| 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...
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