Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

shRNA-triggered RNAi inhibits expression of NDV NP gene in chicken embryo fibroblast

RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a methodological platform for RNAi studies in CEF,three plasmid vectors expressing short hairpin RNAs(shRNAs)targeted against the Newcastle disease virus(NDV)NP gene were constructed.One of them,ndv1,was proven effective on blocking viral replication in CEF and chicken embryos.Four hours prior to infection with NDV,the CEF was transfected with the plasmids by Silent-fect.An unrelated shRNA sequence(HK)was used in mock transfection.The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 fold decreases in NP gene expression at 3,6 and 9 h post infection in CEF,respectively.The ndv1 was able to completely inhibit the replication of the virus in CEF within 48 post infection.Furthermore,the pathological changes in CEF caused by NDV were delayed,and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndv1.When the complex of shRNASilent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 10^(5) or 10^(6) ELD50 NDV,NDV replication was decreased by 94.14% and 62.15% after 17 h,respectively.These findings suggest that the newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.

利用CRISPR/Cas9编辑系统构建ACTA1基因敲除的PEFs细胞系

【目的】利用CRISPR/Cas9技术获得α-肌动蛋白1(actin alpha 1,ACTA1)基因敲除的猪胎儿成纤维细胞(porcine embryo fibroblasts, PEFs),为后续在细胞水平上探究猪ACTA1基因功能及发掘友好基因座位奠定基础。【方法】通过实时荧光定量PCR检测ACTA1基因在巴马小型猪心脏、肝脏、脾脏、肺脏、肾脏、背部皮下脂肪和背最长肌7种组织中的表达情况;利用CRISPOR在线网站在猪ACTA1基因第7外显子区域设计3条向导RNA(single guide RNA,sgRNA),并将其连接至pX330载体;通过T7E1酶切检测不同sgRNA活性,选择效率较高且符合目标的载体质粒共转染至PEFs中;利用有限稀释法筛选单克隆细胞,并对获得的单克隆细胞进行基因型鉴定和脱靶分析。【结果】实时荧光定量PCR结果表明,ACTA1基因在背最长肌中的表达量极显著高于其他组织(P<0.01)。设计的3条sgRNA的基因编辑效率分别为24.87%(sgRNA1)、39.59%(sgRNA2)、36.93%(sgRNA3),综合切割位置和基因编辑效率,选用sgRNA1和sgRNA2共转染细胞。基因型鉴定结果表明,获得的69株单克隆细胞中有20株单克隆细胞发生了编辑,其中单等位基因片段敲除细胞3株,双等位基因片段敲除细胞1株,片段敲除效率为5.8%(4/69)。脱靶分析结果显示,在预测的脱靶位点未检测到脱靶效应。【结论】本研究利用CRISPR/Cas9基因编辑系统成功构建了ACTA1基因纯合敲除的PEFs,研究结果为进一步探究ACTA1基因对猪骨骼肌发育调控提供了技术支撑和理论基础,同时为挖掘猪组织特异性表达的友好位点提供新思路。

龙岩山麻鸭胚胎成纤维细胞的培养和鉴定

为培养龙岩山麻鸭鸭胚成纤维细胞。本研究选择龙岩山麻鸭10日龄鸭胚组织,采用组织块法和酶消化法进行成纤维细胞培养,利用免疫荧光法对培养的成纤维细胞进行鉴定。结果表明,培养的鸭胚成纤维细胞12h后呈典型的长梭形,48h后,细胞轮廓清晰,核仁明显,传至第3代的成纤维细胞经DAPI和绿色荧光标记染色后均呈阳性,具有成纤维细胞的特征。本研究成功从龙岩山麻鸭鸭胚组织中分离培养出成纤维细胞,为后期龙岩山麻鸭开展分子生物学和细胞生物学等研究提供良好的基础。

Adaptability of Fowlpox Virus in Chicken Embryo Passage Cells

[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.

QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

Objective To quantify proline 4-hydroxylase, alpha polypeptide Ⅱ (P4HA2) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E+04 copies/mL of P4HA2 mRNA, corresponding to 103 fibroblasts. In addition, existence of 8.67E+06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13.76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

抗鸭坦布苏病毒感染的中草药筛选及其作用效果的初步研究

为筛选抗鸭坦布苏病毒(DTMUV)的中草药并探究其抗病毒作用效果,本研究通过检测12种中草药对DTMUV感染鸭胚成纤维细胞细胞(DEF)形态学与病毒抑制率的影响,以及对DTMUV病毒滴度与病毒载量的影响,筛选在DEF中抗DTMUV效果较明显的中草药;采用鸭胚接种法测定筛选出的3种药物(板蓝根、醋五味子、酒黄芩)对DTMUV感染鸭胚死亡率的影响;并选择效果最好的酒黄芩验证其对DTMUV感染雏鸭死亡率、临床症状、体重及组织(心脏、脾脏)中病毒载量的影响。结果显示:鱼腥草、淫羊藿、金银花、板蓝根、绵马贯众、黄芪、酒黄芩、茵陈、甘草、连翘、醋五味子、白芍水提物在最佳作用浓度时均能减轻DTMUV感染DEF的CPE,并对DTMUV具有不同的抑制率,其中酒黄芩效果最佳;淫羊藿、白芍、板蓝根、黄芪、酒黄芩、茵陈、甘草、醋五味子、连翘能显著降低DEF中DTMUV的病毒滴度(P<0.05);板蓝根、醋五味子、酒黄芩能显著降低DEF中DTMUV的病毒载量(P<0.05);板蓝根、酒黄芩能起到治疗效果,且显著降低鸭胚死亡率(P<0.05),而酒黄芩、醋五味子还能预防病毒感染,且显著降低鸭胚死亡率(P<0.05);酒黄芩能够减轻病鸭临床症状,显著提高病鸭体重(P<0.05),显著降低病鸭死亡率及组织中病毒载量(P<0.05)。综上结果证明板蓝根、酒黄芩、醋五味子在细胞和鸭胚内具有一定抗DTMUV的作用,其中酒黄芩效果最佳,且酒黄芩对DTMUV感染的雏鸭具有治疗作用,可以成为抗DTMUV的潜在药物。

基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系

为研究甲基胞嘧啶双加氧酶2(tet methylcytosine dioxygenase 2,TET2)及其介导的羟甲基化修饰在调控天然免疫反应中的作用,利用CRISPR-Cas9基因编辑技术构建TET2基因敲除的鸡胚成纤维细胞系。针对鸡TET2 mRNA的2种剪接变体的共有CDS序列设计4组sgRNA,构建sgRNA表达质粒,连同Cas9-T2A-GFP质粒共转染DF-1细胞,筛选高切割效率的sgRNA;将转染该sgRNA的DF-1细胞,进行流式细胞分选,获取表达绿色荧光蛋白(green fluorescent protein,GFP)的细胞,再通过有限稀释法筛选单克隆细胞,并用DNA测序进行鉴定。单克隆敲除细胞扩大培养后,Western blot和Dot blot检测TET2蛋白的表达水平和羟甲基化(5hmC)水平。DNA测序结果表明,TET2-gRNA-2靶位点附近分别发生2和28 bp的缺失突变,引起TET2蛋白移码突变,将该细胞株命名为DT-6。DT-6细胞传代15次以上,细胞形态稳定。Western blot结果显示,与DF-1细胞相比,DT-6细胞几乎不表达TET2蛋白。Dot blot结果显示,与DF-1细胞相比,DT-6细胞全基因组羟甲基化水平显著降低。利用CRISPR-Cas9技术成功构建了敲除TET2基因的鸡胚成纤维细胞系,为研究鸡TET2及其介导羟甲基化修饰参与调控天然免疫反应的途径和分子机制奠定基础。

先导编辑在鸡胚成纤维细胞系中的效率研究

为探究先导编辑在鸡细胞系中的基因编辑效率,研究通过点突变和重组连接的方式构建先导编辑的表达质粒,使用在线软件设计并化学合成靶向OVM、MSTN和NHE1基因的先导编辑引导RNA(pegRNA)质粒,脂质体转染上述质粒至鸡胚成纤维细胞系(DF-1)中并进行药物筛选,PCR产物TA克隆测序检测并统计各位点的编辑效率。结果显示:测序和PCR鉴定结果表明先导编辑表达质粒构建成功;DF-1中OVM基因经先导编辑后成功产生错义突变,其中精确编辑效率为41.10%、错误插入与缺失(indel)效率为6.67%,均显著优于对照组的常规基因编辑(P<0.05);MSTN基因的先导编辑精确编辑效率为10.24%、indel效率为13.57%,NHE1基因的先导编辑精确编辑效率为24.88%、indel效率为7.18%。结果验证了先导编辑在鸡细胞系中的有效性和普适性,为其进一步应用于基因编辑鸡的制备奠定基础。

不同胰蛋白酶在制备腮腺炎减毒活疫苗中的效果比较

目的比较基因重组胰蛋白酶和猪源胰蛋白酶在腮腺炎减毒活疫苗中的使用效果。方法使用浓度为10%、15%、20%基因重组胰蛋白酶制备鸡胚成纤维细胞,通过细胞密度、细胞活性、24 h以及48 h细胞贴壁状态等指标确定基因重组胰蛋白酶使用浓度;与猪源胰蛋白酶进行比较,通过细胞密度、细胞活性、48 h细胞贴壁状态以及病毒滴度等指标比较两种胰蛋白酶的使用效果;使用基因重组胰蛋白酶制备3批腮腺炎减毒活疫苗,检定疫苗的稳定性;规模化放大验证该工艺的稳定性。结果通过研究发现基因重组胰蛋白酶的使用浓度为15%;与猪源胰蛋白酶进行比较,两种胰酶制备的细胞密度水平一致,无显著性差异(P>0.05),48 h后细胞均生长至单层,细胞状态良好,按照同一MOI进行接毒,病毒滴度无显著性差异(P>0.05);使用CF-10进行试验,病毒滴度也无差异(P>0.05);连续3批使用基因重组胰蛋白酶制备的疫苗热稳定性良好,且该工艺可以规模化放大。结论在制备腮腺炎减毒活疫苗中可以使用浓度15%的基因重组胰蛋白酶替代猪源胰蛋白酶,作为鸡胚成纤维细胞消化液使用。

黄芪多糖、淫羊藿多糖和淫羊藿总黄酮对新城疫病毒感染细胞的影响

将黄芪多糖 (APS)、淫羊藿多糖 (EPS)、淫羊藿总黄酮 (EF)分别与新城疫病毒 (NDV)Ⅰ、Ⅳ系以 3种顺序加入到培养 2 4h的鸡胚成纤维细胞 (CEF)中 ,即先加中药后接种病毒、先接种病毒后加中药、病毒和中药混合感作后同时加入。于病毒接种后 72h测定NDVⅠ系的半数组织培养感染剂量 (TCID50 )和NDVⅣ系的血凝效价 ,以评价 3种中药成分对NDV感染细胞的影响。结果表明 ,APS和EPS仅在先于病毒加入时对NDV有抑制作用 ,而EF无论以何种方式给药均呈抑制作用。提示它们有一定的抗病毒作用 。

10种中药成分对单层鸡胚成纤维细胞增殖的影响

将安全浓度范围内的黄芪多糖、当归多糖、蜂胶多糖、淫羊藿多糖、板蓝根多糖、黄芪黄酮、蜂胶黄酮、淫羊藿黄酮、黄芪皂苷和人参皂苷等 10种中药成分分别加入已培养 2 4h、刚形成单层的鸡胚成纤维细胞 (CEF)中 ,在加药后12 ,2 4 ,36 ,4 8和 6 0h用MTT法测定它们对CEF增殖的影响。结果表明 ,黄芪皂苷、黄芪多糖、板蓝根多糖和蜂胶黄酮主要表现为促进增殖 ;淫羊藿多糖主要具抑制效应 ;其他 5种中药成分在某些浓度和时间点能促进增殖 ,而在某些浓度和时间点则抑制增殖 ,并有一定的量效和时效关系。

黄芪多糖和淫羊藿多糖对培养细胞增殖和衰老的影响

将安全浓度范围内的黄芪多糖(astragaluspolysaccharide,APS)和淫羊藿多糖(epimediumpolysaccharide,EPS)分别加入到培养12h和24h的鸡胚成纤维细胞(CEF)中,测定加药后CEF增殖率和成层率的动态变化。结果表明,2种多糖均能促进细胞增殖和延缓细胞衰老,EPS的作用强于APS,具有一定的量效、时效关系。

几种天然药物成分在体外CEF中最大安全浓度的测定

采用组织细胞培养方法 ,测试了板蓝根多糖、当归多糖、黄芪多糖、黄芪皂甙、人参皂甙、淫羊藿多糖、蜂胶多糖、蜂胶黄酮、黄芪黄酮、淫羊藿黄酮共 1 0种天然药物成分在鸡胚成纤维细胞 ( CEF)体外培养中的最大安全浓度。结果表明 :黄芪多糖、当归多糖对鸡胚成纤维细胞毒性最低 ,最大安全浓度达 1 562 .5μg/ m L ,其后依次是板蓝根多糖、人参皂甙、黄芪黄酮、黄芪皂甙、蜂胶多糖、淫羊藿多糖、淫羊藿黄酮和蜂胶黄酮 ;蜂胶黄酮的毒性最大 ,其最大安全浓度仅 4.883 μg/ m L。

昆明鼠胚胎干细胞的分离培养与鉴定

目的:从昆明系小鼠的早期胚胎分离和培养胚胎干细胞(ES细胞).方法:收集小鼠3.5 d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,5-6d后取隆起生长的内细胞团块分离后再培养,观察集落的生长情况并通过碱性磷酸酶染色、原位杂交、细胞核型分析等对细胞集落进行鉴定.结果:ES细胞集落性生长,符合小鼠胚胎干细胞的一系列特性.结论:昆明系小鼠囊胚在胚胎成纤堆细胞饲养层上可以发育成ES细胞,并能进行传代培养.

4种典型纳米材料对小鼠胚胎成纤维细胞毒性的初步研究

为探讨不同种类纳米材料对原代培养小鼠胚胎成纤维细胞(Mouse embryo fibroblasts,MEF)的毒性效应及作用机制,选择4种典型的纳米材料(纳米碳、单壁碳纳米管、纳米氧化锌、纳米二氧化硅)制备颗粒悬液,设立5个剂量组(5、10、20、50、100μg·mL-1)对BALB/c小鼠MEF细胞进行24、48、72h染毒培养,利用细胞形态学观察和噻唑蓝实验(MTT比色法)检测上述4种纳米材料对MEF细胞活性的影响,同时,测定染毒24h后细胞培养液上清中乳酸脱氢酶(LDH)活性以探讨纳米颗粒对细胞膜完整性的影响.结果显示:1)4种纳米材料均能明显影响MEF细胞的生长形态.染毒24h后,MEF细胞发生不同程度的回缩变形,细胞间隙增大,排列稀疏,胞内颗粒物增多,细胞透明度下降.2)纳米碳、纳米氧化锌、纳米二氧化硅对MEF细胞增殖的抑制作用和对细胞膜完整性的损伤作用均随染毒剂量的升高而增强,具有明显的剂量-效应关系,其半数致死浓度(24h-IC50)分别为21.85、21.94、461.10μg·mL-1;碳纳米管组的剂量-效应之间不呈对数线性关系,未能得出其24h-IC50.3)在不同染毒剂量水平上,4种纳米材料的毒性对比差异显著:低剂量水平上纳米碳与碳纳米管的毒性强于纳米氧化锌和纳米二氧化硅,随着剂量的升高纳米氧化锌的细胞毒性升高最为显著.结果提示,纳米材料能够对MEF细胞造成毒性损伤,破坏细胞膜的完整性可能只是作用途径之一;纳米材料的毒性可能受粒径、形状、化学组成等许多因素的影响.

鸡胚成纤维细胞原代培养及应用

原代培养是指在体外模拟体内生理环境,使从体内取出的组织细胞生存、生长和传代,并维持原有组织细胞的结构和功能特性。鸡胚成纤维细胞具有相对容易获得、增殖能力强、适应性强、良好的耐受性、性状比较稳定等特点,使得其被广泛地应用于分子和细胞生物学研究的许多方面。论文综述了近年来有关鸡胚成纤维细胞的培养技术,如鸡胚的取材、细胞的分离、纯化和培养,同时介绍了应用进展和未来研究方向。

胚胎大鼠脑和脊髓神经干细胞的分离和培养

研究采用显微解剖、无血清细胞培养和免疫荧光细胞化学染色等实验技术 ,成功地建立了胚胎大鼠脑和脊髓神经干细胞 (NSCs)的分离和培养方法。结果显示 ,( 1)在含成纤维细胞生长因子 2 (FGF 2 )和表皮生长因子(EGF)的无血清培养液中 ,两种来源的NSCs经体外培养 8- 10代后 ,其细胞数呈指数级增加 ,其中脑来源的NSCs数由原代培养时的 1× 10 6 增加至 1× 10 12 ,脊髓来源的NSCs数从 1× 10 6 增加至 1× 10 11。增殖的细胞表达神经上皮干细胞蛋白 (nestin) ;( 2 )在含 1%胎牛血清 (FBS)的培养条件下 ,它们都能被诱导分化为神经元、少突胶质细胞和星型胶质细胞。但其分化比例可随细胞传代次数的增加而改变 ,其中 ,大脑来源的NSCs分化为神经元的比例从第二代 (P2 )的 11 95± 2 5 %下降至第五代 (P5)的 1 97± 1 16% (P <0 0 1) ,而少突胶质细胞的分化比例则基本保持不变 ,这一分化格局同样可在脊髓来源的NSCs中发现。结果表明 ,我们所分离和培养的细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能 ,它们都表达nestin 。