Effect of EGFP gene transfection on the cell cycle distribution of primary cultured human chondrocytes     

JIANG Xun  ZENG Yao-ying  HE Xian-hui  XU Li-hui  DI Jing-fang  FENG Zheng  ZHAO Jing-xian  WANG Qing  WANG Tong  SHI Jian-bo 《园艺学报》2004,20(6):924-928

AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35.37％ at 48 h. It didn't affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn't affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage.  相似文献   

Astrocyte proliferation in the rat hippocampus after middle cerebral artery occlusion     

ZHANG Liang  WANG Wei  RUAN Xu-zhong 《园艺学报》2004,20(9):1553-1556

AIM: To study rat astrocyte proliferation in ipsilateral hippocampus following focal cerebral ischemia. METHODS: Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). In hippocampus of rats at 3, 7 and 30 days after MCAO, the numbers and anatomic distribution of glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The protein expression of GFAP and proliferating cell nuclear antigen (PCNA) in the ipsilateral hippocampus were analyzed by Western blot analysis. RESULTS: Astrocytes appeared hypertrophic, with increased process thickness and numbers at 7 days after MCAO, and the highest density of astrocytes were seen at 30 days in the CA1, CA2 regions of the ipsilateral hippocampus. Western blot analysis revealed that GFAP levels were normal at 3 days, but increased by 7 days and remained elevation at 30 days. Western blot analysis of PCNA protein also revealed identified upregulation PCNA at 3 days after MCAO and the expression peaked at 7 days. CONCLUSION: This study demonstrates that focal cerebral ischemia in the rat results in a rapid response, a process often referred to as reactive astrogliosis or glial scarring, from resident astrocytes of the ipsilateral hippocampus to the side of ischemia.  相似文献   

Effects of β-ME and RA on expression of GFAP in mesenchymal cells derived from mouse fetal liver     

LIU Ping-ping  ZHANG Yuan 《园艺学报》2004,20(11):2053-2057

AIM: To investigate the effects of β-mercaptoethanol (β-ME) and all-trans rentinal acid (RA) on glial fibrillary acidic protein (GFAP) expression in mesenchymal cells derived from mouse fetal liver in vitro. METHODS: Cells suspension from 14.5-days-old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20％ FCS and mesenchymal cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β-ME and RA. The characteristics of treated cells were assayed by immunocytochemistry staining at 5 hours and 5 days after induction. β-actin as an internal control, GFAP gene expression of mesenchyal cells was detected with semi-quantitative RT-PCR. RESULTS: After being inducted by β-ME and RA, 80％ approximately of the cells exhibited typical neural morphology and about 85％ expressed GFAP phenotype. Semi-quantitative RT-PCR showed that mRNA expression of GFAP increased in treated cells versus untreated cells (P<0.01). CONCLUSION: GFAP expression in mesenchymal cells derived from mouse fetal liver in vitro increases after being treated with β-ME and RA.  相似文献   

Changes of MAPKs expression in rat hippocampal neurons after sleep deprivation     

ZHANG Qian  CHENG Jiang-tao  WANG Shu-chun  QIAO Peng  WANG Yi-ling  WU Jing-lan  WANG Yu-ruo 《园艺学报》2004,20(10):1874-1877

AIM: To explore the change and the possible role of MAPKs in rat hippocampus neuron after sleep deprivation. METHODS: The morphology of hippocampus neuron after sleep derivation was observed by TUNEL and HE staining, the activity of ERK was assayed by β-liquid scintillation counting and the expression of JNK was detected by Western blot. RESULTS: In paradoxical sleep deprivation (PSD) group, the number of apoptotic cells in hippocampus was increased. The scores of ERK activity were 1 764.00±941.56. Compared with control groups, the ERK activity was obviously decreased (P<0.05). The JNK expression was 87.5%, which was higher than that in control group. CONCLUSION: These results provide some important evidences that the sleep deprivation could cause changes in MAPKs activity, which may be related to the mechanism of hippocampus neuron apoptosis.  相似文献   

新扬州鸡IGF-1基因多态性与早期生长速度关系的研究   总被引：9，自引：2，他引：9  

王志跃  范刚  杨海明  汪张贵  盛东风  刘桂琼 《中国家禽》2004,26(24):9-12

以150只非同胞新扬州鸡为材料，采用PCR—RFLP法检测了该基因5’调控区DNA序列多态性，并运用线性模型统计方法分析了多态性与初生重和12周龄体重的关系。结果显示：新扬州鸡IGF-1基因5、调控区自然存在两种不同DNA序列，经。PstⅠ酶切后出现3种基因型(“-／-”、“-／ ”、“ ／ ”)，基因型分布符合哈代一温伯格定律。各基因型个体的初生重、12周龄体重的最小二乘均数存在“-／-”＞“ ／-”＞“ ／ ”的趋势，且“-／-”型个体的12周龄体重显著高于“ ／ ”型个体(P＜0．05)。  相似文献   

家鸡Leptin成熟肽cDNA的克隆、重组蛋白表达及纯化   总被引：4，自引：0，他引：4  

施振旦  邵西兵  方梅霞  于迎春  刘颖  陈楠 《中国兽医学报》2004,24(3):258-260

从 18周龄鸡卵巢组织中抽提总 RNA,使用六聚体随机引物反转录后 ,用鸡 L eptin(瘦素 )特异性引物扩增出鸡L eptin编码区第 5 2～ 4 6 0 bp的长度为 4 0 9bp的 c DNA片段。根据鸡 L eptin的 3′端第 4 6 0～ 4 92 bp序列设计了 3条部分相互重叠并且顺序串联延伸的下游反义引物 ,并在最后 1条引物 3′端连接上 Eco R 切点 ;在以上用于反转录扩增的 5′端引物的 5′端连接一 Bam H 切点。用该 5′端引物分别与 3个 3′端引物配对 ,利用反转录扩增出的 4 0 9bp的L eptin c DNA片段作为第 1模板进行扩增 ,扩增产物再作为模板与下一引物对再次扩增。经 3次扩增得到编码鸡 L ep-tin成熟肽的全长 4 5 1bp的 c DNA序列。将该 4 5 1bp的 L eptin c DNA序列经 Bam H 和 Eco R 双酶切后 ,克隆入表达质粒 p RSET A的 Bam H 和 Eco R 两酶切位点之间 ,构建成表达质粒 p L ep- SCAU。转化有重组表达质粒 p L ep-SCAU的大肠杆菌 BL 2 1(DE3)在 L B培养基中培养后 ,经 IPTG诱导表达出相对分子质量为 2 0 10 0的鸡 L eptin融合蛋白和少量 4 0 2 0 0的 L eptin融合蛋白。L eptin融合蛋白的表达在 IPTG浓度为 0 .0 5 mmol/ L 时达到最高 ,占总菌体蛋白的 32 .6 %。用 Ni- NTA凝胶从 7L 发酵培养菌裂解液中纯化出 180 m g左右  相似文献   

禽大肠杆菌外膜蛋白基因C(ompC)的序列分析   总被引：1，自引：0，他引：1  

曹素芳  黄青云  张煜坤  温纳相  陈红英 《中国兽医学报》2004,24(4):332-334

根据 Gen Bank中人源大肠杆菌 K- 12外膜蛋白基因 C(omp C)的核苷酸序列设计引物 ,应用 PCR方法从禽大肠杆菌 O2 、O78株及它们的融合双价弱毒菌株 O2 ,78(Norr Chlr)中分别扩增得到 omp C基因 ,序列测定及分析比较发现 ,3个菌株的 om p C基因均由 170 2 nt组成 ,核苷酸序列完全相同 ,只有 1个大的开放性阅读框 (ORF) ,长 10 92 bp,编码由 36 3个氨基酸组成的前 Om p C蛋白 ,前 2 1个氨基酸残基组成信号肽 ,成熟的 Omp C蛋白由 342个氨基酸残基组成 ,Mr为 4 0 0 0 0。其氨基酸序列也完全相同。从基因水平上证明了禽大肠杆菌 O2 、O78株及融合双价弱毒菌株 O2 ,78(NorrChlr)存在相同的外膜蛋白 C抗原 ,从而为进一步研究 Omp C蛋白的免疫原性奠定了基础  相似文献   

三江白猪线粒体DNA-RFLP的分析     

杨隽李馨  耿忠诚 《黑龙江畜牧兽医》2004,(3):11-13

采用22种限制性内切酶分析了三江白猪线粒体DNA的多态性。结果表明：在60头个体中检出30种限制性态型．归结为7种基因单倍型；其间的差异来自少数限制性位点的突变。单倍型间平均遗传距离为0．35%，群体遗传多态程度为0．044％。三江白猪遗传多样性非常贫乏，说明三江白猪与地方种猪起源于一个共同祖先，在品种形成早期受到创立者效应的制约。  相似文献   

尿素水平对青山羊血液生化指标的影响   总被引：2，自引：0，他引：2  

方希修  王冬梅  黄秀明  杨在宾  孙存孝 《饲料工业》2004,25(5):20-23

应用饲养试验与氮平衡试验,研究了不同尿素水平的日粮对青山羊血清总蛋白(STP)、血清清蛋白(ALB)、血清球蛋白(GLB)、血清清球蛋白比值(A/G)、血清尿素氮(SUN)及血清谷丙转氨酶的含量变化的影响。试验采用4×4拉丁方试验设计,试验分4个阶段,在基础日粮一致的情况下,采用4、9、14、0(对照)g/kg·DM4种不同的处理,试验结果表明:不同尿素水平极显著影响ALB、SUN浓度和GPT活性浓度(P<0.01),对血清清球比(A/G)无显著差异(P>0.05),尿素水平与STP、GLB浓度存在负相关;血清含氮生化指标STP、ALB、GLB、A/G、GPT、SUN随时间呈动态变化,出现高峰的时间比瘤胃氨浓度出现的时间平均推迟1～2h。进食尿素后,血液生化指标由平稳逐渐缓慢升高到高峰,随后又下降并趋于平衡。  相似文献   

Effects of protein deficiency on the mRNA levels of insulin-like growth factors and myostatin in skeletal muscle of weaned lambs     

Shizuka HIRAI  Hiroyuki KAWACHI  Tohru MATSUI  Hideo YANO 《Animal Science Journal》2004,75(3):207-212

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