从C57BL/6J小鼠胚胎中分离ES细胞系(英文)

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｓ）是来源于小鼠早期胚胎的细胞系,它可以在体外大量培养而不失去其发育的多潜能性。ＥＳ细胞不仅可以用来制作转基因动物,而且能够作为载体进行基因打靶等多种研究。目前,国际上常用的胚胎干细胞系都是来源于１２９小鼠的胚胎。因而,有必要探讨用其它品系小鼠建立ＥＳ系。１９９１年,Ｌｅｄｅｒｍａｎｎ等人首次报道从Ｃ５７ＢＬ／６Ｊ小鼠胚胎中建成了ＥＳ细胞系,但是没有对建立的细胞系进行特性分析。国内柴桂萱等人虽然做了特性分析,但是他们所建细胞系的细胞直径较大,生长速度较慢,不同于常见的ＥＳ细胞系。我们从Ｃ５７ＢＬ／６Ｊ品系小鼠胚胎中共分离了四个ＥＳ细胞系,分别命名为ＣＥ１、ＣＥ２、ＣＥ３、ＣＥ４（Ｆｉｇ．１ａ＆ｂ）。这四个细胞系核型正常率均达到７０％以上。我们检查了ＣＥ２细胞的分化能力,当将ＣＥ２细胞注入同基因型小鼠的皮下后,获得的畸胎瘤（Ｆｉｇ．３）组织切片检查的结果表明：该细胞能够分化成多种组织（Ｆｉｇ．２）。我们也研究了ＥＳ细胞的嵌合能力,用ＩＣＲ小鼠胚胎作为受体胚胎,采用囊胚显微注射法构建嵌合鼠。在幸存的幼鼠中我们获得了来源于ＣＥ２细胞的嵌合鼠（Ｔａｂｌｅ１,Ｆｉｇ．４）。综     

pINC－lacZ逆转录病毒载体的构建及其在小鼠胚胎干细胞中的表达

小鼠胚胎干细胞系（ＥＳ）是从囊胚的内细胞团中建立起来的多潜能胚胎干细胞系。ＥＳ细胞系目前正广泛用于将基因打靶后的突变和其它遗传变化导入小鼠的种系中。其中,嵌合鼠的获得是非常必要的一步。这就需要用一个可以广泛表达的基因对ＥＳ细胞进行标记。大肠杆菌β－半乳糖苷酶（β－ｇａｌ）基因的表达在细胞水平就能很容易地观察到,而且对哺乳动物细胞没有任何毒害作用,因而是一个被广泛地用于各种细胞基因表达研究中的报导基因。猿类巨细胞病毒早期（ＳｉＣＭＶＩＥ）启动子是一个广泛用于转基因小鼠研究中的强启动子。然而,该启动子在ＥＳ细胞方面应用的报道尚未见到。本研究用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,从ｐｓｖ－β－ｇａｌａｃｔｏｓｉｄａｓｅ中得到３．７Ｋｂ的ｌａｃＺ基因片断,将其插入到ｐＩＮＣ载体上（Ｆｉｇ．１）,得到ｐＩＮＣ－ｌａｃＺ（Ｆｉｇ．２）。用ＮｏｔⅠ线性化ｐＩＮＣ－ｌａｃＺ后,电击导入ＭＥＳＰＵ－１３细胞中。ＭＥＳＰＵ－１３为本实验室从１２９／Ｔｅｒ品系小鼠的囊胚中建立的一个ＥＳ细胞系。转化细胞在含有２５０μｇ／ｍｌＧ４１８的培养基上培养两周,进行ＭＥＳＰＵ－１３细胞稳定转化子的筛选。在一次转化实验中,从１ｘ１０７个转化的ＭＥＳ     

LIF基因转染的ES细胞生长与分化特性的研究

我们将人Ｄ型ＬＩＦｃＤＮＡ以正反两种方向分别克隆到载体ＰＫＣＲ３,并引入ｎｅｏ基因,构建成ｐＳＶＬＤ和ＰＳＶＬＤ质粒,按磷酸钙沉淀法分别转染ＥＳ－５胚胎干细胞,,经Ｇ４１８和没浓度ＬＩＥ条件培液共同筛选,,Ｎｏｒｔｈｅｒｎ和Ｓｏｕｔｈｅｒｎ分析以及ＥＳ－５细胞集落分化抑制能力测定,建立了过度表达分泌ＬＩＦ的ＥＳＬ（＋）细胞株和表达外源反义ＬＩＦＲＮＡ的ＥＳＬ（－）细胞株。我们发现,我们发现,ＥＳＬ     

外源TGF—β1基因在小鼠ES细胞中的表达及其对细胞分化的影响

本文利用逆转录病毒载体Ｄｏｌ,在其ＢａｍＨＩ酶切位点插入猪ＴＧＦ－β１ １．７ｋｂｃＫＮＡ,构建成表达质粒,并用磷酸钙沉淀法将该质粒ＤＮＡ转染到小鼠ＥＳ－５细胞,经Ｇ４１８筛选获得抗Ｇ４１８的ＥＳ－５细胞克隆（ＥＳ－Ｔ）,经ＲＮＡ点杂交,Ｎｏｒｔｈｅｒｎ印迹杂交证明有６个细胞克隆能表达外源猪ＴＧＦ－β１的ｍＲＮＡ,其中两个杂交信号较强的克隆进一步用Ｓｏｕｔｈｅｒｎ印迹杂交,也证明猪ＴＧＦ－μ     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

小鼠ES细胞种系嵌合体的获得

种系嵌合体的获得是实现ＥＳ细胞介导的转基因途径的决定步骤,ＥＳ细胞种系分化能力的保持是决定种系嵌合的前提条件,而事体的主种系嵌合体的获得则是判定ＥＳ细胞系是否具有种系分化能力的唯一方法,为考察本室新近建立的３种小鼠ＥＳ细胞系ＭＥＳＰＵ２１．ＭＥＳＰＵ２２和ＭＥＳＰＵ２９的种系分化能力,选用近交系Ｃ５７ＢＬ／６Ｊ及远交系ＫＭＷ和ＩＣＲ为受体胚胎提供者,分别通过囊胚注射法和８细胞期桑椹胚注射法进行了嵌     

一个高效表达,快速纯化大肠杆菌精氨酰—tRNA合成酶的 …

编码大肠杆菌精氨酸－ｔＲＮＡ合成酶的基因ａｒｇＳ被克隆到ｐＭＦＴ７－５载体上。将此质粒转化的大肠 力ＪＭ１０９（ＤＥ３）中,该转化子粗抽液的比活是宿主菌的２５００倍。通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ ＦａｓｔＦｌｏｗ和ＢｌｕｅＳｅｐｈａｒｏｓｅ ＣＬ－６Ｂ两步柱层析在一天内即可将精氨酰－ｔＲＮＡ合成酶纯化至电泳一条带,比活为３６０００ｕ／ｍｇ,总收率可达６９％。     

p35Nck5a基因重复性打靶载体的构建和ES细胞基因打靶研究

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复,采用常规的分子克隆技术,构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞,经过Ｇ４１８和ＧＡＮＣ分组药物选择,获得２４５个 双药物抗性的细胞克隆,细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定,２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复,同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。     

ES细胞分化为血管样结构的机理探讨—TGF—β1在血管形成中的作用

本文利用小鼠ＥＳ细胞的拟胚一培养和三维胶原蛋白培养系统研究了外源ｒｈＴＧＦ－β１对ＥＳ细胞分化为血管样样结构的影响。结果发现,远方介培养中添加外源ｒｈＴＧＦ－β１或细胞经基因转染面有过度表达的ＴＧＦ－β１的ＥＳ－Ｔ６细胞分化为血管样结构的频率明显地受到抑制,相似于其亲本ＥＳ－５细胞的分化水平。在Ｉ型胶原三维培养系统中的单层ＥＳ－５细胞培液中添加ｒｈＴＧＦ－β１时,明显地促进ＥＳ－５细胞形成血管样结     

影响火炬松胚性悬浮细胞原生质体的胚胎发生的因素(英文)

火炬松（ＰｉｎｕｓｔａｅｄａＬ．）是我国亚热带和部分热带地区最重要的绿化和造林树种。它的生长周期长,杂合程度高,难以用常规的杂交方法进行品种改良。建立火炬松的原生质体胚胎发生体系,有可能、进而以遗传转化为基础进行火炬松等针叶树的品种改良。本研究以湖南省绍阳市的火炬松成熟种子为材料,建立胚性细胞悬浮系。将其培养至对数生长期,用１％ＲＳ、２．５％Ｒ１０和０．２％Ｙ２３的酶混和液分离原生质体,活力达９０％以上（Ｆｉｇ．１）。纯化原生质体,在再生培养基上培养２天后,细胞壁再生（Ｆｉｇ．２）;;６天后,６５％的原生质体第１次分裂（Ｆｉｇ．３）;培养３周后,形成小细胞团（Ｆｉｇ．４）;６周后,形成大细胞团（Ｆｉｇ．５）;８周后,形成胚性胚柄细胞团（ＥＳＭ）（Ｆｉｇ．６）;１０周后,形成早期体细胞胚（ＥＳＥ）（Ｆｉｇ．７）;１２周后,形成后期体细胞胚（ＬＳＥ）（Ｆｉｇ．８）火炬松原生质体胚形成过程中的关键结构是ＥＳＭ,ＥＳＥ和ＬＳＥ。它们形成的最佳基本培养基是１．２５ＬＰ培养基。再生培养基中高浓度的ＢＡ和低浓度的肌醇有利于ＥＳＭ的形成,但不利于ＥＳＥ和ＬＳＥ的形成。反之,低浓度的ＢＡ和高浓度的肌醇不利于ＥＳＭ的形成,     

FSH通过SATB1调控上皮性卵巢癌ES-2细胞的增殖和侵袭活性

目的:探讨特异AT序列结合蛋白1(specialAT-rich sequence-bindingprotein,SATB1)在卵泡刺激素(Follicle stimulating hormone,FSH)诱导的上皮性卵巢癌ES-2细胞增殖和侵袭中的作用。方法:以Real-time PCR检测不同浓度FSH(0、10、20、40、80mIU/ml)处理后SATB1基因mRNA表达水平的变化。实验分4组:①siCon组,转染si-阴性对照(si-Negative contro1)序列的实验组,对SATB1无干扰作用;②siSATB1组:转染特异性干扰下调SATB1的siSATB1序列;③FSH+siCon组:以FSH处理的siCon组;④FSH+siSATB1组:以FSH处理的siSATB1组。MTT法检测4组细胞的增殖情况,Western blotting技术检测4组细胞细胞周期蛋白(CyclinD1),基质金属蛋白酶2(MMP-2)的蛋白表达情况,Transwell侵袭实验检测4组细胞侵袭能力的变化。结果:1.FSH+siCon组的细胞增殖能力明显高于siCon组的细胞增殖能力,FSH+siCon组的Cyclin D1蛋白相对表达量0.90±0.08明显高于siCon组的0.37±0.01(P均<0.01),提示FSH具有促进ES-2细胞增殖的作用。2.FSH+siCon组的穿膜细胞数(302 12)个明显高于siCon组(139 19)个,FSH+siCon组的MMP-2蛋白相对表达量0.40±0.01明显高于siCon组的0.28±0.02,提示FSH具有促进ES-2细胞侵袭能力的作用。3.随着FSH浓度的增高,SATB1mRNA的表达量逐渐增加,分别为1,1.66±0.04,1.79±0.21,2.31±0.03,以FSH浓度为80mlU/ml时最显著(P<0.05)。4.FSH+siSATB1组的细胞增殖能力明显低于FSH+siCon组的细胞增殖能力,FSH+siSATB1组的Cyclin D1蛋白相对表达量0.22±0.02明显低于FSH+siCon组的0.90±0.08(P均<0.01);FSH+siSATB1组的穿膜细胞数(52 16)个低于FSH+siCon组的(302 12)个,FSH+siSATB1组的MMP-2蛋白相对表达量0.15±0.00明显低于FSH+siCon组的0.40±0.01(P均<0.01),FSH促进ES-2细胞增殖和侵袭的能力由于SATB1基因表达的下降而被阻断。结论:SATB1是FSH作用的重要靶分子,介导FSH对上皮性卵巢癌ES-2细胞系增殖、侵袭活性的调控。     

FSH通过SATB1调控上皮性卵巢癌ES-2细胞的增殖和侵袭活性

目的：探讨特异AT序列结合蛋白1（special AT-rich sequence-bindingprotein,SATB1）在卵泡刺激素（Follicle stimulating hor-mone,FSH）诱导的上皮性卵巢癌ES-2细胞增殖和侵袭中的作用。方法：以Real-timePCR检测不同浓度FSH（0、10、20、40、80mlU／ml）处理后sATB1基因mRNA表达水平的变化。实验分4组：①siCon组,转染si-阴性对照（si-Negativecontrol）序列的实验组,对SATBl无干扰作用;②siSATB1组：转染特异性干扰下调SATB1的siSATB1序列;（3）FSH＋siCon组：以FSH处理的siCon组;（4）FSH＋siSATBl组：以FSH处理的sisATB1组。MTT法检测4组细胞的增殖情况,Westernblotting技术检测4组细胞细胞周期蛋白（CyclinDl）,基质金属蛋白酶2（MMP．2）的蛋白表达情况,Transwell侵袭实验检测4组细胞侵袭能力的变化。结果：1．FSH＋siCon组的细胞增殖能力明显高于siCon组的细胞增殖能力,FSH＋siCon组的CyclinD1蛋白相对表达量0．90＋0．08明显高于siCon组的0．37＋0．01（P均〈0．01）,提示FSH具有促进ES．2细胞增殖的作用。2．FSH＋siCon组的穿膜细胞数（30212）个明显高于siCon组（13919）个,FSH＋siCon组的MMP．2蛋白相对表达量0．40＋0．01明显高于siCon组的0．28＋0．02,提示FSH具有促进ES．2细胞侵袭能力的作用。3．随着FsH浓度的增高,SATBlmRNA的表达量逐渐增加,分别为1,1．66±0．04,1．79±0．21,2.31±0．03,以FsH浓度为80mlU／m1时最显著（P〈0．05）。4．FSH＋siSATBl组的细胞增殖能力明显低于FSH＋siCon组的细胞增殖能力,FSH＋siSATBl组的CyclinD1蛋白相对表达量0．22±0．02明显低于FSH＋siCon组的0．90±0．08（P均〈0．01）;FSH＋siSATB1组的穿膜细胞数（5216）个低于FSH＋siCon组的（30212）个,FSH＋siSATB1组的MMP-2蛋白相对表达量0．15±0．00明显低于FSH＋siCon组的0．40±0．01（P均〈0．01）,FSH促进ES-2细胞增殖和侵袭的能力由于SATB1基因表达的下降而被阻断。结论：SATBl是FSH作用的重要靶分子,介导FSH对上皮性卵巢癌ES-2细胞系增殖、侵袭活性的调控。     

大鼠内细胞团和胎儿神经干细胞构建嵌合体的潜力

嵌合体大鼠是研究人类疾病的重要动物模犁.用囊胚注射法研究了大鼠内细胞团(ICM)和胎儿神经干细胞(FNS)构建嵌合体的潜力.结果发现来自黑色(DA)大鼠第5天(D5)和第6天(D6)囊胚的ICM细胞注入D5 Sprague-Dawley(SD)大鼠囊胚后得到3只嵌合体大鼠:D5 SD大鼠ICM细胞注射入D5 DA囊胚后得到4只嵌合体大鼠:而体外培养的DA或SD人鼠ICM细胞注射后均未能获得嵌合体大鼠.本研究用大鼠胎儿神经干细胞(rFNS)和LacZ转染的rFNS构建嵌介体,未能获得嵌合体人鼠:但在LacZ转染的SD rFNS注射到DA大鼠囊胚后发育来的41只胎儿中,有2只胎儿其组织切片中发现少量LacZ阳性细胞.结果表明DA和SD大鼠ICM具有参与嵌合体发育的潜力,但ICM细胞经体外培养后构建嵌合体的潜力显著F降(P＜0.05);大鼠胎儿神经干细胞构建嵌合体的潜力较低,可能仅具有参与早期胚胎发育的潜力.     

Stra 8基因的激活与精原干细胞的特异性分化研究

视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8基因的表达特性,我们从小鼠的基因组中克隆了Stra8基因的启动子序列(1.4kb)。将Stra8基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8的转录,还有生殖细胞特异表达基因CyclinA8和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

维生素A酸受体γ基因表达及其对ES细胞分化和凋亡的影响

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.     

Low-copy-number human transgene is recognized as an X inactivation center in mouse ES cells, but fails to induce cis-inactivation in chimeric mice

X chromosome inactivation is initiated from a segment of the mammalian X chromosome called the X inactivation center. Transgenes from this region of the murine X chromosome are providing the means to identify the DNA needed for cis inactivation in mice. We recently showed that chimeric mice carrying transgenes from the human X inactivation center (XIC) region also provide a functional assay for human XIC activity; approximately 6 copies of a 480-kb human transgene (ES-10) were sufficient to initiate random X inactivation in cells of male chimeric mice (Migeon et al., 1999, Genomics, 59, 113-121). Now, we report studies of another human transgene (ES-5), which contains less than 300 kb of the human XIC region on Xq13.2 including an intact XIST locus and which has inserted in one or two copies into mouse chromosome 6. The ES-5 transgene is recognized as an X inactivation center in mouse embryonic stem cells, but is not sufficient to induce random X inactivation in somatic cells of highly chimeric mice. Human transgenes in chimeric mice provide a means to uncouple the key steps in this complex pathway and facilitate the search for essential components of the human XIC region.     

PIWIL4在人卵巢癌中的表达及其功能研究

采用半定量RT-PCR方法检测人卵巢透明细胞癌ES-2细胞中PIWIL1、PIWIL2、PIWIL3、PIWIL4 mRNA表达水平,以及PIWIL4在卵巢癌组织和癌旁正常组织中的表达情况．设计并化学合成针对PIWIL4的siRNA,用脂质体转染法将其转入ES-2细胞内,通过MTT和克隆形成实验,观察PIWIL4-siRNA对ES-2细胞生长活性和增殖能力的影响．半定量RT-PCR实验结果发现,ES-2细胞中,PIWIL4相对PIWIL1、PIWIL2、PIWIL3,其表达水平最高(P < 0.05),而且PIWIL4在卵巢癌组织中的表达明显高于癌旁正常组织(P < 0.01)．MTT实验和克隆形成实验结果显示,转染PIWIL4-siRNA的ES-2细胞生长活性和克隆形成率明显低于对照组(P < 0.05)．由此得出结论：PIWIL4在卵巢癌细胞系ES-2细胞表达较高,且它在卵巢癌组织中的表达明显上调,同时PIWIL4-siRNA可有效抑制ES-2细胞的生长活性和增殖能力,提示PIWIL4可能与卵巢癌发生、发展相关．     

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.     

ES-62, an immunomodulator secreted by filarial nematodes, suppresses clonal expansion and modifies effector function of heterologous antigen-specific T cells in vivo

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes, which has previously been shown to possess a range of immunomodulatory capabilities. We now show, using a CD4+ transgenic TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag (OVA)-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a Th2-biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a. Consistent with this, Ag-specific IFN-gamma produced was suppressed by pre-exposure to ES-62 when T cells were rechallenged ex vivo. However, the response observed was not classical Th2, because although Ag-specific IL-5 production was enhanced by pre-exposure to ES-62, IL-13, and IL-4 were inhibited when T cells were rechallenged ex vivo. Moreover, such T cells produced lower levels of IL-2 and proliferated less upon Ag rechallenge ex vivo. Finally, pre-exposure to ES-62 inhibited the clonal expansion of the transferred Ag-specific CD4+ T cells and altered the functional response of such T cells in vivo, by modulating the kinetics and reducing the extent of their migration into B cell follicles.