东方田鼠胚胎成纤维细胞永生化细胞系的建立

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。     

建立鸡EPGCs单细胞克隆株初探

目的探索鸡EPGCs单细胞克隆建立细胞系的可能性,并对其生物学特性进行鉴定。方法采取19期和28期的鸡EPGCs,体外培养传至第4代的细胞聚合体,用胰酶消化将细胞分散成单细胞悬液,将单个细胞接种至96孔板,每个孔内接种1个细胞,生长出的细胞集落用胶原酶消化传代,细胞化学法和免疫荧光法检测多向分化细胞的表面标志物;常规染色体核型检查。结果接种的288个单细胞中有9个扩增,其中7个传至第2代,2个传至第4代,克隆形成率为3.1%。扩增出的2~4代单细胞克隆能稳定增殖不分化,具有正常二倍性染色体核型、碱性磷酸酶阳性、阶段特异性胚胎抗原(SSEA)-1等特征性细胞表面标志物呈阳性;离体情况下具有形成类胚体和类上皮样细胞的能力。结论建立鸡EPGCs单细胞克隆细胞系是可行的,其生物学特性稳定。     

建立人类异常染色体核型的成纤维细胞系及滋养层细胞系

从自然流产绒毛或者中期妊娠羊水中分离细胞体外培养, 建立人类异常染色体核型成纤维细胞系及滋养层细胞系.中期妊娠羊水染色体诊断或自然流产绒毛染色体诊断过程中, 行G显带细胞核型分型, 对于发现异常核型的细胞, 进行分离、培养、传代、冻存、复苏及鉴定, 建立细胞系, 用PowerPlex 16系统行DNA-STR基因型检测.建立1株来自羊水的21三体成纤维细胞系, 以及7株来自绒毛的滋养层细胞系, 核型分别为47, XX 21; 69, XXX; 69, XXY; 47, XY 12; 47, XX 5; 48, XY 21, 22; 47, XY 18.所有细胞系体外传代均超过10代, 冷冻复苏率大于50%, 核型维持稳定, DNA-STR基因检测能对人细胞系进行个体识别.人异常染色体核型的成纤维细胞系和绒毛膜滋养层细胞系的建立,可以为探讨异常染色体产生机制及相关的分子遗传学研究提供细胞来源.     

人体膀胱移行细胞癌细胞系KGBC的建立及其生物学特性

本文报告建立了一株人膀胱移行细胞癌细胞系,命名为KGBC。KGBC细胞已在体外培养了10个月,传代65代。组织培养中,KGBC细胞形态为上皮样,呈单层或多层生长,接触抑制消失。光镜和电镜下,KGBC细胞的形态结构与膀胱移行细胞癌的形态结构相似。第27代和第42代细胞异种移植,能在小鼠体内生长,组织相与原发癌相似。第9代和第21代细胞的染色体数从65到190条,众数为90—95条。第35代细胞倍增时间为28小时。细胞经液氮保存,复苏良好。这些表明已建立了一株能在体外稳定生长的人膀胱癌细胞系。     

中华鲟精巢细胞系的建立和鉴定

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。     

一株棕尾别麻蝇胚胎细胞系的建立及其特性分析

双翅目昆虫细胞系广泛应用于遗传学、发育生物学、分子生物学、人和动物体病原学以及昆虫抗微生物肽的研究。本研究建立了一株新的棕尾别麻蝇Sarcophaga peregrina胚胎细胞系。该细胞系的原代培养始于2008年11月17日, 取材于棕尾别麻蝇晚期胚胎组织, 在Shields & Sang M3昆虫培养基中于28℃恒温培养, 在第26天进行第1次传代, 至今已历时21个月, 传代72次, 生长状态稳定, 被命名为Sp-E-HNU11。该细胞系的细胞形态主要呈梭形和近圆形, 杂以少量巨型细胞, 紧密贴壁生长。细胞群体倍增时间为42 h。染色体数目一般为10条或12条, 为二倍体或亚二倍体细胞系; 除一对颗粒状微型染色体外, 其他染色体呈短杆状。细胞系的β-萘酯酶和谷草转氨酶同工酶谱上分别显示出1条和3条酶带。随机引物扩增多态性 (random amplified polymorphic DNA, RAPD) 分析结果显示, 该细胞系与小菜蛾细胞系Px-E-HNU12、草地贪夜蛾细胞系IPLB-Sf-9和家蚕细胞系Bm-21E-HNU5呈现明显不同的带型特征。 Sp-E-HNU11细胞系的建立为昆虫抗微生物肽及其他相关的研究工作增添了新的研究工具和生产载体。     

小鼠原生殖细胞建系过程及其分化特性的研究

以小鼠8.5dpc、10.5dpc、12.5dpc胚胎为材料,分离其中包含PGC的胚胎组织,使其生长于饲养层细胞上,在生长因子LIF、SCF和bFGF的共同作用下存活增殖,形成PGC克隆,经过几次分散转移至新的饲养层细胞,产生稳定增殖的EG干细胞克隆,共建成5株EG细胞系,AKP染色以及oct-4基因表达产物的免疫荧光检测均显示阳性。EG1、EG2、EG3、EG4、EG5,分别来自8.5、10.5dpc的胚胎,没有得到长期培养的12.5dpc的EG细胞系。EG细胞系在有饲养层细胞或添加LIF的环境中可稳定传代,保持不分化状态,至少15代内正常核型细胞所占比例80%以上。去除抑制分化因素的前提下,悬浮培养的EG细胞形成胚体,分化出类似胚胎内胚层和外胚层的细胞结构;贴壁生长的胚体能产生不同类型的分化细胞,包括上皮细胞、成纤维细胞、神经细胞等。EG细胞在裸鼠体内形成畸胎瘤。以上结果证实我们建立的EG细胞系具发育多能性,为研究早期胚胎和生殖细胞生长分化提供了模型。     

家兔(Oryctolagus curiculus)角膜内皮细胞系的建立

为了建立兔角膜内皮(RCE)细胞系, 以家兔角膜为实验材料对RCE细胞的体外培养及其细胞系的建立进行了研究. 取活家兔角膜, 采用内皮面朝下进行贴板培养, 并利用时间梯度连续揭膜法筛选出仅含有纯净RCE细胞的培养孔, 收集纯一的RCE细胞悬浮于含有硫酸软骨素氧化降解物、牛眼生素、表皮细胞生长因子、碱性成纤维细胞生长因子、家兔角膜基质细胞培养液、羧甲基壳多糖、N-乙酰葡萄糖盐酸盐和氨基葡萄糖盐酸盐的DMEM/F12培养液(20%胎牛血清)中, 置CO2培养箱中启动原代培养. 启动培养的RCE细胞呈四角形或多角形, 3周后即可长成单层. 在随后的继代培养中, RCE细胞的形状逐渐由多角形变为成纤维样. RCE细胞生长状态稳定, 现已被传至67代, 已经建成了一个新的连续性RCE细胞系. 经鉴定, 该细胞系细胞的群体倍增时间约为53.8 h; 第67代培养细胞的特征性染色体数目为44条, 但出现了染色体的非整倍性. 综合特征性染色体数目、抗神经元特异性烯醇化酶(NSE)抗体的免疫细胞化学染色、血管内皮生长因子(VEGF165)实验和形态回复实验的研究结果, 证明该细胞系确为家兔角膜内皮细胞系, 为哺乳动物角膜内皮细胞的理论研究及人造角膜内皮的研制奠定了基础.     

慢病毒介导的稳定表达bFGF的胎儿肝脏基质细胞株的建立

通过重组慢病毒系统感染人胎儿肝脏基质细胞(fetalliverstromalcell,FLSC),建立了能够稳定、高效表达碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)的细胞株bFGF/FLSC.从流产胎儿肝脏组织分离富集基质细胞,对其进行了生长特性和表面标志的鉴定,其在体外维持传代35代,依然保持正常的染色体核型.从胎儿骨髓间充质干细胞中克隆得到bFGF基因,构建重组慢病毒载体,感染FLSC,根据荧光表达强弱进行流式分选,获得能够继续稳定传代的低表达和高表达bFGF的两株细胞,RT-PCR和蛋白质印迹证实,细胞株中bFGF基因的稳定表达.RT-PCR结果显示,弱荧光和强荧光表达细胞的bFGF,在mRNA水平的表达分别是转染空载体细胞的2.33倍和6.19倍;蛋白质印迹结果显示,在蛋白质水平表达分别是1.76倍和5.05倍.用建立的bFGF/FLSC作饲养层细胞体外培养人胚胎干细胞(humanembryonicstemcells,hES),结果证明,其能在无或少量添加外源bFGF的条件下,维持人ES细胞增殖及其干性达20代.bFGF/FLSC细胞株的建立,将为构建低成本、安全高效的人胚胎干细胞的培养体系及研究造血细胞的发育分化提供适宜的微环境.     

稳定表达牛催乳素基因的小鼠乳腺上皮细胞系的建立

在乳腺上皮细胞体外培养时,为了使其状态接近泌乳期,需要在培养基中添加催乳素.由于催乳素价格昂贵,使得乳腺上皮细胞的体外培养成本颇高.建立在体外培养过程中不需要添加催乳素蛋白的乳腺上皮细胞系,能为在细胞水平研究乳腺细胞相关基因提供诸多方便.利用慢病毒载体的整合特性,建立稳定整合了牛催乳素cDNA(bPRL)表达盒的小鼠乳腺上皮细胞系(HC11细胞系).经10代次以上的传代以后,通过定量PCR检测,证明平均每个细胞中含有2.6个外源的bPRL基因,其表达量为HC11细胞中管家基因β-肌动蛋白(β-actin)表达量的14%左右.另外,先前的研究结果表明催乳素能在HC11和泌乳期的小鼠乳腺上皮组织中有效促进山羊β-酪蛋白启动子启动外源基因的表达.之后的实验证实整合了催乳素基因的HC11细胞(bPRL-HC11细胞系)也有此功能.因此,bPRL-HC11细胞系可以为体外研究乳腺生物反应器提供良好的细胞模型.     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

The immunohistochemical characterisation of an SV40-immortalised human corneal epithelial cell line

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.     

Characterization of an HPV-negative cell line (FR-car) derived from a cervical squamous intraepithelial lesion

Summary A new cell line, FR-car, has been established from a biopsy of a low-grade human cervical squamous intraepithelial lesion (SIL). We confirmed the epithelial origin of the cells by keratin staining using polykeratin, AE1/AE3 and CAM 5.2 antibodies. Sixty percent to 80% of the cultured cells stained positive for proliferative cell nuclear antigen (PCNA) and Ki-67. There was no overexpression of p53. Karyotyping revealed that the cell line was hypodiploid with clonal abnormalities on chromosome 6 and 16. Sections of a biopsy adjacent to the lesion from which the culture was initiated tested positive for human papillomavirus (HPV) 18 DNA by the polymerase chain reaction, but cultured cells tested at several passages were HPV-negative by either type-specific or consensus PCRs. This HPV-negative SIL line may be useful in studies into the cell biology of dysplastic epithelium.     

Hypoxia affects in vitro proliferation and differentiation of mouse corneal epithelial progenitor cell

This study was to investigate the proliferation and differentiation of mouse corneal epithelial progenitor cell in hypoxic airlift culture. Mouse corneal epithelial progenitor cell line progenitor cells were cultured under airlift with normoxic and hypoxic conditions for various durations up to 2 wk. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of mouse corneal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The mechanism by which hypoxia prevents abnormal differentiation may involve downregulation of Wnt signaling pathways, which were inhibited in cells cultured with hypoxic airlift technique. In conclusion, hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal epithelial cells by blocking Wnt/β-catenin signaling pathway.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Generation of transgenic chicks using an oviduct-specific expression system

We successfully replaced the ovalbumin gene of a magnum region in chickens with a human plasminogen activator. We constructed pL-eGFP, pL-tPAGFP and pL-2.8OVtPAGFP vectors and cultured 293FT chicken embryo fibroblasts, chicken primordial germ cells, Hela C127 cells, and oviduct epithelial cells. All vectors were expressed in the transfected cells, except pL-2.8OVtPAGFP vector, which was only expressed in oviduct epithelial cells. A lentivirus with pL-2.8OVtPAGFP was injected in fertilized eggs; 11 chicks hatched in the G? generation, four of them carried the tPAGFP. Two cockerels from the G? generation were crossed with four wild-type hens. Three chicks in G? carried the tPAGFP. We concluded that by using an oviduct-specific vector for transfection, human recombinant plasminogen activator protein can be expressed in the oviducts of laying hens. This character is inherited and can be reproduced with a need for repeated transfection.     

EB病毒潜伏膜蛋白1基因对上皮细胞增殖的影响

为了研究ＥＢ病毒潜伏膜蛋白１（ＬＭＰ１）基因对上皮细胞增殖的影响,探索ＬＭＰ１在上皮细胞肿瘤发生中所起的作用。用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测了转染细胞中ＬＭＰ１的表达,观察细胞在软琼脂中的集落形成能力、ＭＴＴ吸收能力以及ＰＣＮＡ的表达情况。结果显示,被ＬＭＰ１基因转染的细胞生长旺盛,能在软琼脂中形成多个集落,ＭＴＴ吸收能力增强,ＰＣＮＡ的表达水平增高。因此认为ＬＭＰ１基因能明显改变上皮细胞的生物学行为,促进细胞的生长、增殖和转化,使转染的上皮细胞获得肿瘤细胞的生长特征     

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6⁺/K12⁺ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.     

Expression of proliferating cell nuclear antigen in migrating retinal pigment epithelial cells during wound healing in organ culture.

Although 12-day-old chick embryo retinal pigment epithelial (RPE) cells in situ do not express the proliferating cell nuclear antigen (PCNA) which is known to function as an auxiliary protein of DNA polymerase delta, they do so when cultured on glass. Conversely, PCNA was not expressed by RPE cells of the same age maintained in organ culture. If, however, the organ cultures were wounded, allowing the RPE cells to spread and migrate over the exposed basal lamina, the nuclei of cells along the wound edge were stained for PCNA. The time required for cells to express PCNA was longer in organ culture than in tissue culture. This time lag in the expression of PCNA was independent of the time in culture prior to wounding and occurred regardless of whether or not the continuity of the epithelial sheet was reestablished. In organ culture, the staining did not persist as long as in tissue culture. We found that only in wounds exceeding 125 +/- 48 microns did the RPE cells along the wound edge express PCNA. This suggests that a certain degree of either spreading or migration is required for PCNA expression in the wounded region.     

Regulation and function of the two-pore-domain (K2P) potassium channel Trek-1 in alveolar epithelial cells

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.