不同血清对山羊成纤维细胞传代的影响

考察不同血清对山羊成纤维细胞传代培养的影响．第6～8代细胞分别在添加10％Hyclone和10％四季青胎牛血清的DMEM中传代培养，实验发现，前者平均增殖倍数和群体倍增次数均高于后者．随着传代次数的增多，细胞在四季青血清培养液中的生长行为发生明显变化，细胞不能倍增且生长曲线呈不规则波动状．结果表明，细胞传代过程中使用不同血清会改变细胞生物学特性，降低细胞活力．用这种方式传代的细胞不宜用作核移植供体细胞及其他实验研究．     

TALENs和CRISPR/Cas9诱导绵羊成纤维细胞基因组定点双链断裂提高Rad51基因表达量

为检测不同限制性人工核酸内切酶对绵羊成纤维细胞Rad51基因m RNA转录和蛋白表达的影响,本实验将有效切割MSTN(myostatin)基因位点的TALENs和CRISPR/Cas9人工核酸酶电转染到体外培养的绵羊成纤维细胞中诱导产生DNA双链定点断裂,与正常和电转染p Max绿色荧光蛋白报告基因质粒的绵羊成纤维细胞进行对照,采用实时荧光定量PCR和Western Blot方法对不同处理的绵羊成纤维细胞Rad51基因m RNA转录和蛋白质的表达情况进行研究。结果表明:TALENs和CRISPA/Cas9人工核酸酶诱导产生的MSTN基因组DNA双链定点断裂能提高绵羊成纤维细胞Rad51基因表达量。     

原代和传代培养的人输卵管上皮细胞对小鼠胚胎体外发育的影响

体内精卵结合、早期胚胎发育是在输卵管中进行,人们将配子、早期胚胎进行输卵管内移植（GIFT、TET）,发现胚胎发育率和妊娠率都提高。体外模仿输卵管内环境的最好方法是将胚胎与输卵管上皮细胞或组织共培养。本实验用已建立的原代和传代培养的人输卵管上皮细胞分别与小鼠早期胚胎共培养,观察其对早胚发育的影响。胚胎与不同培养时间的人输卵管壶腹部上皮细胞共培养的发育状况见表1。表1人输卵管上皮细胞对小鼠胚胎发育的影响P：原代培养第5－6d细胞;S1－S5：传代1－5代培养第4－5d细胞;T2、T4：传代第2和第4代培养第id细胞,设此…     

小鼠体细胞核移植及ES细胞样集落分离

利用小鼠皮肤成纤维细胞为核供体进行体细胞核移植并从重构胚中分离胚胎干细胞(ES)样集落,以便对体细胞核移植重构胚来源的ES细胞样集落进行研究.结果显示,小鼠皮肤成纤维细胞作为核供体,核移植重构胚激活率为60.48%(254/420),囊胚发育率为6.90%(29/420),6个囊胚中分离出ES细胞样集落,分离率为1.43%(6/420),3个ES细胞样集落能够稳定传代,至第5代时核型正常率分别为77.84%,75.18%,77.20%.分离出的ES细胞样集落具有岛屿状团状隆起结构,碱性磷酸酶染色呈阳性,体外可自发分化成上皮样或梭形细胞.实验证实小鼠唇部皮肤成纤维细胞能够支持体细胞核移植重构胚发育至囊胚,并能分离出可以稳定传代的ES细胞样集落.     

昆明小鼠胚胎成纤维细胞的分离与体外培养

研究了昆明小鼠胚胎成纤维细胞的分离、培养和生长特征,建立了快速、稳定的优质饲养层细胞培养体系.从不同日龄的胎鼠均分离到胚胎成纤维细胞,但最佳分离时间为13.5~14.5d;三种分离原代胚胎成纤维细胞的方法中胰酶消化法效果最好,能在较短的周期内获得大量原代及传代细胞;MEF细胞形态以小梭形为主,呈漩涡状、火焰状生长;增殖速度较快,每1~2d可传一代,按1∶3比例常规传代;5代以内适宜制作饲养层用,5代以后细胞开始变形呈现典型的衰老特征.     

小鼠胚胎成纤维细胞的分离培养

目的探讨小鼠胚胎成纤维细胞(MEF)的分离与培养。方法取BALB/c小鼠胚胎分离成纤维细胞,利用体外培养体系,对MEF的生长形态进行观察,并对传代细胞培养液、胚胎胎龄、胰蛋白酶浓度进行筛选。结果 MEF在体外为贴壁生长型细胞,第三、四、五代细胞纯度较高且增殖最为旺盛;添加血清的M199和DMEM均能较好的满足原代细胞的生长,两种培养液中细胞增殖的速度无明显差异;11.5～16.5d胎龄的小鼠胎儿MEF分离效果最好;0.1%胰蛋白酶消化MEF时间以8～11min为宜。结论通过对MEF分离培养影响因素的筛选,为MEF的进一步研究奠定了基础。     

Follistatin-Like 1单倍体敲除促进肺纤维化小鼠成纤维细胞自噬

Follistatin-like 1(Fstl1)是促纤维化因子,在成纤维细胞中通过调节TGF-β信号参与肺纤维化发生过程.自噬是维持细胞稳态的过程,在细胞衰老和分化中起到重要的作用.在特发性肺纤维化全肺中自噬不足.通过博来霉素处理Fstl1^+/-和Fstl1^+/+小鼠构建肺纤维化模型,研究在肺纤维化发展过程中,Fstl1对肺纤维化的效应细胞即成纤维细胞中的自噬的调节.Western blot检测自噬标记蛋白LC3和p62的表达,q RT-PCR检测自噬相关基因ATG5,ATG7,ATG12的表达.在肺纤维化发展的不同时期成纤维细胞的自噬处于动态变化过程,在第7 d时成纤维细胞自噬降到最低,随后逐渐恢复.在未经肺纤维化诱导的小鼠中,Fstl1单倍缺失不影响成纤维细胞自噬;在博莱霉素处理的小鼠中,Fstl1单倍缺失提高了成纤维细胞自噬.     

原代和传代培养的人输卵管上皮细胞对小鼠胚胎体外发育的影响

为了检测人输卵管上皮细胞对早期胚胎体外发育的影响，将小鼠２细胞胚胎与人输卵管上皮细胞进行体外共培养．结果显示：无论原代培养还是传代培养的人输卵管上皮细胞都可使胚胎发育率提高，发育速度加快．经传代４次以后的细胞对胚胎发育的促进作用有下降的趋势，所以传代第１至第４代是应用于共培养的最佳选择．同一代细胞中已贴壁稳定生长的细胞对胚胎发育的促进作用比刚经传代尚未贴壁的细胞大．     

丹参酮ⅡA磺酸钠对培养人肾间质成纤维细胞的影响

探讨丹参酮ⅡA磺酸钠对培养的人肾间质成纤维细胞增殖、凋亡及分泌胶原蛋白的影响,为肾脏疾病的临床防治提供理论依据.体外培养的成纤维细胞第2~4代,经不同浓度的丹参酮ⅡA磺酸钠及不同时间培养后,采用免疫细胞化学技术和TUNEL法检测其PCNA、p27、Ⅲ型胶原蛋白的表达及凋亡情况.丹参酮ⅡA磺酸钠能抑制肾间质成纤维细胞表达PCNA和Ⅲ型胶原蛋白,促进p27蛋白的表达和细胞凋亡,在一定浓度、时间范围内具有量效、时效关系(P＜0.05).丹参酮ⅡA磺酸钠能够在体外发挥抗肾间质纤维化的作用,其机理可能与抑制细胞增殖、诱导细胞凋亡及减少胶原蛋白的合成等有关.     

转基因猪细胞中CMV启动子区域甲基化的研究

为研究转基因猪外源基因表达与启动子区域甲基化的关系,利用流式细胞仪检测体外传代至第5、10和15代的转基因猪成纤维细胞中EGFP荧光强度的变化情况;采用亚硫酸盐测序的方法对这3代细胞CMV IE启动子区域30个Cp G岛的甲基化程度进行测定。结果显示细胞从第5代培养到第10代时荧光变弱,但是当继续培养到15代时EGFP荧光又渐渐变强,但EGFP阳性细胞的比例没有发生变化,均为100%。第5、10、15代细胞启动子区域的Cp G岛甲基化阳性率分别为3.64%±1.25、2.97%±1.03和1.65%±0.74,统计结果表明3代细胞间的甲基化阳性率差异不显著(P0.05)。表明转基猪成纤维细胞中CMV启动子区域的甲基化程度随着体外培养时间的变化不明显,与EGFP的表达水平变化无明显相关。     

银杏叶提取物对大鼠真皮FB增殖及胶原蛋白分泌的影响

原代培养大鼠真皮成纤维细胞（Fibroblast,FB）传代培养后,以不同浓度的银杏叶提取物（EGb761,20、50、100mg／L）对其生长进行干预,四甲基偶氮唑盐（MTT）法检测药物干预后24、48、72h时FB细胞活力变化,碱水解法测定药物干预后3、6d时培养液中羟脯氨酸含量变化,以分别反映EGb761对大鼠真皮FB增殖及胶原蛋白分泌的影响。研究表明EGB761在一定范围内能促进大鼠真皮FB增殖及及胶原蛋白分泌,且呈一定的剂量、时间相关性。     

TGFβ1shRNA靶向抑制离体胎鼠肺成纤维细胞TGFfβ1基因表达

目的:探讨自行设计的TGFβ1shRNA对离体胎鼠肺成纤维细胞TGFβ1基因表达的干扰作用,为研究纤维化病变的基因治疗提供技术基础和依据.方法:原代培养胎鼠肺成纤维细胞,并建立细胞高氧损伤模型.针对大鼠TGFβ1基因mRNA序列,设计、合成携带3条TGFβ1shRNA绿色荧光蛋白融合表达质粒载体,并设阴性质粒组和空白组为对照,通过JetPEI包裹分别转染上述高氧损伤的胎鼠肺成纤维细胞.转染后24、48和72 h收集细胞,在荧光显微镜下观察干扰效果,采用实时荧光定量PCR检测TGFβ1基因表达情况,并计算干扰效率.结果:①成功培养胎鼠肺成纤维细胞,并建立细胞高氧损伤模型;②荧光显微镜下观察,可见转染后24、48和72 h TG-Fβ1shRNA质粒组细胞绿色荧光强度均明显弱于阴性质粒组细胞,空质粒载体组未产生绿色荧光;荧光定量PCR检测转染后胎鼠肺成纤维细胞TGFβ1 mRNA表达量,转染后24、48和72 hTGFβ1shRNA质粒组TGFβ1 mRNA表达量均显著低于阴性质粒组(P<0.01),其基因干扰效率则依次递减,分别为97.3%、96.9%和71.7%.结论:本研究证明自行设计的TGFβ1shRNA转染胎鼠肺成纤维细胞后24、48和72 h均能够高效干扰TGFβ1基因的表达,其基因干扰效率呈现一定的时间依赖性.     

小鼠LEAP-2基因的克隆及其真核表达质粒的构建

从小鼠肝中提取总RNA,采用反转录聚合酶链式反应（RT-PCR）方法,获得了mLEAP-2基因编码区的cDNA,扩增出小鼠肝表达抗菌肽-2（mLEAP-2）成熟肽基因片段,重组入克隆载体pUCm-T,经DNA测序,该基因为120 bp,编码40个氨基酸。用限制性内切酶切下目的基因,插入毕赤酵母表达载体pPIC9中,构建成表达载体pPIC9-LEAP-2。重组毕赤酵母表达载体pPIC9-LEAP-2的结构,可望获得超量表达的高活性肝表达抗菌肽-2,为研制具有抗菌活性的新型基因药物奠定基础。     

RHD基因转录子的选择性剪切

通过RT-PCR技术分析红系细胞和非红系细胞RHD的转录情况,同时进一步比较不同D表达个体的RHD的转录关系.采用逆转录PCR（RT-PCR）技术检测HL-60,K562,Jurkat,THP-1,胚肺成纤维细胞系（HECF）,10名不同Rh表型个体的网织红细胞（CcDEe 3名,CCDEe 2名,CCDee 2名,CcDee 2名和CCDuee 1名）以及10名不同Rh表型（2 CCDEe,2 CCDee,2 ccDee,2 CcDee,ccDEe和CcDEe）个体的白细胞的RhD mRNA,然后进行cDNA测序分析.结果表明,HL-60,Jurkat,THP-1,胚肺成纤维细胞系（HECF）以及不同Rh表型个体的外周血有核细胞中除网状红细胞外皆不存在RhD mRNA,K562具有一个正常的RHD基因转录本,而不同表型个体的网织红细胞具有复杂的RhD cDNA形式,有的缺少RHD基因的外显子7,有的缺少外显子7～9或外显子4～9,但这些个体都有一个正常形式的RhD mRNA.由此得出结论,选择性剪切使RHD基因的产生多种形式的转录子,但这些不同形式的转录子仅来自红系的血细胞如网织红细胞和K562细胞系,白细胞、单核细胞、T淋巴细胞以及胚肺成纤维细胞都不具有RHD的mRNA.     

制备条件对固体超强酸SO_4~(2-)/ZrO_2性质的影响

较详细地研究了固体超强酸催化剂制备条件与催化剂性质的影响。试验发现制备固体超强酸酸浓度和焙烧温度对催化剂的硫含量、酸强度、形态结构和晶相转变都有影响。     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.     

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.     

Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.     

Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains

We have identified a group of 8 (among 39) human tumour cell strains deficient in the ability to support the growth of adenovirus 5 preparations treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but able to support the growth of non-treated adenovirus normally. This deficient behaviour defines the Mer- phenotype. Strains having the Mer- phenotype were found to arise from tumours originating in four different organs. Relative to Mer+ strains, Mer- tumour strains showed greater sensitivity to MNNG-produced killing, greater MNNG-stimulated "DNA repair synthesis and a more rapid MNNG-produced decrease in semi-conservative DNA synthesis. Here we report that (1) Mer- strains are deficient in removing O6-methylguanine (O6-MeG) from their DNA after [Me-14C]MMNG treatment (Table 1); (2) Mer- tumour strains originate from tumours arising in patients having Mer+ normal fibroblasts (Fig. 1a, b); (3) SV40 transformation of (Mer+) human fibroblasts often converts them to Mer- strains (Fig. 1c, d); (4) MNNG produces more sister chromatid exchanges (SCEs) in Mer- than in Mer+ cell strains (Fig. 2).