用显微注射法建立转bcl—x1基因小鼠

建立转bcl-x1基因小鼠，继而传代建系，用于缺血性脑血管疾病的研究。采用显微注射法，将人bcl-x1基因注入昆明小白鼠受精卵获得子代鼠,然后作PCR，Southern-blot,mRNA,Western-blot检测以获得阳性鼠。实验中注射受精卵1654枚，移植卵数1448枚，受体鼠49只，怀孕鼠7只，子代鼠13只，整合数4只并均有表达，植入受精卵的存活率83％，受精卵的总存活率0．78％，移植鼠的怀孕率14．3％，整合效率30．7％，总有效率为0．24％。初步获得了转bcl-x1基因小鼠。     

用显微注射法建立转bcl-x1基因小鼠

建立转bcl-x1基因小鼠,继而传代建系,用于缺血性脑血管疾病的研究.采用显微注射法,将人bcl-x1基因注入昆明小白鼠受精卵获得子代鼠,然后作PCR,Southern-blot,mRNA,Western-blot检测以获得阳性鼠.实验中注射受精卵l 654枚,移植卵数1 448枚,受体鼠49只,怀孕鼠7只,子代鼠13只,整合数4只并均有表达,植入受精卵的存活率83%,受精卵的总存活率0.78%,移植鼠的怀孕率14.3%,整合效率30.7%,总有效率为0.24%.初步获得了转bcl-x1基因小鼠.     

转突变型淀粉样蛋白前体双荧光融合基因阿尔茨海默病小鼠模型的建立及初步鉴定

目的 探索建立转突变型淀粉样蛋白前体(APP)双荧光融合基因(CFP-54 bp-YFP-C99)转基因小鼠模型的可行性和基因整合效率.方法 采用显微注射法,将构建的突变型APP双荧光融合基因注入昆明小白鼠的受精卵,获取子代鼠,通过聚合酶链反应(PCR)方法初步鉴定基因的整合情况,以获得阳性鼠.结果 注射昆明小白鼠受精卵共2202枚,移植1806枚.受体鼠56只,怀孕鼠13只,共生产52只子代鼠,其中存活32只.PCR初步检查整合有突变型APP双荧光融合基因的阳性鼠2只,均为活鼠.移植鼠的总怀孕率为23.2%(13/56),突变型APP双荧光融合基因的整合效率为3.9%(2/52).结论 显微注射法建立转突变型APP双荧光融合基因的阿尔茨海默病小鼠模型是可行的,但转基因效率较低.     

基于一个伴发脑血管狭窄的NF1家系的点突变小鼠建模

目的利用CRISPR/Cas9技术构建Nf1基因Q181X定点突变的小鼠模型,为研究该点突变与脑血管狭窄的关系提供实验工具。方法根据Nf1基因点突变的位置和基因组结构设计针对Nf1基因Q181X位点的g RNA,经活性检测后,将有活性的gRNA、Cas9 mRNA以及含有点突变序列的donor DNA通过显微注射到小鼠受精卵中,并移植至假孕鼠体内。获得F0代小鼠后,通过PCR和基因测序技术对Nf1基因进行检测和鉴定。对所获的F0代基因突变阳性小鼠继续繁育后获得F1、F2代目标基因阳性小鼠。结果成功构建Nf1-Q181X基因点突变F0代杂合型小鼠3只,F1代杂合型小鼠2只,F2代杂合小鼠6只,经PCR及基因测序鉴定可稳定遗传Nf1-Q181X点突变。结论 H通过CRISPR/Cas9技术成功获得Nf1基因Q181X点突变小鼠模型,为研究该特定点突变与脑血管异常的关系提供了实验工具。     

视神经脊髓炎转基因(2D2)鼠的繁殖和鉴定

目的建立视神经脊髓炎转基因(2D2)小鼠的繁育方法,并对其子代鼠进行基因鉴定。方法将雌雄2D2转基因小鼠各1只与C57BL/6野生型小鼠(1∶1)进行交配,由鼠尾血提取基因组DNA,应用PCR技术扩增,电泳后观察2D2转基因传代小鼠的基因表型。结果共繁育产生56只子代小鼠,其中36只子代小鼠基因组DNA扩增出657bp的2D2条带,阳性率为64.3%。结论成功利用杂交方式有效繁殖2D2转基因鼠和应用PCR技术进行基因鉴定,为后续视神经脊髓炎的实验研究奠定了基础。     

同基因造血干细胞移植治疗EAE小鼠脾脏Treg细胞的变化

目的 探讨Treg细胞在实验性变态反应性脑脊髓炎(EAE)中变化和同基因造血干细胞移植治疗EAE小鼠巾的作用.方法 以MOG35-55免疫C57BL/6小鼠建立EAE模型,分为病鼠-病鼠移植组、正常鼠-病鼠移植组和病鼠对照组.两移植组由EAE鼠和正常鼠做供体行移植后分3组,分别于移植后40d、80d、120d处死;病鼠对照组分4组,于移植前1d、移植后40d、80d、120d处死.流式细胞仪测定小鼠脾脏Treg细胞比例;实时定量聚合酶链反应测定脾脏Foxp3 mRNA水平;western blot法测定Foxp3蛋白水平.结果 移植前的病鼠对照组脾脏Treg细胞(3.3%±1.6%)较正常对照组(6.8%±1.7%)降低(P<0.05),移植后80d,两移植组Treg细胞(20.12%±2.67%、16.34%4±1.48%)上升至峰值(P<0.05);移植前的病鼠对照组Foxp3 mRNA相对表达量为0.48±0.19,移植后40d、80d、120d,两移植组Foxp3 mRNA均高于病鼠对照组(P<0.05);Foxp3蛋白水平为:移植前的病鼠对照组<正常对照组<移植后120d的移植组.结论 Treg细胞的数量减少和功能缺失可能与EAE发生发展有关;其数量增加和功能恢复可能是同基因造血干细胞移植治疗EAE的机制之一.     

超氧化物歧化酶基因突变子对阿尔茨海默病转基因小鼠β-淀粉样蛋白表达影响的实验研究

β 淀粉样蛋白 (Ａβ)在阿尔茨海默病 (ＡＤ)发病过程中起关键作用已被研究证实。本研究应用Ａβ在海马神经元表达的转 β 淀粉样蛋白前体蛋白 (ＡＰＰ Ｃ10 0 )基因小鼠 ,转入铜 /锌超氧化物歧化酶 (Ｇ93ＡＳＯＤ1)基因来探讨ＳＯＤ1基因表达对Ａβ代谢的影响。对象和方法 :转ＡＰＰ Ｃ10 0基因鼠和转Ｇ93ＡＳＯＤ1基因鼠通过杂交育种产生四种基因型小鼠 ,用ＳｏｕｔｈｅｒｎＢｌｏｔ确认ＡＰＰ Ｃ10 0基因和聚合酶链反应确认Ｇ93ＡＳＯＤ1基因的存在。年轻 (2～ 3个月龄 )和年长 (8～ 9个月龄 )的转双基因雄性杂合子小鼠各 4只作为研究组 …     

NAAG肽酶基因剔除小鼠模型的建立及鉴定

目的 建立N-乙酰天冬氨酰谷氨酸(NAAG)肽酶基因剔除小鼠模型,为在体研究NAAG肽酶基因的生物学功能并揭示其在脑损伤后继发性脑损害进程中的所起的作用创造条件.方法 根据小鼠NAAG肽酶基因组的序列,设计基因剔除策略,构建基因剔除载体NAAG-KO-pBR322,以电穿孔方法将基因剔除载体导入胚胎干细胞(ES),应用G418和更昔洛韦进行正负筛选,获得双抗性克隆,聚合酶链式反应(PCR)鉴定并测序获得正确同源重组的ES细胞克隆.结果 同源重组的ES细胞注入小鼠囊胚后获得11只嵌合率＞50％嵌合体雄性小鼠,嵌合体小鼠与C57 BL/6J雌鼠交配后获得11只杂合子小鼠,其中雄性7只,雌性4只.在雌、雄杂合子小鼠交配的后代中获得7只纯合子小鼠,PCR鉴定其基因型,逆转录PCR (RT-PCR)提示该基因鼠未表达NAAG肽酶.结论 我们成功建立了NAAG肽酶基因剔除小鼠模型,其中纯合子小鼠未出现胚胎致死现象;初步的表型观察未发现NAAG肽酶基因剔除小鼠出现异常改变.     

杆状病毒修饰后的脂肪干细胞移植治疗mdx鼠的实验研究

目的利用经杆状病毒基因载体系统进行micro-dystrophin基因修饰后的脂肪干细胞(ADSCs)移植治疗Duchenne型肌营养不良症模型(mdx)鼠,探讨ADSCs移植治疗DMD的安全性及可行性。方法 Mdx鼠60只,分为mdx对照组(30只)和mdx移植组(30只);正常C57小鼠为C57对照组(30只)。体外分离培养小鼠ADSCs,利用杆状病毒基因载体进行micro-dystrophin基因修饰;将基因修饰后的ADSCs经尾静脉移植到mdx鼠体内。于移植后检测mdx鼠的运动功能(采用主动牵引实验和被动转棒实验)、血清CK水平、肌肉病理改变以及肌肉micro-dystrophin表达水平。结果经micro-dystrophin基因修饰的ADSCs移植后,能够重建mdx鼠的micro-dystrophin表达,一定程度上减轻并逆转肌肉的病理损害,进而降低血清CK水平,mdx鼠整体运动功能也有一定改善。结论 ADSCs治疗mdx鼠后,可部分重建模型鼠的dystrophin表达,改善肌肉的病理损害,表明ADSCs是有希望治愈DMD的方法之一。     

移植物化学修饰减轻小鼠半相合骨髓移植后的急性移植物抗宿主病

背景: 甲氧基聚乙二醇是一种无免疫原性的兼性聚合分子，可以共价结合方式修饰各种蛋白质，减少其免疫原性。因此，在进行半相合造血干细胞移植时，若能用甲氧基聚乙二醇对移植物的T细胞进行化学修饰，阻断其激活的途径，就可能减轻移植物抗宿主病反应，提高移植成功率。 目的：构建小鼠半相合骨髓移植模型，观察甲氧基聚乙二醇修饰移植物对小鼠H2半相合骨髓移植后移植物抗宿主病的影响。 设计、时间及地点：随机化配对设计实验，于2003-03/11在解放军总医院医学动物实验中心和小儿内科实验室完成。 材料：纳入20只8~10周龄雄性近交系BALB/cH-2d小鼠作为供鼠，60只8~10周龄杂交获得的雌性CB6 F1 代H-2d/b小鼠作为受鼠。甲氧基聚乙二醇为美国Sigma公司产品。 方法：常规制备供鼠骨髓及脾细胞混合悬液。受鼠行总剂量8.0 Gy 60Co γ射线全身均匀照射，照射后随机分为3组，每组20只。照射对照组每只受鼠经尾静脉输入RPMI-1640培养基0.5 mL，单纯移植组照射后12 h内经尾静脉输入未处理的半相合供鼠骨髓及脾细胞混合悬液0.5 mL，修饰移植组照射后12 h内经尾静脉输入甲氧基聚乙二醇修饰的半相合供鼠骨髓及脾细胞混合悬液0.5 mL。 主要观察指标：移植后观察动物存活率、急性移植物抗宿主病理表现及存活小鼠的染色体核型。 结果：照射对照组小鼠均在2周内死亡；修饰移植组小鼠30 d存活率显著高于单纯移植组（75%，40%，χ2=5.01，P=0.025）。取死亡小鼠的爪垫皮肤、肝及小肠肠管作病理组织学检查，修饰移植组急性移植物抗宿主病病理表现减轻，Thomas 病理分级轻于单纯移植组。移植后75 d应用染色体C带显色法检测存活受鼠的嵌合体，证实为完全供者型植入。 结论：甲氧基聚乙二醇修饰移植物可明显减轻急性移植物抗宿主病，提高半相合骨髓移植存活率。     

Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.     

Cre重组酶敲除小鼠大脑脚背盖背侧核lacZ基因研究

目的 观察Cre重组酶能否敲除双报告Z／EG转基因成熟小鼠大脑脚背盖背侧核局部lacZ基因。方法 用PCR技术对双报告Z／EG转基因鼠系进行基因鉴定，利用立体定位微量注射技术将含有Cre重组酶的病毒载体注射到双报告Z／EG转基因成熟小鼠左侧大脑脚背盖背侧核，将鼠脑冷冻切片行抗绿色荧光蛋白免疫组织荧光染色及PicoGreen对照染色，于Confocal显微镜下观察绿色荧光蛋白的表达。结果 双报告Z／EG转基因鼠所获PCR产物为631bpI未作任何染色切片在Confocal显微镜下可观察到注射Cre重组酶病毒载体左侧大脑脚背盖背侧核区域有弱绿色荧光，经抗绿色荧光蛋白免疫组织荧光染色后发现此区域有红色荧光。结论 Cre重组酶成功地敲除了双报告Z／EG转基因成熟小鼠大脑脚背盖背侧核局部lacZ基因。     

A novel transgenic mouse model of hyperfibrinogenemia

Hyperfibrinogenemia is a risk predictor in several diseases, including cardiovascular disease. Nevertheless, it remains unknown whether elevated fibrinogen has an etiologic role in or is a reflection of disease pathogenesis, or both. To examine this question, we generated a mouse model of hyperfibrinogenemia. We isolated the mouse fibrinogen locus, containing the three fibrinogen genes, in a single P1 clone. This approximately 100 kb clone was injected into C57Bl/6J zygotes. Three transgenic lines were identified, two with elevated fibrinogen, 1.4- and 1.7-fold relative to normal. We characterized the line with the higher level. Northern blots of total RNA showed transgene expression was liver specific, and the message levels were 2- to 3-fold enhanced. Fibrinogen in transgenic mice was normal in both immunologic and clotting assays. Our data indicate that over-expression of all three fibrinogen genes is necessary to achieve hyperfibrinogenemia. We saw no increase in mortality or morbidity, no gross abnormalities in the organs, and no histologic differences in lung, liver, spleen or kidney, in transgenic mice relative to normal littermates. We conclude that elevated fibrinogen did not cause disease in mice. We anticipate that breeding these mice to other mouse models of disease will demonstrate whether hyperfibrinogenemia has a role in the initiation or progression of symptomatic disease.     

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as "fountain of microglia", where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.     

Elevated interferon-gamma in CNS inflammatory disease: a potential complication for bone marrow reconstitution in MS

Bone marrow transplantation (BMT) is increasingly used to treat Multiple Sclerosis (MS) a CNS inflammatory disease with elevated CNS and systemic IFNgamma levels. We wished to determine the effect of IFNgamma on BM graft survival in a transgenic mouse model for chronic MS. BM transplantation into transgenic mice which express elevated levels of IFNgamma in the CNS was unsuccessful. By contrast, there was 100% survival of even fully allogeneic, T-depleted transplants to transgenics that over express TNFalpha in the CNS, using the same MBP promoter. IFNgamma was detectable in spleen of irradiated mice but levels were higher in IFNgamma transgenics. BM transplantation into IFNgamma-deficient recipients also had a high failure rate. Transplants of BM from mice lacking expression of IFNgamma-receptor failed, whereas IFNgamma-deficient grafts survived, suggesting that IFNgamma response status of the graft can also positively influence survival. IFNgamma therefore has a dual role in BM transplantation and the outcome will depend on relative levels of cytokine expression.     

缓释BCNU对GL261胶质瘤细胞凋亡作用机制的研究

目的 研究BCNU-PLGA 对GL261 胶质瘤细胞凋亡的作用机制.方法 利用溶剂挥发萃取法制备BCNU-PLGA 缓释微球.将24 只C57BL/6 小鼠随机等分为治疗组与非治疗组,借助动物立体定向仪,将体外培养小鼠GL261 胶质瘤细胞接种于小鼠颅内,行MR 检查掌握颅内成瘤情况.治疗组于术后第2 周立体定位植入BCNU-PLGA 缓释片剂,观察小鼠生存期,MRI 了解瘤体变化.获取标本行HE 染色,并行GFAP、Bax、Bcl-2 免疫组化检查检测其表达情况,相关数据采用统计学方法 进行分析.结果 治疗组与对照组生存期有明显差异(P ＜0.05),治疗组与对照组凋亡相关基因Bcl-2 免疫组化检查提示明显差异性(P ＜ 0.05),Bax 基本无改变,Bax/Bcl-2 比值明显增加.GFAP 表达为阳性.结论 BCNU-PLGA 局部缓释制剂通过下调Bcl-2 蛋白表达,上调Bax/Bcl-2 的比值,从而使胶质瘤细胞发生凋亡.     

Bcl-2和Bax蛋白在人脑星形细胞瘤中的表达及意义

目的　研究 Ｂｃｌ２ 和 Ｂａｘ 蛋白与人脑星形细胞瘤发生的关系。方法　收集人脑星形细胞瘤标本５８ 例, 行 Ｓ Ｐ 染色。结果　 Ｂｃｌ２ 在星形细胞瘤中阳性表达率为５６９ ％ , 且表达率随肿瘤恶性程度的增加而增高。 Ｂａｘ 的表达率为５５２ ％ , 显著高于对照组。 Ｂｃｌ２ 表达阳性的肿瘤中 Ｂａｘ 蛋白表达率（６６７ ％ ） 显著高于 Ｂｃｌ２ 表达阴性的肿瘤（４００ ％ ） 。结论　 Ｂｃｌ２ 和 Ｂａｘ 蛋白的表达与星形细胞瘤的发生和发展有较密切的关系, 且它们在星形细胞瘤的发生中可能存在相互作用的关系。     

缓释BCNU对GL261胶质瘤治疗作用分析

目的 研究卡氮芥( BCNU)聚乳酸-羟基乙酸(PLGA)缓释微球(BCNU-PLGA)对GL261胶质瘤模型的治疗作用及化疗耐药机制.方法 利用溶剂挥发萃取法制备BCNU-PLGA缓释微球.将36只C57BL/6小鼠随机等分为假手术组、治疗组与非治疗组,借助动物立体定向仪,将体外培养小鼠GL261胶质瘤细胞接种于小鼠颅内,治疗组于术后第2 w立体定位植入BCNU-PLGA缓释片剂,观察小鼠生存期,MRI了解瘤体变化.获取标本行HE染色,并行胶原纤维酸性蛋白(GFAP)、甲基鸟嘌呤DNA甲基转移酶(MGMT)免疫组化检查检测其表达情况,相关数据采用统计学方法进行分析.结果 治疗组与对照组生存期有明显差异(P＜0.05),治疗组与对照组凋亡MGMT无明显差异(P＞0.05).GFAP表达为阳性.结论 BCNU-PLGA局部缓释制剂可延长荷瘤小鼠生存期,且不改变其MGMT表达.