Src基因研究进展

Src是非受体蛋白酪氨酸激酶家族成员之一,属原癌基因,Src表达及活性异常往往引起乳腺癌、结肠癌、胰腺癌、前列腺癌、肺癌等某些肿瘤发生、发展.最近研究表明Src基因与乳腺的提前退化及泌乳失败相关.文从Src基因的定位、蛋白质结构、分布及功能等几方面进行简要的介绍.     

牛TNS1基因的克隆和生物信息学分析

为了克隆牛TNS1基因序列,试验以人TNS1基因序列为探针,通过NCBI在线网站BLAST获得同源性较高的牛表达序列标签(ESTs)及部分mRNA序列,采用RT-PCR方法从牛肌肉组织克隆cDNA序列并与牛ESTs和mRNA序列进行拼接,获得牛TNS1基因序列。结果表明:克隆获得的牛TNS1基因含有32个外显子和31个内含子,编码区长度为5 148 bp,编码1 715个氨基酸。该蛋白含有149个磷酸化位点,分别位于丝氨酸(Ser)、酪氨酸(Tyr)和苏氨酸(Thr)残基上,二级结构以无规则卷曲为主,含有PTEN_C2、Src同源结构域2(SH2)和磷酸化酪氨酸结合区域(PTB)3个功能结构域。牛TNS1蛋白线粒体靶向肽及分泌通路信号肽所占的比例很小,大部分分布于微体和细胞核内,不属于分泌蛋白。     

催乳素受体的研究进展

介绍催乳素受体的结构、功能,催乳素受体基因的定位、表达及调节,催乳素受体基因的多态性与生产性状和疾病的关系等。提示催乳素受体基因可作为生产性状及疾病的一个候选基因。     

催乳素受体的研究进展

介绍催乳素受体的结构、功能,催乳素受体基因的定位、表达及调节,催乳素受体基因的多态性与生产性状和疾病的关系等.提示催乳素受体基因可作为生产性状及疾病的一个候选基因.     

影响猪瘦肉率遗传因素的研究进展

本文综述了影响猪瘦肉率的遗传方面的因素,包括品种(系),主效基因(氟烷基因,肥胖基因及其受体基因、胰岛素生长因子Ⅱ基因、肌肉生长和抑制素基因、脂肪酸结合蛋白基因及其它一些基因如肌浆蛋白基因、垂体特殊转录因子基因等)性别以及其它有关遗传因素。     

动物基因免疫研究及其应用

本文对基因免疫机制与方法、基因免疫安全性及效率等方面进行综述,并对基因免疫应用的有关问题进行了讨论。     

哺乳动物早期胚胎中的基因表达

本文总结最新的相关研究资料,对影响早期胚胎发育中的基因表达进行综述,主要包括原癌基因、Xist基因、性别决定基因、生长因子、生长发育及凋亡调节基因、白血病抑制因子等。     

哺乳动物性别决定相关基因研究进展

性别控制技术在畜牧生产中具有很强的应用性,其基础主要来源于性别决定。哺乳动物性别决定是一个复杂的过程,其主要以Sry基因为主效基因,同时,还受其他基因级联作用调控。主要性别决定的基因有Sf1基因、Wt1基因、Wnt4基因、Dax1基因等,对哺乳动物性别决定机制及其相关基因研究进展进行综述,以期为哺乳动物性别决定相关基因及分子机理研究提供参考。     

家蚕细胞周期素家族基因的克隆及结构特征与表达分析

细胞周期素(cyclin)是推进细胞周期进程最主要的调控因子之一,并与个体发育及肿瘤形成等有关。为研究细胞周期素家族基因在家蚕组织中的表达情况,利用家蚕基因组数据库信息设计引物克隆了家蚕细胞周期素家族的5个基因,对基因序列结构的分析结果表明,cyclinA、cyclinB、cyclinB3基因分别有7个外显子,cyclinE基因有8个外显子,cyclinL1基因只有1个外显子;cyclinA及cyclinE基因存在较多的剪切方式,cyclinA主要包含大小为2 141和2 173 bp的2种转录本,其变化在第7外显子,cyclinE则含有1 422、1 290及1 194 bp等大小不同的序列,变化集中于第5～7外显子,而cyclinB、cyclinB3及cyclinL1基因则相对稳定。克隆的家蚕细胞周期素家族基因的组织表达存在差异:cyclinA基因只在精巢中表达,cyclinB3基因只在精巢和卵巢中有明显的表达,cyclinE基因在丝腺、脑、脂肪体、中肠、马氏管、精巢、卵巢及血液8种组织都有表达,cyclinB和cy-clinL1基因在除丝腺或血液外的其他7种组织检测到表达。研究结果为进一步探究不同细胞周期素在昆虫蜕皮、变态等生理过程中的功能提供了参考。     

铬对基因表达的影响研究

铬是人和动物必需的微量元素之一,在机体许多生理机能中起着重要作用。本文主要论述了铬的营养功能及对最近国内外有关铬对糖代谢基因、脂肪代谢基因、蛋白质代谢基因、核酸代谢基因及能量代谢基因等几个方面的相关研究进行综述,旨在为研究铬对各种基因的表达提供理论依据。     

Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches

Heat shock proteins have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study was to investigate heat shock protein expression in correlation with other neoplasm traits such as: histological type, differentiation grade, proliferative activity, estrogenic receptor expression, and cyclooxygenase-2 and p53 proteins. Material for the investigation comprised 133 tumors of the mammary gland collected from bitches. In total 14 adenomas, 66 complex carcinomas, 47 simple carcinomas and 6 solid carcinomas were collected. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Expression of heat shock protein 70 was observed in all types of evaluated neoplasms. A higher average number of cells undergoing expression of heat shock protein 70, which was statistically insignificant, was established in complex and simple cancers and in cancers with the 1st and the 2nd degree of histological malignancy. Expression of heat shock protein 90 was observed in all studied neoplasms; it was very insignificant in adenomas, compared to cancers, and the highest expression was established in the solid cancers, as well as in cancers with the 2nd degree of histological malignancy. This high expression of heat shock protein 90 was correlated with proliferative activity. The results suggest that heat shock protein 90 is involved in canine mammary gland carcinogenesis. The results also suggest that heat shock protein 90 may be a prognostic factor, but this requires detailed clinical confirmation.     

A novel small molecule Met inhibitor, PF2362376, exhibits biological activity against osteosarcoma

The receptor tyrosine kinase Met is dysregulated in several human cancers including osteosarcoma (OSA) in which overexpression is a negative prognostic indicator and enforced Met expression in normal osteoblasts leads to genomic instability and malignant transformation. Met is also known to be inappropriately expressed in canine OSA tumour samples and cell lines. The purpose of this study was to evaluate the potential utility of an orally bioavailable small molecule Met inhibitor, PF2362376, against canine OSA cell lines as a prelude to future clinical work. PF2362376 inhibited phosphorylation of Met, Gab‐1, Erk and Akt, but not of Src or STAT3. Furthermore, PF2362376 inhibited proliferation of canine OSA cell lines and induced cell death at biologically achievable concentrations. Last, activities associated with Met signalling including migration, invasion, branching morphogenesis and colony formation in soft agar were blocked by PF2362376. These studies support the notion that Met is a relevant target for therapeutic intervention in OSA.     

Detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway –p53 gene mutation or MDM2 overexpression

In human and canine cancers, the inactivation of p53 protein as well as p53 gene mutation and MDM2 overexpression result in centrosome amplification that in turn contributes to chromosomal instability. To explore the usefulness of the detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway, we systematically analysed centrosome amplification, p53 overexpression, p53 gene mutation and MDM2 overexpression in canine tumours. Centrosome amplification was detected in 16 of 51 (31%) naturally developing tumours in dogs. All the tumour specimens with aberrations in the p53 pathway, including p53 overexpression, p53 gene mutation or MDM2 overexpression, showed centrosome amplification, suggesting that the detection of centrosome amplification could serve as a preliminary surrogate marker of dysfunction in the p53 pathway.     

p53 expression in canine lymphoma

The tumour suppressor p53 plays a key role in DNA damage and repair. It is the most frequently altered gene in human cancers and these mutations may implicate the genesis and/or progression of tumours. Mutations of the p53 gene were also found in a number of canine cancers, although it is poorly estimated in canine lymphomas. Thus, the aim of this study was to investigate the p53 status in these types of tumours. We have shown that the expression of p53 in canine lymphomas is rare, however significantly differs between lymphomas of T- and B-cell origin.     

CRISPR/dCas9技术在基因表达调控中的研究进展

CRISPR/dCas9是在CRISPR/Cas9基因编辑系统的基础上改造升级建立起的一种用于调控基因组转录与表观遗传修饰的系统,它不仅继承了CRISPR/Cas9系统的精准性,同时还展现出了良好的作用效果。在该系统中dCas9蛋白保留了Cas9蛋白结合DNA的能力而切割功能不复存在。将dCas9蛋白与不同的激活、抑制效应子域和表观遗传调控酶偶联,可以对基因表达与表观遗传修饰进行精确调控。组蛋白乙酰化、组蛋白甲基化、DNA甲基化等表观遗传修饰过程是基因表达的基础,对整个生命过程作出了巨大的贡献,同时表观遗传与多种疾病和癌症都存在因果关系,因此以CRISPR/dCas9系统为框架的不同表观遗传修饰系统在人类疾病治疗和癌症研究领域具有重要的研究价值。笔者简要介绍了CRISPR/Cas9系统的发现过程以及作用原理,主要总结了以CRISPR/dCas9系统为框架的不同调控系统在基因表达调控和表观遗传调控中的应用以及优化过程,以期为从事相关领域的科研工作者提供一些参考。     

柞蚕杆状病毒诱导鸡异嗜白细胞脱粒效应的信号转导通路

通过探讨柞蚕(Antheraea pernyi)杆状病毒诱导鸡异嗜白细胞的信号转导途径,确证其是否具有活化鸡异嗜白细胞的作用。采用β-葡萄糖苷酸酶释放法检测柞蚕杆状病毒诱导的鸡异嗜白细胞脱颗粒反应,并通过应用蛋白酪氨酸激酶(src、syk)、磷脂酰肌醇3-激酶(PI3-K)以及丝裂原活化蛋白激酶(ERK、p38 MAPK、JNK)的特异性抑制剂,分析各蛋白酶通路在柞蚕杆状病毒诱导鸡异嗜白细胞脱粒反应中的作用。结果表明,柞蚕杆状病毒可以显著提高鸡异嗜白细胞的脱粒反应,其中src、PI3-K、JNK的特异性抑制剂能够抑制柞蚕杆状病毒诱导的鸡异嗜白细胞的脱粒反应,而syk、ERK、p38 MARK的特异性抑制剂则对鸡异嗜白细胞脱粒不起作用,说明柞蚕杆状病毒可通过src→PI3-K→JNK信号转导通路来诱导鸡异嗜白细胞的脱粒反应。     

基于CRISPR/Cas9技术的法尼醇X受体缺失株的构建及其对HeLa细胞增殖的影响

试验旨在利用CRISPR/Cas9基因编辑技术构建法尼醇X受体（FXR）基因稳定敲除细胞株,并考察其对HeLa细胞增殖的影响。根据CRISPR/Cas9靶点设计规则,设计3对特异性识别FXR基因第1外显子相关序列的上下游小向导RNA（small guide RNA,sgRNA）,以PX459质粒为载体,构建真核重组表达质粒。酶切和测序鉴定后将重组质粒转染至HeLa细胞中,用嘌呤霉素进行阳性细胞筛选,再用实时荧光定量PCR检测HeLa细胞FXR基因敲除稳定株中FXR的表达量,并用Western blotting方法鉴定HeLa细胞FXR基因敲除效果。最后用结晶紫染色试验检测FXR基因敲除对HeLa细胞增殖的影响。结果显示,经测序后,3对sgRNA位置与方向均正确插入到PX459质粒载体内,成功构建出重组表达质粒PX459-sgRNA;实时荧光定量PCR和Western blotting检测结果显示,转染后筛选出的细胞中FXR蛋白不表达,表明构建出稳定敲除FXR基因的HeLa细胞株;结晶紫试验结果发现,FXR基因敲除的HeLa细胞染色深度明显低于正常HeLa细胞,FXR基因敲除HeLa细胞孔D_{595 nm}值极显著低于野生型HeLa细胞孔（P<0.01）,说明HeLa细胞FXR基因敲除株的增殖能力较正常HeLa细胞显著降低。本试验利用CRISPR/Cas9技术成功获得了内源FXR基因敲除细胞株,且FXR基因敲除对癌细胞增殖具有显著抑制作用,为研究FXR的功能和作用机制提供了细胞模型,并为研究FXR在相关癌症发生发展中的作用奠定了基础。     

Survivin: a bifunctional inhibitor of apoptosis protein

Survivin is a recently discovered protein belonging to the inhibitor of apoptosis (IAP) gene family. IAP molecules are characterized by both the presence of a zinc-binding fold termed the baculoviral IAP repeat and the ability to suppress apoptosis. In addition to inhibiting apoptosis, survivin is essential for proper cell division. Survivin is expressed during embryonal development but is absent in most normal, terminally differentiated tissues. Survivin is also upregulated in a variety of human cancers, and its expression in tumors is associated with a more aggressive phenotype, shorter survival times, and a decreased response to chemotherapy. The exact mechanism behind the ability of survivin to inhibit apoptosis is still unclear. Furthermore, it is not known why this protein is upregulated in cancer. The purpose of this article is to provide an overview of the current knowledge of survivin, including its role in cell division and its expression in normal and neoplastic tissues. Although much of the current research in this field is focused on human medicine, this area also has potential significance for veterinary species.     

Apoptosis induction in BEFV-infected Vero and MDBK cells through Src-dependent JNK activation regulates caspase-3 and mitochondria pathways

Our previous report demonstrated that bovine ephemeral fever virus (BEFV)-infected cultured cells could induce caspase-dependent apoptosis. This study aims to further elucidate how BEFV activates the caspase cascade in bovine cells. BEFV replicated and induced apoptosis in Vero and Madin-Darby bovine kidney (MDBK) cells, and a kinetic study showed a higher efficiency of replication and a greater apoptosis induction ability of BEFV in Vero cells. Src and c-Jun N-terminal kinase (JNK) inhibitor, but not extracellular signal-regulated kinase (ERK) or p38 inhibitor, alleviated BEFV-mediated cytopathic effect and apoptosis. In BEFV-infected Vero and MDBK cells, BEFV directly induced Src tyrosine-418 phosphorylation and JNK phosphorylation and kinase activity, which was inhibited specifically by SU6656 and SP600125, respectively. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP) and DFF45, were also activated simultaneously upon BEFV infection. In addition, cytochrome c, but not Smac/DIABLO, was released gradually from mitochondria after BEFV infection. SU6656 suppressed Src, JNK, and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage; SP600125 reduced JNK and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage. Taken together, these results strongly support the hypothesis that a Src-dependent JNK signaling pathway plays a key role in BEFV-induced apoptosis. The molecular mechanism identified in our study may provide useful information for the treatment of BEFV.