摘要
程序性细胞死亡(programmed cell death,PCD)是一主动性细胞“自杀”过程,是细胞内部基因引导的一系列精密生化级联反应的结果。凋亡(apoptosis)是PCD的形态学指征,探讨何种基因并以何种途径诱发PCD是当前研究的热点,也是本研究的动机。基因转导是从分子水平研究基因调控的一种手段。本研究采用基因转导法结合电镜观察细胞凋亡,选择的目的基因分别是构建在真核表达载体pMAMneo中人TGF-β1cDNA基因全长,采用磷酸钙共沉淀法,体外转染贴壁生长的人肺巨细胞癌PLA-801D株细胞,以及构建在逆转录病毒载体pDORneo中的反义c-myc、反义bcr/abl基因片段,采用Lipofectin体外转染悬浮培养的人白血病HL-60或K562细胞,除进行一系列分子细胞生物学指标的检测外,重点对转染后不同阶段细胞进行了透射电镜观察。结果发现转染TGF-β1的人肺癌细胞多呈现典型的“凋亡”改变;核异染质凝聚呈“半月形”、细胞出芽(budding),形成大量“凋亡小体”,胞浆细胞器尚完好,凋亡小体内亦见完整压扁的细胞器,并可见吞噬有染色质凝块的癌细胞,而转染反义c-myc,反义bcr/abl的HL-60或K562细胞更多出现核“新月体”,亦见“球形”、“面包圈”状凝聚异染质,并见吞噬“凋亡小体”现象。转染TGF-β1细胞DNA电泳呈现的“DNA梯带”
Apoptosis is the terminal phenomenon of gene-induced biological cascade reaction and a morphological term for programmed cell death (PCD). Our present study is to clarify whether several negative regulator genes can induce PCD, using electron microscopy in the cells transferred with genes of transforming growth factor-β1 (TGF-β1), antisense transcripts to c-myc and ber / abl genes in vitro. A pMAMneo-TGF-β1 recombinant eukaryotic expression vector inserted with a full length (1.3kb) human TGF-β1 cDNA is transferred by calcium phosphate precipitation into a human lung gi-ant cell carcinoma cell line PLA-801D. Under transmission microscope the celis transferred with TGF-β1 showed its nuclear chromatin condensation and concentrated cytoplasm. The organelles kept intact and budding into several membrane-encapsulated bodies-the typical feature of apoptosis body (APO body). A retrovirus vector pDORneo inserted a antisense transcript to c-myc or ber / abl is transferred into K562 or HL-60 leukemic celis in vitro by Lipofectin. The nuclear chromatin of transferred celis condensed into 'crescent' or 'halfmoon body'. Nuclei were clapped into a lot of small chromatin balls. Phagocytosis of APO bodies by neighbor celis was seen often. In addition, the PLA-801D cell transferred by TGF-β1 showed their DNA ladder bands in electrophoresis, the characteristics of the internucleosomal fragmentation in apoptosis. It is suggested that apoptosis induced by transduction of negative regulator genes in an in vitro experimental condition may mimicked the process in vivo.
出处
《中国实验血液学杂志》
CAS
CSCD
1996年第2期152-157,共6页
Journal of Experimental Hematology
