人端粒酶逆转录酶(hTERT)基因对山羊胎儿成纤维细胞的影响

利用包含人端粒酶逆转录酶(hTERT)基因的真核表达载体pCI-neo-hTERT转染山羊胎儿成纤维细胞,筛选阳性克隆扩大培养,并对转染阳性细胞分别进行RT-PCR检测,倍性分析,细胞周期检测和细胞凋亡检测,以观察该基因对山羊胎儿成纤维细胞的影响.试验结果表明,筛选出的阳性细胞现已传至第50代;RT-PCR检测,端粒酶基因成功整合到山羊胎儿成纤维细胞并持续表达;倍性分析结果显示,转基因第50代细胞呈正常二倍体;对细胞周期进行分析,结果显示转基因第50代细胞较未转染第30代细胞有较高的S期,说明该细胞DNA合成旺盛,具有很强的增殖能力;细胞凋亡检测结果发现,转基因第50代细胞中凋亡细胞的比例明显少于转染第30代山羊胎儿成纤维细胞.这些试验结果均表明,hTERT能增加山羊胎儿成纤维细胞体外培养的代数,并保持细胞良好的形态及较强的增殖能力.     

人端粒酶逆转录酶(hTERT)基因对山羊胎儿成纤维细胞的影响

利用包含人端粒酶逆转录酶（hTERT）基因的真核表达载体pCI-neo-hTERT转染山羊胎儿成纤维细胞,筛选阳性克隆扩大培养,并对转染阳性细胞分别进行RT-PCR检测,倍性分析,细胞周期检测和细胞凋亡检测,以观察该基因对山羊胎儿成纤维细胞的影响。试验结果表明,筛选出的阳性细胞现已传至第50代;RT-PCR检测,端粒酶基因成功整合到山羊胎儿成纤维细胞并持续表达;倍性分析结果显示,转基因第50代细胞呈正常二倍体;对细胞周期进行分析,结果显示转基因第50代细胞较未转染第30代细胞有较高的S期,说明该细胞DNA合成旺盛,具有很强的增殖能力;细胞凋亡检测结果发现,转基因第50代细胞中凋亡细胞的比例明显少于转染第30代山羊胎儿成纤维细胞。这些试验结果均表明,hTERT能增加山羊胎儿成纤维细胞体外培养的代数,并保持细胞良好的形态及较强的增殖能力。     

转hTERT基因的山羊胎儿成纤维细胞的生物学特性及其表达分析

用脂质体介导法将hTERT基因转染到山羊胎儿成纤维细胞中,以期获得永生化的山羊胎儿成纤维细胞系。对转染后的阳性细胞进行细胞学和分子学相关检测。结果显示,导入hTERT的阳性细胞形态正常,并已传至第58代;未转染组细胞经长期传代后（30代）,增殖缓慢,部分细胞表现衰老和凋亡的迹象。RT-PCR检测转染组细胞中有hTERT基因表达。生长曲线绘制结果显示转染组（30代和50代）山羊胎儿成纤维细胞生长速度稍快于未转染组（5代）细胞,但差异不显著（P〉0.05）。转染组细胞生长旺盛,细胞接种后第6天,基本覆盖培养孔。而未转染组（30代）细胞一直生长缓慢,差异显著（P〈0.05）。未转染组（30代）山羊胎儿成纤维细胞处于S期细胞和G1期细胞分别为28.9%和60.8%,而转染组（30代）分别为45.2%和45.0%。说明外源性hTERT基因可促进山羊胎儿成纤维细胞由G1期向S期转变,并提高细胞增殖能力。转染组（50代）山羊胎儿成纤维细胞染色体核型正常,未发生变化。未转染组（30代）细胞凋亡率和死亡率分别为39.7%和29.4%,而转染组（30代）分别为11.0%和12.7%,差异极显著（P〈0.01）。hTERT蛋白在转染组（30代）山羊胎儿成纤维细胞中表达,蛋白相对分子质量为127 000。说明外源性hTERT基因可以延长胎儿成纤维细胞在体外培养的寿命,降低细胞凋亡率。     

镉对鸡胚成纤维细胞增殖和凋亡的影响

以鸡胚成纤维细胞为研究对象,探讨镉影响细胞增殖及凋亡的作用。应用酶标仪、倒置相差显微镜、激光共聚焦显微镜及流式细胞仪,分析细胞毒性、细胞形态、凋亡率、细胞周期与线粒体膜电位及胞内钙离子稳态。结果表明:镉处理后,细胞活性下降,呈现凋亡形态学变化,细胞周期阻滞于G1期,细胞凋亡率呈浓度和时间依赖性,细胞线粒体膜电位下降,胞内游离钙离子浓度增大。研究发现,镉可通过阻滞细胞周期,降低线粒体膜电位,影响胞内钙离子稳态来诱导鸡胚成纤维细胞凋亡。     

MSTN基因敲低绵羊成纤维细胞株的筛选

为了获得绵羊MSTN基因敲低成纤维细胞株,克隆获得MSTN基因序列构建其真核表达载体;设计合成并筛选3种MSTN基因干涉序列(M1、M2和M3),构建相应的3种RNA干涉载体;将MSTN基因真核表达载体单独或分别与3种RNA干涉载体共转染绵羊成纤维细胞后,分别检测其对MSTN基因表达的干涉效率以获得最佳的MSTN基因RNA干涉载体;将筛选的RNA干涉载体线性化后转染绵羊成纤维细胞,通过筛选获得转基因细胞株。结果显示,成功克隆得到与GenBank中序列同源性为100%的绵羊MSTN基因序列,并获得真核表达载体pcDNA3.1(+)-MSTN;构建获得MSTN基因RNA干涉载体pRNAT-M1、pRNAT-M2和pRNAT-M3;与单独转染pcDNA3.1(+)-MSTN后293GP细胞中MSTN基因的相对表达量相比,转染干涉载体后MSTN基因相对表达量明显下降,其中pRNAT-M1干涉效率最佳;将pRNAT-M1线性化后转染绵羊成纤维细胞并利用G418筛选获得转基因阳性细胞;获得的转基因阳性细胞的细胞形态、生物学特性均呈现正常;PCR检测结果显示,pRNAT-M1已整合到细胞基因组中。上述结果表明,成功获得了MSTN基因敲低的绵羊成纤维细胞株。     

鸭病毒性肠炎病毒致鸭胚成纤维细胞发生凋亡作用的研究

研究通过HE染色镜检发现,鸭病毒性肠炎病毒CH强毒株(DEV-CHv)可致鸭胚成纤维细胞(DEF)出现染色质颗粒化、细胞核变形等显著凋亡特征;TUNEL试验显示,接毒组细胞核内有多量棕黄色DAB显色颗粒,表明细胞出现了凋亡现象;DNA Ladder检测表明,接毒组细胞具有分子量分别为180～200bp及其整数倍的凋亡梯带电泳图谱特征;电镜观察发现,接毒组细胞具有染色质浓缩、边移,胞浆严重空泡化,细胞核严重变形,形成凋亡小体等典型凋亡特征.上述研究结果表明鸭病毒性肠炎病毒具有显著的致鸭胚成纤维细胞发生凋亡的作用.     

用转染或非转染的胎儿成纤维细胞克隆侏儒山羊

本文对山羊胎儿成纤维细胞经体外转染 ,核移植后核的发育潜能性进行了研究。1　材料和方法1.1　分离供体细胞利用外科手术从侏儒山羊 BEL E品系 (配种早 ,泌乳早 ) 2 7～ 30 d胎儿分离出成纤维细胞。将去头和内脏的胎儿机械分离 ,于 DMEM+2 0 % FBS+2 0 mg/m L庆大霉素、38℃和 5 % CO2 条件下培养。培养中成纤维细胞生长占优势 ;当细胞生长丰度达 70 %时转移到 0 .0 5 %胰蛋白酶 - EDTA中处理 ,记数。一半冷冻于10 % DMSO+90 % FBS中 ,另一半放入有孔培养板中作遗传学分析。解冻后染色体正常的细胞用于核移植或转染 DNA以繁殖…     

转pK2.10-拟蜘蛛拖丝蛋白基因绵羊成纤维细胞株的筛选

为了筛选转pK2.10-拟蜘蛛拖丝蛋白基因绵羊成纤维细胞株,本研究参照GenBank上蜘蛛拖丝蛋白基因序列(AY555585和AH015065)设计并人工合成拟蜘蛛基因四聚体(4S),与pcDNA3.1载体(K2.10启动子)相连构建真核表达载体(pcDNA3.1-K2.10-4S),采用脂质体介导法转染绵羊成纤维细胞,通过药物筛选、阳性细胞鉴定及体外培养等方法,获得了转蜘蛛拖丝蛋白基因的绵羊成纤维细胞株.结果,用pcDNA3.1-K2.10-4S表达载体转染冷冻复苏的绵羊成纤维细胞后,利用G418筛选法获得neo基因的抗性细胞,即阳性细胞;采用PCR方法对阳性细胞鉴定显示,所构建的载体整合到了细胞基凶组中;对阳性细胞的染色体核型分析显示,阳性细胞核型稳定,与正常细胞的核型无差异;阳性细胞经冷冻复苏后仍能保持原有的特性.该细胞株的建立为进一步开展转拟蜘蛛拖丝蛋白基因的克隆羊研究奠定了基础.     

二甲基砷酸对辽宁绒山羊皮肤成纤维细胞的毒性作用以及诱导细胞凋亡的机制

旨在用类金属毒性物质——砷对辽宁绒山羊皮肤成纤维细胞的染毒试验,探索砷对绒山羊皮肤成纤维细胞毒性作用和其诱导细胞凋亡的机制,为后续研究砷对绒山羊皮肤细胞生长的影响提供理论依据.本研究随机选取1只7月龄各项机能正常,体重为25 kg左右的雄性辽宁新品系绒山羊,取其腹部皮肤,进行细胞培养.将二甲基砷酸药物配制成11个不同浓...     

绵羊耳成纤维细胞的体外培养

用组织块法和冷消化法对绵羊耳成纤维细胞在含血清培养液(RPMI—1640)中进行原代和传代培养，探讨动物体细胞培养模式，并对正常细胞形态进行观察。结果表明组织块法和冷消化法培养可获得比较均一、稳定的细胞群，细胞呈典型的成纤维形态，并成功进行传代培养，建立了绵羊耳成纤维细胞系。     

牛碱性成纤维细胞生长因子基因在大肠杆菌中的表达及其表达产物的生物学活性

采用巢式PCR方法克隆了牛18ku-bFGF基因完整的编码序列,并构建了原核表达载体pET-28a-bFGF,将其转化大肠杆菌BL21,在25℃低温条件下,用0.5 mmol/L IPTG诱导表达5 h,用Ni-NTA亲和纯化细胞裂解上清液,经Western-blotting检测,结果显示,在特定的诱导条件下,重组牛bFGF基因在大肠杆菌中获得了表达,并且主要以可溶性状态存在于细胞中。经检测,纯化后的重组蛋白能显著促进成纤维细胞的增殖（P〈0.05）,其活性与商品用重组人18ku-bFGF没有差异（P〉0.05）。表明,所获得的可溶性重组牛18ku-bFGF蛋白具有较高的生物学活性,可用于后续研究工作。     

陆川猪耳部成纤维细胞的体外培养

本研究建立陆川猪耳部成纤维细胞的体外培养体系,采用组织块培养法可以获得陆川猪耳部成纤维细胞。用0.25%胰蛋白酶＋0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,能很好的支持陆川猪耳部成纤维细胞的生长。传3代后,观察到培养的细胞形态逐渐均一,为典型的成纤维细胞,绝大部分呈梭形或不规则三角形...     

CPEB3 regulates the proliferation and apoptosis of bovine cumulus cells

Cytoplasmic polyadenylation element‐binding protein 3 (CPEB3) is a member of the Cytoplasmic polyadenylation element‐binding family, which has been found to regulate the translation of dormant and masked mRNA in Xenopus oocytes and plays potential roles in regulating biological functions in cells and tissues. However, its role in cumulus cells is not clear. In this study, the mRNA expression of CPEB3 in bovine cumulus cells was inhibited with small interfering RNA. Cell cycle progression, proliferation, and apoptosis were measured after inhibition of CPEB3. Subsequently, changes in intracellular Reactive oxygen species content, mitochondrial membrane potential and expansion‐related gene expression were examined. The results showed that after CPEB3 inhibition, cumulus cells had an abnormal cell cycle, the numbers of cells in the S and G2/M phases were significantly increased, cell proliferation was increased and apoptosis rates were decreased. These effects were likely due CPEB3 inhibition‐induced decreases in intracellular Reactive oxygen species levels; increases in mitochondrial membrane potential; decreases in apoptosis; downregulation of CCNA, CCND, CCNE, CDK2, CDK4, CDK6, p21, and p27 mRNA expression; and upregulation of CCNB, CDK1, HAS2, PTGS2, PTX3, and CEBPB mRNA expression. Therefore, CPEB3 plays potential roles in regulating the biological and physiological functions of bovine cumulus cell.     

表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定

为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。     

Effect of caspase inhibitor Z-VAD-FMK on bovine sperm cryotolerance

The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.     

奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。     

Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

Background

Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described.

Findings

Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells.

Conclusions

Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.     

维生素E对牛核移植供体细胞凋亡率的影响

采用光镜观察、DNA琼脂糖凝胶电泳、Hoechst33342荧光染色及流式细胞仪分析等方法,检测了不同浓度的维生素E对新生牛耳缘成纤维细胞凋亡的影响。结果表明,不同浓度的维生素E均可显著降低牛耳缘成纤维细胞的凋亡率,与对照组相比差异极显著(P<0.01),以100μmol/L为最佳浓度。DNA琼脂糖凝胶电泳检测,100、150μmol/L维生素E处理组电泳结果未见梯状条带,对照组和50μmol/L维生素E处理组则出现典型的梯状条带。