Abstract:目的探讨心脏近端流出道隔的融合和心肌化过程及其机制。方法选用胚胎(embryonicday,ED)11.5至出生后1dC57BL6小鼠(简称C57小鼠)。采用免疫组化法测定横纹肌肌动蛋白αSCA,神经嵴细胞的标记物AP2及HNK1,凋亡相关分子activecaspase3的表达;采用TUNEL法原位检测细胞的凋亡程度。结果①近端流出道隔的融合及心肌化的时空模式:近端流出道心内膜垫于ED11.5开始融合,ED13.5基本完成融合;心肌化约从ED12.5开始,至ED15.5基本完成。整个心肌化过程呈现心肌从流出道隔由外向内的内向性生长趋势。②近端流出道隔HNK1的表达:从ED11.5到生后1d近端流出道隔极少或几乎无HNK1表达。③近端流出道隔AP2的表达:ED11.5几乎无表达,ED12.5、ED13.5可见少量表达,从ED14.5以后消失。④近端流出道隔细胞的凋亡:TUNEL和caspase3的检测均表明近端流出道隔的凋亡集中出现在ED12.5和ED13.5,一部分凋亡细胞可能为AP2阳性的神经嵴源性细胞。结论C57小鼠心脏近端流出道隔在胚胎发育早中期开始融合,并逐渐完成心肌化过程,心脏神经嵴细胞可能通过凋亡途径参与了该过程。

Abstract:目的建立两种激光光凝的恒河猴慢性高眼压性青光眼模型,评价模型眼的相关生物学特性。方法成年恒河猴15只,分别采用半导体倍频532激光和氩激光,在房角镜下对功能小梁网区行360°光凝。对其中7只恒河猴分别采用A超、视网膜断层成像仪和视网膜血流仪进行模型眼和另侧对照眼的眼球及视盘形态、血流参数的检测。结果两种光凝模式相比,眼压升高后第4周,倍频532激光组平均眼压为48.4±10.3mmHg,氩激光组平均眼压为44.2±7.0mmHg,倍频532激光组与氩激光组的三次光凝成功率的差异无显著性意义。除视盘面积外,恒河猴模型眼的视杯形态指数、杯盘面积比、盘沿面积、视网膜神经纤维层的平均厚度,与对照眼相比差异有极显著性意义。眼轴和前房深度与对照眼相比差异有显著性意义。筛板血流量、血流速度和红细胞移动速率与对照眼相比差异无显著性意义。结论两种激光光凝恒河猴小梁网均可用以建立慢性高眼压性青光眼模型,模型眼出现青光眼眼底特征性的形态学改变。

Abstract:Objective In order to establish a monitoring system in transgenic models of controllable temporal and spatial expression in endothelial cells. Methods\ Two binary transgenic mouse models were constructed. One was a spatial specificity expressing model by using tissue specific promoter, and the other was an artificially controlled temporal expressing model using tet off system. Results\ Endothelial cell specific driver mice VE cadherin:tTA was created by cloning the tetracycline regulated transcriptional activator (tTA) under the control of the endothelial cell specific VE cadherin promoter. TET:myrAkt1 transgenic mice were constructed using full length Akt1 with a c Src myristoylation sequence under the control of the tet operon promoter. The binary transgenic mouse was produced by crossing with transgenic mice, and an inducible expression of endothelial cell Akt1/PKB can be controlled by administration with tetracycline. Conclusions\ Temporal and spatial expression of target gene in endothelial cells can be controlled artificially by using VE cadherin promoter and tet off system.

Abstract:目的通过剑尾鱼属内鱼类的杂交,观察黑色素瘤在其杂交后代的形成情况。方法白体剑尾鱼(♀)与黑体剑尾鱼(♂)杂交,子一代(F1)和子二代(F2)分别自交,观察其后代外部特征变化和黑色素瘤形成的情况;对黑色素瘤进行组织病理观察。结果剑尾鱼杂交种的F2代开始出现性状分离,F3中有较高的黑色素瘤发生率,为20.5%。HE染色和Lillie黑色素染色证实增生组织为黑色素瘤。结论通过不同剑尾鱼的杂交和自交,后代有黑色素瘤形成,可望进行黑色素瘤模型的构建。

Abstract:目的为建立SHIV艾滋病动物模型提供毒力较强的病毒株,将新合成的SHIV XJ02170病毒适应猴体,并增强其毒力。方法实验前采集猴血清并进行血清学检查和PCR检测。选出13只无SIV,STLV1,SRV D和B病毒感染的猴。第一批实验,将SHIV前病毒DNA质粒经肌肉注射到猴体内,每只500μg;SHIV病毒液,经静脉注射到猴体内,每只2ml。病毒质粒和病毒液各接种2只猴。当第一批猴体检出病毒后,10ml感染猴的全血,抗凝后静脉注射到第二批猴体内,当第二批猴检出病毒后再将10ml感染猴的全血静脉注射到第三批猴体内,连续传代4次。每批实验均定期采集血液标本,分别用肝素和EDTA抗凝,进行病毒分离;病毒基因PCR检测;CD4,CD8测定;病毒抗体检测。结果SHIV XJ02170病毒和SHIV XJ02170前病毒DNA质粒在猴体内的传代中均能分离出病毒;从传代猴的血浆和外周血单核细胞(PBMC)中检出了病毒DNA和RNA基因;CD4,CD8测定结果显示有暂时性倒置现象,后变为正常倒置与正常交替出现;在传代的猴血清中检测出特异性HIV病毒抗体。结论SHIV XJ02170病毒与SHIV XJ02170前病毒DNA质粒,均能在恒河猴体内复制。

Abstract:Objective (1) To develop a new RT PCR method to assess the viral RNA in the plasma of SIV infected monkeys and to qualitatively compare the sensitivity of RT PCR and traditional methods in detection of virus isolated from plasma. (2) To develop a DNA PCR technique for measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs). (3) To verify the practicability and maneuverability of DNA PCR and RNA PCR, techniques used in SAIDS monkey models. Methods\ 9 rhesus monkeys (Macaca mulatta) were inoculated with SIVmac251 intravenously. Blood samples were colleted and the virus RNA was extracted from the plasma and the RNA was amplified by RT PCR. High molecular weight genomic DNA was extracted from PBMCs and amplified with specific nested DNA PCR. The PCR products were assessed by electrophoresis in 1 5% agarose gel. Results\ A 477bp specific fragment was amplified with nested DNA PCR and RNA PCR respectively. It was verified by sequencing. 7 days after challenging, among the 9 monkeys, the positive rate was 7/9 assessed by RNA PCR and 100% (9/9) by DNA PCR. At the same time the positive rate was only 5/9 assayed by virus isolation from the plasma. From then until 42 days after inoculation, the results of RNA PCR and DNA PCR were always 100% positive, whereas the positive rate of plasma virus isolation method went down to 4/9 at 35 days and 0 at 42 days postinoculation. Conclusions\ PCR method is more sensitive than virus isolation. Especially, it is true for DNA PCR. It can be used not only to detect infected cells with active virus replication, but also infected cells at transcription latent period. So it may become an important and necessary technique in SAIDS models, especially in the early stage of infection or in the middle and late stages but the viral load in the plasma is rather low or the virus is in a latent period. It should be more sensitive than virus RNA detection in plasma. scriptionlatentperi finfectionorinthe dbemoresensitive

Abstract:Objective To establish a reproducible rat model of chronic diabetic ulcers and investigate their characteristics and major pathological features. Methods\ The rat model of chronic diabetic ulceration was induced by repeated cyclic magnetic compression on the skin. The skin injury was measured grossly and tissue samples were taken for histological examination and biochemical assay. Results\ The model generates reproducible skin injury as characterized by tissue necrosis, leukocytes inflitration and high concentration of advanced glycation endproducts (AGEs). Conclusion\ A rat model of diabetic ulcers was established by an ischemia reperfusion method. The models present virtually all the pathological characteristics found in human diabetic ulcers. Therefore, it is a useful animal model in investigation of pathogenesis and therapy of diabetic ulcers.

Abstract:Objective To establish standardized maintaining methods for mouse models of human diseases and long term conservation of their genetic characteristics. Methods\ Three strains of mice were selected as experimental objects: FVB/ TgN, MRL/ Mpj and SAMP1/ Ka. Comparing to specific controls, considerable differences were detected in growth curves, RAPD, isoenzyme electrophoresis and behavior tests and thus established a set of detecting methods to provide evidence of maintenance. Result\ The growth curves of MRL/Mpj mice differed statistically from those of controls. The RAPD diagrams of FVB/TgN, MRL/Mpj and SAMP1/Ka mice differed each other in their positive primers and bands and may provide as another item of characteristic indexes. The critical loci of isoenzyme can be indicated by isoenzyme electrophoresis. Moreover, behavior tests revealed in a more direct way multiple aspects of differences of these strains. Conclusion\ The study demonstrated that special measurements such as growth curves, RAPD, isoenzyme electrophoresis and behavior tests are needed in addition to routine examinations to determine specific characteristics of particular mouse strains in time, providing evidence of strain maintenance and avoid occurrence of genetic drift.

Abstract:Objective To explore whether repeatedly administered multiple doses of scopolamine to rats is suitable for establishment of an animal model for senile memory deficits or Alzheimer disease research. Methods\ 14 rats were randomly divided into two groups (n=7): normal control group and scopolanmine treated group. In the experimental group, the rats received subcutaneous injection of scopolamine in a dose of 2mg/kg, twice a day for 21 consecutive days. The normal rats received the same volume of saline. Then reference memory testing in Morris water maze(MWM)was followed. Nissl staining was adopted to count the number of pyramidal cells in hippocampal CA1 and CA3 areas under light microscope. Ultrastructural changes of CA1 cells were examined by transmission electron microscopy. Results\ In the experimental group, escape latencies were statistically not significantly different from those of normal control rats on day 1, 2, 4 and 5, except day 3 in place navigation trials. In spatial probe trials, there were no significant differences between two groups. The quantity of pyramidal cells in hippocampal CA1 and CA3 areas was not significantly different between two groups. Ultrastructure of the pyramidal cells including nuclei and cytoplasmic organelles was not much changed. However, the neurons showed a decreased number of synaptic vesicles and reduced post synaptic density (PSD) in scopolamine treated rats in comparison with those of control rats. Conclusions\ Scopolamine given to rats in repeated doses may produce mild impairment of reference memory in Morris water maze. The number and ultrastructure of pyramidal neurons in CA1 and CA3 areas are not seriously changed, but some abnormalities of synapses in hippocampal CA1 cells may occur. Those findings indicate that scopolamine given in repeated doses to rats is not an ideal approach to establish an animal model for Alzheimer disease or senile memory deficits research.

Abstract:目的对比观察正常大鼠和心梗大鼠离体心脏动作电位和有效不应期的特点。方法用离体灌流吸附电极记录单向动作电位,常规电生理方法测量最大动作电位幅度(APA)、复极90%(MAP90)、复极50%(MAP50)、复极20%(MAP20)、有效不应期(ERP)。结果(1)和正常大鼠相比,心梗大鼠离体心脏左心房电生理参数MAP90(56.3±2.7vs.64.5±8.7,P<0.05)显著延长,ERP MAP90(0.89±0.2vs.0.78±0.3,P<0.05)减小,基础周期为250ms;右心室的电生理参数MAP90(67.6±14.1vs.134.1±26.7,P<0.001),ERP(55.0±3.53vs.69.0±8.9,P<0.05)明显延长,ERP MAP90(0.79±0.1vs.0.60±0.1,P<0.05)减小,基础周期为250ms;左心室的电生理参数MAP90(87.2±15.7vs.168.8±31.2,P<0.001)也呈显著延长,ERP(59.0±4.2vs.90.0±17.7,P<0.001),ERP MAP90(0.65±0.081vs.0.54±0.090,P<0.05)呈显著减小基础周期为250ms;(2)与正常大鼠相比,心梗大鼠的MAP90离散度[(LVMAP90-RVMAP90)(17.0±6.5vs.51.4±28.7,P<0.001)]、ERP离散度[(LVERP-RVERP)(4.0±2.2vs.20.0±7.9)P<0.001]显著增加,基础周期为250ms。结论心梗大鼠心脏不同部位的MAP的复极时间都显著性延长,MAP90和ERP离散度增加,这些电生理特点是促进折返形成、造成心律失常的主要原因。

Abstract:Objective To further discuss the indexes which can be used to objectively appraise the rat myocardial ischemia models. Methods To observe the effects of intraperitoneally injected isoprenaline in a dose of 10 mg/kg b.w. on the changes of electrocardiogram (ECG) and to assay the serum content of creatine kinase (CK), lactate dehydrogenase (LDH) and free fatty acids (FFA), and to measure the necrotic area of myocardia, the superoxide dismutase (SOD) and malondialdehyde (MDA) contents in cardiac muscle of Sprague_Dawley rats, compared with those of control rats received saline injection. Results The J point of ECG in myocardial ischemia model rats descented 30s after injection of isoprenaline (10 mg/kg) and recovered slowly after 2 min, but could not return to the original state within 20 minutes, and the J points at each time points within 20 min were significantly different from those in control rats ( P <0 05). T waves of ECG in myocardial ischemia model rats were also decreased since 30 s after s.c. injection of isoprenaline and were significantly different from those in control rats within 10 min. ( P <0 05). T waves of ECG in myocardial ischemia model rats at 30 s and 5 min after s.c. injection of isoprenaline were significantly different from those of control rats ( P <0 01 and P <0 05, respectively). The content of CK, LDH, FFA in serum and MDA in cardiac muscle of model rats were significantly higher than those of the control rats and the area of myocardial necrosis was positively correlated with those alterations. But SOD in cardiac muscle was negatively correlated with them. Conclusions The J point of ECG can be considered as a main index and T waves as an adjuvant index to evaluate this animal model, and the changes should be monitored within 20 min after drug administration. Some biochemical indexes such as CK, LDH, FFA, SOD or MDA are also helpful in studying the mechanisms and effects of anti_myocardial ischemia drugs.

Abstract:Objective Intratesticular injection of pEGFP-N 1 was used to investigate its integration in spermatozoa and expression in early embryos . Methods\ Four local male goats were selected and different doses of pEGFP N 1 plasmid DNA were injected into the bilateral testes. The integration of pEGFP N 1 in the sperms was determined by PCR and Southern blotting. Results pEGFP N 1 was integrated into spermatozoa, and the transfection rate peaked 81% at 40 d after intratesticular injection. Expression of green fluorescent protein was found in spermatozoa and early goat embryos with a maximum positive rate of 66 7%. Conclusion\ Intratesticularly injected pEGFP N 1 can be integrated into spermatozoa and then green fluorescent protein can be detected in early embryos produced by in vitro fertilization. Intratesticular injection may be a feasible and simple approach to produce transgenic goats.

Abstract:目的探讨颈内动脉内皮不同损伤强度在不同时间出现狭窄的相关性。方法由颈外动脉向颈内动脉插入相同规格气囊,分别充入1、2、3Pa的压力,反复重复3次,分别在造型1、2、3周时各处死一批家兔,观察造型家兔血管内皮的病理学改变及相关活性因子的变化。结果实验结果表明,在血管内膜损伤强度为2Pa,球囊直径为3.0mm,球囊长度为20mm,插入深度为30mm的条件下,在造型时间为3周时,造型家兔血管内皮可呈现典型狭窄性病理变化,血浆TXB2、GMP-140、ET-1、CGRP含量、全血粘度明显增高,红细胞聚集指数、聚集面积增加,红细胞变形指数、变形面积、血浆6-keto-PGF1α含量明显减少。结论在球囊压力为2Pa,造型时间为3周时可得到与临床极为相似的狭窄性动物模型,血管内膜、中膜明显增厚,相关活性因子变化显著,能满足药理学研究的需要。

Abstract:目的探讨人地中海贫血β654基因为外源基因用于转基因小鼠制作时,外源基因的制备方法,为进一步制作转基因小鼠打下基础。方法采用反向点杂交法确诊人地中海贫血β654纯合子DNA,以PCR法扩增获得人地中海贫血β654基因,分离纯化后,通过连接酶反应将其克隆入含βLCR调控序列的基础载体pBGT51质粒中,经PCR扩增、酶切、反向点杂交及DNA测序鉴定重组质粒,用EcoRⅤ酶切获得转基因所用的外源基因。结果将人地中海贫血β654基因作为外源基因克隆入基础载体pBGT51质粒中构建了重组质粒βBGT51,用EcoRV酶切获得含人β654基因及βLCR调控序列的6.5kb的DNA片段,制备了可用于显微注射转基因的外源基因。结论采用该方法构建的含人地中海贫血β654基因的重组质粒,可获得用于显微注射转基因的外源基因。

Abstract:Objective To investigate the effects of ofloxacin on mouse embryonic development and to determine wether ofloxacin is of reproductive and teratogenic toxicity. Methods\ Ofloxacin was administered orally by gavage to groups of inseminated mice for 3 days (day 0 to day 2 post insemination) and for 10 days (day 0 to day 9 post insemination), and to male mice for 10 days at a daily dose of 0, 36, 72 and 360 mg/kg b.w. respectively. The mouse fetuses were taken for examination at 16d of pregnancy. Results\ The results indicated that ofloxacin did not show effects on the number of living fetuses, dead fetuses and absorbed fetuses. The body weight, placenta weight of fetuses in all treatment groups had no significant difference (p>0 05) as compared with those of the control. Ofloxacin did not produce significant difference on the development of pre implantation embryos in female mice, but significant difference was observed in male mice. Conclusion\ The results of this study showed no evidence for reproductive and teratogenic toxicity of ofloxacin.