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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~3H-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。
In this study, the functional activities of Xenopus laevis oocytes on the nuclei of mammalian erythrocytes and the tumor cell nuclei (Sp 2/0 and HeLa cells) cultured in vitro were studied by means of nuclear microinjection and molecular biology. Regulatory effect. The experimental results showed that: (1) When the erythrocyte nuclei of different physiological states were injected into the eggs, only part of them (about 1/3) survived and were activated, with the appearance of nuclear enlargement, loose chromatin, clear nucleation and other activated forms Feature, and has the ability to combine ~ 3H-uridine to synthesize RNA. Most of the nuclei of erythroblasts, which were originally in the late stage of differentiation or nuclear pyknosis, failed to be activated by oocyte, and appeared deep staining, condensation and lysis, or tended to degenerate and disappear. In the poorly differentiated and proliferative Sp2 / 0 and HeLa cell nuclei, almost all of them were activated, the nucleus was dilated (the volume increased tens of times to more than 200 times), the chromatin was loose and the nuclei were clear , Nucleolus developed, activated to a greater extent than the erythrocyte nucleus more obvious. Isoelectric focusing electrophoresis analysis showed that oocytes implanted into erythrocytes nuclei showed a band with isoelectric point (pI) of 6.28 on the electrophoresis map, which was similar to that of rat bone marrow erythrocyte electrophoresis band Quite equivalent but failed to detect hemoglobin. Indicating that the gene activity of the implanted nucleus has been rearranged for the egg, the original active globin gene is turned off, and the gene selectively activated to express the egg is expressed accordingly. 4. After the purified globin mRNA polyribosomes were injected into oocytes, hemoglobin synthesis could be detected. The above results indicate that the oocyte has the condition of faithful translation and regulation or reprogramming of nuclear gene expression to polysome mRNA. Regulatory effect due to different structural levels vary. Which is closely related to the original physiological state of the implanted nucleus and the ability of sensing the egg. The originally implanted nucleus in an actively proliferating state is much more regulated than the terminal differentiated nucleus.