科技图书

<正>现代基因治疗分子生物学(第二版)成军主编科学出版社2014年9月出版《现代基因治疗分子生物学》是成军教授《基因治疗》(1993年)的再版,旨在对近十年来国内外基因治疗的新认识、新观点、新技术和新成就进行系统、集中展示,并对基因治疗研究中的难点和热点问题进行归纳、总结。内容包括基因治疗概论、基因克隆、基因的人工合成、基因的PCR扩增、基因转移的靶细胞、干细胞与基因治疗、基因的转移方法;基因     

基因治疗与人类健康

随着分子生物学及基因工程技术的迅猛发展 ,基因治疗已经成为治疗人类疾病的重要方法之一 ,同时也是维护人类健康最有发展前景的手段之一。诸如遗传病、肿瘤、和传染病与心血管病的基因治疗。遗传免疫方面 ,病毒性疾病和肿瘤的基因治疗 ,如将病毒抗原基因 (HBsAg)及一些肿瘤抗原基因 (CEA)直接注入人体内而产生抗体 ;人类亚健康状态 ,如肥胖、秃顶、疲劳、衰老等的基因治疗。然而基因治疗目前仍面临着许多困扰 ,如基因治疗的有效性、安全性、及社会伦理等诸多问题 ,因此在临床实际应用中要慎之又慎。只有对基因治疗合理规范和正确引导并遵循伦理原则 ,才能最终推动现代医学的发展。     

1型糖尿病的基因治疗进展

尽管皮下注射胰岛素、口服降糖药等可以缓解糖尿病患者的高血糖,但是这些治疗措施只是暂时性的,并不能从根本上彻底治疗糖尿病以及阻止其他并发症的发生。随着人们对糖尿病本质的深层次揭示和现代分子生物学手段的发展,针对由胰岛素分泌缺乏引起的1型糖尿病(T1D)基因治疗手段逐渐丰富。总结了胰岛素替代基因的直接导入,刺激新的β细胞再生以及阻止胰岛β细胞的自身免疫,抑制胰岛β细胞的凋亡等1型糖尿病的基因治疗新进展,并展望其未来发展方向。     

胰岛素和瘦素抵抗的重要调控因子:细胞因子信号转导抑制因子-3

细胞因子信号转导抑制因子-3(Suppressor of Cytokine Signaling-3,SOCS-3)是细胞信号转导生理抑制剂家族重要成员之一,主要参与负调控生长激素、白介素(IL),肿瘤坏死因子(TNF)等细胞因子信号转导.最近发现,SOCS-3在胰岛素和瘦素抵抗中具有重要调控功能,且与糖尿病关系密切,是未来糖尿病基因治疗的新靶点.该文综述了近年来SOCS-3结构及其对胰岛素和瘦素抵抗的调控机制研究进展,并探讨SOCS-3在糖尿病中的作用.     

基因转移指日可待

对基因的定点转移,既可增加基因治疗成功的机率,也将为研究基因在发育过程中的作用提供一条新途径。学者们正研究把外源基因引入他们所要引入的特定基因位点,而不是基因组的任一位置。这一新的把基因引入染色体特定位点的能力,可提高对人类遗传疾病如镰状细胞性贫血(SCA)进行有效基因治疗的机会。在此之前,要控制被转移基因在基因组中的终止位置是不可能的。这些引入的基因可进     

心血管疾病的基因治疗

近年来 ,随着分子生物学技术和载体技术的发展及目的基因不断被克隆 ,基因转移为心血管疾病提供了新的治疗途径 ,常被用于治疗重要蛋白质的过度表达和矫正基因的缺陷。如血管内皮生长因子 (ＶＥＧＦ)和成纤维细胞生长因子 (ＦＧＦ)等基因转移 ,促进血管新生 ,改善了缺血性心肌的供血和冠脉侧枝循环形成 ;而反义性基因治疗 ,在转录或翻译水平上关闭或抑制某些生长因子的基因表达 ,抑制其合成 ,从而降低血管内膜厚度 ,减轻再狭窄程度 ,为经皮冠状动脉腔内成形术(ＰＴＣＡ)和冠脉内支架植入后再狭窄的防治带来新的希望[1,2 ] 。1 .基因治疗的载…     

基因治疗的应用研究进展

基因治疗 (ｇｅｎｅｔｈｅｒａｐｙ)是向靶细胞引入正常有功能的基因 ,以纠正或补偿致病基因所产生的缺陷 ,从而达到治疗疾病的目的 ,通常包括基因置换、基因修正、基因修饰、基因失活等。 80年代初 ,Ａｎｄｅｒｓｏｎ首先阐述了基因治疗的概况 ;1990年美国国立卫生研究院的Ｂｌｅａｓｅ等[1] 成功地进行了世界上首例临床基因治疗 ,即腺苷脱氨酶 (ａｄｅｎｏｓｉｎｅｄｅａｍｉｎａｓｅ ,ＡＤＡ)缺陷病的人体基因治疗 ;1991年我国首例基因治疗Ｂ型血友病也获得成功。近年来 ,这一领域的研究取得了重大进展 ,基因治疗作为一种全新的疾病治疗…     

AMPK及脂肪细胞因子调节胰岛素抵抗的研究进展

腺苷酸激活蛋白激酶(AMP-activated Protein Kina,AMPK)信号通路是调节细胞能量状态的中心环节,被称为"细胞能量调节器",在增加骨骼肌对葡萄糖的摄取、增强胰岛素(Insulins,Ins)敏感性、增加脂肪酸氧化以及调节基因转录等方面发挥重要作用.在整体水平,AMPK通过激素和脂肪细胞因子如瘦素、脂联素和抵抗素等调节能量的摄入和消耗.多种脂肪源性细胞因子表达异常与胰岛素抵抗(Insulin Resistance,IR)密切相关,而胰岛素抵抗又是Ⅱ型糖尿病发生的基础,并贯穿于Ⅱ型糖尿病发生发展的全过程.研究AMPK及脂肪细胞因子与胰岛素抵抗的关系,将为AMPK作为防治肥胖和Ⅱ型糖尿病提供新的药理学靶点.     

cDNA RDA在类似普通2型糖尿病大鼠肾脏组织基因差异表达筛查中的应用

应用代表性差异分析 (cDNARDA)技术 ,对类似普通 2型糖尿病大鼠肾脏组织基因差异表达进行筛查 ,初步探讨类似普通 2型糖尿病大鼠肾脏损害发病的分子机制 .首先以类似普通 2型糖尿病大鼠肾脏组织作为实验组 (Tester) ,正常大鼠肾脏作为对照组或驱动组 (Driver)通过cDNARDA进行基因差异表达筛查 ;最终的差异产物亚克隆到Puc 18载体 ,测序及并进行生物信息学分析 ;半定量RT PCR对筛查到新的基因进行初步的鉴定 .结果发现 9个新ESTs ,2个新基因 .这 2个新基因分别与人及小鼠的丝氨酸蛋白酶抑制因子F ,及真核细胞转录启动因子 3亚单位 5 (EIF 3epsilon)基因有高度的相似性 (>90 % )并在类似普通 2型糖尿病大鼠肾脏组织表达上调 .推测 2个新基因分别是大鼠的丝氨酸蛋白酶抑制因子F及真核细胞转录启动因子 3亚单位 5 .两个新基因在类似普通 2型糖尿病大鼠肾脏组织表达上调 ,可能与类似普通 2型糖尿病大鼠肾脏损害相关 .同时 ,对新基因RS91进行了全长cDNA克隆     

四氧嘧啶诱导糖尿病小鼠的胰岛素原基因治疗

为了解决基因治疗中的安全性问题 ,和寻求一种简便而又经济的糖尿病基因治疗模式 ,采用腹腔注射四氧嘧啶的方法 ,以昆明小鼠为实验对象 ,成功地建立了糖尿病小鼠模型 .通过电穿孔的基因转移 ,将含有胰岛素原cDNA的质粒pCMV IN转移到这些小鼠的股四头肌 .反转录PCR的结果表明 ,转基因在转染部位有转录活性 ,而放射免疫分析结果却表明转基因的表达产物分泌进了小鼠的血循环系统之中 .血糖浓度的变化说明 ,这些产物具有明显的降血糖效果 .在电基因转移前对转移部位用透明质酸酶处理 ,使电激时的使用电压降低 ,增加了转移效果 ,也更加安全 .     

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment.     

Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells

Islet cell replacement is considered as the optimal treatment for type I diabetes. However, the availability of human pancreatic islets for transplantation is limited. Here, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) could be induced to differentiate into functional insulin-producing cells by introduction of the pancreatic duodenal homeobox-1 (PDX-1). Recombinant adenoviral vector was used to deliver PDX-1 gene into hMSCs. After being infected with Ad-PDX-1, hMSCs were successfully induced to differentiate into insulin-secreting cells. The differentiated PDX-1+ hMSCs expressed multiple islet-cell genes including neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide in a weak glucose-regulated manner. After the differentiated PDX-1+ hMSCs were transplanted into STZ-induced diabetic mice, euglycemia can be obtained within 2 weeks and maintained for at least 42 days. These findings validate the hMSCs model system as a potential basis for enrichment of human beta cells or their precursors, and a possible source for cell replacement therapy in diabetes.     

SHB and angiogenic factors promote ES cell differentiation to insulin-producing cells

The potential use of embryonic stem (ES) cells for cell therapy of diabetes requires improved methods for differentiation and isolation of insulin-producing beta-cells. The signal transduction protein SHB may be involved in both angiogenesis and beta-cell development. Here we show that cells expressing the pancreatic endodermal marker PDX-1 appear in the vicinity of vascular structures in ES cell-derived embryoid bodies (EBs) cultured in vitro. Moreover, overexpression of SHB as well as culture of EBs in presence of the angiogenic growth factors PDGF or VEGF enhanced the expression of PDX-1 and/or insulin mRNA. Finally, expression of GFP under control of the PDX-1 promoter in EBs allowed for the enrichment by FACS of cells expressing PDX-1, C-peptide, and insulin as determined by immunofluorescence. It is concluded that SHB and angiogenic factors promote the development of cells expressing PDX-1 and insulin in EBs and that such cells can be separated by FACS.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.