猪成纤维细胞细胞周期同期化对核移植胚胎发育的影响

供体细胞所处的细胞周期及细胞周期同期化的方法对于体细胞核移植（somatic cell nuclear transfer,SCNT）的成功非常重要,本研究对血清饥饿培养处理与培养至完全汇合后的猪成纤维细胞周期同期化水平进行了检测。利用不同方法对猪成纤维细胞同期化处理后,通过流式细胞仪对细胞的细胞周期分布比率进行了检测。将细胞进行血清饥饿24⁷2h,显著地增加了G₀/G₁期的细胞百分率（92.2%⁹3.7%vs.77.8%,P<0.05）。将细胞培养至完全汇合后再培养24⁴8h,G₀/G₁期的细胞比例类似于血清饥饿法（94.4%,89.6%）。血清饥饿24h后,置换为10%FBS能逆转至生长期。用这两种不同方法处理后的体细胞作为核移植的供体构建重构胚,分裂率与囊胚率差异不显著（P>0.05）。结果表明,猪成纤维细胞通过血清饥饿法或者培养至汇合完全均能有效地将细胞周期同期化至G₀/G₁期,且均可作为体细胞核移植的供体细胞。     

西藏小型猪胚胎成纤维细胞的分离培养及性别鉴定

目的探索和建立西藏小型猪胚胎成纤维细胞体外分离培养及性别鉴定的技术方法。方法取35d的西藏小型猪胚胎分离胚胎成纤维细胞,进行体外原代培养及传代培养,观察细胞成纤维细胞的形态和生长状况。根据猪Y染色体上性别决定基因SRY设计引物进行性别鉴定,同时以β珠蛋白作为内参基因,建立PCR反应体系鉴别胚胎的性别。结果西藏小型猪胚胎成纤维体外分离后,呈贴壁生长,快速增殖。PCR性别鉴定结果表明雄性胚胎细胞可扩增出一特异性SRY基因条带,而雌性则没有。该法可快速鉴定胚胎的性别,可用于体细胞克隆动物早期性别鉴定。结论研究结果表明利用胶原酶消化法所获得的西藏小型猪胚胎成纤维细胞可在体外稳定培养并传代,利用PCR鉴定猪胎儿性别具有简单、快速、准确的特点,可应用于克隆猪研究中体细胞系的早期性别鉴定。     

绵羊胎儿成纤维细胞体外培养及转基因研究

目的用增强型绿色荧光蛋白(EGFP)基因转染体外培养绵羊胎儿成纤维细胞,探讨绿色荧光蛋白对绵羊胎儿成纤维细胞生物学特性的影响.方法体外分离培养绵羊胎儿成纤维细胞,经脂质体介导EGFP基因转染第一代成纤维细胞,G418筛选10～12*!d,挑选转基因单克隆细胞,传代培养,进行细胞形态观察、生长曲线以及染色体核型分析,并进行了培养细胞性别鉴定.结果整合有EGFP基因的绵羊胎儿成纤维细胞生物学行为与未转染外源基因的细胞无明显差别,根据荧光强度可直接反应外源基因的表达量.结论 EGFP基因作为体内报告基因可用于转基因细胞的研究,并将整合有EGFP基因的转基因细胞为克隆动物提供核供体奠定了基础.     

血清饥饿、汇合培养及放线菌酮对奶牛成纤维细胞周期的影响

在动物克隆研究中,研究者普遍认为位于细胞周期的G0+G1期的二倍体细胞对于核移植中供核细胞的重新程序化是必需的。本文探讨了血清饥饿、汇合培养及放线菌酮（CHX）处理对不同传代次数的体外培养奶牛成纤维细胞周期分布的影响。流式细胞仪分析结果显示：第3代和第13代细胞经血清饥饿处理72h后,细胞周期分布与对照差异显著(P<0.05);汇合培养可以显著增加奶牛成纤维细胞处于G0+G1期细胞数。CHX处理第3代细胞经CHX处理后处于G0+G1期的细胞数差异不显著,而第13代细胞处理后差异显著。结果表明体外培养奶牛成纤维细胞高代对血清饥饿、汇合培养及CHX处理更敏感。     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

家兔供体细胞的发育周期与重构胚发育的关系

采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明：培养基中血清含量在0．5％的情况下,G0／G1期的细胞比例由正常培养条件下(培乔液中含有10％FCS)的73．2％明显地增加到86％以上。饥饿1～3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。     

奶牛体细胞核移植胚胎产业化生产条件优化北大核心CSCD

通过优化高产奶牛体细胞克隆胚胎体外生产技术条件,制备高质量的奶牛克隆胚胎,旨在提高奶牛体细胞核移植产业化应用效率。就受体卵母细胞去核方法、不同年龄供体牛细胞来源、血清饥饿与否以及不同气相组成培养等条件对奶牛体细胞克隆胚胎生产效率的影响进行了研究和探讨。结果表明,虽然荧光染色辅助去核和盲吸法的去核率、囊胚发育率分别为100%、24.83%和92.44%、28.26%,两者之间无显著差异(P>0.05),但盲吸法操作简单、效率高;不同年龄来源供体牛的细胞系构建的克隆胚胎的囊胚发育率分别为31.43%、25.68%,两者之间没有显著差异(P>0.05);经血清饥饿和未饥饿供体细胞重构的克隆胚胎囊胚发育率分别为24%、29.9%,两者之间没有显著差(P>0.05);富氧和低氧气相培养的克隆胚胎的囊胚发育率分别为28.26%、31.55%,两者之间差异不显著(P>0.05),低氧气相组成更有利于囊胚的发育。根据上述结果,奶牛体细胞核移植胚胎(克隆胚胎)的产业化生产条件为:供体细胞无需进行同期饥饿处理,直接注入到盲吸去核后的受体卵子透明带下构建克隆胚胎,融合后的克隆胚胎在密封的混合三气(5%CO2-5%O2-90%N2)的气相组成下进行体外培养,能保持稳定的囊胚发育率。     

不同供体细胞及其处理对猪核移植重构胚体外发育的影响

系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40~44 h的猪卵母细胞去核后, 将经血清饥饿(0.5%FBS)培养2~9天、0.1 mg/L Aphidicolin(APD)培养+0.5% FBS培养2~9天或一般培养法(10% FBS)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞, 直接注射到去核的卵母细胞质中, 或注射到卵周隙中, 再经电融合(100 V/mm, 30 [mu]s, 电脉冲1次)构建重构胚。重构胚以钙离子载体A23817 或电脉冲结合6-DMAP 激活处理, 体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1 mg/L APD + 0.5% FBS培养处理后的重组胚卵裂率, 均高于血清饥饿和一般培养处理的同种供体细胞(P<0.01)。卵丘细胞、颗粒细胞经0.1 mg/L APD + 0.5% FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P<0.05)。以猪颗粒细胞为核供体时, 电融合法的重构胚分裂率显著高于胞质内注入法(P<0.05), 但囊胚发育率无显著差异(P>0.05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞, 其具有正常二倍染色体的细胞比例均无显著差异(P>0.05); 以这2种细胞不同培养代数做供体进行核移植时, 各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P>0.05)。这些结果表明: (1) 猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定; (2) 0.1 mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果, 血清饥饿培养则无明显效果; (3) 猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞, 核移植后都能得到体细胞克隆的囊胚, 但前者的效果略优于后者, 且其核移植效果不受供核细胞培养代数的影响; (4) 电融合核移植胚胎的发育率高于胞质内直接注入法, 但两者的总体效率相近。     

慢病毒直接诱导形成猪iPS细胞无需限定因子

为了证实慢病毒对细胞具有遗传修饰和重编程作用,在本实验中使用慢病毒感染猪胎儿成纤维细胞.结果显示:慢病毒介导的EGFP在猪胎儿成纤维细胞中稳定和高效表达,使用添加LIF和bFGF的细胞培养液,部分猪的胎儿成纤维细胞逐渐改变原有的纤维状形态,形成圆形的细胞,细胞逐步增殖形成细胞集落,细胞集落边界清晰,在饲养层上细胞集落生长迅速,具有稳定的生长性能和正常核型,细胞碱性磷酸酶染色为阳性,表达干细胞特有的标记Oct4、Nanog和SSEA1,在体外能够形成拟胚体,在体内分化形成包含三个生殖层的畸胎瘤.作为核移植的供体细胞,克隆胚的卵裂率为53.33%、桑椹胚率为9.03%、囊胚率为2.07%、孵化囊胚的总细胞数为26.5,在桑椹胚率和囊胚率方面显著低于猪普通胎儿成纤维细胞核移植克隆胚的发育能力(P<0.05).结果证实慢病毒能够直接使猪的胎儿成纤维细胞转变成iPS细胞,因此慢病毒将成为一种理想的材料和工具用于细胞的遗传修饰和细胞重构等方面的研究.     

牛胎儿成纤维细胞的组织块贴附法分离培养与电穿孔法基因转染

为获得转基因克隆牛的供体细胞,采用组织块贴附培养的方法分离培养牛胎儿皮肤成纤维细胞,经2～3次传代纯化,绘制生长曲线,分别分析体外传代培养10代以内和20代以上细胞的核型特征。分别采用800、900、1000V／cm和1、5、10、15和20ms的参数组合,将线性化的带有新霉素抗性和绿色荧光蛋白双重筛选标记的人胰岛素原乳腺特异表达载体pNEI电穿孔转入体外培养的牛胎儿成纤维细胞,经800μg／mLG418筛选2周,继续以300μg／mLG418扩大培养2～3代,取部分筛选后的细胞进行PCR检测结果表明,体外培养的牛胎儿成纤维细胞生长旺盛,体外传代20次后核型未发生改变;转染后24～48h在荧光镜下检测各组均可观察到绿色荧光表达,筛选后各组克隆形成数以900V／cm和5ms组最多;PCR检测得到了预期条带,说明目的基因已经成功导入。分离得到的牛胎儿耳成纤维细胞有可能作为体细胞核移植的供体,进行转基因克隆研究。     

不同方法处理供核细胞对细胞周期和重构胚发育的影响

利用流式细胞仪和细胞染色体核型分析技术,比较奶牛的转基因体细胞和正常细胞经血清饥饿、抑制培养周期同步化处理后的G0/G1期细胞比例;并将同步化处理的核供体细胞进行核移植,然后统计囊胚发育率.结果表明,血清饥饿和抑制培养均能获得较高比例的G0/G1期细胞,两组间差异不显著(P>0.05),但均显著高于未处理对照组(P<0.05);血清饥饿组的囊胚率显著高于抑制培养组和非处理对照组(P<0.05);但细胞同步化处理6 d后细胞染色体核型异常率增加.因此,要获得正常核型的G0/G1核移植供体细胞和较高的囊胚率,同步化处理时间以不超过4 d为宜.     

Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts

The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.     

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.     

Inhibition of apoptosis in serum starved porcine embryonic fibroblasts

In nuclear transplantation, serum starvation is a general method to synchronize donor cells at the quiescent stage (G(0)) of the cell cycle. However, serum starvation during culture of mammalian cells may induce cell death, especially through apoptosis, thus contributing to the low efficiency of nuclear transplantation. This study was performed to characterize apoptosis during serum starvation and to determine the effects of apoptosis inhibitors such as a protease inhibitor [alpha(2)-macroglobulin (MAC)] and antioxidants [N-acetylcysteine (NAC), glutathione (GSH)] on serum starved porcine embryonic fibroblasts (PEF). PEF, collected from day 25-30 porcine fetuses, were cultured for 5 days in media containing 0.5% FBS to induce quiescence. Serum starved PEF showed typical morphology of apoptotic cells and stained for DNA fragmentation by TUNEL assay (26.7%). All apoptosis inhibitors tested in this study significantly (P < 0.05) reduced apoptosis of serum starved PEF, with antioxidants having better results (MAC: 7.4% vs. NAC: 1.0%, and GSH: 0.8%). Equally and importantly, the treatment with apoptosis inhibitors did not change the proportion of G(0)/G(1) stage cells. Therefore, the addition of MAC and antioxidants during serum starvation of PEF reduces apoptosis of quiescent fibroblasts and may contribute to increasing the efficiency of nuclear transplantation by improving the quality of donor nuclei.     

Cell synchronization for the purposes of nuclear transfer in the bovine

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.