Basic Experimental Study Of Monitoring The Genetically Modified Stem Cells In The Treatment With Radionuclide Reporter Gene Imaging

PurposeConstructing recombinant eukaryotic plasmid expression vector pcDNA3.1-TK-IRES-BDNF and recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, to assess the multiplication capacity, differentiation potential, uptake of 131I-FIAU capacity of rat BMSCs transfected with recombinant vectors, finally selecting a appropriate vector for further monitoring the genetically modified BMSCs in vivoMethods1. Construct different vectors and culture rat BMSCs1.1. Construct and identify vector pcDNA3.1-TK-IRES-BDNFThe primers of HSV1-TK, IRES and BDNF were designed and sythesised according to Gene bank. Restriction enzyme sites were added into the both ends, which were matched with the multiple cloning sites (MCS) of eukaryotic expression plasmid vector pcDNA3.1+. The cDNA of target genes HSV1-TK and IRES were obtained from recombinant plasmid pcDNA3.1-TK, pIRES-EGFP by polymerase chain reaction (PCR) separately. The cDNA of target gene BDNF was obtained from a week-old SD rat brain tissue by RT-PCR technology. Three target genes were restricted with respective enzymes, and subcloned into the eukaryote plasmid pcDNA3.1+restricted with the same enzymes. Restriction analysis and sequencing were used to confirm the recombinant plasmid vector pcDNA3.1-TK-IRES-BDNF.1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFP The plasmid vector, pDC316-TK-IRES-BDNF-EGFP, and the virus vector, E1/E3-deleted replication-defective recombinant adenovirus type 5 (Ad5-TK-IRES-BDNF-EGFP), carrying the HSV1-TK-IRES-BDNF coupling gene under the transcriptional control of the cytomegalovirus (CMV) promoter, were constructed and purified in the Vector Gene Technology Company Ltd. in Shanghai.1.3. Culture rat BMSCsThe femur and tibia were isolated from health SD rat aged 4-6 weeks, the bone marrow were washed out with DMEM-F12 containg 15%FBS, and operated into the cultured bottles after density gradient centrifugation. BMSCs were cultivated and expanded in vitro. The cell morphology were observed under inverted microscope at different times; expression of surface antigens CD44 and CD34 were detected by immunocytochemistry.2. BMSCs were transfected with different recombinant vectors in vitro2.1. The transfection efficiency for BMSCs with different vectorsBMSCs were transfected with recombinant plasmid vector pcDNA3.1-EGFP, enhanced green fluorenscent protein (EGFP) expression were observed under fluorescence microscope, transfection efficiency was analyzed with flow cytometry (FCM). BMSCs were transfected with recombinant vector Ad5-TK-IRES-BDNF-EGFP at different multiplicity of infection (MOI), from 0,50,100,150,200 and 250, transfection efficiency was assayed by fluorescence microscope.2.2. The proliferation and differentiation of genetically moditified BMSCs with different vectorsAfter BMSCs were transfected with different vectors, morphology and density changes of genetically moditified BMSCs were observed under inverted microscope at different times. MTT were used to detect the proliferation, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were added into the genetically moditified BMSCs, the morphology and the potential of differentiation were observed under inverted microscope.2.3. The expression of target genes When BMSCs were transfected with plasmid vector pCDNA3.1-TK-IRES-BDNF, RT-PCR and Western blot analysis were used to detect the expression of target genes in BMSCs. Real-time quantitative polymerase chain reaction (RQ-PCR) and Western blot were used to detect the expression of target gene in BMSCs transfected with different MOI of recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP.3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors3.1. The radioiodination of FAUUsing solid phase oxidation with Iodogen, FAU was labeled with 131I. The product, 131I-FIAU, was purified on a reverse-phase Sep-Pak C-18 column and the labeling efficiency was then assayed. Moreover, the peak fractions were identified for the radiochemical purity assessment. To study the stability in vitro, the 131I-FIAU was incubated in serum at 37℃for 4,12 and 24 hours before the aliquots were removed for radiochemical purity analysis.3.2. Uptake of 131I-FIAU for transgenic BMSCs by gamma counterBMSCs were transfected with different vectors,48h later, the purified 131I-FIAU were added into the genetically modified BMSCs. The radiocounting of cell culture medium and digested cell were counted by gamma counter separately, uptake rate= C digested cell/(C digested cell+C cell culture medium)×100%.PartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in a certain period of time, the control group was pcDNA3.1-EGFP.PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed at the same time, control group was Ad5-EGFP.PartⅢ, the best MOI was obtained from the above-mentioned experiments, and then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP in the best MOI. Uptake of 131I-FIAU was observed in a certain period of time, the control group was Ad5-EGFP.4. Statistical methodsData were pcocessed by SPSS 11.0 software.Results1. Construct different vectors and culture rat BMSCs1.1. Construct and identify plasmid vector pcDNA3.1-TK-IRES-BDNFThe identification of recombinant eukaryotic expression plasmid vector pcDNA3.1-TK-IRES-BDNF by PCR, enzyme digestion and sequence analysis, and the result was matching with expectations.1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFPThe recombinant plasmid, pcDNA3.1-TK-IRES-BDNF, were identified by PCR,restriction enzyme digestion and sequencing. The titer of packaged recombinant adenovirus, Ad5-TK-IRES-BDNF-EGFP, was 2.0×1010 PFU/ml; A replication-defective adenovirus type 5 (Ad5-EGFP) was used as a control with the titer of 3.6×1010PFU/ml.1.3. Culture and identify rat BMSCsBMSCs can obtain high purity and good proliferation by gradient centrifugation. The surface antigen CD44 were observed in more than 95% of the BMSCs, while CD34 were no observed by immunohistochemistry.2. BMSCs were transfected with different recombinant vectors in vitro2.1. The transfection efficiency for BMSCs with different vectorsThere was high transfection efficiency for BMSCs transfected with recombinant plasmid pcDNA3.1-EGFP, when the ratio between liposome (μl) and the recombinant plasmid (μg) was 2.5:1. When the MOI was 150, transfection efficiency for BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP can reach peak, at the 48h, the transfection efficiency was more than 90%.2.2. The proliferation and differentiation of transgenic BMSCs with different vectorsBMSCs transfected with recombinant plasmid pcDNA3.1-TK-IRES-BDNF, the cell density were increased seriously and there have many floating cells, death cells were confirmed by placenta blue staining. BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately. Little floating cell were observed under inverted microscope, when the MOI was 250. There have little toxicity for BMSC transected with recombinant adenovirus by MTT assay. The cell survival rate was more than 98% when the MOI was 150.After BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, non-transfected recombinant adenovirus group was control, adding bFGF and EGF separately. The morphology change of genetically moditified BMSCs were observed under inverted microscope in a certain period of time, and the difference was no founded between adenovirus transfection group and non-transfected recombinant adenovirus group. Transgenic BMSCs remain fine ability of differentiation into neuron-like cells.2.3. The expression of target gene were detectedThe expression of TK and BDNF were detected with RT-PCR and Western blot separately, when BMSCs were transfected with recombinant eukaryotic expression vector pcDNA3.1-TK-IRES-BDNF,48h later. BMSCs were transfected with different MOI, the mRNA of TK and BDNF were deteted by RQ-PCR, expression of target gene upgrade with the increased MOI for a range. There was a strong relation between the mRNA expression of TK and BDNF for transfected BMSCs(r=0.973,p<0.05, n=3). The expression of target gene was detected by Western blot also.3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors 3.1. Radiolabeling and stabilityFAU could be labeled efficiently by solid phase oxidation with Iodogen. The radiolabeling efficiency of 131I-FIAU was (64.35±4.89)%(n=5). After purification on Sep-Pak C-18 column, the radiochemical purity of the final product 131I-FIAU was (98.62±0.62)%(n=5). The radiochemical purity of 131I-FIAU remained above 95% after incubated in serum at 37℃for 4,12 and 24 hours, which indicated 131I-FIAU was stable in serum.3.2. Uptake of 131I-FIAU for genetically modified BMSCs by gamma counterPartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in 5 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with time extension in 3 hours, there has significant increase compared with the control.PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed in 3 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with the MOI raising, uptake was reach peak at 3h point, when the MOI was 150.PartⅢ, the best MOI was obtained from the above-mentioned experiment, the best MOI was 150. Then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP at MOI was 150. Uptake of 131I-FIAU was observed in 5 hours, uptake of 131I-FIAU for genetically moditified BMSCs was increased with time extension in 3 hours. There has significance between genetically modified BMSCs and control BMSCs for uptake of131I-FIAU at all the time points(t=20.74-98.16, p<0.05, n=3).ConclusionRecombinant adenovirus can contain more bases, get higher transfection efficiency, lower toxic for BMSCs, and remain fine differentiation into neuron-like cells. There has a strong relation between the mRNA expression of HSV1-TK and BDNF in genetically modified BMSCs. The expressions of upstream reporter gene can indirect response to the downstream therapeutic gene by IRES. FAU could be labeled efficiently by solid phase oxidation with Iodogen; the high radiochemical purity for product can obtain by Sep-Pak-C-18 reversed-phase chromatography. BMSCs transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP can express HSV1-TK possessed fine activity, effective uptake of 131I-FIAU, there has significance between genetically modified BMSCs and control BMSCs for uptake of 131I-FIAU at all the time points. This study indicates that recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP may be a suitable gene vector for research tracing genetically modified stem cells.