TY - JOUR
T1 - Construction of immortalized rat astrocyte strain genetically modified by rat preprogalanin gene for the analgesia research
AU - An, Ke
AU - Tian, Yu Ke
AU - Yang, Hui
AU - Gao, Feng
AU - Wang, Peng
PY - 2005/4/14
Y1 - 2005/4/14
N2 - Aim: Transgenic cell transplantation for analgesia is a new direction to treat chronic pain. This paper aims to construct an immortalized rat astrocyte strain (IAST) genetically modified by rat preprogalanin (GAL) gene in order to provide theoretical bases for analgesia by GAL genetic transplantation. Methods: The experiment was completed in the Institute of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from May 2003 to October 2004. A cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was cloned into eukaryotic expression vector pcDNA3.1(+) by DNA recombinant technology, and the restriction enzyme digestion and DNA sequencing were carried out to evaluate this recombinant plasmid. The pcDNA3.1(+) -GAL and pcDNA3.1(+) construction were transfected into IAST by standard lipofectamine techniques respectively and the population of cells which stably integrated was selected with 600 mg/L G418. Then, individual clone was screened and expanded into clonal cell strain. Immuncytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion of GAL in the transfected rats. Detection of Neo gene was used to validate successful transfection. Results: After yhe recombinant plasmid constructed by restriction enzyme digestion and DNA sequencing, 5.4 kb and 521 bp fragments were found, and their sequencing analysis accorded with that reported in related literatures, indicating that recombinant plasmid pcDNA3.1(+) -GAL was subcloned successfully. Detection of Neo gene showed that the pcDNA3.1(+)-GAL and pcDNA3.1(+) have been transfected into IAST successfully. After selected by G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunohistochemically stained positive for GAL, but the GAL average absorbance value of IAST/GAL was significantly higher than that of IAST/Neo and IAST(P < 0.001). The level of GAL mRNA expression and the supernatant concentration of GAL secretion in cultured IAST/GAL were higher significantly than those of IAST and IAST/Neo (P < 0.001), but there was no significant differences between the IAST and IAST/Neo(P > 0.05). Conclusion: An immortalized rat astrocyte line modified genetically by rat preprogalanin gene(IAST/GAL) is constructed successfully, which provides a research basis for transgenic cell transplantation for analgesia.
AB - Aim: Transgenic cell transplantation for analgesia is a new direction to treat chronic pain. This paper aims to construct an immortalized rat astrocyte strain (IAST) genetically modified by rat preprogalanin (GAL) gene in order to provide theoretical bases for analgesia by GAL genetic transplantation. Methods: The experiment was completed in the Institute of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from May 2003 to October 2004. A cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was cloned into eukaryotic expression vector pcDNA3.1(+) by DNA recombinant technology, and the restriction enzyme digestion and DNA sequencing were carried out to evaluate this recombinant plasmid. The pcDNA3.1(+) -GAL and pcDNA3.1(+) construction were transfected into IAST by standard lipofectamine techniques respectively and the population of cells which stably integrated was selected with 600 mg/L G418. Then, individual clone was screened and expanded into clonal cell strain. Immuncytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion of GAL in the transfected rats. Detection of Neo gene was used to validate successful transfection. Results: After yhe recombinant plasmid constructed by restriction enzyme digestion and DNA sequencing, 5.4 kb and 521 bp fragments were found, and their sequencing analysis accorded with that reported in related literatures, indicating that recombinant plasmid pcDNA3.1(+) -GAL was subcloned successfully. Detection of Neo gene showed that the pcDNA3.1(+)-GAL and pcDNA3.1(+) have been transfected into IAST successfully. After selected by G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunohistochemically stained positive for GAL, but the GAL average absorbance value of IAST/GAL was significantly higher than that of IAST/Neo and IAST(P < 0.001). The level of GAL mRNA expression and the supernatant concentration of GAL secretion in cultured IAST/GAL were higher significantly than those of IAST and IAST/Neo (P < 0.001), but there was no significant differences between the IAST and IAST/Neo(P > 0.05). Conclusion: An immortalized rat astrocyte line modified genetically by rat preprogalanin gene(IAST/GAL) is constructed successfully, which provides a research basis for transgenic cell transplantation for analgesia.
UR - https://www.scopus.com/pages/publications/27744567495
M3 - Article
AN - SCOPUS:27744567495
SN - 1671-5926
VL - 9
SP - 109
EP - 111
JO - Chinese Journal of Clinical Rehabilitation
JF - Chinese Journal of Clinical Rehabilitation
IS - 14
ER -