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1.
采用组织块移植培养技术,对来源于匙吻鲟鳍条组织的细胞进行原代培养,建立了匙吻鲟鳍条组织细胞系,已稳定传代培养80多代,定名为PF-Fin。匙吻鲟鳍条组织细胞的形态为成纤维样细胞,其最佳培养基为M199,最适血清浓度为10%,最适培养温度为25℃。液氮冷冻保存8个月后的细胞经台盼兰染色检验,约87.68%±3.61%仍保持活性,细胞复苏后培养生长旺盛。匙吻鲟鳍条组织细胞的集落形成效率为13.75%±2.28%;细胞染色体分析结果表明,第10代传代细胞的染色体数目为正常二倍体2n=120,第59代传代培养细胞的染色体众数为90。病毒敏感性试验结果表明,PF-Fin细胞对草鱼呼肠孤病毒(GCRV)、斑点叉尾鮰呼肠孤病毒(CCRV)和鲤鱼春季病毒血症病毒(SVCV)敏感,可产生典型细胞病变效应(CPE)。  相似文献   

2.
为了开展中华鲟(Acipenser sinensis)种质资源保存及其精原干细胞体外培养的研究, 采用蛋白酶消化法, 对中华鲟精巢组织细胞进行原代培养, 建立了中华鲟精巢细胞系(Acipenser sinensis testicular cell line, AST), 经352d传代培养, 已稳定传至80代。中华鲟精巢细胞系形态主要呈类纤维状, 培养基为DMEM, 培养温度为25℃, 最适血清浓度为15%。正常传代的AST细胞冻存、复苏后, 经台盼蓝染色, 约(81.36±1.13)%的细胞具有活性, 复苏后细胞仍生长旺盛。染色体核型分析结果显示, 第30代中华鲟精巢细胞系染色体数目分布在142—310, 众数为264。通过RT-PCR检测发现, 在P0和P1细胞中, Sertoli细胞特异表达基因(amh和gsdf)、Leydig细胞特异表达基因(cyp17a1)和生殖细胞特异表达基因(dazl、dnd和vasa)都有表达, 且表达量与精巢中的相似; 在P15、P30和P60细胞中, 只有amh和vasa基因有微弱的表达, 说明细胞系传代到了后期, 只含有极少量的Sertoli细胞和生殖细胞。通过脂质体转染法将pEGFP-N3质粒转入AST细胞中, 可表达增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)。AST细胞系的建立为中华鲟种质资源的保存、精原干细胞的体外增殖与分化、基因功能等研究提供了重要的实验材料。  相似文献   

3.
我们从1983年9月至1985年1月,将草鱼尾鳍组织细胞在体外培养17个月,传代100次,确立为建立的细胞系(established cell lioe),命名为GCCF-2。1.细胞系的建立无菌获取草鱼尾鳍组织,采用组织块培养法置TC-199培养液中进行初代培养。20代之前培养液中小牛血清含量为20%;20代之后,小牛血清含量降为15%。培养温度为28—30℃。1∶4传代培养,4—5天传代一次。2.细胞形态由于在初代培养及以后的传代培养过程中未加任何选择,GCCF-2为上皮样细胞和成纤维样细胞的混合细胞系。但上皮样细胞占多数。绝大多数细胞为单核,二、三及多核细胞很少。3.细胞生长速度以10~5细胞/ml的浓度接种培养,第二天细胞数即有所增加,第五天达最  相似文献   

4.
为开展鳗鲡病毒性疾病流行病学调查和病原生物学研究, 进行鳗鲡病毒的分离、培养和鉴定, 研究采用组织块培养法, 以欧洲鳗鲡肾脏组织为材料, 建立了欧洲鳗鲡肾脏细胞系(European eel kidney cell line, EEK), EEK形态呈类纤维状, 经过约12个月的培养, 已成功传至38代。通过对其培养液、血清浓度和培养温度等条件进行优化, 发现DMEM/F12、L15培养液均适合其正常生长和增殖, 而在MEM培养液中无法正常生长; 在5%—15%FBS(Fetal Bovine Serum)浓度范围内, 其生长速度随FBS浓度的升高而增快, 当FBS浓度高于20%或过低于5%时, 其生长速率有所减慢; 在22—27℃时生长良好, 但当温度低于17℃和高于32℃时细胞生长速度明显减慢。对EEK细胞进行鳗鲡疱疹病毒(Anguillid herpesvirus, AnHV)敏感性实验, 结果表明该细胞系对鳗鲡疱疹病毒敏感, 可产生明显的细胞病变。EEK的建立丰富了鱼类细胞系的种类, 为鳗鲡病毒性疾病诊断、病毒性病原学研究和病毒疫苗研制提供了重要实验材料。  相似文献   

5.
以背角无齿蚌(Anodonta woodiana)血细胞(含颗粒细胞和透明细胞)、消化腺细胞(含上皮样细胞和圆形小细胞)、外套膜细胞(含上皮样细胞、透明细胞、圆形大细胞和圆形小细胞)、斧足细胞(含上皮样细胞、透明细胞、圆形大细胞和圆形透亮细胞)为材料,比较了上述各类细胞的原代培养特征以及不同条件下的细胞存活率,优化了细胞制备及细胞活性鉴定的方法。结果显示,上述各类组织细胞存活率受不同培养温度、培养时间和培养基种类的影响,其中,血细胞96 h在20℃下、培养基2中的存活率最高,为94.67%±0.47%;消化腺细胞48 h在20℃下、培养基3中的存活率最高,为93.67%±1.70%;外套膜细胞48h在15℃下、培养基1中的存活率最高,为93.67%±1.7%;斧足细胞48h在15℃下、培养基1的存活率最高,为94.33%±0.94%。相较于25℃,在20℃和15℃下,4种组织细胞96h的存活率更为理想。本研究旨在为背角无齿蚌组织细胞系的建立及贝类细胞水平毒理学的研究提供基础数据。  相似文献   

6.
采用组织块培养法启动二倍体、三倍体和四倍体泥鳅鳍组织细胞原代培养,采用胰蛋白酶消化法传代,目前二倍体、三倍体和四倍体细胞已经分别传至59代、68代和68代。三种细胞均为成纤维细胞样细胞。鳍组织无菌处理方法是:先用质量浓度为10%的碘伏浸泡15min;后用青霉素和链霉素的混合液(500 IU/mL青霉素,500μg/mL链霉素)浸泡30min;原代培养和早期传代培养所用的培养基为DMEM/F12,添加体积分数为20%的胎牛血清、10 ng/mL人碱性成纤维细胞生长因子(bFGF)、20 ng/mLI型胰岛素样生长因子(IGF-I)以及10μg/mL硫酸软骨素。30代以后所用培养基为20%FBS-DMEM/F12。细胞培养在25℃、5%CO2培养箱中。在此条件下,二倍体、三倍体和四倍体的倍增时间分别为48.43h、36.01h和41.45h;二倍体、三倍体和四倍体的特征染色体数目分别为50条、75条和100条;测定的二倍体、三倍体和四倍体细胞及核的体积比分别为1︰1.37︰2.37和1︰1.53︰1.97;细胞经液氮冷冻保存60d后,解冻复苏后测定存活率分别为(80.88±1.38)%、(84.48±1.13)%、(81.57±1.28)%。不同倍性的泥鳅细胞系的建立丰富了鱼类细胞系的种类,为揭示多倍体鱼类生长、遗传等机制打下基础。  相似文献   

7.
用逆转录病毒载体将单纯疱疹病毒苷激酶基因(HSVtk)导入恶性肿瘤细胞,随后可应用药物9(1,3-二羟基-珍氧基-甲基)鸟嘌呤选择性地杀死肿瘤细胞,将HyTK基因替换逆转录病毒载体GlNa中的neo基因,构建成重组逆转病毒载体GTK,转染混合包装细胞(双噬性PA317细胞和单噬性GP+E-86细胞),通过“乒乓效应”获得高滴度重组病毒,用该重组病毒转染不鼠恶性黑色素瘤细胞系B16细胞,用ygrom  相似文献   

8.
采用组织块培养法启动二倍体、三倍体和四倍体泥鳅鳍组织细胞原代培养,采用胰蛋白酶消化法传代,目前二倍体、三倍体和四倍体细胞已经分别传至59代、68代和68代。三种细胞均为成纤维细胞样细胞。鳍组织无菌处理方法是:先用质量浓度为10%的碘伏浸泡15min;后用青霉素和链霉素的混合液(500 IU/mL青霉素,500μg/mL链霉素)浸泡30min;原代培养和早期传代培养所用的培养基为DMEM/F12,添加体积分数为20%的胎牛血清、10 ng/mL人碱性成纤维细胞生长因子(bFGF)、20 ng/mLI型胰岛素样生长因子(IGF-I)以及10μg/mL硫酸软骨素。30代以后所用培养基为20%FBS-DMEM/F12。细胞培养在25℃、5%CO2培养箱中。在此条件下,二倍体、三倍体和四倍体的倍增时间分别为48.43h、36.01h和41.45h;二倍体、三倍体和四倍体的特征染色体数目分别为50条、75条和100条;测定的二倍体、三倍体和四倍体细胞及核的体积比分别为1︰1.37︰2.37和1︰1.53︰1.97;细胞经液氮冷冻保存60d后,解冻复苏后测定存活率分别为(80.88±1.38)%、(84.48±1.13)%、(81.57±1.28)%。不同倍性的泥鳅细胞系的建立丰富了鱼类细胞系的种类,为揭示多倍体鱼类生长、遗传等机制打下基础。  相似文献   

9.
将HIV-1中国株42(B亚型)gag基因及gag与gp120 V3区的嵌合基因gag V3插入腺病毒伴随病毒(AAV)表达载体(pSNAV)质粒中,构建重组质粒pSNAV-gag及pSNAV-gagV3;采用脂质体转染的方法分别将重组质粒转入BHK细胞,G418筛选得到转入重组质粒并能表达外源基因的细胞系,命名为BHK-gag及BHK-gagV3。用具有重组腺病毒伴随病毒(rAAV)包装功能的一种重组单纯疱疹病毒(rHSV)分析感染这两株细胞系,纯化后得到rAAV,电镜观察可见到大量实心病毒颗粒,核酸杂交检测重组病毒滴度达到10^12病毒颗粒/ml,重组病毒感染293细胞,ELISA检测有gag及gagV3基因的表达。用重组病毒免疫Balb/C小鼠,检测抗体及细胞免疫水平,证明重组病毒可以在小鼠体内诱导产生细胞及体液免疫。  相似文献   

10.
藏羚羊(Pantholops hodgsonii)是我国特有的国家Ⅰ级重点保护野生动物,生存在高海拔低氧环境下,具有高原生物的细胞遗传特性。实验采用组织块贴壁培养法进行原代培养,后经差异贴壁法联合消化排除法纯化,成功构建了藏羚羊成纤维细胞系,并对培养不同代次细胞的形态、生长动力学、染色体核型等进行了研究。结果表明,培养得到的藏羚羊细胞为典型的成纤维细胞,第7、10、15代细胞群体倍增时间分别为26、48、50 h。细胞的生长均为"S"型。染色体中二倍体染色体(2n=60)占主体,所占比例为65%~91%,包括性染色体X、Y在内均为端着丝粒类型,染色体组的总相对长度为193.45,平均相对长度为3.22,未发现随体、次缢痕等特殊标志性特征。  相似文献   

11.
The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

12.
The first continuous cell line from the embryo of Gampsocleis gratiosa (Orthoptera: Tettigoniidae), designated as RIRI-GG1, was established. This cell line was serially subcultured in modified Grace medium. The cells were grown adherent to a culture flask and had spindle-like and polygonal shapes. The chromosome number ranged from 26 to 79 at the 50th passage, and 68% of cells had a diploid chromosome number. The growth rate was determined at the 53rd passage, and the population doubling time was calculated to be 122.1 h. The rDNA internal transcribed spacer and the mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-GG1 cell line was derived from G. gratiosa. This cell line had no apparent susceptibility to Autographa californica nucleopolyhedrovirus and Bombyx mori nucleopolyhedrovirus.  相似文献   

13.
A continuous Puntius sophore caudal-fin (PSCF) cell line of the pool barb Puntius sophore, an important freshwater food and ornamental fish of Asia and South East Asia, was developed from the caudal fin for the first time. The cell line was optimally maintained at 28° C in Leibovitz-15 (L-15) medium supplemented with 10% foetal bovine serum (FBS). The cytogenetic analysis revealed a diploid count of 50 chromosomes at 25th and 50th passage and 52 chromosomes at passage 70, 85 and 100. The viability of the PSCF cell lines was 75% after 6 months of storage in liquid nitrogen (-196° C). The most striking feature of the PSCF cells was its high increased growth ratio as evident from the population doubling time of 25 h at passage 100. The origin of the cell lines was confirmed by the amplification of 581 and 655 bp fragments of 16 S rRNA and cytochrome oxidase subunit I (COI) of mitochondrial DNA (mtDNA) genes, respectively. The PSCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the potential utility of the cells in gene expression studies.  相似文献   

14.
Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm3 for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.  相似文献   

15.
P. Li  L. Zhou  S. Wei  M. Yang  S. Ni  Y. Yu  J. Cai  Q. Qin 《Journal of fish biology》2017,90(5):1944-1959
A cell line derived from the head kidney of golden pompano Trachinotus ovatus (TOHK) was established and characterized in this study. The TOHK cells grew most rapidly at 28° C and the optimum foetal bovine serum concentration in L‐15 medium was 10%. The TOHK cells have a diploid chromosome number of 2N = 54. The transfection efficiency of TOHK cells was 7·5% at the 15th passage and 72% at the 40th passage. The transfection efficiency in TOHK cells was high, so these cells are suitable for foreign gene expression. The cytotoxic effects of heavy metals and extracellular products from Vibrio anguillarum and Vibrio alginolyticus were demonstrated in TOHK cells, so this TOHK cell line could also be applied in environmental monitoring of heavy metals and pathogenic bacteria. TOHK cell line showed high virus susceptibility, such as grouper nervous necrosis virus (GNNV) and Singapore grouper iridovirus (SGIV). Then, TOHK cell line could be used for the study of viral pathogenesis and the development of antiviral strategies.  相似文献   

16.
目的:改进现有的细胞冷冻保存方法,建立一个不舍二甲基亚砜(DMSO)和血清(FBS)的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法:海藻酸微囊包埋鼠胚成纤维细胞(STO细胞)后用不含DMSO和FBS的冷冻保存液进行冷冻保存。,设四个对照组:添加10%DMSO和20%FBS的组、仅添加10%DMSO的组、仅添加20%FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物(EthD)、钙黄绿素-AM(Calcein—AM)进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果:冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论:使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMS0和FBS的高效冷冻保存方法。  相似文献   

17.
A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.  相似文献   

18.
目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。  相似文献   

19.
The cell line designated HCS-2 established from a squamous cell carcinoma of the uterine cervix has been subcultivated 77 times since Nov. 8, 1983. The cultured cells appear epithelial in shape, with a pavement-like arrangement and grow without contact inhibition. In electron microscopy, the cells are characterized by desmosomal cell contacts and a few tonofilaments. The cells are transplanted subcutaneously to nude mice and produce tumor which resembles the original tumor of large cell non-keratinizing squamous cell carcinoma. The growth rate of subculture has increased gradually, and population doubling time of cells at 17th passage was about 65 hours. The chromosome studies show aneuploidy and chromosomal number was mainly from hypertriploid to hypotetraploid range. The modal number of cells at 33rd passage was 81. Specific marker chromosome is not realized. The production of SCC antigen is detected from the cells and the amount of SCC antigen in cultured media was recorded from 1.5 to 2.0 ng per 1 x 10(4) cells for 48 hours. CEA synthesis is also confirmed immunohistochemically.  相似文献   

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