Abstract:目的建立稳定整合Tet-on基因的ES-D3细胞系,用于人胰岛素基因(pTRE2-human-Ins)在ES-D3细胞中诱导表达调控的研究。方法通过电穿孔转染的方法,将pTet-on质粒导入ES-D3细胞中,利用G418的药物选择特性,对转染的ES-D3细胞进行压力筛选,用PCR和Southern blot方法进行DNA整合鉴定,并用瞬时转染荧光素酶报告基因(pTRE-Luc)对筛选的Tet-on阳性克隆株的功能进行鉴定。结果经400μg/mL的G418压力筛选后,获得了11个细胞克隆株,特异性核苷酸引物检测细胞基因组DNA,有9个ES-D3细胞株可以扩增出相应的核苷酸片段,部分Tet-on阳性ES-D3株Southern blot鉴定结果表明Tet-on基因片段已整合入ES-D3细胞基因组DNA,荧光素酶报告基因功能鉴定获得1株诱导表达倍率为21.31的ES-D3细胞株,且Tet-on基因阳性ES-D3细胞的形态和生长速度与正常ES-D3细胞没有差异。结论通过电穿孔转染的方法成功地获得1株诱导表达倍率为21.31的高表达ES-D3细胞株,为进行目的基因(pTRE2-human-Ins)在ES-D3细胞中转染和诱导表达打下了基础。

Abstract:目的既往研究表明LRP16基因是一个雌激素反应基因,其表达水平与乳腺癌细胞增殖及侵袭密切相关。为对该基因进行生理功能研究,本文构建了针对小鼠LRP16基因的打靶载体,转染胚胎干细胞(embryonicstem cell,ES cells)并筛选同源重组克隆。方法PCR方法筛选129品系小鼠基因组文库中LRP16克隆,在第5外显子内插入SA-RIES-βgeo序列,构建插入失活型打靶载体并转染ES细胞,经G418筛选,挑取抗性克隆,Southern blot方法鉴定同源重组ES细胞克隆。结果将包含第5至11外显子的LRP16基因组片段亚克隆到pBluescript SKⅡ+载体,SA-IRES-βgeo序列的正确插入第五外显子中,打靶载体成功转染ES细胞,Southern blot结果显示具有一个打靶序列同源重组型插入的ES细胞克隆。结论成功构建了第五外显子插入失活型LRP16基因打靶载体并筛选到同源重组型ES细胞,为下一步建立LRP16缺失型小鼠并为从整体水平研究LRP16基因的生理功能奠定基础。

Abstract:目的建立一种稳定、持久的大鼠慢性高眼压模型,为青光眼视神经损伤及保护机制的研究提供基础资料。方法雄性SD大鼠(300±20 g)65只,分为改良实验组60只,经结扎上巩膜静脉联合术后球结膜下注射5-Fu;对照实验组5只,单独结扎上巩膜静脉。观察改良实验组模型建立后1周、4周、6周1、0周视网膜神经节细胞(retinal ganglion cells,RGCs)数量的变化情况。结果改良实验组可诱导较长时间(>10周)稳定高眼压,眼压升高1周4、周、6周、10周后,视网膜神经节细胞的存活率分别为:90.16%,83.50%,75.01%,62.37%;但对照实验组眼压升高仅维持2周左右。结论本模型具有操作简单,重复性好,成模率高,可稳定维持较长时间等优点,是较为理想的大鼠慢性高眼压模型。

Abstract:目的观察血液磁极化疗法(简称血磁治疗)对糖尿病(DM)的治疗效果和对胰岛功能的影响。方法制备了四氧嘧啶DM兔模型20只,随机分为两组,即DM模型血磁治疗组及DM模型未治疗组,同时设立正常对照组10只,分别测定治疗前后空腹血糖(FPG)、糖化血红蛋白(HbA1c)、胰岛素(INS)及C-肽(C-P),并于观察期结束后处死动物取胰腺组织常规石蜡包埋切片,行HE及Mallory三色法特殊染色观察3组胰岛形态学变化。结果血磁治疗显著降低DM兔的FPG(P<0.001)。DM治疗组INS及C-P水平较治疗前明显升高(P<0.01),与未治疗组相比差异显著(P<0.05)。胰岛病理改变可见治疗组β细胞数量明显多于DM未治疗组,提示血磁疗法具有促进胰岛修复,改善胰岛功能的作用。结论血磁疗法能够促进DM时受损胰岛组织的修复,改善胰岛β细胞分泌功能,降血糖作用明显。

Abstract:Objective The purity of DNA for microinjection is a crucial factor for preparing transgenic animals.The aim of this study is to develop an improved DNA purifying method for preparation of DNA fragments in high purity,feasible for routine transgenic animal research work.Methods DNA fragments containing lumbrokinase gene were extracted and purified by phenol-chloroform repeated extraction method and routine gel extraction kit.The purified DNA was microinjected into pro-nucleus of mice fertilized eggs and the injected living eggs were transplanted into the oviducts of pseudo-pregnant mice.The offspring after transplantation were identified by Southern blot.The two purifying methods were compared according to the results of transgenic experiments.Results Transgenic mice could be obtained with DNA purified by both two methods.There was no notable difference in DNA purity and viability of injected eggs,but higher birth rate of transplanted eggs and transgenic positive rate were obtained by the extraction method than those obtained by the kit method.Conclusion Our findings indicate that the extraction method can be used in purifying DNA for microinjection,replacing the kit method,with advantages such as lower cost,simplified experimental procedure,and improved transgenic positive rate in preparation of transgenic animals.

Abstract:Objective To explore the expression of myogenic regulatory factors MyoD and myogenin in DMD mouse model mdx mice at different age.Methods The gastrocnemius muscles of mdx and normal C57 mice at different age were collected and cut into frozen sections and stained by H.E.Pathologic changes of the muscles were examined by light microscopy.The expression of myogenic regulatory factors MyoD and myogenin were detected by SABC-DAB staining.Results The degree of muscle degeneration and regeneration was different in mdx mice at different ages.The expression of MyoD and myogenin was strongest in 1 month old mdx mice.The expression of MyoD and myogenin could be ever detected in 13 months old mdx mice and couldn't be detected in normal C57 mice at the same age.Conclusions MyoD and myogenin play a role in muscle regeneration after muscle injury.They can be used as an index to identify muscle precursor cells and reflect muscle regeneration.

Abstract:Objective To analyze the phylogenetic relationship among four groups of Microtus fortis by gene distribution characteristics of ten biochemical loci.Method Ten isozymes and proteins of Microtus fortis were investigated by cellulose acetate membrane electrophoresis.Result and Conclusion The electrophoretic patterns of the small voles from Heilongjiang province revealed great divergence in comparison with the other three groups.The small voles from Heilongjiang had their particular alleles at six loci and the phenotypes of them were completely different at three of these loci.The genetic distances among the voles from Hunan,Ningxia and the big voles from Heilongjiang ranged from 0.0633 to 0.2107,while that between the small voles from Heilongjiang and the other three groups ranged from 0.7068 to 0.8953.The phylogenetic tree constructed by UPGMA showed that the voles from Hunan,Ningxia and the big voles from Heilongjiang could be gathered into one group,but the small voles from Heilongjiang were in another group alone.Those results indicated that the phylogenetic relationship among the voles from Hunan,Ningxia and the big voles from Heilongjiang is closer than that between them and the small voles from Heilongjiang.

Abstract:Objective To establish a model of brain tumor by VX2 carcinoma implantation and observe the growth characteristics of the tumor.Methods VX2 tumor mass was implanted into the right parietal lobe brain 24 New Zealand rabbits.B-mode ultrasonography was proceeded to observe the growth and the volume of tumors.Animals were sacrificed at day 13, 17,19,21,23 and 25 after tumor implantation,respectively.Sections were cut for histopathological examination.The survival time and significant neurological alterations such as difficult feeding and hemiplegia were recorded.Results The median survival time of rabbits with implanted VX2 tumor was 24.5 days and the average was 24.8 days.The log-transformed plots of tumor volume approximate a straight line.17 days after tumor implantation,the VX2 tumors showed a brown coloration,appeared like fish and metastasized to the abdomen.The tumor cells infiltrated into the surrounding brain tissues and peri-tumor edema appeared since day 17 after tumor implantation.Conclusion Implantation of VX2 tumor into the rabbit brain is an useful model for human brain tumor research.

Abstract:目的制备视网膜中央静脉阻塞的动物模型,为视网膜中央静脉阻塞疾病的研究提供稳定的模型。方法采用氩激光直接光凝法封闭兔眼视网膜静脉建立视网膜静脉阻塞模型,利用HE染色法观察正常兔及造模后3周兔视网膜组织中视网膜新生血管的增生情况,利用免疫组化法观察正常兔及造模后3周兔视网膜组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,并检测造模前及造模后3周闪光视网膜电流图(flash electroretinogram,FERG)。结果兔视网膜静脉阻塞(retinal vein occlusion,RVO)眼可见明显的视网膜新生血管增生,缺血视网膜及新生血管组织中VEGF不同程度表达增强,FERG-b波的振幅明显下降(P<0.01)。结论采用氩激光直接光凝法建立视网膜静脉阻塞模型是成功的。

Abstract:Objective To extend the survival time of diabetic rat in order to observe the formation and development of diabetic retinopathy(DR).Method 70 SD rats were divided randomly into control group(n = 20) and DR group(n = 50).Streptozotocin(STZ) in a dose of 60 mg/kg was injected into peritoneal cavity to induce diabetes mellitus.DR group rats were killed at 6~(th),9~(th) and 12~(th) month,respectively.Retina microvasculature was digested,stretched,PAS stained and used to examine the DR microvascular alteration.Result Diversified lesions occurred with the progressing of survival time.The most serious arteriolosclerosis occurred at 12~(th) month.Conclusion The prolongation of survival time of diabetic rats is beneficial for investigation of the progression of diabetic retinopathy.

Abstract:目的探索大鼠左冠状动脉前降支不同结扎处理后,对心肌形态学及心功能的影响,以建立适合移植干细胞再生修复心肌梗死研究的稳定、可靠和更合乎发病机制的动物模型。方法雄性SD大鼠70只,随机分为五组。即:结扎(15、30、456、0 min)再灌、结扎非再灌。于处理后1 d、1周2、周或4周动态观察心肌梗死变化,并于处理一月后测量动脉收缩压(ASP)、动脉舒张压(ADP),左室收缩压(LVSP),左室舒张末压(LVEDP)及左室压力上升及下降最大速度(±dp/dtmax)。结果引起明显的心肌梗死至少需要结扎30 min。结扎(456、0 min)再灌、结扎非再灌的心肌梗死明显,并观察到梗死区域心肌已绝大部分纤维化,且梗死面积变化较恒定。同时测定不同结扎时间心功能的变化发现,结扎(456、0 min)再灌或结扎非再灌各组ASP、DAP、LVSP、±dp/dtmax显著下降,LVEDP明显升高。并见不同结扎时间处理后,大鼠心功能的变化与心肌梗死后的梗死面积变化密切相关。结论建立了实验大鼠左冠状动脉前降支中上1/3处结扎45 min以上的大鼠心肌梗死模型。不仅合乎临床心肌梗死的发病机制,而且梗死部位、梗死区域面积稳定,适合于移植细胞再生修复心肌梗死的研究。

Abstract:目的研究两种金属催化剂Ag/Al2O3和Cu/Al2O3对SARS冠状病毒和其他呼吸道疾病传播的阻断作用。方法100μL 106TCID50/mL SARS-CoV,100μL 106PFU/mL。表达重组仓鼠朊蛋白的杆状病毒和106大肠杆菌分别缓慢地滴加至两种圆形金属催化剂的表面,吸附5~20 min。使用不同培养液洗涤金属催化剂表面,检测洗涤物的病毒感染性和细菌的增殖。结果病毒和细菌分别吸附5和20 min后,SARS-CoV和杆状病毒的病毒滴度下降到非常低的水平,甚至难以检测到;大肠杆菌组经培养后无克隆生长。5 min吸附后,杆状病毒表达的重组PrP的表达下降到21.8%;20 min吸附后,没有PrP的表达。电镜检测发现大肠杆菌的细胞壁崩解,细胞破碎。结论与金属催化剂Ag/Al2O3和Cu/Al2O3表面接触后,破坏了SARS-CoV,杆状病毒和大肠杆菌的复制增殖能力。两种金属催化剂利用空气中的氧分子有效的杀灭吸附于其表面的病毒和细菌,有希望用来作为医院、家庭和公共场所的空气消毒。

Abstract:目的建立家兔子宫内膜炎模型,观察模型的临床病理学变化,为家畜子宫内膜炎的诊断和防治研究提供材料。方法造模通过子宫灌注病原菌的方法,临床病理学观察通过临床症状、体温、血常规、子宫分泌物和尿液检查、子宫剖检、子宫内膜的显微和超微结构观察。结果模型兔精神变差、采食量减少,阴门肿胀,流出脓性分泌物;每次灌注细菌后8 h内体温升高0.5℃左右,血液中性粒细胞的比例升高;子宫分泌物中混有大量白细胞、脓球和少量脱落的子宫内膜上皮细胞;子宫显著肿大,子宫内膜溃疡和出血,上皮结构不完整,细胞变性、坏死,微绒毛和纤毛脱落,上皮下出血、瘀血、水肿和炎性细胞浸润。结论通过子宫灌注病原菌的方法可以成功制备家兔子宫内膜炎模型,模型的临床病理变化以局部炎性为主。

Abstract:Objective To establish a rapid hyperlipidemic gerbil model.Methods Ten rats,ten mice and ten gerbils were fed with semipurified diet containing 1% cholesterol.Serum total cholesterol,triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in the rats of the three groups were measured at the end of first,second,third and the fourth week and the data were compared among the 3 groups.Results Serum TC,LDL-c and HDL-c in gerbils reached a stable high level in the first week and were kept up to the fourth week,however,serum TG did not change significantly.Serum TC,TG,LDL-c and HDL-c in rats and mice were not changed.Conclusion Rapid hyperlipidemic model can be created in gerbils.

Abstract:Objective To explore the cataract development and its influence on the living status of BY-F swordtail fish.Methods To notice the development of opaque appearance in the eyes of swordtail fish and to examine the external appearance and behavior changes of the blind fish.The eyeballs were taken at different development stage of the disorder and examined by histopathology.Results The blind fish often became dark-colored.Varying opacities were seen in the fish eyes.Red hyperplastic lesions covering the cornea surface were found in some blind fishes.Most pathological changes were found in the lens.Conclusion The blind fish were suffering from cataract.The cataract can cause other eye complications in BY-F swordtail fish.The BY-F swordtail fish are susceptible to develop cataract.

Abstract:Objective To study the pathological changes of relapsing-remitting experimental allergic encephalomyelitis(EAE) in Wistar rat.Method\ Tissue sections were processed with HE staining,Weil myelin staining and modified Bielschowsky staining.The expression and distribution of MMP-2 and-9 were detected with the method of IHC.Result There were many perivascular cuffings in tissues accompanied by large pieces of demyelination and inflammatory cells infiltrating into partial demyelinated tissues,axons with some vesicular loss,strong postive GFAP around the old lesion,and MMP-2 and-9 were expressed in vascular endothelial cells,meninges,ECM and inflammatory cells.Conclusion(1) Pathological changes of relapsing-remitting EAE in rat were in coincidence with the clinical manifestations of patients and there were active and inactive lesions in the tissues.(2) MMP-2 and-9 were expressed in the active lesions and they participated in the pathogenesis.(3) Pathological changes of relapsing-remitting EAE were similar to multiple sclerosis and such rats are an ideal animal model.

Abstract:Objective Several methods of preparing heart failure animal model have been established.Since animal heart failure induced by continuously rapid pacing much resembles human being heart failure in the hemodynamic,neurohormonal and pathological changes.Moreover,pigs hold many common features with humans in physiologic function and anatomic structure of circulatory system.Therefore,this rapid pacing induced heart failure pig model is an ideal model and more suitable for research work.This article reviews recent advances in research on preparation of heart failure pig model induced by rapid pacing.

Abstract:With the development of transmitochondrial technique,we have successfully establisheda a transmitochondrial mouse model carrying mutated mitochondrial DNA.Now there are three methods to produce transmitochondrial mice.One is direct mitochondrial microinjection.The second is by fusing cytoplasts produced by enucleation of cells(donor) derived from patients or controls with undifferentiated female mouse embryonic stem cells and then injecting the embryonic stem cells into blastocysts.The third is direct by fusing cytoplasts with one-cell mouse embryo.With the discovery of a number of mitochondrial related diseases,tramsmitochondrial mice containing human mitochondrial disorders will have a great value to investigate mtDNA mutation and mitochondrial defect pathogenesis,evaluate gene therapy of diseases caused by these mutations and screen drugs of treating mitochondrial diseases.

Abstract:It is generally accepted that dendritic cells are the most powerful professional antigen-presenting cells in vivo.They play a major role in the induction of primary immune responses.The most important feature of those cells is to activate naive T cells to proliferate dramatically.In recent years,rapid progress in research on murine dendritic cells has been acquired.This article will review the significant advances in understanding the relationship between murine dendritic cell subsets and T cells.