| PrefaceType 1 diabetes mellitus is caused by severe insulin deficiency secondary to the autoimmune destruction of pancreatic b cells, which is incompletely treated by exogenous insulin administration. Recent advances in molecular and cell biology may allow for the development of new strategies for the treatment and cure of type 1 diabetes. Diabetes mellitus is caused by deficient production of a single protein, insulin. So, it is logical for gene therapy,which purpose is to reconstruct the capacity of insulin secretion. The Methods involve the introduction of a foreign gene into somatic cells and the reconstruction of insulin -producing cells in vitro followed by their implantation into body.Besides pancreas b cell, hepatocyte is the only somatic cell containing Glut2 and GK. Moreover, many specific gene of liver are regulated by physiological glucose level. Thus, more and more people pay attention on hepatocyte. Hepatoma cells are also ideal cells for gene therapy. In normal individual, conversion of proinsulin to insulin is mediated by two endoproteases, PC2 and PC3. There are no PC2 and PC3 but furin in hepatoma cells. It should be noted that furin are required to recognize the sequence of Arg-X-Lys-Arg. By introduction of the sequence into the conjunction of B - C and C - A of human proinsulin gene, it remains possible that the mutations needed to make proinsulin sensitive to cleavage by furin may make insulin produced.Recombined retrovirus vectors encoding a genetically modified human proinsulin were constructed and subsequently introduced into hepatoma cell line HepG2 by liposomes. After G418 selection and cloning, chromosomal DNA was isolated from transfected HepG2 cells and PCR was performed. PCR products were analyzed by electropho-resis to confirm the integration of reporter gene. Besides, insulin level of culture mediums were monitored by radioimmuniassay. The study provides experimental data for the following recombination of regulatory expression vector and animal experiment.MaterialRecombined retroviral vector encoding genetically modified human proinsulin (pLXSN-MpINS ) was constructed by prophase of experiment. Heptoma cell line HepG2 was from Cell Biology Research Institution of Shanghai. LIPOFECTAMINTM, G418 and DMEM were from GIBCO BRL Corporation of America. Regents of PCR were from Takara Corporation of Dalian.Method1. Extraction of recombined retroviral vector encoding genetical human proinsulin.Preparing the plasmids of pLXSN - MpINS with the method of alkali splitting. Measuring the concentration and purity of DNA by UV absorption.2. Transfecting pLXSN - MpINS into heptoma cells HepG2 by li-posome.In a six - well or 35 mm tissue culture plate, seed 2x 105 cells per well in 2 ml Basal Medium containing 10% FBS 24 hours before transfection. The next day, transfect pLXSN and pLXSN-MpINS to HepG2 cells. Divide cells into untransfected group, pLXSN transfect-ed group and pLXSN - MpINS transfected group. Incubate cells for 72h at 37 C in a CO2 incubator.3. G418 screening and cloning.Besides the first well, add G418 into the wells( total concentration is 300g/ml). Observe growth of cells each day and change mediums and G418 every two days. Two weeks after trasfection, clones appear in wells. Transfer positive cell colons to two flasks of 50ml and culture them continually, which are used for extracting DNA.4. Samples of cultured cells harvesting and detecting.Monitor insulin concentrations in culture mediums of pro - transfection and post - transfection ( once every 3 days) and detect intra-cellular insulin level before transfection and on the 24th day after transfection.5. extraction of cellular chromosome DNA.With method of trizol, individually extract cellular chromosome DNA from untransfected HepG2 cells, positive cell clones of pLXSN vector transfected HepG2 cells and pLXSN - MpINS transfected HepG2 cells.6. PCR amplification and product analysis by electrophoresis DNA was amplified in 50l PCR reaction. Samples were amplified... |
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