Abstract:目的建立SYBR Green I荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数。方法巢式RT-PCR扩增SIV病毒RNAgag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEMT载体上,构建pGEM-SIVgag477质粒。该质粒经限制性内切酶NotⅠ酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL。结论制备的RS外标准品纯度高,SYBR Green I荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数。

Abstract:目的 利用PCR技术克隆了人类α-actin 基因的启动子(约450 bp).方法 将PCR产物连接到pMD-18T载体上,酶切鉴定后测序,并进行软件分析.结果与结论 序列分析表明,扩增片段虽然与GenBank里登陆的序列同源性仅为72%,但包含有完整的启动子元件和转录专一调节因子相应的识别序列.用去掉启动子的pEGFP-N1作为框架结构,尝试构建真核表达载体,并获得了含人类心肌α-actin启动子的真核表达载体pEGFP-N1-α-actin-P.

Abstract:Objective To analyze single nucleotide polymorphism(SNP) of inbred mouse genes by a lately established method-single-tube bi-directional allele specific amplification(SB-ASA).Method 16 SNP were detected by the SB-ASA method in 5 inbred mouse strains.We also designed a double-blind experiment and sequencing to validate the reliability of this method,and assessed its influence on the results of PCR system and amplification conditions.Results SB-ASA was successfully used to type 5 inbred mouse strains for 16 SNP loci,and the results were completely consistent with those obtained by sequencing.Five inbred mouse strains were successfully distinguished with a double-blind experiment by just 3 SNP loci.Conclusion SB-ASA can be used to SNP genetic monitoring of inbred mice,and it can be popularized and applied as a new molecular biological method of genetic monitoring.

Abstract:目的通过对人低分化胃癌细胞系SGC-7901状态和接种细胞数目的研究,建立良好的皮下接种胃癌的动物模型。方法采用腹腔注射SGC-7901细胞,使裸鼠形成腹水;光镜及电镜观察细胞状态;将购买的SGC-7901细胞以及形成腹水后的肿瘤细胞分别以1×108、1×107和1×106个进行裸鼠皮下接种,每组接种5只。观察其肿瘤形成时间、大小、状态及病理学变化。结果SGC-7901细胞接种裸鼠形成腹水后进行培养的肿瘤细胞增殖状态发生改变;购买的SGC-7901细胞以1×108及1×107接种裸鼠,在第21天肿瘤组织中央均出现出血和坏死;1×106的裸鼠第21天未见肉眼可见的肿瘤形成。腹水培养的肿瘤细胞,接种1×108的裸鼠在第21天肿瘤组织中央可见大面积的出血和坏死;接种1×107及1×106的裸鼠在第21天均未见出血和坏死,接种1×107的肿瘤组织体积较大。结论SGC-7901细胞接种裸鼠形成腹水后的细胞,更容易建立SGC-7901细胞皮下接种的胃癌动物模型,其中以接种细胞数为1×107的肿瘤生长较好,更适用于胃癌的实验研究。

Abstract:Objective To compare the therapeutic effects of retinal detachment operation performed at different stages on the visual function,and to provide theoretical and experimental evidence for choosing the best operation time and recovery of visual function.Methods 15 colored rabbits were used for rhegmatogenous retinal detachment.The animals were for retinal reattachment after 1,7 and 14 days,respectively.Multifocal eletroretinography was performed and tissue samples were taken for light and electron microscopic examinations.Results The average response densities of P-wave at the first day detached retina after rhegmatogenous retinal detachment were significantly longer than those at seventh and fourteenth days,respectively,and the latencies of N-wave and P-wave were significantly smaller than those in other groups. Both the light and electron microscopic examinations depicted morphological alterations in the rhegmatogenous retinal detachment eyes followed by reattachment surgery.Conclusion The results demonstrated that there is a negative correlation between the recovery of retinal cells function after reattachment surgery and the post-operation time after rhegmatogenous retinal detachment.Multifocal eletroretinography may have significant value in evaluation of topical visual function of the retina.Histopathological examination may provide important evidence for explaination of the alterations of visual function.

Abstract:Objective To develop a DNA vaccine against canine parvovirus(CPV) infection.Methods The VP2 gene of CPV with or without CpG motif was cloned by PCR.The eukaryotic expression vector pcDNA3 was modified by restriction digestion to remove its neo~r gene and to replace its amp~r with kan~r to conform gene therapy requirements.The recombinant plasmids were constructed by subcloning the VP2 gene with or without CpG motif into the modified vector pcDNAK and injected into BALB/c mice and Beagle dogs.Both primary and secondary antibody responses were detected by hemagglutination inhibition(HI) assay.Results Injection of the recombinant vector pcDNA3-VP2C1 containing one copy of the consensus CpG motif into BALB/c mice lead to more elevated antibody response than the empty vector pcDNAK and the recombinant vector pcDNAK-VP2 without CpG motif.Gene immunization experiments in dogs preimmunized with an inactivated vaccine showed that the pcDNAK-VP2C2 vector containing two copies of canine-specific CpG induced higher secondary immune response than that of the pcDNA3-VP2C1 vector.Conclusion These data indicate that the pcDNAK-VP2C2 vector containing canine-specific CpG is highly antigenic and could be used for further studies for development of gene vaccines against canine parvovirus disease.

Abstract:Objective To study the different role of 5 behavioral tests on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson's disease(PD) in C57BL mice.Methods The PD models were formed with intraperitoneal injections of MPTP.The effects of MPTP on behavioral exhibition,number of dopamine(DA) neurons in the substantia nigra and density of tyrosine hydroxylase-immunoreactive(TH-ir) nerve fibers in the striatum,level of DA in the striatum were studied by comparing 5 behavioral tests(open field,rotarod,swim test,pole test,grid test),immunohistochemical analysis and fluorospectrophotometry.Results The score of behavioral tests were declined after MPTP.But there was no efficient result in pole test.At the same time,there were high coefficient of variation(CV) and accidental result in grid test.The CV was intermediate and the result was moderately ascending in rotarod.In open field and swim test,the CV was low and the result was obviously downtrend.The number of DA neurons in the substantia nigra were decreased about 58%.The density of TH-ir nerve fibers was also showed to be reduced.The level of DA in the striatum were decresased about 88%.Conclusion The lesions induced by MPTP in C57BL mice are similar to PD in humans.Open field and swim tests are better than others in behavioral tests of studying MPTP-induced PD in C57BL mice.

Abstract:目的 建立SYBR Green Ⅰ荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数.方法 巢式RT-PCR扩增SIV病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒.该质粒经限制性内切酶Not I酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品.结果 应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL.结论 制备的RS外标准品纯度高,SYBR Green Ⅰ荧光染料实时定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)RNA拷贝数.

Abstract:Objective To compare the reliability of genetic analysis by biochemical markers and microsatellite DNA in Mongolia gerbil colonies.Method To analyze 27 biochemical marker loci and 13 microsatellite DNA loci of two Mongolia gerbil colonies and compare the results obtained by the two techniques.Result 13 biochemical marker loci of the 27 loci showed polymorphism in the whole population,and the polymorphism rate was 48.1%.11 microsatellite DNA loci were polymorphic in the whole population,and the polymorphism rate was 84.6%.The average effective allelic number derived from biochemical marker analysis was similar to that from microsatellite DNA analysis.Percentage of polymorphic loci and average homozygosity of microsatellite DNA were higher than those obtained by biochemistry marker analysis.But the results of biochemistry marker and microsatellite DNA analyses were coincident in genetic diversity and Hardy-Weinberg equilibrium.Conclusion Both biochemistry maker and microsatellite DNA marker analyses are good methods for analyzing the population genetics of Mongolia gerbil colonies.

Abstract:Objective To establish a method for monitoring H-2 gene in inbred strains of mice.Methods two pairs of specific primers were designed to amplify mice H-2 alleles.Through the specific PCR and polyacryl amide gel electrophoresis(PAGE) to get the H-2 genotype of inbred strains of mice.Results From the PCR products we were able to distinguish the H-2 genotype of different strains.Different inbred strains of mice were distinguished.Conclusion Using this PCR technique,it is possible to monitor the H-2 gene and to supplement the shortage of existing methods.This method is rapid,sensitive and simple to use compared with previous methods.

Abstract:Objective To evaluate the therapeutic effect of calcitonin on osteoporosis in ovariectomized rats,and to investigate the association between calcitonin and IGF-1 and vitamin D.Methods 40 female 10-month old SD rats were randomized into 4 groups: sham operation group,ovariectomized(OVX) group,calcitonin(CT) group,Raloxifene hydrochloride group.The experimental model of osteoporosis was established by bilateral ovariectomy.Given CT 60 mg/kg body weight subcutaneous injection for 8 weeks,bone mineral densities of global and the lumber spine and femur neck were measured with Hologic dual energy x-ray bone densitometer in rat.Femur trabecular area,bone mineral deposition were measured by morphometry after the completion of therapy.Serum IGF-1 levels and serum 25OHVitD concentrations and lymphocyte vitamin D receptor contents were determined by ELISA.Results BMD of the lumber spine and femur neck in CT group and Raloxifene group were higher than that in OVX control group(P<0.01).Calcitonin increased trabecular area and bone deposition compared with that in OVX group.The serum IGF-1 and 25-OHVitD concentration in CT group and Raloxifene group were significantly higher than that in OVX control group(P<0.01).Conclusion Our findings indicate that the use of calcitonin demonstrated definite effect on IGF-1 levels and vitamin D metabolism and could be an effective therapy for osteoporosis.

Abstract:目的建立SRV-1巢式PCR检测方法并进行初步应用。方法针对SRV-1env基因的保守区序列,设计特异性引物,以感染SRV-1 Raji细胞提取出的含有前病毒DNA的基因组DNA为模板,进行巢式PCR反应。扩增产物测序后与GenBank报道的序列进行同源比对。将DNA样本进行10倍梯度稀释,以检测巢式PCR反应的灵敏度。使用该方法对正常Raji细胞以及感染SIV、STLV的外周血淋巴细胞DNA样本进行扩增,检测该方法的特异性。用建立的巢式PCR方法检测40份储存猴血标本。结果使用巢式PCR扩增出的特异片段经测序分析,结果证实与GenBank报道的序列一致。所建立的巢式PCR检测法检测限度可达1.5×10-3ng/μL,而且方法特异。用此方法检测40份猴血标本,未检测到阳性标本。结论初步建立SRV-1的巢式PCR检测方法,该方法灵敏、特异,为SRV-1的检测提供了一个快速、有效的手段。

Abstract:目的小鼠窦前卵泡体外培养得到成熟卵母细胞,观察卵母细胞的染色体和纺锤体形态,分析其变化及原因。方法完整小鼠窦前卵泡培养12 d,得到的卵母细胞进行免疫荧光染色,共聚焦显微镜观察纺锤体和染色体的形态和分布。结果经过体外培养,得到GV、M I、MⅡ期卵母细胞分别占总数27.9%、37.2%、34.9%;GV期卵母细胞存在完整的染色质圆环,11.8%的M I期卵母细胞显示正常纺锤体和染色体;38.5%的MⅡ期卵母细胞显示正常的纺锤体和染色体。结论小鼠窦前卵泡经体外培养后能够得到成熟的MⅡ期卵母细胞,但是其效率较低。原因可能是卵母细胞骨架结构的异常使染色体分离障碍,部分卵母细胞停留于M I期;同时成熟卵母细胞的受精率低也与纺锤体异常有关。

Abstract:目的 阐明含有去整合素和金属蛋白酶结构域的跨膜蛋白19(ADAM19)在小鼠睾丸发育中的作用.方法 采用半定量RT-PCR和免疫组化两种实验方法,分别检测ADAM19 mRNA和蛋白质在小鼠睾丸发育中的时空表达.结果 ①最早在胚胎发育的15.5 d才能检测到ADAM19 mRNA的表达,后其表达随着胚胎发育天数的增加而逐渐升高,到围产期表达水平达到最高.出生后,ADAM19 mRNA的表达呈现显著下降的趋势,到成体睾丸中就几乎检测不到ADAM19的表达.②和其mRNA表达变化趋势一样,ADAM19蛋白也是首次在胚胎发育的15.5 d被检测到,一直持续存在到出生后一周,一周后则几乎检测不到;阳性表达信号主要定位在睾丸的曲细精管(睾索)中.结论 ADAM19 在小鼠睾丸中的表达具有显著的发育依赖性.

Abstract:目的测定早期断奶仔猪血常规和血清生化参数、氨基酸浓度以及脏器指数常值,以供相关研究参考。方法分别采用全血细胞计数分析仪、全自动血液生化分析仪和全自动氨基酸分析仪等。结果断奶后第0、71、4、28天仔猪的血常规和血清生化参数、氨基酸浓度以及脏器指数均取得平均值和标准差。随着日龄的增加,WBC总数和分类计数不断增加,然后趋于稳定;MCV和PDW先降低后升高,第7天时最低;MCH和MCHC不断升高,第14天时最高。TP、LAC和Mg2+的浓度有升高趋势;P、BUN、TCHO和GLU的浓度以及LPS、CK和GPT的活性先降低后升高;AMM的活性和Zn2+、Na+、Cl-的浓度先升高后降低,第7天时最高,然后趋于稳定;TG的浓度在第14天时开始显著下降,然后趋于稳定;ALP的活性先升高后降低,第14天时最高。Asp的浓度不断增加,Cys的浓度不断降低,Ser、Ala、Leu、Phe、Gly、Val、Met、Lys、His、Pro、BCAA、AAA和总氨基酸的浓度先升高后降低,第7天时的浓度最高。肾脏指数不断下降,至第28天时趋于稳定;胃、颌下淋巴结和肝脏指数第7天时最大,脾脏指数第14天时最大。结论随着日龄的增加,早期断奶仔猪多数血常规和血清生化参数、氨基酸浓度及脏器指数表现不同的变化规律。

Abstract:目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。

Abstract:Objective To characterize the growth and development traits of the first generation young Tibet mini-pigs.Methods The measurements of body weight,body length,chest girth,wither height,rectal temperature,respiratory rate,heart rate,blood physiological and biochemical traits of the first generation young Tibet mini-pigs were carried out at a breeding farm in Guangzhou.Results The Tibet mini-pigs in Guangzhou area had smaller size,stable growth performance and the same growth rate as that reared in original area.It demonstrated that rectal temperature,respiratory rate and heart rate were similar among the first generation and generations raised in original area.Meanwhile,no significant difference was found between boar and gilts(P>0.05).Except basophils,monocytes and serum BUN,other parameters of the F1 male and female pigs were also approximate and close to that of human beings.Conclusion The present study indicates that Tibet mini-pig is an ideal experimental animal for medical researches due to its smaller size,stable growth performance and physiologically and biochemically closely related to human beings.

Abstract:Objective The aim of this study was to determine the blood physiological and biochemical parameters of Tibet mini-pigs raised in subtropical environment.Methods Nine physiological and 9 biochemical blood parameters in original and F1 generations of Tibet mini-pigs raised in subtropical area were measured and the differences between generations and between both sexes were analyzed.Results Among the biochemical indexes,there was a significant difference in AST between male and female pigs of original generation,and TG,ALB,BUN and CHOL between male and female pigs in the first generation.In the blood physiological indexes,there was no significant difference between male and female pigs of original generation,but there was significant difference in MONO between male and female pigs of the first generation.Conclusion Tibet mini-pigs are adapted to the subtropical environment through local domestication over the past two years.Compared with other animals and human beings,Tibet mini-pigs as laboratory animals are more closely resembling human beings than dogs and monkeys in regards of some physiological and biochemical parameters,and more suitable to be used in experimental studies instead of other animals such as dogs and monkeys.

Abstract:目的对广州地区西藏小型猪的繁殖性能和繁殖行为进行了观察研究。方法以广州地区雌雄西藏小型猪为研究对象,根据其繁殖卡的详细资料进行分类统计。结果雄性西藏小型猪阴茎首次伸出日龄为(39.50±2.63)d,此时体重为(3.26±0.87)kg;其射精日龄为(65.22±3.69)d,体重为(5.34±1.03)kg。雌性西藏小型猪初情日龄为(142±5.3)d,成年母猪发情周期为(20.35±0.72)d,发情持续期(4.2±0.31)d;其妊娠期约为114 d左右。初产雌性西藏小型猪产仔数为(2.30±0.82)头,2月龄断乳存活率为86.5%;而经产雌性西藏小型猪产仔数为(5.70±1.00)头,其2月龄断乳存活率为91.4%。哺乳期雌性西藏小型猪母性强,护仔行为强烈。结论广州地区西藏小型猪生长发育繁殖正常,产仔率和2月龄存活率等繁殖性能指标与原产地一致。

Abstract:Objective To study the genetic polymorphism of mtDNA D-loop and microsatellite loci in Tibet mini-pigs in order to evaluate their background and lay a molecular basis.Methods Differential primers to amplify the mtDNA D-loop and ten microsatellite loci in the Tibet mini-pigs were used.Then mtDNA Dloop from gel was purified and sequenced.PAGE was used to separate the alleles of microsatellite loci in the Tibet mini-pigs.Results Analysis of polymorphism of mtDNA D-loop in Tibet mini-pigs showed that there was no change in the mtDNA D-loop.The polymorphism of 6 microsatellite loci in 30 Tibet mini-pigs was high with a mean polymorphism information content(PIC) of 0.584 and mean rate of heterozygosity(H) of 0.573.Conclusion MtDNA D-loop in Tibet mini-pigs presents no diversity which proves that it is consistent with other seeds of pigs in evolution and inheritance.The average rate of microsatellite loci in Tibet mini-pigs proves that this seed is pure.