p110δ突变失活上调血管MMPs表达并引发血管壁结构损伤

 目的:研究p110δD910A 基因突变失活 (p110δ-mutant)对动脉血管结构的影响及机制。方法: 选取8周龄雄性的野生型（WT）小鼠与p110δ-mutant小鼠（n=20;20~25 g）,取胸主动脉进行石蜡包埋, HE染色,采用免疫组化、免疫荧光以及real-time PCR等方法,研究p110δ突变对小鼠动脉血管形态变化的影响及机制。结果: 与WT相比,p110δ突变失活后小鼠主动脉血管壁变薄,管腔变大。 p110δ突变失活后主动脉血管壁形态紊乱,弹力纤维局部断裂,血管中内皮细胞凋亡。p110δ突变失活后主动脉血管α平滑肌肌动蛋白（α-SMA）表达量、胶原Ⅰ和胶原Ⅳ含量减少。 p110δ突变失活后主动脉血管中的基质金属蛋白酶-3（MMP-3）和MMP-9的mRNA水平明显升高。结论: PI3K活性在维持小鼠动脉血管的正常形态中起重要作用,p110δ突变失活后可能通过上调基质金属蛋白酶的表达促进血管壁结构的破坏。     

Pkhd1基因的缺失可促进ApcMin/+小鼠肠道肿瘤的发展演进

目的 确定人类常染色体隐性遗传性多囊肾病(ARPKD)致病基因Pkhd1在ApcMin/小鼠肠道肿瘤发生中的作用.方法 Pkhd1基因敲除小鼠模型(Pkhd1-/-)与肠道肿瘤模型ApcMin/+小鼠杂交后,比较不同月龄(1、3、6月)单一ApcMin/+小鼠与缺失Pkhd1的ApcMin/+小鼠(Pkhd1-/-;ApcMin)肠道肿瘤的数目及大小,并观察其肠道肿瘤组织病理的变化.结果 与单一的ApcMin/+小鼠相比,Pkhd1-/-;ApcMin/+小鼠在1月龄时,肠道肿瘤数目及大小均无显著差异.但该小鼠在3月龄和6月龄时肠道肿瘤数目及大小均显著增加(P=0.017、P=0.022).此外,在6月龄时,Pkhd1-/-;ApcMin/+小鼠病理表型有向恶性转化的明显趋势.结论 具有Pkhd1缺失的Apc Min/+小鼠与单一的ApcMin/小鼠相比能够显著促进肠道肿瘤的发生和发展,并具有促进其肿瘤恶性转化的趋势.     

APCMin/+结直肠癌癌前病变小鼠模型的生物学特性

目的 研究C57BL/6J-APCMin/+小鼠肠道腺瘤的生物学特性.方法 记录APCMin/+小鼠的繁育情况,通过统计不同周龄小鼠小肠及大肠腺瘤的数目和总体积,观察其肠道腺瘤的发生、发展规律;通过病理组织学切片,观察腺瘤的病理学变化;通过免疫组织化学染色,观察WNT信号通路相关基因的表达情况.结果 APCMin/+小鼠9周龄时肠道开始有腺瘤发生,24周龄时多数小鼠出现死亡.从9～24周龄,腺瘤体积持续增大,但小肠腺瘤数目不再增加.免疫组化染色显示腺瘤中β-catenin入核,启动WNT信号通路下游原癌基因cyclinD1活化.结论 APCMin/+小鼠是良好的结直肠癌前病变动物模型.     

The role of PSGL-1 in the growth of intestinal tumor in ApcMin/+ mice

目的 研究P-选凝素糖蛋白配体1(P-selectin glycoprotein ligand 1,PSGL-1)在ApcMin/ +小鼠肠道肿瘤中的作用.方法 PSGL-1基因缺失的基因工程小鼠和肠道肿瘤模型ApcMin/ +小鼠杂交后,统计ApcMin/ +小鼠与(ApcMin/ +;PSGL-1-/-)杂交小鼠小肠及大肠肿瘤的数目和总负荷,观测其肠道肿瘤的发生变化.结果 相比ApcMin/ +小鼠,(ApcMin/ +;PSGL-1-/-)杂交小鼠在18周龄时肠道肿瘤数目与总负荷有明显增加.结论 PSGL-1的缺失促进了ApcMin/ +小鼠肠道肿瘤的发生,PSGL-1在人类肠道肿瘤中可能发挥着抑制肿瘤形成的作用.     

胰岛β细胞Metrnl基因敲除小鼠的鉴定及初步表型分析

目的：基于Cre-LoxP重组酶系统，构建胰岛β细胞Metrnl基因敲除小鼠(Metrnl-KO)，为进一步探究Metrnl基因在糖尿病疾病中的发病机制提供动物模型。方法：将雌雄基因型均为Metrnl^floxp/+的小鼠合笼杂交，筛选出基因型为Metrnl^floxp/floxp的小鼠，将该基因型小鼠与胰岛β细胞特异性表达Cre重组酶(Ins-2)工具鼠进行杂交繁育，获得基因型为Metrnl^floxp/+;Cre+的小鼠；再将Metrnl^{floxp/+；Cre+}小鼠与Metrnl^floxp/floxp小鼠杂交获得Metrnl^{floxp/floxp;Cre+}小鼠，基因型为Metrnl^{floxp/floxp;Cre+}的小鼠即为目的鼠。采用RT-q PCR检测Metrnl和胰岛素(Ins-1和Ins-2)的m RNA水平；免疫组化和免疫荧光染色观察胰岛组织Metrnl和胰岛素的蛋白表达情况；记录小鼠体重，同时测定小鼠糖耐量和胰岛素耐量。结果：...     

P-选择素对ApcMin/+小鼠肠道肿瘤的作用

目的: 研究P-选择素(P-selectin)在Apc^Min/+小鼠肠道肿瘤中的作用。方法: 采用P-selectin 基因缺失的基因工程小鼠和肠道肿瘤模型Apc^Min/+小鼠杂交,计数Apc^Min/+小鼠与Apc^Min/+ P-selectin ^-/-杂交小鼠小肠及大肠肿瘤的数目,并测量其肿瘤体积,研究P-selectin对Apc^Min/+小鼠肠道肿瘤的作用。结果: 与Apc^Min/+小鼠相比,Apc^Min/+P-selectin^-/-杂交小鼠在9周龄时肠道肿瘤数目与总负荷明显减少。结论: P-selectin 缺失能够显著抑制Apc^Min/+小鼠肠道肿瘤的生长。     

48例广西结直肠癌p53基因突变分析

目的： 探讨广西地区结直肠癌p53基因突变范围。方法: 应用聚合酶链反应—单链构象多态技术和基因测序技术检测48例结直肠癌标本的p53基因,包括外显子2-11及外显子内含子剪切位点。结果: 31.25％(15/48)结直肠癌存在p53突变,共有13种突变类型,分别是：c.370T>G,c.524G>A,c.528C>G, c.529-546del18,c.536A>G,c.736A>G,c.743G>A,c.770-771del2,c.772G>T,c.814G>A, c.949delC, c.782+1G>A, 和c.919+1G>C。c.524G>A是最常见的突变类型,占18.75%（3/16）。60岁以上结直肠癌p53基因突变率是45.83%,明显高于60岁以下的16.27%(P<0.05)。直肠癌p53基因突变率是47.83%,明显高于结肠癌的16.00%(P<0.05)。结论: IARC TP 53突变数据库中（2006版本）未见c.370T>G突变,c.370T>G是个新突变。c.528C>G、c.949delC、c.782+1G>A 和c.919+1G>C突变在IARC TP 53结直肠癌突变数据库中（2006版本）未见报道,这些突变首次在结直肠癌中被发现。广西p53基因突变与其它地区有所不同。     

Gab2 在SHP-2酪氨酸磷酸酶激活突变所致小鼠髓系异常增殖中的作用

 目的：观察SHP-2信号通路中关键接头蛋白Gab2对SHP-2激活突变引发的小鼠髓系异常增殖是否具有调控作用。方法：用Gab2-/-和SHP-2D61G/+模型小鼠建立4种基因型(SHP-2+/+、Gab2-/-、SHP-2D61G/+和SHP-2D61G/+/Gab2-/-)小鼠,解剖分析其脾大小,外周血白细胞计数,流式细胞术检测外周血及骨髓髓系细胞表面标志分子Mac-1和Gr-1并计数Mac-1和Gr-1阳性髓系细胞比例,骨髓造血干/祖细胞集落形成实验检测小鼠造血干细胞或祖细胞对细胞因子反应性,Western blotting和免疫沉淀实验检测骨髓来源肥大细胞经IL-3刺激后磷酸化蛋白激酶B (p-Akt)和磷酸化胞外信号调节激酶(p-ERK)的活化水平,以及Gab2与SHP-2蛋白的结合情况。结果：敲除Gab2后显著减轻SHP-2激活突变导致的小鼠髓系增殖表型,主要表现在：脾指数减小,外周血白细胞减少,小鼠髓系来源的Mac-1和Gr-1阳性细胞比例降低。与SHP-2D61G/+小鼠相比,经IL-3刺激后,骨髓细胞的集落形成能力显著降低;骨髓来源肥大细胞内p-ERK和p-Akt表达明显下调,SHP-2D61G/+/Gab2-/-小鼠肥大细胞内无Gab2与SHP-2结合。结论：敲除Gab2可以明显减轻SHP-2D61G/+激活突变导致的小鼠髓系异常增殖,这种减轻作用可能与SHP-2无法与Gab2结合而导致下游信号途径ERK和Akt活化减弱有关。     

RER+结直肠癌组织转化生长因子βⅡ型受体的突变

目的　检测转化生长因子 βⅡ型受体 (TGF βRⅡ ) (A) 10 、TGF βRⅡ (GT) 3、TGF βRⅡ 452 /454、TGF βRⅡ 53 3、hMSH3 (A) 8、hMSH6(C) 8、Bax (G) 8、胰岛素样生长因子Ⅱ型受体 (IGFⅡR) (G) 8、IGFⅡR (CT) 3 等微卫星突变热点及TGF βRⅡ点突变在RER 结直肠癌中的突变率、突变发生在肿瘤进程的哪一阶段。方法　采用聚合酶链反应 单链长度多态性分析 (PCR SSLP)、微切割 PCR SSLP、PCR 单链构象多态性分析、克隆测序、免疫组织化学对 76例结直肠癌标本进行分析。结果　RER (replicationerrorpositive)肿瘤TGF βRⅡ (A) 10 突变率约为 3 / 9,突变可发生于重度不典型增生腺瘤。其余位点未见突变。RER 结直肠癌多见于男性 ,发病年龄较早 (P <0 0 5) ;肿瘤多位于结肠 (P <0 0 5)。结论　RER 结直肠癌多见于较年轻男性 ,好发于结肠 ,仅约三分之一病例存在TGF βRⅡ(A) 10 突变 ;TGF βRⅡ (A) 10 突变发生于重度不典型增生腺瘤 ,可能对RER 腺瘤进展为癌起重要作用。RER 结直肠癌在临床病例特征上与西方发达国家的病例存在差异 ,可能有不同的发病机制     

大肠埃希菌对结直肠癌小鼠免疫相关细胞因子表达的影响

目的研究大肠埃希菌对结直肠癌小鼠免疫相关细胞因子表达的影响并探讨其作用机制。方法运用AOM-DSS建立结直肠癌小鼠模型,标记为AOM-DSS组,大肠埃希菌与AOM-DSS共喂养小鼠,标记为AOM-DSS+Escherichia coli组,未做任何处理组标记为Control组;ELISA实验检测血清中炎性因子IFN-γ、IL-6、COX-2、iNOS的含量;Western blot实验检测组织中Vasohibin-1、p65蛋白表达。结果与Control组相比,AOM-DSS组、AOM-DSS+Escherichia coli组IFN-γ、IL-6、COX-2、iNOS含量均显著升高,Vasohibin-1蛋白表达量均显著升高,p65蛋白表达量均显著升高,小鼠体质量均显著降低(P0.05);与AOM-DSS组相比,AOM-DSS+Escherichia coli组IFN-γ、IL-6、COX-2、iNOS含量均显著降低,Vasohibin-1蛋白表达量显著降低,p65蛋白表达量显著降低,小鼠瘤质量显著升高(P0.05)。结论大肠埃希菌可抑制Vasohibin-1的表达,而后可抑制结直肠癌小鼠免疫相关细胞因子的表达,发挥抑癌作用,其机制与部分失活NF-κB有关,将可为结直肠癌的治疗提供实验依据。     

The modifier of Min 2 (Mom2) locus: embryonic lethality of a mutation in the Atp5a1 gene suggests a novel mechanism of polyp suppression

Inactivation of the APC gene is considered the initiating event in human colorectal cancer. Modifier genes that influence the penetrance of mutations in tumor-suppressor genes hold great potential for preventing the development of cancer. The mechanism by which modifier genes alter adenoma incidence can be readily studied in mice that inherit mutations in the Apc gene. We identified a new modifier locus of ApcMin-induced intestinal tumorigenesis called Modifier of Min 2 (Mom2). The polyp-resistant Mom2R phenotype resulted from a spontaneous mutation and linkage analysis localized Mom2 to distal chromosome 18. To obtain recombinant chromosomes for use in refining the Mom2 interval, we generated congenic DBA.B6 ApcMin/+, Mom2R/+ mice. An intercross revealed that Mom2R encodes a recessive embryonic lethal mutation. We devised an exclusion strategy for mapping the Mom2 locus using embryonic lethality as a method of selection. Expression and sequence analyses of candidate genes identified a duplication of four nucleotides within exon 3 of the alpha subunit of the ATP synthase (Atp5a1) gene. Tumor analyses revealed a novel mechanism of polyp suppression by Mom2R in Min mice. Furthermore, we show that more adenomas progress to carcinomas in Min mice that carry the Mom2R mutation. The absence of loss of heterozygosity (LOH) at the Apc locus, combined with the tendency of adenomas to progress to carcinomas, indicates that the sequence of events leading to tumors in ApcMin/+ Mom2R/+ mice is consistent with the features of human tumor initiation and progression.     

Tensin4 (TNS4) is upregulated by Wnt signalling in adenomas in multiple intestinal neoplasia (Min) mice

Apc^Min/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. Apc^Min/+ mice (n = 11) were sacrificed at approximately 120 days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n = 14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of β‐catenin activity with MSAB or its increase by transfection with a Flag β‐catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT‐qPCR analysis showed approximately 5‐fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, β‐catenin inhibition resulted in reduced TNS4 expression, and conversely, β‐catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the Apc^Min/+ mouse represents a good model to study this.     

Abnormal craniofacial growth and early mandibular osteoarthritis in mice harbouring a mutant type II collagen transgene

Skull morphology and histology in the heterozygous offspring of a transgenic founder mouse Del1, harbouring 6 copies of deletion mutation in Col2a1 gene, were compared with those in normal siblings. On visual observation and roentgenocephalometric examination the heads of heterozygous Del1 mice were smaller than normal. Histologically the sizes of cartilaginous structures of the cranial base were reduced. Severe defects were seen in the temporomandibular joint as progressive osteoarthritic lesions. These observations elucidate the relationship between the genotype and phenotype and demonstrate that heterozygous Del1 mice are a useful model for studies on a genetic disturbance where 'clinical' manifestations are not evident until adult age.     

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.     

Tumor suppression by miR-26 overrides potential oncogenic activity in intestinal tumorigenesis

Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apc^min/+ mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.     

PTEN loss and KRAS activation leads to the formation of serrated adenomas and metastatic carcinoma in the mouse intestine

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co‐occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal‐specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild‐type (WT) mice and mice with a predisposition to adenoma development (Apc^fl/+). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life‐span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk     

Spontaneous aberrant crypt foci in Apc1638N mice with a mutant Apc allele

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.