜡切片,按TUNEL原位杂交试剂盒推荐方法进行细胞凋亡检测。5.统计学分析:应用SPSS12.0统计学软件进行统计处理,计量数据均用均数±标准差（（?）±s）表示,两组均数的比较采用t检验,多样本均数的比较采用方差分析,检验水准α=0.05。结果一、SV40T基因胃壁细胞特异性表达载体的构建1.pMT/HK质粒的构建:小鼠肝组织基因组DNA用引物P₁、P₂扩增得到H⁺/K⁺ATPaseβ亚基启动子,扩增产物经10g/L琼脂糖凝胶电泳可见约1060bp的DNA条带,与预期长度吻合。构建的pMT/HK质粒用XbaⅠ、BamHⅠ双酶切电泳,可见到约1.06kb与2.6kb的两条DNA条带,与预期结果一致。2.H⁺/K⁺ATPaseβ亚基启动子DNA测序:pMT/HK测序显示,PCR扩增的H⁺/K⁺ATPaseβ亚基启动子长度为1057bp。测序结果与lorenz发表序列经NCBI网站的Blast 2 sequences对比分析,发现-501bp与-634bp之间均存在碱基重复序列,并且不同小鼠扩增产物测序以及与文献序列之间比对发现存在1到2个重复序列的不同,推测此段序列可能为多态性位点。3.pcDNA3.1/HKSV质粒的鉴定:pcDNA3.1/HKSV用XbaⅠ、BamHⅠ双酶切电泳,可见到约1kb、2.7kb与5.4kb的三条DNA条带;用XbaⅠ、KpnⅠ双酶切电泳,可见到约3.7kb与5.4kb的两条DNA条带;用BamHⅠ单酶切电泳,可见到2.7kb与6.4kb的两条DNA条带;用EcoRⅠ单酶切,只见到约9.1kb的一条DNA条带,酶切电泳结果均与设计一致。4.pcDNA3.1/SV表达载体的鉴定:pcDNA3.1/HKSV用BamHⅠ单酶切电泳,可见到约2.7kb与6.4kb两条DNA条带;pcDNA3.1/SV用BamHⅠ单酶切电泳,可见到约2.7kb与5.4kb两条DNA条带;pcDNA3.1用BamHⅠ单酶切电泳,仅见到一条5.4kb的DNA条带,酶切电泳结果均与设计一致。5.SV40T基因测序结果:经DNA测序分析验证了pcDNA3.1/SV2是SV40T基因以正确方向插入而构建的重组质粒,且pcDNA3.1/HKSV质粒中SV40T基因测序结果与NCBI公布的NC₀01669序列完全一致。二、特异性表达SV40T基因转基因小鼠的建立1.显微注射DNA的回收纯化:XbaⅠ、KpnⅠ双酶切表达载体pcDNA3.1/HKSV,回收约3.7kb的DNA片段,溶于显微注射用TE缓冲液,将其终浓度稀释至3ng/μl。2.转基因小鼠的制备:共对150只小鼠进行超排卵,有96只小鼠合笼后产生阴栓,得到受精卵2500余枚,平均超排卵26枚/只（2500/96）。选取发育正常、原核清晰的受精卵548枚用于显微注射,短暂培养后取422枚健康完好受精卵移植16只受体鼠,生崽77只,移植成功率为18.2%。三、特异性表达SV40T基因转基因小鼠的鉴定与繁殖1.转基因小鼠的PCR检测:PCR检测77只G₀代小鼠,共有10只小鼠扩增出特异性条带,分别为2^#、4^#、8^#、16^#、24^#、51^#、57^#、61^#、68^#、73^#,且阳性对照条带明显,阴性对照无条带。转基因鼠的阳性率约为13.0%（10/77）。2.首建鼠的繁殖与F1、F2代仔鼠的PCR检测:将首建雌鼠与B6雄鼠1∶1、首建雄鼠与B6雌鼠1∶2合笼。发现除68^#不育外,其他9个品系共生出99只仔鼠。8^#品系所生的23只仔鼠PCR检测均为阴性,2^#、4^#、16^#、24^#、51^#、57^#、61^#、73^#所生的76只仔鼠检测到31只阳性鼠,阳性鼠比例为40.8%（31/76）。将F1代阳性鼠与B6鼠合笼,4^#、16^#、24^#、51^#、57^#、73^#品系共生出51只F2代仔鼠,PCR检测到27只阳性鼠,阳性鼠比例为52.9%（27/51）,F1、F2代雌雄鼠比例基本为1∶1。3.转基因小鼠的Southern blot检测:将4^#、16^#、24^#、57^#、73^#1～2只PCR检测阳性F1代转基因小鼠处死,取其肝脏提取基因组DNA,BamHⅠ过夜消化电泳检测。地高辛随机引物标记与检测试剂盒I标记SV40T基因PCR 618bp扩增产物为基因探针,与4^#、16^#、24^#、57^#、73^#PCR阳性小鼠的基因组DNA进行杂交,5个品系小鼠在2.7kb和3.8kb处均出现明显的杂交带,而未转基因B6鼠无。4.转基因小鼠的RT-PCR检测:分离处死的68^#不育鼠、F1代阳性鼠与未转基因鼠的胃、食管、肝、脾、肾、心脏、胰腺、肺、十二指肠、盲肠、空肠、骨骼肌、睾丸或卵巢等组织,提取总RNA进行RT-PCR与电泳分析。各组织均扩增出443bp的内参GAPDH条带,68^#不育鼠与F1代阳性鼠的胃组织扩增出272bp的SV40T mRNA转录条带,而68^#不育鼠与F1代阳性鼠的食管、肝、脾、肾、心脏、胰腺、肺、十二指肠、盲肠、空肠、骨骼肌、睾丸或卵巢及未转基因鼠胃组织均未扩增出SV40T特异性基因条带。5.转基因小鼠的免疫组化检测:F1代阳性鼠与未转基因B6鼠胃、食管、十二指肠、盲肠、小肠、肝脏、心脏、肺、肾、脾、脑等组织免疫组化显示,SV40T抗原仅在F1代转基因阳性鼠的胃组织中表达,而在转基因阳性鼠食管、十二指肠、盲肠、空肠、肝脏、心脏、肺、肾、脾、脑及未转基因B6鼠胃组织均不表达。四、特异性表达SV40T基因转基因小鼠的生物学特性1.转基因鼠胃组织形态学观察:转基因鼠的胃粘膜明显增厚,胃小凹与胃底腺拉长,胃底腺中成熟壁细胞数目减少,未成熟前体细胞数目显著增加。2.转基因鼠胃粘膜厚度的改变:出生后35d、60d、120d转基因阳性鼠相对于同龄未转基因鼠胃粘膜厚度明显增加,分别为393±59μm、495±68μm、715±79μm与243±47μm、300±62μm、315±56μm。3.电镜观测胃粘膜壁细胞亚显微结构的变化:转基因鼠的壁细胞相比于未转基因对照鼠体积缩小,线粒体数目减少,胞内微管泡系统不发达甚至缺如。4.小鼠胃液酸度测定:对未转基因阴性鼠与转基因阳性鼠的胃液酸度进行测定,其PH值分别为2.06±0.5与7.1±0.4,二者经统计学分析具有显著性差异（P＜0.01）。5.TUNEL实验检测细胞凋亡:正常B6对照鼠凋亡细胞多仅见于胃粘膜上皮的表面粘液细胞,而转基因鼠除表面粘液细胞发生凋亡外,在胃底腺处尚存在部分凋亡细胞。未转基因对照鼠与转基因阳性鼠胃粘膜总的细胞凋亡率分别为2%与15%,而在胃底腺处,未转基因对照鼠与转基因阳性鼠的细胞凋亡率分别为0.5%与12%,二者相比具有显著性差异（P＜0.01）。6.转基因鼠胃粘膜组织病理学改变:转基因阳性鼠均表现为腺上皮增生,有些品系转基因鼠尚出现萎缩性胃炎,局部囊性扩张,腺体数量减少,坏死和炎性渗出等病理学改变。7.不育首建鼠的检测:68^#雌鼠出生后发育明显落后于同龄其它首建鼠,并且不育。被毛稍稀疏,行动迟缓,腹部膨大。出生后150d将其处死解剖,发现胃体出现蕈伞形肿瘤,腹腔中尚存在直径约8mm的腹膜瘤。而该鼠食管、肝、脾、肾、心脏、胰腺、肺、十二指肠等组织未见明显病变。结论1.构建了胃壁细胞H⁺/K⁺ATPaseβ亚基启动子驱动下表达SV40T抗原基因的真核表达载体,完成了对H⁺/K⁺ ATPaseβ亚基启动子的测序分析,发现存在的重复序列多态性位点。2.建立了胃壁细胞中特异性表达SV40T基因的转基因小鼠,RT-PCR与免疫组化证实该基因在转基因鼠的胃粘膜特异表达,转基因鼠呈现胃粘膜增厚,胃底腺拉长,成熟壁细胞数目明显减少等形态学改变。3.转基因鼠壁细胞微管泡系统不发达,线粒体数目减少,胃酸分泌显著降低,细胞凋亡率增加。转基因鼠可发生萎缩性胃炎、局部囊性扩张等病理学改变。4.不育转基因首建鼠存在蕈伞状胃部肿瘤和腹腔肿瘤,为转基因胃癌动物模型的建系与胃癌发病机理的研究提供了十分有用的实验动物模型。更多还原

【Abstract】 Gastric cancer is one of the most familiar malignant cancers in the digestive tracts,and its mortality ratio is the primacy in all malignant cancers.The basic and preventive researches have made great progress recently following the rapid developments of molecular biology,immunology,cell biology and pathology.But the most of these researches come from clinical retrospections to the patients with gastric cancer,so there are much localization of the time and space.If we would like to verify the effects of carcinogens,illustrate the mechanisms of gastric carcinogenesis,and discuss the role of anti-tumor drugs,animal models of gastric carcinoma must be established.Human diseased animal models have been playing great roles in medical development.The carcinogens have been used to establish tumor animal models for many years,but its application has been limited because of its disadvantages,such as uncertainty of inducement,randomicity of carcinogenesis,and no biological effects in the whole level.Transgenic animal models are able to overcome these shortcomings mentioned above,the biological effects of transfered genes can be observed in animal whole level.The tissue specific expression and controlling process can be studied. The transgenic animal models have provided valuable data for the researches of functionary mechanisms of oncogene,carcinogenesis and developing process of cancer cells,gene expression and regulation.The transgenic models have initiated a new scientific research approach for animal models of human diseases. Transgenic animal is such an animal -that foreign gene has been injected into its germ cells or embryo,inserted in its chromosome genome and can be transfered to its offspring stably.Transgenic technique is one of the greatest progresses in biology field and is in possession of the infinite foregrounds in the researches of medicine, molecular biology,molecular genetics,animal and veterinary science.At the present time,the foreign genes are often transferred into zygotes by microinjection or electricity transfer.The operation includes the construction of recombinant,collection of zygotes,microinjection of foreign genes,transplantment of injected zygotes and detection of transgenic animals.Transgenic animal models of human genetic diseases and human oncogene have been built abroad,and they have been used in cancer and genetic research.But the study in this aspect in our country is still in initial stage.SV40 belongs to polyomavirus and papovaviridae,used widely in transgenic animal models.The two transfer proteins,large T antigen（Tag） and small t antigen （tag）,had been encoded before virus DNA replication.Tag was a phosphoprotein, which was indispensable to cell transfer and played a decisive role in this process,tag was a non-phosphoprotein and it wasn’t necessary to cell transfer,but it could enhance this action.SV40T gene had been used widely in establishment of transgenic tumour animal models.For example,Santarelli had transmitted SV40T gene in mouse cells and constructed mouse breast cancer models,Brister transferred SV40T gene and metallothionein gene fusion plasmid into mouse germ line to establish transgenic mice which produced brain venation papilloma,Hochman constructed eye tumor transgenic mouse models.The gastric parietal cells are the kind of main types in fundic gland.They can synthesize and secrete hydrochloric acid which stimulates the secretion of pancreatin and endocrine cells in gastric intestine tracts.H⁺/K⁺ATPase gene is expressed in gastric parietal cells specifically and connect to the synthesis and secretion of hydrochloric acid directly.Gordon had established transgenic animal models specifically expressing human growth hormone（hGH） and intrinsic factor（INF） in gastric parietal cells.This model had made foundation for the research of proliferation, evolvement process in gastric mucous cells.We had constructed the eukaryotic specific expression vector SV40T controlled by H⁺/K⁺ATPaseβunit promoter and injected it in mouse zygotes to establish transgenic mice.SV40T gene has been specifically expressed in gastric parietal cells.They would induce the hyperplasia of pre-parietal cells,and pathologic changes in gastric mucosa.These models would be very useful experimental animals for the researches on the molecular mechanisms of gastric carcinogenesis,diagnosis and therapy of gastric cancer,and special drug choice.MethodsPartⅠConstruction of specific expression vector of SV40 large T antigen in gastric parietal cell1.Amplification of H⁺/K⁺ATPaseβunit promoter.The genome DNA was extracted from mouse liver using hydroxybenzene-chloroform method. H⁺/K⁺ATPaseβunit promoter was amplified by polymerase chain reaction（PCR） and its product was named as HK.2.Construction of pMT/HK.The PCR product was purified and ligated to pMT18-T vector.The recombinants were transformed to competent cells DH5α.8 clones were picked from the transformed plate and incubated at 37℃overnight. Plasmid DNA was extracted from the bacteria.Positive recombinants were identified with XbaⅠand BamHⅠdigestion and sequenced.They were named as pMT/HK.3.Construction of pcDNA3.1/HK.1060bp DNA fragment was cleaved from pMT/HK with XbaⅠand BamHⅠrestricted enzyme digestion and connected to pcDNA3.1（-） which had also been digested with XbaⅠand BamHⅠ.The recombinants were transformed to competent cells and clones were picked from the transformed plate.Plasmid DNA was extracted and the positive recombinants were identified with restricted enzymes digestion.They were named as pcDNA3.1/HK.4.Construction of pcDNA3.1/HKSV.2700bp DNA fragment was cleaved from pLITAg（including SV40T gene） with BamHⅠrestricted enzymes digestion and ligated to pcDNA3.1/HK which had also been digested with BamHⅠ.The recombinants were transformed to competent cells and clones were picked from the transformed plate.Plasmid DNA was extracted and positive recombinants were identified with restricted enzymes digestion.The inserted orientation and sequence of SV40T gene were verified by sequencing.The correct orientation recombinants were named as pcDNA3.1/HKSV.5.Construction of pcDNA3.1/SV.2700bp DNA fragment was cleaved from pLITAg with BamHⅠrestricted enzymes digestion and ligated to pcDNA3.1（-） which had also been digested with BamHⅠ.Plasmid DNA was extracted and recombinants were identified by restricted enzymes digestion.The inserted orientation was verified by sequencing.The correct orientation recombinants were named as pcDNA3.1/SV.PartⅡEstablishment of transgenic mice expressing SV40T specifically1.Preparation of transgenic DNA.The plasmid DNA pcDNA3.1/HKSV was extracted and digested with XbaⅠand KpnⅠ.The reaction products were electrophoresed on 0.7%agarose gel.3.7kb H⁺/K⁺ ATPaseβpromoter/SV40T fragment was collected and purified by using agarose extraction kit for DNA microinjection.2.Preparation of ligated males,donative females,pseudopregnant females.The DNA fragments were injected into oocyte pronucleus.Injected eggs were transferred into the oviduct of pseodopregnent females using standard techniques.PartⅢIdentification and proliferation of transgenic mice expressing SV40T specifically1.Detection of transgenic positive founders and F1 offspring using PCR.The genome DNA was extracted from mouse tails.The transgenic founders and F1 positive mice were detected by PCR.2.Detection of F1 transgenic mice using Southern blot.The genome DNA was extracted from the livers of F1 PCR positive mice and detected by Southern blot.3.Detection of SV40T mRNA.The total RNA was extracted with Trizol reagents from the stomach,esophagus,liver,spleen,kidney,heart,pancreas,lung, duodenum,cecum,jejunum,skeletal muscle and testicle of 68^# founder mouse,F1 positive mice and non-transgenic B6 mice.The mRNA expression was measured by RT-PCR.4.Detection of SV40T antigen expression by immunohistochemistry.The tissue sections of stomach,esophagus,liver,spleen,kidney,heart,pancreas,lung, duodenum,cecum,jejunum,skeletal muscle and testicle of F1 positive mice and non-transgenic B6 mice were prepared.SV40T antigen expression was detected by immunohistochemistry.PartⅣThe biological characters of transgenic mice expressing SV40T specifically1.Gastric mucosa observation of transgenic mice.The tissue sections of transgenic mice and non-transgenic mice stomach were prepared and stained with HE.The changes of gastric mucosa morphology were observed and the thickness of gastric mucosa was measured.2.Observation of parietal cell’s ultrastructure using transmission electron microscopy The stomachs of transgenic mice and non-transgenic mice were fixed, dehydrated,embedded,polymerized,sliced up and stained.The parietal cell’s ultrastructure was observed using transmission electron microscopy.3.Measurement of gastric acidity.Eight wild-type and 8 transgenic mice were denied access to food 24h.Gastric fluid in the stomach was collected after polyrus ligation.The PH of the gastric fluid was measured after centrifugation.4.Detection of cell apoptosis by TUNE1 assays.Stomachs of wild-type and transgenic were embedded in paraffin and sections were prepared from the zymogenic zone.TUNEL assays were performed as described in the method recommended by the kit.5.Statistical analysis.The SPSS 12.0 statistical software was used for statistical analysis of the data.To numerical variable,the data were expressed with（?）±s, the t-test and ANOVA were used to compare these data.The level of significant isα=0.05. ResultsPartⅠConstruction of specific expression vector of SV40 large T antigen in gastric parietal cell1.The construction of pMT/HK.H⁺/K⁺ATPaseβunit promoter was obtained by PCR using the mouse liver genome DNA and P₁,P₂ primers.1060bp fragment could be seen in 10g/L agarose gel and its length fitted the anticipation,pMT/HK plasmid was digested with XbaⅠand BamHⅠ,two DNA fragments（1kb and 2.6kb） could be observed and their length accorded to prospective results.2.The sequencing of H⁺/K⁺ ATPaseβuint promoter.The sequencing of pMT/HK showed that the length of H⁺/K⁺ ATPaseβpromoter PCR products was 1057bp.The sequencing result was compared with the sequence reported by Robert Levenson using Blast 2 sequences software.Base repeated sequence existed at -501bp to -634bp,and there was 1 to 2 different repeated sequence among different mice or reported sequence,so this region might be polymorphism loci.3.Identification of pcDNA3.1/HKSV.When pcDNA3.1/HKSV was digested with XbaⅠand BamHⅠ,three DNA bands could be observed.Their length was 1 kb,2.7kb and 5.4kb respectively.When it was digested with XbaⅠand KpnⅠ,two DNA bands could be seen.One was 3.7kb fragment,the other was 5.4kb.When pcDNA3.1/HKSV was digested with BamHⅠ,2.7kb and 6.4kb DNA bands could be observed.If it was digested with EcoRⅠ,9.1kb DNA band could be detected.The results of restricted enzymes digestion accorded to anticipation.4.Identification of pcDNA3.1/SV,when pcDNA3.1/HKSV was digested with BamHⅠ,2.7kb and 6.4kb DNA bands could be observed.While pcDNA3.1/SV was digested with BamHⅠ,2.7kb and 5.4kb DNA bands could be seen.When pcDNA3.1 was digested with BamHⅠ,only one 5.4kb DNA band could be detected.All these results also accorded to design.5.Sequencing result of SV40T.pcDNA3.1/SV2 was verified to be the recombinant in which SV40T had inserted into pcDNA3.1 at the correct orientation.SV40T sequence in pcDNA3.1/HKSV was the same as NC₀01669 completely.PartⅡEstablishment of transgenic mice expressing SV40T specifically1.The purification of injection DNA.pcDNA3.1/HKSV was digested with XbaⅠand KpnⅠ.3.7kb DNA fragment was collected and purified with agarose gel extraction kit and dissolved in TE buffer for microinjection.Its concentration should be 3ng/μl.2.Establishment of transgenic mice.150 mice were superovulated and pessaries were observed in 96 mice.2500 zygotes were collected and the number of average superovulation was 26/mouse（2500/96）.548 zygotes in which pronucleus were clear were used for microinjection and 422 integral germ cells were transplanted to 16 psuedopregnant mice.77 offspring were born and the success ratio of transplantation was 18.2%.PartⅢIdentification and proliferation of transgenic mice expressing SV40T specifically1.Detection of transgenic mice using PCR.10 offspring were verified to be positive transgenic mice from 77 young mice by PCR.They were 2^#,4^#,8^#,16^#, 24^#,51^#,57^#,61^#,68^# and 73^#.The positive ratio of transgenic mice was 13.0% （10/77）.2.Reproduction of founder mice and detection of F1、F2 offspring.One founder female and one B6 male,or one founder male and two B6 females were put in one cage to reproduce.68^# female mouse was sterile,and 99 mice were born by other 9 pedigrees.None positive mouse was found in 23 offspring of 8^# pedigree.31 mice were proved to be positive by PCR in 2^#,4^#,16^#,24^#,51^#,57^#, 61^#,and 73^# offspring.The positive rate was 40.8%（31/76）.F1 offspring were mated with B6 mice to produce F2.There are 51 mice were born in 4^#、16^#、24^#、51^#、57^#、73^# pedigrees,and 27 mice were proved to be positive by PCR. The positive rate was 52.9%（27/51）.The proportion between male mice and female mice in F1 and F2 offspring was round about 1:1. 3.Southern blot result.1～2 F1 transgenic mice which had been measured in 4^#、16^#、24^#、57^#、73^# pedigree by PCR were sacrificed.Genome DNA was extracted from their livers and digested with BamHⅠovernight.SV40T 618bp PCR products were labeled with Gigoxingenin and hybridized with genome DNA of 4^#, 16^#,24^#,57^# and 73^#F1 mice.2.7kb and 3.8kb DNA hybridized bands could be observed in these pedigrees,but there was no band in non-transgenic mice.4.RT-PCR detection.The Stomach,esophagus,liver,spleen,kidney,heart, pancreas,lung,duodenum,jejunum,caecum,skeletal muscle and testicle were separated from 68^# sterile mouse,F1 transgenic mice and non-transgenic mice and stored at -80℃.The total RNA was extracted from these tissues and subjected to reserve transcription.443bp GAPDH bands could be amplified in every tissue. 272bp specific SV40T mRNA fragments were amplified in the stomach of 4^#、16^#、24^#、57^#、73^# pedigree’s offspring and 68^#,but not in non-transgenic mice’s stomachs.RT-PCR results of esophagus,duodenum,jejunum,caecum,liver,heart, lung,kidney,spleen,skeletal muscle,testicle from positive mice showed that there was no SV40T 272bp fragment in these tissues.5.Detection of immunohistochemistry.SV40T antigen only existed in the stomachs of F1 transgenic mice,but not in non-transgenic B6 mice stomachs or other tissues of transgenic mice,such as esophagus,duodenum,jejunum,caecum, liver,heart,lung,kidney,spleen,skeletal muscle,testicle etc.PartⅣThe biological characters of transgenic mice expressing SV40T specifically1.Stomach mucosa observation of transgenic mice.In transgenic mice,the thickness of gastric mucosa was increased,gastric pit and fundic gland were elongated.The number of mature parietal cells decreased and the number of progenitor cells increased greatly.2.The thickness change of gastric mucosa in transgenic mice.The Thickness of gastric mucosa in transgenic mice on postnatal day 35,60,120 was increased greatly than that of non-transgenic mice on same postnatal day.The thickness of gastric mucosa in P35,P60,P120 transgenic mice was 393±59μm、495±68μm、715±79μm respectively,while that was 243±47μm、300±62μm、315±56μm in non-transgenic mice.The thickness of gastric mucosa in several transgenic mice pedigrees had no significant difference.3.Observation of parietal cell’s ultrastructure using transmission electron microscopy. The volume of parietal cells in transgenic mice was smaller than that in non-transgenic mice.The number of mitochondria was decreased and tubulovesicular system was underdeveloped or scarce in transgenic mice.4.Measurement of gastric acidity.The PH of gastric juice from transgenic mice and non-transgenic mice was 2.06±0.5 and 7.1±0.4 respectively.There was significant difference between them（P＜0.01）.5.Detection of cell apoptosis by TUNE1 assays.The apoptotic cells in non-transgenic B6 mice were only observed in the surface mucous cells.But in transgenic mice,the apoptotic cells can be found in fundic glands excepting in the surface mucous cells.The total ratio of cell apoptosis in non-transgenic mice and transgenic mice was 2%and 15%respectively.The ratio of cell apoptosis in fundic glands was 0.5%and 12%respectively.There was significant difference between them（P＜0.01）.6.The changes of pathology in transgenic mice gastric mucosa.The gland epithelial cell in transgenic mice increased greatly.In some pedigree,atrophic gastritis,local vesicle expanse,necrosis,and inflammation exudation can be observed and the number of gland decreased.7.Observation of sterile founder mouse 68^# female founder grew slower than other founders and was sterile.Its hair was sparse,spirit was weary,abdomen enlarged.68^# was killed on 150 days old and polypoid carcinoma could be found in its stomach.Peritoneum carcinoma existed in its abdomen,which diameter was about 8mm.There were no significant changes in esophagus,duodenum,jejunum, caecum,liver,heart,lung,kidney,spleen etc. Conclusions1.The eukaryotic specific expression vector of SV40 large T antigen controlled by H⁺/K⁺ATPaseβsubunit promoter had been established.H⁺/K⁺ ATPaseβsubunit promoter was analyzed by sequencing and repeated polymorphism loci was found in this region.2.The transgenic mice expressing SV40T gene in gastric parietal cells had been established.The gastric specific expression of transgenic mice had been verified by RT-PCR and immunohistochemistry.The thickness of gastric mucosa in transgenic mice was increased,gastric pit and fundic gland elongated and the number of mature parietal cells decreased greatly.3.The ultrastructure of parietal cells in transgenic mice changed.The number of mitochondria decreased and tubulovesicular system was underdeveloped or scarce in transgenic mice.The secretion of gastric acid was decreased. SV40T-induced pre-parietal proliferation was accompanied by apoptosis. Atrophic gastritis,local vesicle expanse can be observed in some pedigree.4.Gastric polypoid tumour and peritoneum carcinoma had been found in transgenic sterile mouse.The transgenic mice would be useful animal models for the research of gastric cancer.更多还原