人胚细胞提取物抗突变作用的研究

本文应用体外培养人淋巴细胞和小鼠骨髓微核与染色体畸变检测,研究了人胚细胞水提物(胚提物)的遗传毒性和抗突变效应。结果表明,(1)胚提物(3和30μg/ml,下同)可显著抑制培养人淋巴细胞的自发和γ-射线诱发的微核形成;(2)胚提物对培养淋巴细胞的自发染色体畸变无明显影响,但可显著抑制丝裂酶素C(MMC)诱发的染色体畸变; (3)胚提物显著抑制环磷酰胺诱发的小鼠骨髓多染性红细胞微核(PCE-MN)的增加; (4)胚提物的上述抗突变效应呈剂量依赖性增加。因此,作者认为,人胚细胞水提物是一种抗突变剂, 可望用于肿瘤的化学预防,并可用作为肿瘤放疗和化疗的辅助药物,以减少毒付反应和二次肿瘤的发生率。 Abstract:Genetially toxic and antimutagenic effects of the aqueous extracts of human fetal cells(HFCAE) against mutagen-induced chromosomal aberrations and micronucleus formation in human lymphocytes in vitro and bone marrow of mice were studied.HFCAE (3 and 30 μg/ml)significantly inhibited spontaneous and γ-rays-induced micronucleus formation and MMC-induced chromosomal aberrations in human lymphocytes in vitro.HFCAE (3 and 30μg/ml) strongly suppressed also micronucleus formation induced by CP in PCEs of mice.These antimutagenic effects of HFCAE were dose-dependent.Our results suggest that HFCE might be an antimutagen and have potential value in its clinical application.     

利用CRISPR/Cas9技术对miRNA-125a进行基因编辑的实验研究

近年来, CRISPR/Cas9技术被迅速应用于生物学研究的各个领域。miRNA-125a已被证实与生殖密切相关,研究者在自然流产的患者中发现了异常的miRNA-125a。该研究利用CRISPR/Cas9技术在人绒毛膜滋养层细胞HTR8-S/Vneo中对miRNA-125a进行了定点编辑。对于定点敲除,我们首先在pX458表达载体的基础上额外引入一组启动子和sgRNA骨架序列用于片段敲除,此后分别设计用于单点敲除和片段敲除的sgRNA进行表达载体的构建,表达载体转染HTR8细胞后通过流式分选和T7EI酶切鉴定阳性单克隆;对于定点敲入,我们在体外构建供体载体,与CRISPR表达载体共转染HTR8细胞,通过流式分选和PCR鉴定阳性单克隆。DNA测序结果显示,敲除及敲入阳性单克隆细胞系在指定区域的碱基序列发生了改变; RT-qPCR结果显示,阳性单克隆细胞系中mi RNA-125a-5p和mi RNA-125a-3p的表达量较野生型也发生了相应变化;体外实验还显示, miRNA-125a有抑制HTR8-S/Vneo细胞系增殖及迁移的功能。研究证实, CRISPR/Cas9技术在细胞系中编辑mi RNAs是有效可行的,且对于miRNA-125a的定点敲除,片段敲除的效果要好于单点敲除。