RESUMO
AIM: To examine the role of nucleostemin in the growth regulation of gastric cancer, liver cancer and other cancers. METHODS: RT-PCR was used to clone the fragment of nucleostemin cDNA from HEK 293 cells. Eighteen kinds of malignant tumor tissues including gastric adenocarcinoma and liver cancer tissues, 3 kinds of benign tumor tissues, 3 kinds of benign hyperplastic tissues and normal tissues were employed to examine nucleostemin gene expression by RT-PCR, Slot blot, Northern blot and in situ hybridization. RESULTS: We successfully cloned a 570 bp fragment of nucleostemin-cDNA from HEK-293 cells. All detected malignant tumor tissues, benign tumor tissues, and benign hyperplastic tissues had high levels of nucleostemin expression. Nucleostemin was also expressed in human placenta tissue at a high level. In terminally differentiated normal human adult kidney and mammary gland tissues, no nucleostemin expression could be detected. CONCLUSION: Nucleostemin can help regulate the proliferation of both cancer cells and stem cells. It might play an important role in the growth regulation of gastric cancer, liver cancer and other cancers.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Feminino , Proteínas de Ligação ao GTP , Regulação da Expressão Gênica , Humanos , Hibridização In Situ , Rim/citologia , Rim/metabolismo , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Distribuição TecidualRESUMO
BACKGROUND: To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro. METHODS: The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond trade mark Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel. RESULTS: The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells. CONCLUSION: hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.
Assuntos
Neoplasias Ósseas/patologia , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Osteossarcoma/patologia , Sialoglicoproteínas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , OsteopontinaRESUMO
Chromosomal DNA in higher eukaryotes is spatially organized into loops by periodic attachment to the nuclear matrix at its base via a specific matrix attachment region (MAR). In order to study the nature of DNA sequences that affixed the loops to the nuclear matrix, we have cloned the MAR DNA from bovine lactating mammary tissues. In vitro binding assay showed that the cloned fragments could be co-complexed with nuclear matrix proteins to form insoluble complex easily removed by centrifugation. Sequences of the two chosen MAR loci are composed of TG-, CA- and GA- blocks, as well as the ATTA motifs. Both the MAR loci show numerous replication/transcription factor binding sites, enhancer motifs, several perfect or imperfect inverted repeats, and sequences sharing the common features of the potential DNA bending core sequence. The possibility that a combination of different elements in the same DNA sequence may function as either positive or negative regulatory elements in controlling a variety of cellular and developmental processes is discussed.
Assuntos
DNA/química , Glândulas Mamárias Animais/metabolismo , Regiões de Interação com a Matriz/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Mapeamento Cromossômico , DNA/fisiologia , Feminino , Regulação da Expressão Gênica , Genes Reguladores/fisiologia , Dados de Sequência Molecular , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismoRESUMO
This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.