Abstract:Objective To establish a rat diabetic model closely resembling the metabolic abnormalities of human type 2 diabetes mellitus and observe its renal and arterial structural changes.Methods 8 weeks old SD rats were injected with low dose streptozotocin(STZ) after high-fat and high-sugar diet given for 4 weeks.The changes of vascular function and renal function indexes were assessed after 4 and 8 weeks,respectively,and the pathological changes of kidney and arteries were examined.Results The model group showed hyperglycemia,hyperlipidemia,insulin resistance,renal and vascular disfunctions.The renal pathological examination revealed hyperplasia of mesangial and endothelial cells,swelling of renal tubules,presence of numerous protein and cellular casts in the renal tubules,and a prominent leukomonocyte infiltration in the renal pelvic tissue.Loss or damages of endothelial cells and increase of the sub-endothelial space in the artery wall were observed by transmission electron microscopy.Conclusion A rat model of type 2 diabetes mellitus has been successfully established by using high fat and sugar diet and low dose STZ injection.Renal and vascular pathological changes occurred in the rats within 4 and 8 weeks after treatment.The established rat models provide an ideal basis for experimental studies on type 2 diabetes mellitus.

Abstract:目的以转hMan2c1基因小鼠为模型,分析hMan2c1基因在转基因小鼠脾脏的蛋白表达和活性,研究hMan2c1基因对于机体免疫系统的影响。方法Western blot方法检测hMan2c1基因在脾脏的蛋白表达并检测小鼠脾脏α-甘露糖苷酶活性;血常规分析外周血中各种血细胞的比例;以BSA作为抗原观察机体的免疫应答;流式细胞技术观察外周血中CD4 、CD8 、B、NK细胞的数量。结果Western blot结果显示,与野生型小鼠比较,hMan2c1基因在转基因小鼠脾脏组织有明显的表达,α-甘露糖苷酶活性明显增高,中性粒细胞明显升高。BSA刺激后,转基因小鼠外周血免疫细胞中的CD8 T淋巴细胞明显高于野生型小鼠。结论hMan2c1基因在小鼠脾脏表达引起α-甘露糖苷酶活性显著升高,并进一步影响淋巴细胞的生成,增加中性粒细胞和CD8 T淋巴细胞对免疫原的应答。

Abstract:Objective It is first and foremost to genotype and determine the transgene copy number for making a transgenic model.Southern blot analysis,the traditional means,is time consuming and laborious.The aim of this study was to built up a novel method to determine transgene copy number by using the absolute quantitative real-time PCR.Methods Taken TG-CARK founder mice as examples,the transgene copy numbers in transgenic mice were determined by quantitative real-time PCR and Southern blot.The conserved mouse housekeeping gene,Fabpi,served as an internal control to calculate transgene copy number.Results The copy numbers of transgenic founders estimated by real-time quantitative PCR were highly correlated with actual copy number based on Southern blot assay.The three TG-CARK founders had the transgene copies of 1,7,45 respectively.Conclusion Real-time PCR is a more efficient and convenient method for estimating copy number in transgenic mice than Southern blot.

Abstract:目的探讨成年小鼠心肌细胞分离培养的方法,评价心肌细胞的存活状况。方法应用改良Langendorff方法进行成年小鼠心脏灌流,分离并培养心肌细胞,评价杆状心肌细胞的比例变化。台盼蓝染色判定心肌细胞活力,应用毒胡萝卜素(thapsigargin)治疗心肌细胞24 h后,WST方法测定细胞生存率。结果在每个成年小鼠心脏分离了40~60万的心肌细胞,台盼蓝染色结果68%为存活心肌细胞。心肌细胞培养1 h2、4 h及48 h后,杆状心肌细胞的比例分别为70%、50%及30%。心肌细胞生存率随着毒胡萝卜素浓度的增加逐渐减低,其中1、5及10μmol/L毒胡萝卜素的生存率明显低于对照组(P<0.05,P<0.01)。结论应用改良Langendorff方法及严格控制心脏灌流培养的条件,可以成功地分离培养成年小鼠心肌细胞,有助于进行细胞分子水平的研究。

Abstract:Objective To establish rabbit models of diabetic and non-diabetic atherosclerosis and to study characteristics of their plaques in the aorta.Methods Diabetes was induced by alloxan injection,atherosclerosis was induced by balloon injury of the abdominal aorta and high lipid feeding for ten weeks(Non-diabetic atherosclerotic rabbits received physiological saline injection instead of alloxan).Angiography and intravascular ultrasound(IVUS) of the abdominal aorta were performed to evaluate plaque morphology.Transverse sections of the aorta were taken to perform H&E staining and immunohistochemistry,through which thickness of the intima,contents of macrophages and smooth muscle cells in the intima were examined.Results Atherosclerosis developed more seriously in abdominal than in thoracic aorta in all rabbits.Significantly more macrophages and fewer smooth muscle cells were observed in plaques of diabetic abdominal aorta,especially near the aortic lumen,than in non-diabetic rabbits.Conclusion Rabbit models of diabetic and non-diabetic atherosclerosis have been successfully established.More active inflammation was present in diabetic than in non-diabetic atherosclerotic plaques,indicating a more unstable nature of the diabetic atherosclerosis.

Abstract:Objective To explore the possibility of use of IVF-20 medium in rat in vitro fertilization(IVF) experiments,and provide a practical reference for embryo conservation.Methods Using IVF-20 as capacitation and fertilization medium,four strains of rats,including SD,Wistar,GK and F344 were chosen for the IVF experiment.The resulting IVF embryos were cultured in vitro with mR1EMC medium.Besides,some of 2-cell embryos were transferred into the oviducts of recipients after induction of pseudopregnancy.Results The cleavage rates of SD,Wistar,GK and F344 rats were 83.2%,72.6%,87.8% and 71.6%,respectively.The blastocyst development rate in SD rat was 16.7%,lower but with no significant difference than that(24.5%) of embryos produced in vivo.Both recipients transferred with embryos from IVF established pregnancy,and delivered 10 pups(12.5%).Conclusion IVF-20 medium used in human in vitro fertilization is also suitable for in vitro capacitation and fertilization in rats,resulting in good cleavage rate,as well as blastocyst development rate,and the 2-cell embryos from IVF can fulfill the complete development after transplantation and give birth to normal pups.

Abstract:Objective To explore the correlation among site of left anterior descending artery(LAD) ligation,infarct size and left ventricular function in establishing myocardial infarction model of swine.Methods LAD was surgically occluded at different percentage point of LAD varying from 30% to 70% in forty-seven mini-swine.Cardiac function was examined by left ventricular ejection fraction(LVEF) using echocardiography before and 1 hour after myocardial infarction(MI).Coronary angiography was performed for confirming the position of ligation after MI and the ischemic area was examined at the 4th week after MI.Linear regression analysis was used to analyse the correlation between LAD ligation point and infarct size or ventricular function. Results Thirty-nine of the 47 pigs survived from ventricular fibrillation.The correlation between percentage point of LAD ligation(x) and infarct size or ventricular function(y) was: y = 0.53x-5.43,r = 0.90(P<0.001) for infarct size(%);y = 65.88-0.55x,r = 0.87(P<0.001) for LVEF post-MI(%);y = 0.12 0.59x,r = 0.78(P<0.001) for decreased LVEF post-MI.Conclusion There is a significant correlation among LAD ligation site,infarct size,and LVEF,which makes it reasonable to determine the ligation site of LAD according to different study purposes in establishing MI model of swine.

Abstract:Objective To study the distribution and excretion of lovastatin in Bama minipigs.Methods The concentration of lovastatin in tissues and body-fluid in 6-month old male Bama minipigs after oral administration were detected by RP-HPLC.The serum protein binding rate in pigs was determined by dialysis method.Results Lovastatin was showed to be widely distributed in many organs after i.g.administration of 45 mg/kg.The higher level of lovastatin was found in the stomach,intestine and liver.The concentrations in stomach,small intestine,liver,heart,adrenal and bladder at 1 h were higher than those at 4 h.7.4% of the given dose was excreted in unchanged form in urine within 96 h.More than 80% was excreted in bile and feces within 96 h.The plasma protein binding ratio was higher than 95%.Conclusion It could be concluded that distribution,excretion and the plasma protein binding ratio of lovastatin in the Bama minipigs are similar to those in humans.

Abstract:Objective To establish an animal model sensitive to SARS-CoV and study the pathogenesis of the disease.Methods Using RT-PCR and immunofluorescence assay(IFA) after virus islation and inoculation, SARS-CoV was examined in the lungs of young and aged ICR mice challenged by SARS-CoV.Pathological changes in the lungs and other organs of the two groups were examinined.The replication sites of virus in the lungs were analyzed by immunohistochemistry staining.Results Aged mice were more susceptible to SARS-CoV than young mice do.More severe diffuse alveolar damages were observed in aged mice.In addition,damages were observed in other organs of two aged mice such as degeneration,focal necrosis and vein dilation and congestion in liver,kidney and brain.Virus antigen was detected in alveolar epithelial and vascular endothelial cells.Conclusion Aged ICR mice may be used as a susceptible animal model for SARS-CoV in studies of mechanisms of SARS and evaluation of the effect of antiviral drugs and vaccines.

Abstract:Objective To analyze the characteristics of multifocal electroretinogram(mfERG) of healthy pigmented rabbit eyes, and assess their retinal function.Methods MfERG were performed in 18 eyes of 9 rabbits,and compared with those of 18 human eyes. Results In mfERG the macular peak arose in three-dimensional picture,composed of P1-wave amplitude density,but is lower than that of humans.In other parts of the retina,the slope composed of higher density in the temporal area and lower in the nasal area.The P1-wave amplitude density, P1-wave amplitude value and N1-wave amplitude value in temporal retina of mfERG are higher than those in nasal retina in the rabbit eyes,and also higher than those in temporal retina in human eyes.In 5 rings mfERG of rabbit eyes,P1-wave amplitude density in the first ring was the highest and declines from the first ring to 5th ring gradually.P1-wave and N1-wave amplitude values in 4th ring and 5th ring were the highest.P1-wave amplitude density and P1-wave amplitude value in the first ring were lower than those in human eyes,but higher in 4th and 5th rings.Conclusion The function of temporal retina of rabbit eyes is superior to that of nasal retina.The rabbit retinal function of central macula is inferior to human one,but the function of para-macular area is superior to that of human eyes.

Abstract:Objective To study the expression and cloning of IL-10 gene in Chinese white rabbits and prepare the polyclonal antibody against recombinant IL-10.Methods The Chinese white rabbit IL-10 gene was amplified by RT-PCR method from the PMBCr stimulated by ConA,and then cloned,sequenced and analysed.The IL-10 gene was subcloned into prokaryotic expressing vector pET28a for expression in E.coli.Results The complete length of white rabbit IL-10 gene was 537 bp,encoding 178 amino acids. The IL-10 of Chinese white rabbit shared higher identities with that of European rabbit,but differed significantly from IL-10 genes of mouse,chicken and zebrafish.After the induction of IPTG,the expressed recombinant IL-10 protein,with a molecular weight of 23.4 kDa,was successfully detected by SDS-PAGE and confirmed by Western blot assay,which amounted to 15.2% in the total protein of the induced bacteria.Conclusion IL-10 has been successfully expressed in E.coli and mouse anti-rabbit IL-10 polyclonal antibody has been prepared by immunization with the purified recombinant IL-10 protein.

Abstract:Objective To investigate the genetic diversity of the original population of Z:ZCLA Mongolian gerbils by biochemical markers.Methods 19 biochemical loci as markers were used to detect the genetic variation in the conventional closed colony of Z:ZCLA Mongolian gerbils.Results The loci Es-1,Car-2,Hbb,Gpi-1,Cs-1,Ce-2,Idh-l,Mod-1 had only one allele,while the loci Es-2,Es-3,Es-4,Es-6,Es-8,Es-9,Es-10,Gpd-1,Pgm-1,Trf,Akp-1 had two to four alleles,respectively.The average heterozygosity,allele number and polymorphism information content were recorded as 0.512,3.0 and 0.455 respectively.Conclusion The analysis of polymorphism information content on 11 biochemical gene marker loci of this conventional closed colony of Z:ZCLA Mongolian gerbils revealed the genetic diversity at a moderate level.

Abstract:Objective In order to improve the preparation efficiency of mouse dormant embryos,the normal superovulation combined with anti-PMSG serum(A-PMSG) was applied to collect the dormant ones.Methods According to the different doses of A-PMSG injected into the mouse after injection of PMSG,the mice were divided into five groups: 1) injecting A-PMSG before mating,2) injecting A-PMSG after finding a vaginal plug,3) injecting A-PMSG after ovariectomy in the morning of day 4(day1= vaginal plug),4) superovulating without A-PMSG,and 5) the control one.Results The average number of recovered embryos(9.4) of the group injected with the A-PMSG after finding a vaginal plug,is significantly higher than the other groups(P<0.01).Conclusion Superovulation combined with anti-PMSG serum can improve the collecting efficiency of murine dormant embryos.

Abstract:Objective To identify the 17 helicobacter isolates isolated from laboratory rodent animals to obtain a strain which possesses typical biological and genetical characteristics as a type strain.Methods To identify the isolates to species level by means of biological characteristics,16S rRNA sequence analysis,phylogenetic tree and ultrastructure analysis.Results the isolates can be classified into two species: one was Helicobacter bilis,another was Helicobacter sp. Conclusions the isolates are Helicobacter bilis and Helicobacter sp.not identified at species level.The 16S rRNA sequences of Helicobacter bilis has 99.6% similarity to the type strain ATCC 51630.Helicobacter bilis can be used as a type strain for surveillance.

Abstract:Objective To establish an experimental model to study the stressralated changes of intestinal mucosa barrier(IMB) following serious traumatic brain injury(TBI) Methods Sixty-four healthy male Wistar rats were randomized into two groups.TBI model group(n=32) treated by TBI by Feeney's method,and control group(n=32)treated by sham operation.Each group was divided into four subgroups taken at 6 h,12 h,24 h,and 48 h after acute brain trauma(n=8,for each subgroup).The ileum tissue was carefully examed by light and electron microscopy.Results The intestinal mucosa was damaged and the intercellular space of intestinal mucosal epithelial cells was increased in TBI rats compared with that of control group.Conclusion This experimental model can be used to study the stress-related changes of IMB after serious TBI.

Abstract:目的探讨自制心包穿刺装置转染心脏的安全性、可行性。方法应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ),将12头中国小型猪分为实验组和对照组,采用球囊堵塞前降支第一对角支远端,心肌梗死模型建立后即刻,采用自制简易心包腔穿刺装置经皮剑突下穿刺,成功后置中心静脉导管于心包腔内并行转染,28 d后处死。实验组:胶原酶1200 U及透明质酸酶3000 U预处理心包后,在心包腔内注射Ad-LacZ基因2.0×109p.f.u;对照组:同样方法预处理心包后,在心包腔内注射生理盐水1 mL。于注射后3、7及28 d分别对缺血心肌进行染色及病理观察。结果冠状动脉造影证实前降支远端完全闭塞,病理显示心肌有缺血和梗死;实验组注射Ad-LacZ基因后第3、第7天及28d后X-gal染色有阳性细胞,以第7天最明显,对照组无阳性细胞。结论自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的,可行的,并且腺病毒可持续表达4周。

Abstract:目的建立一套动物模型检测方案,用于筛选和研究对人类和动物早期妊娠有影响的环境卤代芳烃类污染物。方法利用TCDD作为初始研究标准化合物,以NIH小鼠建立动物早期妊娠模型评估方案,包括剂量-反应评估(DRE)、着床前后毒性比较(PPP)、子宫蜕膜细胞反应(DCR)和胚胎转移速率(ETR)分析。结果DRE发现TCDD剂量依赖性地引起了第9天胚胎数量的显著减少。PPP比较发现100 ng/(kg.d)对胚胎着床前期影响显著大于着床后期;胚胎重量评估发现,TCDD在妊娠早期的不同时间给药均影响了胚胎的发育,造成发育迟缓,重量减轻。DCR检测发现TCDD处理显著抑制(P<0.01)假孕小鼠子宫蜕膜。ETR分析发现TCDD未影响胚胎在输卵管中的转移速度,但造成分裂卵发育迟缓,着床前分裂卵丢失(P<0.05)。对出生前胚胎成活率观察发现,TCDD可造成出生前胎儿死亡,胎儿成活率低于妊娠中期的胚胎成活率。结论TCDD对着床前胚胎的毒性明显大于着床后,并具有持续毒性的特点。其机理涉及抑制子宫蜕膜细胞反应、造成着床前分裂卵的丢失或发育的不同步。

Abstract:目的建立乳鼠成骨细胞体外培养方法,探讨该方法的可行性和应用价值。方法用出生1~3 d乳鼠颅骨,采用多次胶原酶消化法进行细胞体外培养。倒置显微镜观察细胞形态,对其碱性磷酸酶(ALP)活性及矿化能力进行鉴定,并测定细胞生长曲线。结果原代培养24 h后,大量细胞贴壁生长,细胞呈圆形,48 h后,贴壁细胞呈长梭形、三角形或不规则多边形,并且贴壁细胞伸出2~3个突起,胞质透亮、饱满,7 d后细胞铺满整个平皿底面。经鉴定,培养细胞具有体内成骨细胞的生物学特性。细胞接种后第1与第2个24 h为细胞的潜伏适应期,第3与第7个24 h生长曲线基本为线性曲线,是细胞的对数生长期。结论采用胶原酶消化法分离培养成骨细胞的方法切实可行。

Abstract:Animal models of breast cancer are playing an vital role on studying biological behavior and treatment of human breast cancers.According to the method and destination,the animal models of breast cancer include spontaneous,induced,transplanted,transgenic models and bone metastasis models.Each kind has respective characteristics and applicative condition.

Abstract:Chronic obstructive pulmonary disease(COPD) is an important disease of respiratory system.It is characterized by chronic emphysema and(or) bronchitis,combined with airway obstruction.COPD causes high morbidity and mortality and costs much medical expenditure every year globally.The development of studies on COPD presented stagnation because of its complicated pathogenesis and clinical progression.Researchers had begun to focus their research on the pathogenesis,clinical course and pathological alterations by establishing animal models.This review is to sum up the methods of how to establish and evaluate the animal models of COPD,and advances and some new opinions about the animal selection and evaluation.