【作者】 沈新明；

【导师】 姚开泰；

【作者基本信息】 中国人民解放军第一军医大学 ， 病理和病理生理学， 2003， 博士

【摘要】 恶性肿瘤是严重危害人类健康和生命的一类重大疾病，是由于环境中致癌和促癌因子作用于正常细胞，破坏了核质平衡关系，改变了遗传物质组成或基因表达，进而引起细胞癌变、生长和分化失去控制而形成。 以往对恶性肿瘤发病机理的研究包括癌变原理的研究，多集中在正常细胞如何转变为恶性肿瘤细胞方面。近年来，尤其在利用体细胞核移植技术成功获得克隆动物之后，有研究者基于前人的有关工作，提出了“肿瘤细胞恶性表型可以通过核移植逆转”的假说，这为阐明肿瘤的发病机制提出了一条新的思路，对恶性肿瘤的预防和治疗有重要的意义。 我们知道，体细胞克隆动物的成功表明：终末分化细胞(如体细胞)的细胞核移植到去核的卵母细胞中，可以和卵母细胞的胞质相互作用，发生重新编程，使体细胞去分化，恢复全能性。按照上述理论，可以推测，肿瘤细胞作为一类已经分化的细胞，如果通过核移植技术，被移植到去核的卵母细胞中，在适当的条件下，也有可能与细胞质作用而重新编程，从而恢复全能性，改变其恶性表型。 基于对以上“肿瘤细胞恶性表型可以逆转”的假说和体细胞与肿瘤细胞核移植技术的认识和兴趣，我们对经典的去核方法做了改进，并建立了改良的小鼠体细胞核移植技术。在此基础上，用小鼠恶性黑色素瘤B16-F1细胞作为核供体获得了肿瘤细胞的重组胚，并对其融合、激活和体外培养等条件进行了摸索。同时，我们利用了cDNA微阵列技术，对发育到2细胞期的小鼠恶性黑色素瘤细胞的重组胚进行了基因表达谱的分析，并与2细胞期的卵丘细胞重组胚进行了对比，初步研究了肿瘤细胞和体细胞核移植后早期胚胎发育的基因表达规律。实验结果简述如下： 首先，我们改良的小鼠体细胞核移植技术以二步法切开透明带为基础，采用一步法有效去除了卵母细胞的细胞核。用Hoechst33342染色表明可以完全去除MⅡ卵母细胞细胞核，并证实了这种方法的可行性。完成一个完整的去核过程平均约为20秒，去核的成功率为95.5％。随后，我们以c57Bu6)小鼠卵丘细胞作为核移植供体获得重组胚，在cE45。细胞融合仪上进行重组胚胎的融合和激活，重组胚胎的原核形成率为622%。体外培养72小时内，发育到2细胞期、今8细胞期和桑堪胚(16细胞期以上)比例分别为57.5%、39.1%和27.6%。用2个微卫星标记 (D7Mit22和D4Mit204)对来源于卵丘细胞的重组胚进行鉴定，证实重组胚来源于c57BU6)小鼠卵丘细胞。 在上述工作的基础上，我们以c57BL/6)小鼠恶性黑色素瘤B16一F1细胞为对象研究了肿瘤细胞恶性表型逆转的可能性。通过染色体核型分析、裸鼠接种及HE染色证实B16一Fl细胞具有高度的恶性。用0.5%小牛血清培养B16一Fl细胞5一8天诱导静止后，将其作为核移植供体构建重组胚，经摸索确定了该重组胚的合适融合条件为1400v/cm、40一80协s、2次(2次间隔时间为5秒)，且根据重组胚的原核形成率推断，该肿瘤细胞中直径8一10um的细胞是核移植的最佳核供体。重组胚胎的原核形成率为53.6%。体外培养72小时内，发育到2细胞期、小8细胞期和桑堪胚(16细胞期以上)比例分别为51.5%、40.3%和26.3%。由于我们对B16一Fl细胞的遗传背景不清楚，为了对来源于肿瘤细胞的重组胚胎进行鉴定，我们以转染了EGFP基因且有较强绿色荧光表达的B16一Fl细胞为核供体，获得的重组胚可用EGFP引物PCR扩增出目的条带，但胚胎未见荧光表达，这可能是由于重新编程使这个外源性的基因表达失活，或者是延迟表达有关。 为了进一步了解肿瘤细胞核移植后重组胚重新编程并脱离恶性表型的机制。我们利用cDNA微阵列技术，对发育到2细胞期的重组胚进行了基因表达谱的研究。利用SMART cDNA合成技术，首先从上述微量样本中抽提出低于Zng的总RNA，然后反转录获得dsDNA并扩增至1.5一Zug，从而使重组胚的基因表达谱研究成为可能。用AilasTM Mousc 1.2微阵列膜对7个样本进行了基因表达谱的研究，并采用Ailaslmagel.Ola软件分别比较样本的杂交结果。 通过比较我们发现，体细胞核移植后2细胞期重组胚与卵丘细胞和2细胞期受精卵相比较，基因表达主要以上调为主;而肿瘤细胞核移植后2细胞期重组胚与B16一Fl细胞和2细胞期受精卵相比较则以下调为主;肿瘤细胞与体细胞核移植后2细胞期重组胚相比较也以下调为主。 一2-其中，2细胞期卵丘细胞重组胚与卵丘细胞相比较，共有164个基因表达上调，而下调的基因只有2个;与2细胞期受精卵相比较有56个基因表达上调，下调的基因只有8个。2细胞期的B16一F1细胞重组胚与B16一Fl细胞相比较只有18个基因上调，下调的基因则达81个;与2细胞期受精卵相比有36个基因上调，下调的基因高达208个。肿瘤细胞和卵丘细胞2细胞期重组胚比较，差异表达的基因也主要以下调为主，共有63个，上调只有2个。上述表达差异的基因按照它们在重新编程过程中的可能功能主要分为以下几类:编码转录因子、细胞周期调节、瘤基因与抑瘤基因、凋亡与死亡、生长因子/细胞因子/趋化因子和受体、细?更多还原

【Abstract】 Malignant neoplasm is a sort of serious disease that can severely endanger the health and life of human beings. A normal cell is able to become a malignant one under the action of environmental carcinogens and tumor promoters, which can upset the balance between nucleus and cytoplasm, make the genetic component abnormal, disorder the expression of genes, thus induce normal cells to get malignant phenotype, and then uncontrolled in proliferation and differentiation.In the past, the study on the initiation of malignant neoplasm, including the pathogenesis of cancer, was mostly focused on how normal cells transformed to malignant cells. In these years, especially after the success in cloning of animals with somatic cells by nuclear transfer(N-T), that "the malignant phenotype of neoplasm cells could be reversible by N-T" has been gradually brought forward as a hypothesis based on the former studies. This provides a new thought for clarifying the mechanism of carcinogenesis, and is very important for the prevention and therapy of cancer.In evidence, the success of cloning suggested that the nucleus of a cell in terminal differentiation(such as many somatic cells) could interact with oocyte cytoplasm, be induced to reprogram when transplanted to an enucleated oocyte, which would lead the cell to dedifferentiation and restore its totipotency. According to that theory, we can infer that the malignant cells, as a sort of differentiated cell, when transplanted into enucleated oocyte by N-T, would interact with cytoplasm, be induced to reprogram, and restore totipotency, thus reverse its malignant phenotype under an appropriatecondition.Due to the cognizance and interest in the above hypothesis and the N-T technology of somatic cells and malignant cells, we modified the classical enucleating methods and established an improved N-T technology of mouse somatic cell. On the basis of that, we used B16-F1 cells derived from mouse malignant melanoma as nuclear donor and acquired reconstructed embryos, then tested various parameters for electrofusion, activating and cultivating of them in vitro. Meanwhile, we analyzed the gene expression profiling of 2-cell reconstructed embryos from somatic cells and malignant cells by cDNA microarray, preliminarily compared the gene expression patterns early embryos reconstructed with somatic or malignant cell nuclei. The results of the experiment are as follows:First, based on the two-step dissection method of zone pellucida, our modified N-T technique could remove the whole nucleus out of oocyte in one-step. The feasibility of that technique was confirmed by exposing the aspirated karyoplast stained by Hoechst33342 under UV light for checking the result of enucleation. The process of enucleation took an average of 20 seconds, and the number(%) of surviving oocytes after the enucleation is 95.5%. A single cumulus cell was placed below the zone pellucida in contact with each enucleated oocyte, then electrofused and activated. The number(%) of reconstructed embryos that formed pronuclei is 62.2%. The reconstructed embryos were cultured in CZB media. Cultured for 72hrs in vitro, The number(%)of embryos that developed to 2-cell, 4-8 cell and Morula (more than 16 cells) are 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences were amplified with two sets of the primers (D7Mit22 and D4Mit204) for identifying the origin of the reconstructed embryos, and the cumulus cell origin from C57BL/6J mouse was confirmed.Secondly, the reverse of malignant phenotype of tumor cells was studied by transplanting B16-F1 cells of melanoma into the enucleated oocyte. The high malignancy of B16-F1 cells were confirmed by karyotype analysis, oncogenecity in nude mice and HE stain of slides. Subsequently, the B16-F1cells were starved for 5~8 days in medium containing 0.5% PCS to quiescence and then transplanted as nuclear donor to constitute reconstructed embryos. The couplet was electrofused and activated. The best electrofused parameter was 1400V/cm, 40~80ns, twice. (There should b更多还原