Gene Transfer And Expression Of Recombined Human Proinsulin In Rice Hepatoma Cells

PrefaceType 1 diabetes mellitus (T1DM) is caused by severe insulin deficiency seconday to the autoimmune destruction of pancreatic B cells. Since clinical symptoms are caused by diminished production of a single protein, diabetes is a natural candidate for treatment by gene therapy, transfer the modified human proinsulin gene into diabetes mellitus patients which will produce long term,high efficient and physiologically controlled insulin. Then we will get a permanent therapy method of type 1 diabetes mellitus. But before transferring it to diabetes mellitus patients or animal' s body, it must be demonstrated that this recombined human proinsulin be effective in vitro.The purposes of this trial are:1. To master the techniques of cell culture ,anabiosis and frozen.2. To master the methods of gene tranfection and identification.3. To detect the insulin levels of CBRH7919 cells afte transfected with a genetically modified human proinsulin 38h and one month at different glucose levels.4. To master DNA abstraction from cells,PCR amplification and electropho-resis techniques.Materials1. Rice hepatoma cell line CBRH7919 was from Cell Biology Research Institution of Shanghai.2. Recombined retroviral vector encoding genetically modified human proin-sulin(PLXSN-(GLRE)₃BP-1- MpINS₂ and (pLXSN - (GLRE)₃BP - 1 -MpINS₃ was constructed by prophase of experiment.3-D - glucoes,RPMI1640,G418,calf serum , flasks and six - wells were from Beijing HuaMei Corporation. Regents of PCR were from Takara Corporation of Dalian.Methodsl.The PLXSN-(GLRE)₃BP-1 -MpINS₂ and (pLXSN - (GLRE)₃BP -1 -MpINS₃ were transfected into CBRH7919 cells, and regulated glucose levels in culture mediumsIn 6 six - wells ,1 × 10⁵ cells were seeded in per well before transfection. The next day, pLXSN - (GLRE)₃BP-1 -MpINS₂ and (pLXSN - (GLRE)₃BP -1 - MpINS₃ were transfected to CBRH7919 cells according to design. Cells were divided into untransfected group, transfected pLXSN - ( GLRE )₃ BP - 1 -MpINS₂ group and transfected ( pLXSN - ( GLRE)₃ BP - 1 - MpINS3 group. Cells were incubated for 14h at 37℃ in a CO₂ incubator. Then culture medium including different glucose levels was added, and make the termial glucose levels are 0mmol/L,5mmol/L, 15mmol/L,25mmol/L and continue to incubated for another 24h.2. Samples of cultured cells harvesting and detectingMonitored insulin concentrations by chemoluminescence in culture mediums of three groups at different glucose levels after transfection 38h and pro - transfection.3. G418 screening and cloningBesides the untransfected well, added G418 into the wells (total concentration is 400mg/L). Observed growth of cells each day and changed mediums including G418 every three days. 7days after transfection, clones appeared in some wells. Transfered positive cell clones to a twenty -four well, and cultured them continually with selective medium including G418 200mg/L, changed mediums every three days. When the cells were full, transferred them to five flasks of 25 ml and cultured them with selective mediums continually.4. Samples of cultured positive clone cells harvesting and detecting Monitored insulin concentrations by chemoluminescence in culture mediumsof positive cells including pLXSN - (GLRE)3BP - 1 - MpINS2 and (pLXSN -(GLRE) 3 BP - 1 - MpINS3 at different glucose levels after the positive cells could be steadily passaged, (one month or so).5. Extraction of celluar chromosome DNAWith method of hydroxybenzene - chloroform - isoamylanol, extracted cellular chromosome DNA from untransfected CBRH7919 cells, positive cells of pLXSN - (GLRE) 3BP -1 - MpINS2 and (pLXSN - (GLRE)3BP - 1 - MpINS3 transfected CBRH7919 cells individually.6. PCR amplification and product analysis by electrophoresisDNA was amplified in 25 ul PCR reaction, samples were amplified in a thermal cycle with an initial denaturation at 94℃ for 5 minutes, denaturation at 94℃. for 30 seconds, annealing at 55℃ for 30 seconds and extension at 72℃ for 1 minute, with a final 5 minutes extension at 72℃. PCR products were detected by PAGE.7. Data analysisData were analyzed with One way ANOVA of SPSS 11.0 Methods.Results1. Evaluating the expression levels of recombined human proinsulin gene after transfecting 38h.CBRH7919 cells transfected with pLXSN - (GLRE)3BP - 1 - MpINS2 had no insulin detected during 0 -15mmol/L glucose levels,and at 25 mmol/L glucose level,its insulin concentration was (2.10 ±0.23) iu/L.CBRH7919 cells transfected with pLXSN - (GLRE)3BP - 1 - MpINS3 had secreted higher insulin than the cells with pLXSN - ( GLRE)2BP -1 - MpINS2, and its insulin levels increased with the added concentration of glucose. (P <0. 05) (see fig 1)2. Positive cell clones appearingAfter G418 screening ,all of untransfected cells died in 3 days and the cellswith pLXSN - (GLRE)3BP - 1 - MpINS2 and pLXSN - (GLRE)3BP - 1 -MpINS3 grew well and formed some clones. ( see fig. 3)3. Evaluating the expression level of recombined human proinsulin gene after steadily passaged, (one month or so)There was no insulin in culture mediums of CBRH7919 transfected pLXSN - ( GLRE)3BP - 1 - MpINS2 between 0 - 15mmol/L glucose except an average of (2. 05 ± 0. 17) iu/L insulin produced when exposed to 25mmol/L glucose during the same time incubation.Cells including ( pLXSN - ( GLRE) 3 BP - 1 - MpINS3 secreted an average of (3.56 ± 0.21)iu/L when it' s exposed to Ommol/L glucose, and (23.23 ± 1.12) iu/L to 25mmol/L. The correlation between glucose and insulin secretion was dose - dependent. ( see fig 4 )4 Identification of the integration of goal geneBy PCR amplification and electrophoresis analysis, it is successful to confirm that recombined human proinsulin gene pLXSN - ( GLRE) 3 BP - 1 -MpINS2 and (pLXSN - ( GLRE)3BP - 1 - MpINS3 had been integrated with the chromosome of CBRH7919 cells, (see fig. 5)DiscussionGene therapy for type 1 diabetes mellitus requires the design of artificial systems that mimic pancreatic β - cells, and produce physiological levels of mature insulin in response to stimuli. Recent advances in molecular and cell biology make it possible.In this study, we have engineered rice hepatoma cells CBRH7919 by stable transfection with a recombined proinsulin gene including a promoter composed of three copies of a stimulatory glucose responsive element (GLRE) from the rat liver pyruvate kinase (rLPK)gene. Using retroviral vector - mediated gene trans-duction ,we transferred pLXSN - (GLRE)3BP - 1 - MpINS2 and (pLXSN -( GLRE)3BP -1 - MpINS3 gene to CBRH7919 cells and made these cells secret mature human insulin. The use of retroviral vectors is attractive for this purpose because of transgene integration into the host genome and long term stable geneexpression. In addition, Retroviral vectors do not elicit a cytotoxic cellular immune response because no viral genes are contained in the vector.Taking non (3 cells as secreting insulin cells, The following two steps should be successful achieved : the first step is to have appropriate enzyme to produce proinsulin into mature,active insulin. Another is to have a sensitive regulated system which secreting insulin can be stimulated by glucose and inhibited by insulin itself. The first step has been successfully obtained. Using site - directed mutagenesis ,the human proinsulin gene has been modified by introduct-ing new cleavage sites at both the B - C and the C - A junctions, thereby generating suitable substrates for furin------an ubiquitously expressed endoprotease. Efficient processing of the mutated proinsulin in the non - β cells, such as hepota-ma cells. Most researches are based on the above theory of two mutagenesis. Yet we generate a punctual His - to - Asp mutation at position 10 in the B -chain. This has been done previously . In this study,Transferred the two kinds of recombined human proinsulin pLXSN - ( GLRE) 3 BP - 1 - MpINS3 and pLXSN - (GLRE)3BP - 1 - MpINS2 to CBRH7919 ,0ur results showed that it produced insulin nearly 23 times higher than the cells containing pLXSN - ( GL-RE)3BP - 1 - MpINS2. It is conformed with the literature.Another key step is the question of insulin regulated productin . To achieve it, we designed an three copies of a stimulatory glucose responsive element( GLRE ) from the rat liver pyruvate kinase ( rL - PK) gene inserted directly upstream of a promoter composed of insulin regulating system by an inhibitory insulin response element of the insulin - like growth factor binding protein - 1 (IG-FBP - 1) . Glucose - stimulated insulin production was achieved by ( GLRE ) 3 driven insulin expression in vector - transduced CBRH7919 cells. In this study ,The insulin production was shown to be glucose concentration dependent . The insulin levels under no glucose and 25mmol/L glucose conditions after transfec-tion 30 days were (3.57 ± 0.21)iu/L, (23.233 ±1.12)iu/L,respectively.In the current study, Expression of insulin by transfected cells is temporal, but our results indicated that CBRH7919 cells containing genetically modified human proinsulin could produce insulin not only temporal but also constant, under both conditions, The insulin levels increased in response to the increasing...