基因捕获技术

基因捕获技术是一种产生大规模基因突变的便利手段，对于揭示大量基因序列所对应的基因功能具有重要应用价值。本文综述基因捕获技术的基本原理和研究方法、发展现状及远景。     

嘌呤霉素抗性基因捕获载体的构建及在细胞中的功能验证

目的 构建具有嘌呤霉素抗性基因捕获载体,扩大基因捕获载体的应用范围.方法 用经改造的捕获载体(gene trapping vector)稳定转染HepG2.2.15肝癌细胞系,经嘌呤霉素筛选,制作单克隆细胞株.用PCR方法验证该载体的在细胞染色体中的整合,ELISA方法证明捕获载体捕获基因后的细胞的功能改变.结果 嘌呤霉素抗性基因捕获载体整合在HepG2.2.15肝癌细胞的染色体上,并能影响细胞HBsAg和HBeAg的分泌.结论 新构建的嘌呤霉素抗性基因捕获载体能在具有G418抗性的细胞中捕获有意义的目的 基因.     

十二烷基化壳聚糖基因支架血管内基因转运的实验研究

目的评估新型十二烷基化壳聚糖质粒DNA支架向局部动脉血管内转运基因的可行性及效果。方法十二烷基化壳聚糖同质粒DNA按文献制备纳米粒并进行表征。将纳米粒涂布于金属支架表面同时进行电镜观察。分别将十二烷基化壳聚糖质粒DNA支架进行细胞转染(pEGFP-C1)和兔颈动脉在体转染实验(pGL3-control),观察细胞转染效果。结果十二烷基化壳聚糖质粒DNA纳米粒为球形,稳定,平均粒径126nm,Zeta电位( 28±3)mV。该纳米粒涂布于支架表面,电镜下呈片状不规则粒子。细胞转染实验显示,支架表面细胞及支架邻近细胞大量转染,远离支架细胞未见转染。动物实验结果显示,支架植入部位荧光素酶高表达,同时有2只动物肝脏标本有微量表达,其他部位未见表达。结论十二烷基化壳聚糖质粒DNA支架是一种新型有效的血管内基因转运体系,可能对诸如再狭窄等心血管疾病有良好的应用前景,同时,这一基因运载思路可以广泛应用于各种质粒DNA的转运。     

高分子载体的基因释放与组织工程

以高分子为载体释放编码蛋白质质粒DNA实现组织重建的方法是在多学科、基因治疗和组织工程概念的基础上提出的。其技术的核心问题是以生物可降解高分子为载体的基因缓释；本将综述以高分子为载体的基因释放及其在组织工程应用的研究进展，并指出现存的问题。     

HLA-DRA基因逆转录病毒载体重组质粒的构建和包装

用逆转录病毒载体将ＨＬＡ－ＤＲＡ基因转当供体组织,使供受体之间ＨＬＡ－ＤＲ抗原表型趋于一致形成嵌合体,可能诱导免疫耐受,从而减缓受者免疫活性细胞对移植物的识别和攻击,延长移植物存活时间。     

胶原膜用于血管内基因投递的实验研究

目的评价胶原膜上通过抗体结合腺病毒或质粒DNA作为基因投递载体的可行性及效果。方法采用交联剂N-琥珀酰亚胺基3-（2-吡啶二硫基）丙酸酯（SPDP）将抗腺病毒或抗DNA抗体共价键结合在胶原膜上,通过这些抗体将腺病毒载体（Ad-GFP）和质粒DNA（pEGFP-C1）结合在膜上。采用同位素标记的质粒DNA（pDNA）在体外测定基因的释放,用细胞转染试验评价这种新型基因递送体系的功能。结果通过SPDP交联剂成功地将抗体共价键连接在胶原膜上,并结合了表达绿色荧光蛋白（GFP）的腺病毒和质粒DNA。细胞实验发现胶原膜上有大量表达GFP的阳性细胞,而膜以外的区域则没有GFP阳性细胞,表明具有局部递送特征。偶联Ad-GFP的胶原膜最大转染效率达到92.8%,而且在10^7-10^10病毒粒子/mL范围内,转染效率呈正剂量相关性。偶联pEGFP-C1的胶原膜诱导的细胞转染效率约为21.8%,结合32P标记的pDNA的胶原膜体外基因释放持续13天以上。用胶原涂层的不锈钢血管支架进行了同样的试验,在细胞培养中支架表面有大量GFP阳性细胞,其他区域没有GFP阳性细胞。结论胶原膜携带抗体偶联基因是一种新型的基因投递体系,可实现高效和局部定位的基因转染。     

用基因捕获法制作鸟氨酸脱羧酶抗酶抑制子基因"敲出"小鼠

目的分析鸟氨酸脱羧酶抗酶抑制子基因在小鼠生长发育过程中的功能.方法用特殊的捕获载体导入小鼠ES细胞中,5′RACE方法鉴定成功地捕获鸟氨酸脱羧酶抗酶抑制子基因(Oazin)后,由这种ES制作了Oazin"敲出"小鼠.结果 Oazin"敲出"小鼠捕获载体位于Oazin基因启动子上游,Qazin基因的转录被抑制.结论 Oazin"敲出"小鼠为分析Oazin基因的功能提供了有用的实验材料.     

目标序列捕获测序技术及其在疾病相关基因研究中的作用

目标序列捕获测序是指将感兴趣的基因组区域定制成特异性探针与目标基因组DNA在序列捕获芯片(或溶液)进行杂交,将目标基因组区域的DNA片段进行富集后再利用新一代测序技术进行测序,以获得目标基因组序列的研究策略.该项技术具有高度灵活性、特异性及覆盖率,操作便捷等特点,该文就目标序列捕获测序技术的原理及其在疾病相关基因研究中的应用作一简要综述.

Abstract：

Target sequence capture sequencing refers to a sequencing technology that uses the interested genomic region as specific probes, which are attached to chips or beads. The probes then hybridize with free target genomic DNA on sequence capture chip ( or in solution ) to enrich target genomic DNA. The enriched target genomic DNA fragments can then be amplified and studied using the next-generation sequencing technologies. The technology has a high degree of flexibility, specificity and coverage,and easy operation characteristics. The goal of this review is to outline the principle of target sequencing technology and its implication in disease-related gene research.     

双磷酸改性血管支架作为基因载体的研究

目的 将基因通过化学偶联和特异性免疫结合在双磷酸氨基酯改性的316L不锈钢冠状动脉支架上,评价支架改性和负载质粒DNA的效果.方法 金属支架首先利用双磷酸氨基酯改性引入氨基活性基团,化学偶联抗DNA抗体,然后免疫偶联质粒DNA,得到血管支架上负载抗体和基因的模型.结果 利用X射线光电子能谱(XPS)和原子力显微镜(AFM)等分析确定了支架表面磷酸氨基的存在,同位素标记验证了改性后支架表面结合的抗体具有很好的稳定性和结合容量.结论 双磷酸氨基改性的血管支架表层富含可反应氨基,可以作为新型反应界面,与生物活性分子进行偶联反应.     

目标序列捕获测序技术及其在疾病相关基因研究中的作用

目标序列捕获测序是指将感兴趣的基因组区域定制成特异性探针与目标基因组DNA在序列捕获芯片(或溶液)进行杂交,将目标基因组区域的DNA片段进行富集后再利用新一代测序技术进行测序,以获得目标基因组序列的研究策略.该项技术具有高度灵活性、特异性及覆盖率,操作便捷等特点,该文就目标序列捕获测序技术的原理及其在疾病相关基因研究中的应用作一简要综述.     

导入小鼠IFN-γ基因的肿瘤细胞的建立和鉴定

从小鼠脾细胞提取总ＲＮＡ,通过ＲＴ ＰＣＲ得到含信号肽的小鼠γ 干扰素（ｍＩＦＮ γ）全长ｃＤＮＡ,经测序鉴定后将其克隆到含标记基因ＮｅｏＲ的逆转录病毒载体ｐＬＸＳＮ中,采用脂质体法将其导入包装细胞ＰＡ３１７,经过包装,用含ｍＩＦＮ γ基因的缺陷型病毒上清感染小鼠肝癌细胞,经Ｇ４１８筛选建立起ｍＩＦＮ γ基因修饰株。ＰＣＲ、ＲＴ ＰＣＲ、Ｓｏｕｔｈｅｒｎ及Ｎｏｒｔｈｅｒｎ杂交结果显示有ｍＩＦＮ γ及标记基因的转入和表达。生物学活性测定也表明该基因修饰株细胞培养上清中有一定活性的ＩＦＮ γ分泌。     

Identification of MicroRNAs Dysregulated in CD14 Gene Silencing RAW264.7 Macrophage Cells

A cluster of differentiation antigen 14 (CD14) is involved in lipopolysaccharide (LPS)-induced proinflammatory cytokine release and LPS-induced septic shock. MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the epigenetic regulation of cellular process and bacterial infection. Our previous study indicated that siRNA against CD14 effectively inhibited LPS-induced tumor necrosis factor alpha, chemokine (C-X-C motif) ligand 2, interleukin-6 release, and NO production. To identify miRNAs which are affected by CD14 gene silencing and dissect the mechanisms of the attenuating of LPS-induced damaging immune activation more clearly, based on the CD14 knockdown RAW264.7 macrophage cell line established in our previous study, miRNAs expression profiling of CD14 knockdown RAW264.7 cells were analyzed with miRNA microarray and validated by qRT-PCR, the potential targets were predicted and subjected to gene ontology (GO) pathway and biological processes analysis. We demonstrated for the first time that CD14 knockdown significantly changed the expression of 199a-3p, miR-199a-5p, and miR-21-5p in RAW264.7 cells, and significantly enriched GO terms in the predicted target genes of these miRNAs were apoptosis process, immune response, inflammatory response, innate immune response, anti-apoptosis, cytokine production, and cytokine-mediated signaling pathway. These findings may improve our understanding about functional mechanism of miRNAs in the attenuating of LPS-induced damaging immune activation more clearly.     

人GM－CSF基因在昆虫细胞中表达的研究

本工作构建的昆虫表达重组体ｐＡＣ６１０－ＧＭＴ，是在ＡｃＭＮＰＶ的Ｐｏｌｙｈｅｄｒｉｎ启动子控制下，表达去除了信号肽编码顺序的人ＧＭ－ＣＳＦ基因（ｃＤＮＡ）的转染载体。它与野生ＡｃＭＮＰＶ病毒ＤＮＡ共转染Ｓｆ２１细胞，经过筛选得到纯化的可表达人ＧＭ－ＣＳＦ的重组病毒株ｖＡｃＧＭＴ。其感染细胞总ＲＮＡ的Ｎｏｒｔｈｅｒｎ分析结果表明，重组病毒在ｍＲＮＡ水平有人ＧＭ－ＣＳＰ特异性表达，其表达水平在感染后４８ｈ时达高峰，７２ｈ未见明显下降。感染细胞裂解物的Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析和活性测定也证实其蛋白水平的表达，并有人ＧＭ－ＣＳＦ的生物学活性。     

Gene Therapy in Experimental Autoimmune Encephalomyelitis

Gene therapy traditionally has been associated with gene replacement, where exogenous recombinant DNA is introduced ex vivo into somatic cells that are then introduced back into the patient as a way to correct an inherited genetic defect. However, several novel gene therapy strategies for treating autoimmune diseases recently have emerged. Strategies involving the use of several types of DNA vaccines, the application of various viral vectors, and the use of diverse cellular vectors have shown promise in inhibiting autoimmune-mediated inflammation and repairing tissue damaged as a result of autoimmune attack. In the current review, we examine and discuss the development and proposed use of emerging gene therapy strategies for the treatment of autoimmune disease with specific emphasis on experimental autoimmune encephalomyelitis (EAE), an animal model widely used in multiple sclerosis (MS) research.     

Induction of the Matrix Metalloproteinase 13 Gene in Bronchial Epithelial Cells by Interferon and Identification of its Novel Functional Polymorphism

Matrix metalloproteinases (MMPs) are a class of extra-cellular and membrane-bound proteases involved in a wide array of physiological and pathological processes including tissue remodeling, inflammation, and cytokine secretion and activation. MMP-13 has been shown to be involved in lung diseases such as acute lung injury, viral infections, and chronic obstructive pulmonary disease; however, the molecular pathogenesis of MMP-13 in these conditions is not well understood. In this study, we investigated the mechanisms and roles of MMP-13 secretion in human small airway epithelial cells (SAECs) and functional polymorphisms of the MMP13 gene. Polyinosinic–polycytidylic acid (poly(I:C)) and interferon β (IFN-β) stimulated the secretion of MMP-13 from SAECs by more than several hundred-fold. Stimulation of the secretion by poly(I:C) was abolished by SB304680 (p38 inhibitor), LY294002 (PI3K inhibitor), Janus kinase (JAK) inhibitor I, RNA-activated protein kinase (PKR) inhibitor, and Bay 11-7082 (NF-κB inhibitor), while stimulation by IFN-β was inhibited by all except Bay 11-7082. These data suggested that the secretion of MMP-13 was mediated through IFN receptor pathways independently of nuclear factor kappa B (NF-κB) and that poly(I:C) stimulated IFN secretion in an NF-κB-dependent manner from SAECs, leading to IFN-stimulated MMP-13 secretion. Chemical MMP-13 inhibitors and MMP-13 small interfering RNA (siRNA) inhibited IFN-stimulated secretion of interferon gamma-inducible protein 10 (IP-10) and regulated on activation, normal T-cell expressed and secreted (RANTES), suggesting that MMP-13 is involved in the secretion of these virus-induced proinflammatory chemokines. We identified a novel functional polymorphism in the promoter region of the MMP13 gene. The MMP13 gene may play important roles in defense mechanisms of airway epithelial cells.     

Identification of Cells with Monocyte Markers in Panhypogammaglobulinaemia

Nonphagocytic cells bearing receptors for complement and IgGFc in blood samples from four boys with panhypogammaglobulinaemia who lacked B lymphocytes were positively stained for human macrophage antigen (HuMA) and intracellular peroxidase. Similar non-T. non-B cells were found in adult blood preparations but in lower numbers than in the four boys above and in two of three other boys with various immunodeficiencies. Because of their monocyte characteristics. it is improbable that these cells are B-lymphocyte precursors, this is consistent with our view that there is a central failure of B-cell induction in boys with panhypogammaglobulinaemia who lack lymphocytes with surface immunoglobulin.     

抗癌胚抗原单链抗体基因与Mn-SOD基因的融合及在昆虫细胞中的表达

将抗癌胚抗原单链抗体基因与锰 超氧化物歧化酶基因融合 (ScFv Mn SOD )插入昆虫杆状病毒供体质粒pFastBacHTb中 ,经大肠杆菌DH1 0Bac体内转座 ,产生重组杆状病毒pBacHTb Mn SOD ScFv。将其转染粉纹夜蛾Tn 5B1 4细胞 ,经扩增后在细胞内进行表达。SDS PAGE分析结果表明 ,融合基因得到高效表达 ,其表达产物相对分子质量为 40 0 0 0单体和 1 60 0 0 0左右的四聚体。Western印迹分析结果 ,以 6×His单克隆抗体为一抗进行蛋白印迹在相对分子质量 40 0 0 0单体和 1 60 0 0 0四聚体处可见表达条带 ,放射免疫分析表明重组杆状病毒表达ScFv Mn SOD融合蛋白能与CEA有较高的结合力 ,并且此融合蛋白具有特异SOD酶活性 ,酶比活可达 32 6 5u/mg。