Abstract:

Abstract:Objective To develop RT-PCR kits for detection and identification of type A and subtype H5 avien influnza virus (AIV). Methods Two pairs of primes were designed for type A and subtype H5 avian influenza vir us according to the report by Ming-Shiuh Lee. The cDNA of AIV were amplified by one step RT-PCR for AIV type A and subtype H5, and identified by electrophores is analysis. The RT-PCT kits for AIV type A and subtype H5 were assembled. The H1-H15 reference strains and 38 isolated strains were detected using the RT -PCR kits. Results The RT-PCR method for detection of AIV type A and subt ype H5 was established. All stains of AIV were positive using RT-PCR kits for A type. The sensitivity of this kit was 1/1024 hemagglutinin unit. Subtype H5 reference strains and 19 subtype H5 isolated strains were positively detected using the RT-PCR kit for AIV subty pe H5. Subtypes H1-H4 and H6-H15 reference strains, subtypes H7 and H9 isolated strains, and 1 subtype H5 isolated strain showed a negative result. The sensiti vity of this kit was 1/64 hemagglutinin unit for AIV subtype H5. The samples of H5N1 AIV infected chicken were all positively detected using these two types of kits. Conclusion The RT-PCR kits for AIV type A and subtype H5 are excellent in specificity, sensitivity, stability and reproducibility.

Abstract:Objective To investigate the expression of HA1 gene of avian influe nza virus (AIV) subtype H7N2, which can be used for detection of antibodies to H 7 subtype AIV, and for further study of HA1 protein function. Method The gene of H7N2 subtype AIV was amplified by RT-PCR and cloned into pGEM -T-easy vecto r. The HA1 gene expressing plasmids was constructed by inserting the target g ene fragments into pCEX-4T-2 vectors. The proteins expression was induced by IPTG and analyzed by Western blot. An indirect ELISA method was established as to de tect the expression of HA1 fusion protein. Positive sera of H5, H7, H9 subtype A IV were detected by this indirect ELISA. Results HA1 gene of H7 N2 subtype AIV was s uccessfully cloned and sequenced. This gene encoded 322 amino acids. The constru cted HA1 gene expressing plasmids was transformed into E. Coli BL21 competen t ce lls, and the HA1 fusion protein with molecular weight of 61kD was expressed. The expressed HA1 fusion protein showed a positive reaction with H7N2 subtype AIV p ositive serum, and a negative reaction with H5 and H9 subtype AIV positive serum . Conclusion The HA1 gene of H7N2 subtype AIV was expressed in E. Coli BL21. T he expressed HA1 fusion protein shared a positive reaction with H7 subtype AIV p ositive serum, and a negative reaction with H5 and H9 subtype AIV positive serum .

Abstract:Objective To clone and sequence the non-structure (NS) protein gene of avian influenza virus (AIV) isolates and analyze the homology and allele of the NS gene, and to set up a method for detection of antibodies against NS prote in. Method The NS gene of 7 AIV isolates was amplified by reve arse transcription polymerase chain reaction (RT-PCR). The DNA fragments were c loned into pGEM-T vector and sequenced subsequently. The nucleotide sequence wa s compared with GenBank data and the phylogenetic tree of NS gene was generated. Results The entire ORF sequences of NS1 and NS2 protein were obtained. Homology analysis showed that the NS gene of these 3 H9N2 strains exhi bited 96% ~ 98% identity with each other, as to 2 H5N1 strains, 91.6%, while the 2 H7N2 strains showed 98.9% ident ity. The identity between H5N1 and H9N2 isolates was more than 90%, and about 60 % ~ 70% between H7N2 and the other two subtypes. In NS gene phylogenetic tree, t he H5N1 and H9N2 isolates were situated in branch of allele A, while H7N2 isolat es in branch of allele B. The location of the 3 H9N2 isolates was very close, ha ving the same origin with some of the H5N1 isolated in Hong Kong and Guangdong. Conclusion There is a significant defference in the nucleotide sequence identity of NS gene of AIV isolated in China. The NS gene of these sev en AIV isolates belongs to different alleles, allele A and allele B.

Abstract:Objective To investigate the spatio-temporal expression of CX43 i n the developing human and mouse hearts. Methods 63 normal human fetal hearts i n 6-18 gestational weeks and 64 normal mouse fetal hearts in embryonic days 9.5 -16.5 were included in this study. Immunohistochemistry was used to detect th e CX43 expression. Results In early stage of development of hu man fetal heart, no CX43 expression was detected in ventricular myocardium and w eak expression in atrial myocardium. High expression was observed in primitive t rabecular nets. With the fetal development the CX43 expression was enhanced in t he atria and ventricles. Peak expression was revealed in trabecular net at 13 - 14 weeks. Expression was weak in muscular interventricular septum. No expressio n was observed in membranous interventricular septum, atrial-ventricular valves and large arteries. Except for the large arteries, CX43 expression in the devel oping mouse heart was similar to that in human fetal hearts. Conclu- s ion CX43 may play a crucial role in the heart development.

Abstract:目的　通过向大鼠脑内单侧、双点、间隔注射 6 OHDA ,建立PD动物模型。方法　取SD大鼠 4 0只 ,随机分为实验组 35只和对照组 5只。实验组大鼠右侧黑质致密部和内侧前脑束注射 6 OHDA ,两次注射间隔一周 ,对照组大鼠注射人工脑脊液 ,观察经阿朴吗啡诱导后大鼠的行为及黑质DA神经元形态学变化。结果　①实验组有 2 3只恒定左转鼠且旋转圈数 >2 10r 30min ,被认为是成功的PD模型 ,占 6 7 7% ;有 1只动物死亡 ,占 2 9%。②对PD大鼠模型的免疫组化研究发现 ,注射侧黑质区多巴胺神经元较健侧和对照组显著减少。结论　利用向脑内单侧、双点、间隔注射 6 OHDA制备PD大鼠模型 ,结果稳定可靠 ,动物死亡率低 ,为PD动物模型的建立提供了新方法。

Abstract:目的　构建pCR○R3 1 p16真核表达质粒 ,并建立其稳定表达的NIH3T3细胞株。方法　将人的p16cDNA亚克隆至pCR○R3 1,将该重组质粒转化DH5α感受态大肠埃希菌 ,筛选阳性克隆 ,进行EcoRⅠ和XhoⅠ双酶切鉴定及DNA测序 ,将已鉴定的pCR○R3 1 p16经脂质体介导转染至NIH3T3细胞中。结果　重组质粒双酶切后 ,出现约 80 0bp片段 ,提示p16cDNA片断已插入pCR○R3 1多克隆位点内 ,DNA测序显示p16cDNA按照正确方向和阅读框插入表达载体pCR○R3 1。通过pCR○R3 1 p16转染NIH3T3细胞 ,获得 10个G4 18抗性克隆 ,其中 2个为RT PCR阳性。结论　成功构建pCR○R3 1 p16真核表达载体并建立了其稳定表达的NIH3T3细胞株。为建立p16转基因鼠奠定基础。

Abstract:Objective To overcome difficulties in passage for SV40T transgenic mice, two genes containing different fragments of SV40T were constru cted: one gene is called SV40T-DRb which was mutated in Rb motif of SV40 T, another gene is called SV40T-Dp53 which was deprived of p53-binding d omain. Methods U sing recombi nant DNA techniques, the two changed gene fragments of SV40T were sequenced and cloned, and finally the two genes were cloned into the mammary gland-specific e x pression vector-p205C3. Microinjection of the expression vectors into male nucl e i was used to establish transgenic mice. PCR and Southern-blot were used to det e ct the positive transgenic mice, avoiding the occurrence of false positive trang enic mice. Results Six transgenic mice that were positive for b oth gene SV40T-DRb and gene SV40T-Dp53 were detected by PCR. One transgenic mouse that was positive for both gene SV40T-DRb and gene SV40T-Dp53 was detected by Southern blot. Co nclusion The above mentioned findings suggested that the two gene const ructs con taining different fragments are successful and the transgenic mice are surely wi th reduced tumorigenesis of SV40T.

Abstract:Objective To afford an effective method to determine the ovulatory phase in female Macac a fascicularis for its artificial insemination and hybridization. Meth od 30 female Macaca fascicularis at the age of 4-6 years were selec ted to assess the changes of various types of cells in vaginal smears. R esult and Conclusion The major cell types in the Macaca fascicularis vaginal smears included leucocytes, keratinized epithelial cells, intermediat e and basal cells and cell debris. The amount of leucocytes is significantly dif ferent in various phases of the menstrual cycle(p<0.05). The number of kerat inized epithelial cells is the same in both preovulatory and menstrual phases, but is significantly(p< 0.05) different in other phases of the menstrual cycle. There is no significant dif ference in the number of intermediate and basal cells at menstrual and postovula tory phases, but is significantly(p<0.05) different in other phases of the m en strual cycle. The amount of cell debris is similsr to that of keratinized epithe lial cells. The changes of cells profile in various phases were that the number of keratinized epithelial cells increased gradually to tiptop from menstrual pha se to ovulatory phase and then reduced gradually, but the changes of leucocytes, intermediate and basal cells and cell debris are contrary to that of keratinize d epithelial cells.

Abstract:Objective To analyze whether there are sensitive PRNP genes in the r hesuses used in spaceship analog experiments, and to provide a reference for ass essment of pathological brain alterations of these rhesuses tprovoked by exposur e to hypergravity.Method\ Total DNA was isolated from peripheral bl ood ymphocyte s of 17 healthy male adult rhesuses, which would be used in corresponding parabo lic flight experiments. The PRNP gene was amplified by PCR and analyzed.R esults\ The cloned PRNP gene is 762 nucleotides long and contains the en tire PRNP coding sequence, which has no intron and has more than 95% homology w ith the published gene sequence, deducing 253 amino acids and molecular weight ( MW) of 27.6 kd.Conclusion\ There are no sensitive PRNP genes in th e rhesuses, which provide a reference for judgement of pathological brain alterations of these rhesuses as e xposed to hypergravity.

Abstract:目的　建立蒙古长爪沙鼠标准G带染色体的核型 ,提供可靠的细胞遗传学的背景数据。方法　雌、雄各 3只成熟长爪沙鼠 ,外周血淋巴细胞培养 ,制片、镜下观察分裂中期的淋巴细胞。随机计数 10 0个分裂细胞中的染色体数目 ,确立长爪沙鼠体细胞染色体数目。选择 10个典型细胞测量染色体基本数据 ,建立标准G带核型。结果　长爪沙鼠染色体数为二倍体 ,4 4条染色体 ,可划分A、B、C、D、E、F六组。中着丝点区域的 11对 ,主要分布A、C、E三组中 ;近中着丝点区域的染色体 5对 ,主要分布B组中 ;端着丝点区域的染色体 5对 ,主要分布D、F组中 ;x为第 6号中着丝点染色体 ,y为端着丝点的区域染色体大小在 14号与 15号染色体之间。结论　长爪沙鼠染色体为 2n =4 4 =2× 11m +2× 5sm +2× 5t+(x)m +(y)t

Abstract:Objective To establish a typical rat model with syndrome X. Method Adult male SD rats were operated on two kidneys, one-clipped to induce renal h ypertension. After operation, the rats were fed with a common diet for 4 weeks, and subsequently a high fructose diet for 4 weeks to induce syndrome X. Results The systolic blood pressure (SBP) was significantly increased a t 4 weeks after operation, but blood glucose and lipid were not changed markedly . Howevever, after feeding with high fructose diet for 4 weeks, hyperglycemia, h yperinsulinemia, insulin resistance, hypertension and hyperlipidemia occurred in the SD rats. Conclusion On the basis of renal hypertention for med by operation on kidneys, syndrome X can be induced in SD rats by high fructo se diet for 1 month. it may provide an ideal animal model for investigating insu lin resistance and accompanying cardiovascular disorders.

Abstract:Objective To establish a PCR method to dete4ct monkey B virus (BV). Method According to published primers provided by Makoto H, w e used PCR method to amplify BV DNA directly from monkey blood or indirectly from propagated bloo d BV cultured in Vero cells. PCR segments were bound to pGEM-T vector.R esults The primers were able to amplify both blood BV and propagated bl ood BV and give identical results. The amplified segment was sequenced and was 1 00% homologous with E2490 (American BV strain). Conclusion A PC R method has been established to directly detect monkey B virus from blood.

Abstract:Objective In order to set up an accurate and convenient method for genetic assessment of macaca rhesus. Method DNA polymorphic microsatellites of macaca rhesus were analyzed using PCR technique in 20 macaca rhesuses.Res ults\ Nine polymorphic microsatellites loci, 4 non-polymorphic microsatel lites loci, a nd alleles of 2 microsatellites loci in a smaller number were screened. Conclusion\ An efficient, accurate and convenient PCR method charac terizing DNA polymorphic satellites loci had been established which can be used for genetic assessment of macaca rhesus.

Abstract:Gout is a metabolic disease characterized by a significant hyperuric emia. In recent years the incidence of gout is rapidly increasing along with the development of China. Therefore, the prevention and treatment of gout aroused g reat interests among medical researchers. However, there is still a lack of suit able and reliable experimental animal models of gout and hyperuricemia. This art icle is a brief overview of recent advances in how to establish animal models of gout and hyperuricemia, and an introduction of own work and experiences of the authors, which could be beneficial in future research.

Abstract:Objective Melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and body weight, and is asso ciated with human obesity. Since MC4R gene was cloned, there has been a large am ount of research on structure, physiological function, regulatory mechanism of i ts effects and the association between MC4R gene mutation and body weight. The r ecent progress in those aspects is discussed in this review.