成年大鼠胰岛细胞的体外培养

目的： 建立一种高效、实用的成年大鼠胰岛细胞分离、纯化和体外培养的方法。方法：采用胰管内逆行灌注胶原酶溶液消化,密度梯度离心纯化胰岛,显微镜下手挑法捡出全部胰岛。分离后胰岛置胶原中培养，利用RT-PCR检测胰岛细胞特异基因的表达。结果：分离的胰岛细胞呈圆形或椭圆形，双硫腙染色呈猩红色，在胶原中培养能保持形态完整，表达胰岛细胞特异性基因PDX-1和insulin。 结论：本胰岛分离技术已趋于完善,获得的胰岛纯度高，培养效果好。     

携带大鼠酪氨酸羟化酶基因慢病毒载体的构建及转染

目的:构建携带增强绿色荧光蛋白(EGFP)和大鼠酪氨酸羟化酶(TH)基因的慢病毒载体,转染大鼠骨髓间充质干细胞(rMSC),观察TH基因的表达.方法:RT-PCR方法获得大鼠TH基因,将其克隆至慢病毒载体.通过瞬时转染法包装出病毒上清,鉴定滴度.感染rMSCs,荧光显微镜下观察EGFP的表达、转染效率,RT-PCR、免疫印迹法分别检测TH mRNA和蛋白的表达情况.结果:重组慢病毒载体质粒pNL-TH-IRES2-EGFP经双酶切鉴定正确,所获TH基因经测序后与GenBank报道序列完全一致;生产的病毒浓缩后滴度为4.1×10~7TU/ml;感染rMSCs结果显示荧光激发可见绿色荧光,TH-rMSCs、空载体-rMSCs组5d转染效率差异无统计学意义.RT-PCR、免疫印迹法显示TH基因成功在rMSCs中表达.结论:成功构建带有EGFP和大鼠TH基因的慢病毒载体,并获得TH-rMSCs基因工程细胞.     

慢病毒介导EPO基因治疗肾性贫血大鼠的实验研究

目的:探索用慢病毒载体介导方法对肾性贫血基因治疗的可行性及疗效。方法:采用氯化钙法制备含EPO基因的重组慢病毒,以直接肌肉注射法将重组慢病毒(6×107IU/ml)导入慢性肾衰引起贫血的大鼠后,定期检测血浆中CBC、血肌酐和EPO浓度,并与对照组进行比较。结果:注射后第3周,治疗组的血红蛋白和血清EPO浓度分别上升为(1.717±0.117)g/ml、(203.54±1.31)×10-3U/ml,并持续到15周,比对照组有统计学差异。结论:在动物模型中,制备的重组慢病毒直接肌肉注射能有效改善肾性贫血,为其他临床疾病伴随的贫血基因治疗提供理论和实验依据。     

载体与基因转染的研究进展

基因载体是指将基因或其它核酸物质运载到细胞中的工具.其化学本质可以是蛋白质或多肽、核酸、脂类、糖类、其它有机分子或它们的复合物.基因传递系统是基因治疗的重要组成部分,也是目前基因治疗的瓶颈.现有的基因载体包括两类.即病毒载体和非病毒载体.病毒载体转染效率高,但由于其转染具有免疫原性和致突变性限制了它的应用;非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.就各种载体的最新研究进展作一综述.     

β胰岛细胞表面GAD的表达决定NOD小鼠自身免疫型糖尿病的发病

型糖尿病 ,是一种由 T细胞介导的 β胰岛细胞损伤引起的自身免疫型疾病。许多学者认为 GAD是自身抗原最可能的候选者 ,但一直缺少确切的证据。本文作者将 GAD的反义基因转移到人类 型糖尿病的动物模型 NOD小鼠染色体 DNA上 ,建立 β胰岛细胞缺失表达 GAD的 NOD小鼠品系。利用这种转基因小鼠 ,作者研究了 β胰岛细胞上 GAD的表达与 NOD小鼠糖尿病发病的确切关系。作者将两种不同变异型的小鼠 GAD c DNA的反义基因转移到 NOD小鼠的胚系细胞上 ,然后将这些干细胞植入雌性 NOD鼠 ,经传代得到六个表达 GAD反义基因的转基因NOD…     

慢病毒载体的构建及其介导的绿色荧光蛋白在胰岛β细胞中的表达

目的获得能够在大鼠胰岛β细胞中高效表达的慢病毒载体。方法以PCR的方法扩增绿色荧光蛋白(green fluorescent protein,GFP)片段,并将其通过穿梭质粒装入pLenti6/V5表达质粒,然后用脂质体转染试剂将plenti6/V5GFP、pLP1、pLP2以及pLP/VSVG转染入293FT细胞,获得的病毒用人纤维瘤细胞系的HT1080细胞进行滴定。然后用一定滴度的慢病毒转导大鼠胰岛β细胞系INS-1,观察转导效率。结果通过限制性内切酶和琼脂糖凝胶电泳方法,观察到所克隆入pLenti6/V5表达质粒的GFP片段大小正好与PCR扩增出的片段一致。经测序验证,序列与NCBI网站上GFP序列完全一致。转染结果显示,经293FT细胞所产生的慢病毒,转导效率达到80%以上。而在1×106/ml病毒颗粒的情况下,AAV-GFP病毒几乎不能转导β细胞。结论与同一滴度的AAV-GFP病毒相比,胰岛β细胞的转导效率有非常显著的差异。即慢病毒载体表达系统在对胰岛β细胞转导的能力上,明显优于重组腺辅病毒表达系统。表明慢病毒载体在糖尿病基因治疗中,特别是对胰岛β细胞的转基因工作中,有着良好的应用前景。     

重组痘苗病毒载体介导的IL—2基因局部转染对小鼠黑色素瘤的治…

采用编码人ＩＬ－２基因的重组痘苗病毒载体（ｒＶＶ－ＩＬ－２），以ｉｎｖｉｖｏ模式体内局部转染小鼠色素瘤，结果肿瘤生长速度明显减慢，荷瘤小鼠存活明显著延长，体内免疫功能的检测表明，荷瘤小鼠脾细胞的ＮＫ活性及诱导后的ＬＡＫ，ＣＴＬ活性明显高于对照组，表明采用ｒＶＶ－ＩＬ－２载体介导ｉｎ，ｖｉｖｏ的肿瘤基因治疗具有一定的前景。     

实验性糖尿病大鼠胰岛移植的初步研究

实验研究证明,胰岛移植不仅能纠正实验性糖尿病动物的代谢异常,并能防止糖尿病性微血管病变的发生和发展,因此,对胰岛移植的研究具有重要的临床意义。我们自1983年开始,用大白鼠进行胰岛移植的实验研究,初步取得成效,现将观察结果报告如下。     

常用病毒载体转染对树突状细胞的影响

树突状细胞是功能最强的抗原提呈细胞 ,在介导免疫应答和维持免疫耐受中起着极其重要的作用。用携带目的基因的病毒载体转染树突状细胞已广泛应用于抗肿瘤和诱导耐受等方面的治疗。但在病毒载体转染树突状细胞过程中 ,病毒本身常能影响树突状细胞作为抗原提呈细胞的功能。因此 ,了解各种病毒载体的特点和对树突状细胞产生影响的作用机制 ,并选用合适的病毒载体 ,成为针对 DCs进行基因治疗成功的关键     

RGD肽表面修饰壳聚糖载基因纳米粒子的体外实验研究

目的将Arg—Gly—Asp（RGD）肽偶联到壳聚糖（CH）材料表面,并制备成包载质粒DNA的纳米粒子,以未偶连RGD的壳聚糖载质粒DNA作为对照,进行体外内皮细胞转染,观察其是否能提高对内皮细胞的转染效率。方法以1-乙基-3-（3-二甲基氨基丙基）碳化二亚胺盐酸盐（EDC）和N-羟基丁二酰亚胺（NHS）为偶联剂,通过酰胺键将RGD肽偶联到壳聚糖表面,对其进行表征,并以未偶连RGD的壳聚糖作为对照,制备载pEGFP-C1质粒DNA纳米粒子,比较2者对Hy926细胞的转染效率。结果壳聚糖-RGD（CH-RGD）载基因纳米粒子转染Hy926细胞的效率明显高于未偶连RGD的壳聚糖载基因纳米粒子（35．7％VS14．3％．P〈0．001）。结论RGD肽表面修饰壳聚糖载基因纳米粒子可用于体外细胞转染,其对细胞的转染效率明显优于未偶连RGD的壳聚糖。     

慢病毒载体介导2次转基因对人胚胎干细胞特性的影响

目的： 研究2次转基因对人胚胎干细胞（hESCs）多能分化特点和保持自我更新能力的影响。方法：构建携带非转录因子外源基因细胞毒性T细胞相关分子4免疫球蛋白 (CTLA4Ig) 、吲哚胺2,3双氧化酶（IDO）和荧光素酶的慢病毒载体,在hESCs先转导外源基因CTLA4Ig或IDO,完成筛选后转导外源基因荧光素酶。检测hESCs中外源基因的表达和功能,免疫组化和RT-PCR以及流式细胞仪方法检测hESCs细胞表面标记物、类胚体（EB）形成和体内畸胎瘤形成能力。结果：转导的外源基因均能表达并表现相应的功能。2次转导慢病毒载体后hESCs细胞表面标记物肿瘤排斥抗原（Tra-1-60） 和Octomer转录因子-4（OCT-4）染色仍为阳性,能形成EB,RT-PCR检测到甲胎蛋白(AFP)、配对盒基因6（Pax6）和MSI1的表达,异种移植后荧光信号逐渐增强。结论：利用慢病毒2次转导非转录因子外源基因不影响hESCs的未分化状态和多能分化能力,这对hESCs的转基因研究具有借鉴价值。     

Microchip-based engineering of super-pancreatic islets supported by adipose-derived stem cells

Type 1 diabetes mellitus (T1DM) is a chronic disorder characterized by targeted autoimmune-mediated destruction of the β cells of Langerhans within pancreatic islets. Currently, islet transplantation is the only curative therapy; however, donor shortages and cellular damage during the isolation process critically limit the use of this approach. Here, we describe a method for creating viable and functionally potent islets for successful transplantation by co-culturing single primary islet cells with adipose-derived stem cells (ADSCs) in concave microwells. We observed that the ADSCs segregated from the islet cells, eventually yielding purified islet spheroids in the three-dimensional environment. Thereafter, the ADSC-exposed islet spheroids showed significantly different ultrastructural morphologies, higher viability, and enhanced insulin secretion compared to mono-cultured islet spheroids. This suggests that ADSCs may have a significant potential to protect islet cells from damage during culture, and may be employed to improve islet cell survival and function prior to transplantation. In vivo experiments involving xenotransplantation of microfiber-encapsulated spheroids into a mouse model of diabetes revealed that co-culture-transplanted mice maintained their blood glucose levels longer than mono-culture-transplanted mice, and required less islet mass to reverse diabetes. This method for culturing islet spheroids could potentially help overcome the cell shortages that have limited clinical applications and could possibly be developed into a bioartificial pancreas.     

A role of thyrotropin-releasing hormone in insulin secretion by isolated rat pancreatic islets

Insulin-secreting pancreatic cells also express thyrotropin-releasing hormone (TRH). Although the physiological role of TRH in this localization is unclear, its participation in glucoregulation has been implied. To test this hypothesis, we blocked the last step of post-translational maturation of the TRH molecule by disulfiram, which is an active inhibitor of peptide -amidation (PAM) within pancreatic islet cells. The treatment of male rats with 200 mg/kg/day of disulfiram during a 5-day period resulted in a low PAM activity, a high insulin content and its basal secretion from pancreatic islets, and the inability to release insulin in response to glucose (16.7 mM) or hypo-osmotic (30%) challenge in vitro. The addition of TRH (1 nM) to the medium during incubation restored the insulin content and both basal and glucose stimulated insulin secretions to control levels. Conclusion: TRH plays an important role in the mechanism of insulin secretion and its response to glucose stimulation.     

Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin-producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine-induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)-γ + tumour necrosis factor (TNF)-α + interleukin (IL)-1β. Consequently, MIF-deficient [MIF-knock-out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine-induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF-KO mice maintained normal insulin expression and produced less cyclooxygenase-2 (COX-2) than those from wild-type C57BL6 mice. The final outcome of cytokine-induced islet apoptosis in islets from wild-type mice was the activation of mitochondrial membrane pore-forming protein Bcl-2-associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF-KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up-regulation of prosurvival kinase extracellular-regulated kinase 1/2 in MIF-KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor-κB and mitochondria-related proteins.     

Detection of MspI RFLP in human THY1 gene by the polymerase chain reaction

THY1 gene encodes a cell surface glycoprotein predominantly expressed in brain and peripheral nerves. Human THY1 gene region on chromosome 11q23 has been implicated in susceptibility to type 1 diabetes (Wonget al., 1991). Two primers derived from the sequences flanking the polymorphic MspI site in intron 2 of the human THY1 gene (Gattiet al., 1988) were selected for RCP to amplify a 566 bp fragment that spans the MspI polymorphism. Polymorphism was detected by MspI digestion of the PCR product.     

角质细胞生长因子促进大鼠胰腺导管上皮细胞体外增殖

目的 探讨不同浓度的角质细胞生长因子(KGF)对大鼠胰腺导管上皮细胞增殖的影响,寻求最佳浓度.方法 免疫细胞化学染色及RT-PCR方法鉴定SD大鼠胰腺导管上皮细胞;不同浓度的KGF刺激胰腺导管上皮细胞增殖,寻求最佳浓度.结果 大鼠胰腺导管上皮细胞表达Nestin和CK19,不表达Insulin及Glucagon;不同浓度KGF刺激胰腺导管上皮细胞2d后,细胞均有不同程度增殖,20 μg/L KGF作用2d时,细胞数为0.35±0.03,显著高于对照组的0.27±0.02(P ＜0.01).结论 KGF在本实验的剂量范围内可促进胰腺导管上皮细胞增殖,20 μg/L为促进增殖的最佳浓度.     

Polyethylenimine coating to produce serum-resistant baculoviral vectors for in vivo gene delivery

Recombinant baculoviral vectors efficiently transduce many types of mammalian cells. However, their in vivo applications are hampered by the sensitivity of the virus to complement-mediated inactivation. Based on our observation that the surface charge of baculovirus is negative at neutral pH, we developed a procedure to coat baculoviral vectors with positively charged polyethylenimine 25 kDa, a commonly tested non-viral gene delivery vector, through electrostatic interaction. This coating was effective in protecting baculoviral vectors against human and rat serum-mediated inactivation in vitro, providing transduction efficiencies comparable with that generated by the control virus used under a serum-free condition. Enhanced in vivo gene expression in the liver and spleen was observed after tail vein injection of the coated viruses into mice. When injected directly into human tumor xenografts in nude mice, the coated viruses suppressed tumor development more effectively than uncoated viral vectors. These findings demonstrated the usefulness of using a simple coating method to circumvent a major obstacle to in vivo application of baculoviral vectors. The method may also serve as a flexible platform technology for improved use of the vectors, for example introducing a targeting ligand and minimizing immune responses.