非洲爪蟾卵母细胞卵质对哺乳类细胞核功能活动调整作用的研究

THE REGULATORY EFFECT OF XENOPUS OOCYTE CYTOPLASM ON THE FUNCTIONAL ACTIVITY OF INJECTED MAMMALIAN NUCLEI

本文用细胞核微注射技术和分子生物学方法,研究非洲爪蟾卵母细胞对植入的发育不同阶段的哺乳类成红细胞核、体外培养的肿瘤细胞核(Sp 2/0和HeLa细胞)的功能活动调节作用。实验结果表明:1.处于不同生理状态的大鼠成红细胞核注入卵内后,只有部分(约1/3)存活和被激活,出现核膨大、染色质松散、核质清明等被激活的形态特征,且具有并合H-尿嘧啶核苷合成RNA的能力。大部分原来处于分化晚期或核固缩状态的成红细胞核,未能被卵母细胞质激活,而呈现深染、浓缩和裂解,或趋于退化消失。2.处于低分化和增殖活跃状态的Sp 2/0和HeLa细胞核植入后,几乎全部被激活,核膨大(体积增大数十倍至二百倍以上),染色质充分松散、核质清明、核仁发达,被激活的程度较成红细胞核更为明显。3.等电聚焦电泳分析表明,植入成红细胞核的卵母细胞,在电泳图上出现了一条等电点(pI)为6.28的条带,与大鼠骨髓成红细胞电泳条带同源物相当,但未能检测到血红蛋白。表明植入核的基因活动状态已为卵质所重排,原来活跃的珠蛋白基因被关闭,而选择性地激活与卵质相适应的基因使之表达。4.将提纯的珠蛋白mRNA多核糖体注入卵母细胞后,可检测到有血红蛋白的合成。上述结果说明,卵质具备对多核糖体mRNA忠实翻译和调节或重编排植入核基因表达的条件。调节作用因不同结构水平而异。与植入核原来的生理状态及对卵质的感应能力密切相关,原来处于活跃增殖状态的植入核受调节的程度,较终末分化的细胞核显著得多。

The present study was designed to investigate the regulatory effect of Xenopus oocyte cytoplasm on the functional activity of the injected mammalian erythroid nuclei taken from different developmental stages and the tumor cell nuclei from human HeLa and mouse plasmocytoma(Sp 2/0)cell lines.Experimental results demonstrated that:(1)only 1/3 of the injected erythroid nuclei derived from different physiological status survived and-were induced to become enlarged,accompanied by chromatin dispersion,clear nucleoplasm,and with the ability of incorporation of~3H-uridine for the synthesis of RNA,while most of the injected nuclei in pyknosis or in late differentiated stage were inactivated,stained black and degenerated steadily;(2)almost all the"injected tumor cell nuclei originally in active division of Sp 2/0 and HeLa survived and were activated by showing nuclei enlarged to about 10 to 200 times with fully chromatin dispersion,concomitant clear nucleoplasm and well developed nuclcoli.The state of activation is much more pronounced than that in erythroid nuclei;(3)isoelectrophoretic analysis of the oocytes with transplanted nuclei showed a dark band at pi 6.28 which is corresponding to a homologous component,but other than hemoglobin from rat bone marrow erythrocytes.It is suggested that the gene activities of the injected nuclei were reprogrammed by the oocyte cytoplasm,globin gene originally activated were closed and oocyte selected to open genes that suitable to host genetic state;(4)when injected into Xenopus oocyte with purified rat globin mRNA polyribosomes,it translated efficiently into hemoglobin.The above mentioned results could be concluded that the Xenopus oocyte provided favorable conditions for the translation of purified mRNA polysomes as well as to regulate or reprogram the expression of gones in the injected nuclei.The regulatory effect is closely related to the initial physiological status and the competence of the injected nuclei themselves responsed to oocyte cytoplasm,i.e.the efficiency is much more prominent in originally active proliferative nuclei than those in late or terminal differentiation.