神经组织特异表达CTF1转基因小鼠的建立

目的建立神经组织特异表达CTF1的转基因模型小鼠,为研究CTF1生物学功能及与老年痴呆等疾病发病机制的关系提供工具动物。方法把CTF1基因插入神经组织特异的启动子PDGF下游,构建转基因表达载体,显微注射法建立C57BL/6J CTF1转基因小鼠。PCR鉴定转基因小鼠基因型,采用Western Blot方法鉴定CTF1在脑组织中的表达,对转基因小鼠脑组织进行石蜡切片,HE染色,显微镜观察组织结构形态的改变。结果建立了2个不同表达水平的CTF1转基因小鼠品系。转入的CTF1基因在脑组织的表达水平均高于同龄对照小鼠。组织学分析显示CTF1转基因小鼠大小脑组织基本结构形态未见异常。结论成功建立了稳定遗传的神经组织特异表达CTF1转基因小鼠品系,为CTF1的生物学功能及与老年痴呆等疾病发病机制关系的研究提供了有力的模型工具。     

转基因肺癌动物模型研究进展

肺癌是世界各国常见的恶性肿瘤,肺癌动物模型在人类肺癌病因、诊断、治疗等方面发挥重要的作用。其中转基因肺癌模型能够更好的模拟肺癌,更有利于肺癌病因及发病过程的研究。本文介绍了近年来转基因肺癌模型的研究进展。     

HBV转基因小鼠的舌色改变及血流变化

目的:研究HBV转基因小鼠的舌色表现和微血管血流变化.方法:10只C57BL/6J-HBV转基因小鼠和10只正常小鼠(C57BL/6J非转基因小鼠),均雌雄各半,采用小鼠舌象观察装置对小鼠舌色进行观察;使用激光多普勒血流仪进行舌和肝脏微血管血流检测;断头采血,测定血清丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)的含量;HE染色观察肝和舌组织病理变化.结果:HBV转基因小鼠中,6只小鼠舌色呈紫色,4只舌色呈暗红色,而正常小鼠的舌色均为淡红色.HBV转基因小鼠舌色色调(H值)变深,有显著性差异(P<0.01);舌色亮度(V值)变弱,有极显著性差异(P<0.001).和正常鼠相比,紫舌HBV转基因小鼠微血管血流灌注量和血流速度显著降低(0.206±0.13vs0.794±0.13;0.479±0.07vs0.331±0.04,P<0.01,P<0.001),该模型肝脏微血管血流灌注量和血流速度显著降低,HBV转基因小鼠肝脏和舌存在明显的炎性改变.结论:HBV转基因小鼠舌色以紫色多见,其形成机制与微循环障碍有关.该模型存在肝脏和舌的炎症病理,提示炎性微环境是形成其肝脏和舌微循环障碍的重要原因.     

破骨细胞转基因永生化问题的初步探讨

目的：通过转基因技术建立破骨细胞永生化细胞系，方法：经1，25（OH）2D3诱导，获得小鼠骨髓来源的破骨前体细胞，脂质体法（Fugene6）将猿猴病毒40（SV40）和绿色荧光蛋白（GFP）质粒分别转染入破抽前体细胞，G418筛选抗性克隆；同时将GFP转染入逆转录病毒包装细胞（P167）中，作为方法对照，继而，用含有GFP的逆转录病毒感染破骨前体细胞，G418筛选抗性克隆。结果：获得小鼠骨髓来源的破骨前体细胞，Fugene6转染SV40和GFP到破骨前体细胞后，G418筛选未获得阳性克隆，但GFP转染PT67细胞获得阳性克隆和含有GFP的逆转录病毒。将含有GFP的逆转录病毒感染破骨前体细胞，G418筛选未获得阳性克隆，结论：通过破骨细胞转基因永生化，建立破骨或破骨前体系细胞系的方法是一个非常有意义的研究课题，但是难度较大。可以初步认为：脂质体和逆转录病毒载体的方法不是破骨细胞转基因的最佳方法。     

κB抑制蛋白基因对血管内支架植入术后内膜增生抑制作用的实验研究

目的　观察血管内局部转移腺病毒携带的IκBα基因对兔髂动脉内支架植入术后内膜增生的抑制作用。方法　 12只杂种新西兰大白兔 ,2 4支髂动脉 ,随机 (计算机 )分为转基因组、磷酸盐缓冲液 (PBS)对照组和空白对照组 (各组均为 8支髂动脉 )。转基因组髂动脉于支架植入术后 ,经多隧道球囊导管输送腺病毒携带的IκBα基因行局部转基因治疗 ;PBS对照组于髂动脉支架植入术后 ,局部注射PBS ;空白对照组则仅行髂动脉内支架植入术。分别于术后 1周和 4周 6只兔重复髂动脉造影 ,之后再处死动物 ,取支架植入处动脉作病理检查 ,分近、中、远 3段 ,测量新生内膜截面积等。结果术后 1周和 4周造影显示各组的管腔内径间差异均无显著性意义 (F =0 .0 5 ,2 .71;P >0 0 5 ) ,但术后4周 ,各组新生内膜截面积分别为 (2 2 8± 0 14 )mm2 ,(3 2 6± 0 2 5 )mm2 ,(2 80± 0 2 0 )mm2 ,转基因组明显小于对照组 (F =5 .0 7,P <0 0 5 )。结论　血管内局部转移腺病毒携带的IκBα基因可以抑制兔髂动脉内支架植入术后的内膜增生 ,从而可能降低支架植入术后再狭窄的发生率     

研究生实验动物学实验课的教学体会

实验动物学吸纳了生物学、医学、兽医学、遗传学等诸多学科的知识,是一门具有很强的综合性和交叉性的学科。人们普遍认为实验动物是生物医学乃至整个生命科学研究的重要支撑条件之一,在新技术革命蓬勃发展的当今世界中占有重要地位,因而也受到了各个医学院校的高度重视,成为许多学校本科生的选修课和研究生的必修课。实验动物学实验课作为理论知识的验证和补充,也受到学校和学生的高度重视。但由于师资、经费和教学条件的限制,多数学校只能选择性地面向研究生开设实验课。我系对研究生开设实验动物学实验课程已10年有余,对于普及实验动物学的有关知识和研究生的动手能力起到了一定的推动作用。通过长期的实验课教学,作者的有关认识是:     

Murine models for the study of congestive heart failure: Implications for understanding molecular mechanisms and for drug discovery

Congestive heart failure (CHF) is a complex illness of diverse aetiology. Despite the current multiple therapies, the prognosis for CHF patients remains poor, and new therapeutic targets need to be identified. With the advent of the genetic era, the mouse has become an increasingly valuable animal species in experimental CHF research. A large number of murine models of cardiac hypertrophy and CHF have been created by genetic engineering. Meanwhile, traditional CHF models created by coronary artery ligation, cardiac pressure, or volume overload have been adapted to mice. The present review categorises and highlights the value of these murine models of cardiac hypertrophy and CHF. These models, combined with sophisticated physiological measurements of cardiac haemodynamics, are expected to yield more and valuable information regarding the molecular mechanisms of CHF and aid in the discovery of novel therapeutic targets.     

Inactivation of transgenes in Phytophthora infestans is not associated with their deletion,methylation, or mutation

The mitotic and meiotic stabilities of transgenes were evaluated in the oomycete, Phytophthora infestans. Genes encoding -glucuronidase (GUS), neomycin phosphotransferase (NPT) and hygromycin phosphotransferase (HPT), fused to one of six promoters from P. infestans or other oomycetes, were usually stably expressed during continued asexual culture and transmitted to progeny. However, the activity of these genes became undetectable in many strains during asexual or sexual propagation. Over 33 months of growth, transgene expression stopped each month in 1–3% of the transformants. Silencing of the genes was not associated with their deletion, mutation, or hypermethylation. The conformation of the integrated sequences was similar in strains destined to continue or terminate expression of the transgenes. Expression of the genes was not associated with a loss of fitness during growth in vitro and in planta, which might otherwise have selected for silencing events.     

Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13 (1993) 2942-2951; J. Sap, Y.-P. Jiang, D. Friedlander, M. Grumet, J. Schlessinger, Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding, Mol. Cell. Biol. 14 (1994) 1-9]. The homology of RPTP-kappa's ectodomain to neural cell adhesion molecules indicates potential roles in developmental processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene, the consequent loss of RPTP-kappa's enzymatic activity does not produce any obvious phenotypic defects [W.C. Skarnes, J.E. Moss, S.M. Hurtley, R.S.P. Beddington, Capturing genes encoding membrane and secreted proteins important for mouse development, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6592-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells throughout the CNS that display RPTP-kappa promoter activity. Several neural systems highly expressed the transgene-most notably cortical, olfactory, hippocampal, hypothalamic, amygdaloid and visual structures. These well-characterized brain regions may provide a basis for future studies of RPTP-kappa function.     

小鼠受精卵显微注射影响因素的体外研究

目的:利用体外培养探讨受精卵显微注射的影响因素的作用。方法:获取小鼠受精卵,在其他条件固定的情况下,分别比较不同的注射基因浓度,在CZB(加HEPES)操作液中显微操作时间以及注射前受精卵的质量对显微注射后的胚胎体外培养结果的影响。结果:基因浓度越高,受精卵发育越差,操作时间不同的卵发育无明显差异,质量好的受精卵发育较好,质量差的几乎不发育。结论:选用较低的基因浓度,良好的操作介质CZB(加HEPES),使用质量好的受精卵,会提高显微注射的效率。