Study On Exogenous Gene PDX-1 Inducing Differentiation Of The Bone Marrow-derived Nestin～+ Cells Into Insulin Producing Cells

Islet cells replacement is considered as the optimal treatment for typeâ… and parts of type 2 diabetes.In recent years some diabetes' patients received islet celltransplantation through hepatic portal vein.However,the availability of human pancreas islets for transplantation is limited,and immune rejection response is another difficulty for the transplantation treatment.In recent years,attention has been focused on the possibility of gene or cell therapy of diabetes mellitus using artificially prepared non-β-cell-derivedβ-cells.Stem cells,which have self-renewal and pluripotecy,appeal to people for developing new source of islet cell replacement. Cells derived from bone marrow can be cultured and expanded in vitro and retain their ability to differentiate along these multiple mesenchymal pathways.If bone marrow stromal cells(BMSCs)could form new beta cells,they would become a particularly useful target for therapies that aim at beta-cell replacement in diabetic patients,because they are abundantly available in the human bone marrow.In addition,the ability to genetically manipulate these BMSCs,by direct gene transfer, would further enable the selective enhancement of specific differentiation pathways. Thus,differentiation gene transfer techniques are key to the use of BMSCs in tissue engineering protocols. PDX-1,as a important transcription factor in pancreas development, directs the normal development of pancreas and can regulate a number of genes involved in maintaining beta-cell identity and function.These include insulin, GLUT-2,GK and islet amyloid polypeptide.Also regulates gene expression in islet delta-cell and in the developing brain.The ability PDX-1 to activate gene transcription in a tissue specific manner is dependent on its capacity to example,in interact with other transcription factors.Some studies have highlighted the potential usefulness of PDX-1 as a reprogramming factor of non-β-cells towardβ-cell-like cells that can be used in diabetes cell/gene therapy.GLP-1 stimulates insulin secretion and proliferation by islet cells in vitro and in vivo,associated with an activation of pdx-1 function.In addition,it is well-known that glucose stimulates insulin production and secretion in normal beta-cells via the activation ofpdx-1.Study showed vitro high glucose culture is critical for promoting WB-1 cells to undergo further differentiation into functional beta-like IPCs.Another study showed that high glucose concentrations stimulate an increase in pdx-1 transcriptional activity compared to low glucose concentrations in the pancreatic beta-cell line Min6.The current study has established the fact that glucose,GLP-1 and insulin etc positively regulate the pdx-1 gene promoter in pancreatic beta-cells. When the pdx-1 promoter was stimulated with GLP-1 at high glucose concentrations, there was a further increase in transcriptional activity,indicating that glucose and GLP-1 have an additive effect on pdx-1 promoter activity indicating that GLP-1 and glucose may signal through different mechanisms.The effects of GLP-1 and glucose on pancreatic differentiation have been reported in several cell lines such as AR42J, ARIP,Panc-1,IMPE,Capan-1,MIN6 and WB-1.These studies emphasized the effect of GLP-1 and glucose regulation controlling pdx-1 expression.The effect of GLP-1 and glucose on the bone marrow-derived stem cells is not clear when they are treated in conjunction with the gene transfer of pdx-1.In the current study,we investigated effect of the GLP-1 and glucose regulation of insulin secreting in conjunction with pdx-1 gene in the bone marrow-derived stem cells.The results are summaried as follows:Partâ… :Construction of eukaryotie expressing vector pEGFP-PDX-1.ObjectiveTo study the role of PDX-1,the key gene to regulate the development of pancreatic endocrine cells,during the differentiation of bone marrow-derived stem cells into insulin-producing cells,we try to construct a eukaryotic expressing vector carrying PDX-1Methods1.Construction of eukaryotic expressing vector pEGFP-C2-PDX-1.All experiment methods are performed according to the standard protocols of molecular clone technology.1.1 Polymerase chain reaction(PCR)was used to proliferate PDX-1 cDNA from SK900/BLSCRIPT plasmid,producing an 858bp band with Hindâ…¢,Primer:F:5'-CGC AAG CTT CATG AAT AGT GAG GAG CAG T-3'; R:5'-GCG CGA TCC C TCA CCG GGG TTC CT-3'.1.2 Cloned PDX-1 cDNA into the MCS of pEGFP-C2 to get a recombined eukaryotic expressing plasmid pEGFP-C2-PDX-1.1.3 The recombinant vector was identified by enzyme digestion analysis and sequencing.2.Plasmid DNA Purification All plasmid DNA samples used in transfection were isolated and purified by EndoFree Plsmid Kit(Tiangen)following the manufacturer's protocols.Quality of the purified plasmid DNA preparations was assessed by enzymatic restriction amalysis and spectrophotometrically on the basis of A260 to A280.Results1.Enzyme digestion analysis showed that interesting gene had been integrated into recombinant vector.2.Sequencing analysids the recombined plasmid has the same open reading frame as pEGFP and perfect potential to translate。3.Plasmid DNA purification.A260/A280＞1.8 show plasmid DNA was purification.ConclusionPDX-1 was cloned into the C terminus of PEGFP-C2.Under the control of enhancer and promotor of pEGFP-C2,PDX-1 would be expressed as fusions with pEGFP-C2. We constructed successfully an eukaryotic expressing vector pEGFP-PDX-1.Partâ…¡Optimization Protocol and expressing of Gene PDX-1 Transfer into Rat Bone Marrow Stromal Cell by Nucleofection^TM.ObjectiveTo investigate the optimization protocol of a new transfection method for rat MSCs termed the Nucleofector^TMtechnology method,developed by amaxa Biosystems so as to provide a new method of high-efficiency transfection of rat bone marrow-derived cells in vitro.Methods1.Culture of bone marrow-derived stem cells.1.1 Isolation and culture bone marrow stromal cellsBone marrow(BM)was obtained from the long bones of 3-4 week old SD rats.Rat bone marrow-derived stromal cells were isolated by combining gradient density centrifugation with plastic adherence and cultured(37℃,5%CO₂)in 25cm² culture flask L-DMEM with 10%FBS;One week later,adherent cells gaining 80% confluence were passaged by pipetting with the use of trypsin. 1.2 Isolation and culture bone marrow-derived NSCs.Bone marrow(BM)was obtained from the long bones of 3-4 week old SD rats. Gentle pipetting resulted in the generation of a single-cell suspension.There cells were cultured(37℃,5%CO2)in 25cm² culture flask with NSCs culture medium containing 10%FBS,48h later,total medium in the culture were changed included removal the floating cells.The cells have been cultured for 7-10 days.2.Characterization of the cultured cells2.1 Cell morphology:under the phase-contrast microscope.2.2 Polymerase chain reaction for nestin.2.3 Nestin immunocytochemical3.Transfection protocol3.1 All reagents were prepared for Nucleofector transfection as described by amaxa Biosystems.The plasmid pEGFP-C2 5μg was transferred into MSCs or nestin⁺ cells.Electroporation was performed using either Program A33 or A31.Immediately after electroporation,the cells were transferred to six-well plates containing L-DMEM supplemented with 10%FBS.Cells were cultured and observed at 24h posttransfection.The cell expressed GFP can be observed by fluorescence microscope.3.2 The plasmid pEGFP-C2 5μg was transferred into nestin⁺ cells;Electroporation was performed using Program A33.Immediately after electroporation,the cells were transferred to six-well plates containing L-DMEM supplemented with 10% FBS or 20%FBS.Cells were cultured and observed at 24 h posttransfection.3.3 The plasmid pEGFP-PDX-1 5μg,15μg and 30μg was transferred into nestin⁺ cells respectively;Electroporation was performed using Program A33. Immediately after electroporation,the cells were transferred to six-well plates containing L-DMEM supplemented with 20%FBS.Cells were cultured and observed at 24h posttransfection.4.EGFP-positive fluorescent cells were counted to measure the transfection rate 24 hours after transfection by fluorescence microscope.5.Cell viability was determined on the basis of trypan blue dye exclusion.6.Analyze transfection rate 24hours after transfection by FACS.7.Polymerase chain reaction for PDX-1.8.PDX-1 immunofluorescence9.PDX-1 Westem blot.10.Statistical analyses were performed using an independent sample t test.A value of P＜0.05 was considered significant.Results1.Culture of BMSCsMononuclearcell were obtained by gradient density centrifugation and viability＞99%.Cells were plated at density of 2-3 x 10⁶.One week later,adhere cells gaining 80%confluence were passaged by pipeting with the use of trypsin. Following three to four passages,the cells become morphologically homogeneous, with a slim-spindle appearance.2.Culture of bone marrow-derived NSCs.The single-cells from bone man'ow were nucleolus cells mixed with a small number of red blood cells.At 48 hours,the adherent cells proliferated and filled the plate bottom one week later.If cells were cultured with the existence of adherent cells,big and round cells with plasmic granules were found sprouting at 24 hours with plasma granules at the sprouting end disappeared.After that, sprouting end gradually extended and such cells became increased and clustered, which then formed island cell clones that could be induced to differentiate into cells of star shapes cells at 8-10^thdays.3.P4 MSCs unexpressed nestin gene by RT-PCR;At day 6 of culture,NSCs expressed nestin gene by RT-PCR.4.P4 MSCs were nestin negative at the immunocytochemical;At day 6 of culture, NSCs were nestin positive at the immunocytochemical.5.Transfection protocol5.1 EGFP-positive fluorescent cells were counted to measure the transfection rate 24h after transfection by fluorescence microscope.Statistical analyses were performed using t test'(One-way ANOVA)LSD.(F=90.93,P=0.000).The transformed nestin⁺ cells gained higher transfection efficiency than transformed MSCs by both A33(30.56±3.29/3.62±0.95,P＜0.05)and A31(15.1±5.07/2.22±0.64, P＜0.05);The difference between nestin⁺ cells and MSCs is statistically significant. Higher efficiency could be gained by A33 program than A31 (30.56±3.29/15.1±5.07).The difference between program A33and A31 is statistically significant.5.2 We transfected nestin⁺ cells with 5μg EGFP-C2 plasmid by program A33 followed by inoculating in 10%L-DMEM or 20%L-DMEM.Statistical analyses were performed using independent sample t test(Transfection rate:F=0.226, P=0.647;Viablity:F=0.589,P=0.465).Higher transfection efficiency can be gained when nestin⁺ cells inoculated in L-DMEM with 20%FBS than 10%FBS. (38.98±4.0/30.56±3.29,P＜0.05)The difference between 10%and 20%is statistically significant;The cell in medium with 20%FBS obtained higher viability than 10%.(89.18±4.25%/67.04±3.60%,P＜0.05).The difference between 10%and 20%is statistically significant.5.3 The plasmid pEGFP-PDX-1 5μg,15μg and 30μg was transferred into nestin⁺ cells respectively;Electroporation was performed using Program A33. Immediately after electroporation,the cells were transferred to six-well plates containing L-DMEM supplemented with 20%FBS.Transfection efficiency was 33.3%,45.4%及78.6%respectively.6.At day 7 after transferred,nestin⁺ cells expressed PDX-1 gene by RT-PCR.7.At day 7 after transferred,NSCs werePDX-1 positive at the immunofluorescence.8.A single band at 46kDa of the PDX-1 protein was detected in transferred nestin⁺ cells cells,but not in the parental nestin+ cells cells by Western blot.ConclusionWe have enhanced the efficiency of transfection by optimizing the transformation conditions.The expression of PDX-1 gene in the transformed cells was detected by RT-PCR,immunofluorescence and Western blot.It is possible to use the bone marrow-derived nestin⁺ cells as seed cells in tissue-engineering.Partâ…¢Effect of PDX-1 gene,GLP-1 and GLU inducing nestin⁺ cells differentiation into IPCsObjectiveTo induce transferred PDX-1 gene nestin⁺ cells to differentiate into insulin producing cells(IPCs)with GLP-1,GLU and GLP-1+GLU in vitro.To investigate the role and interrelation on GLU,GLP-1 and transcription factor PDX-1 induce bone marrow-derived nestin⁺ cells differentiation and mature into functional IPCs.Methods1.The experimental cells were divided in five groups:A:The nestin⁺ cells transferred with PDX-1 were plated in L-DMEM supplement with no cell factor;B:The nestin⁺ cells transferred with PDX-1 were plated in L-DMEM supplement with GLP-1(100nM); C:The nestin⁺ cells transferred with PDX-1 were plated in L-DMEM supplement with GLU(25mM);D:The nestin⁺ cells transferred with PDX-1 were plated in L-DMEM supplement with GLP- 1(100nM)and GLU(25mM);E:The nestin⁺ cells untransferred were plated in L-DMEM supplement with GLP- 1(100nM)and GLU(25mM);2.Cell morphology:under the phase-contrast microscope.3.The expressions of Isl1,PDX- 1 mRNA were detected by RT-PCR assay on the 7^th day after induction.4.The expressions of PDX-1 was detected by immunofluoreseence and Westernblot on the 7^thday after induction.Each tested with positive control,negative control and blank control.5.The expressions of insulin was detected by immunofluorescence and immunocytochemisty stain.Each tested with positive control,negative control and blank control.6.Enzyme-linked immunosorbent assayInsulin and Copeptide release of pre- and post glucose-stimulating were detected by using an ultrasensitive rat insulin and C-peptide enzyme-linked immunosorbent assay(ELISA)kit(DSL,USA)following the manufacturer's protocols.Serum-free culture medium containing 0.5%BSA was used as a medium control.7.Statistical analyses were performed using One-way ANOVA test.A value of P＜0.05 was considered significant.Result:1.Inverted optical microscopic observation indicated that the inoculated cwlls were slim-spindle shape at 24hour.The proliferation ability of transformed cells was strong and had large nucleus with scant cytoplasm.The cells changed into round with some protrusions.At 6-7^thday,the cells of group B,C,D formed some clusters. The cells of group A and E didn't.2.The expressions of pdx-1 mRNA were both positive detected by RT-PCR on the 7^th day after induction in each group except group E.The expressions of Isll mRNA were both positive detected by RT-PCR on the 7^th day after induction in each group except group A and E.3.The expressions of PDX-1 was positive on the 7th day after induction by immunofluorescence in experiment groups.The expressions of PDX-1 was negative on the 7th day after induction by immunofluorescence in group E.Westernblot for PDX-1 in each was compatible to the results of immunofluorescence detection.4.The expressions of insulin was positive on the 7th day after induction by immunofluorescence in each groups except group A and E;Immunocytochemistry for insulin in nestin⁺ cells cells was compatible to the results of immunofluorescence detection.5.Enzyme-linked immunosorbent assay.Statistical analyses were performed using t test'(One-way ANOVA)LSD.Insulin(pg/ml)and C-peptide(ng/ml)level in PDX-1 transfected nestin cells before glucose-stimulating. Group A:141.99±27.46/0.08±0.02;Group B:331.50±38.96/0.35±0.01;Group C:340.25±46.42/0.33±0.06;Group D:346.50±27.96/0.33±0.04。Medium control:121.93±14.74/0.05±0.03Compared of C-peptide variable groups(F=69.56,p=0.000):The difference between group A and medium control is no statistically significant(P＞0.05); There were significant difference C-peptide level in the group A compared with that of the group B,C,D respectively(P=0.000);There were significant C-peptide level in the group B,C,D compared with medium control(P=0.000); There were no significant difference when C-peptide level in the group B,C,D compared with each other(P＞0.05).Compared of insulin variable groups(F=48.09, P=0.000):The difference between group A and medium control is no statistically significant(p＞0.05);There were significant difference insulin level in the group A compared with that of the group B,C,D respectively(P=0.000);There were significant difference when insulin level in the group B,C,D compared with medium control(P=0.000);There were no significant difference when insulin level in the group B,C,D compared with each other(P＞0.05)Insulin(pg/ml)and C-peptide(ng/ml)level in PDX-1 transfected nestin⁺ cells after glucose-stimulating.Group A:152.00±30.17/0.06±0.01;Group B:332.00±44.02/0.37±0.02;Group C:337.75±81.81/0.37±0.09;Group D:377.75±44.51/0.40±0.09。Medium control:116.50-a:17.74/0.06+0.03.Compared of C-peptide variable groups(F=35.19,P =0.000):The difference between group A and medium control is no statistically significant(P＞0.05);There were significant difference C-peptide level in the group A compared with that of the group B,C,D respectively(P=0.000);There were significant difference when C-peptide level in the group B,C,D compared with medium control(P=0.000);There were no significant difference when C-peptide level in the group B,C,D compared with each other(P＞0.05).Compared of insulin variable groups(F=24.20,P=0.000):The difference between group A and medium control is no statistically significant(P＞0.05);There were significant difference insulin level in the group A compared with that of the group B,C,D respectively(P=0.000);There were significant difference when insulin level in the group B,C,D compared with medium control(P=0.000);There were no significant difference when insulin level in the group B,C,D compared with each other(P>0.05)ConclusionBone marrow-derived nestin⁺ cells can be converted into insulin-producing cells by GLP-1/glucose in conjunction with Nucleofector mediated gene transfer of PDX-1. The results suggest that GLP-1 and glucose may be another important determiner of nestin⁺ cells differentiation as is PDX-1.But the combination of GLP-1 and glucose had no additive effect on insulin release.