| Background and purposeNervous system diseases and trauma seriously affect the health and life quality of human, the efficacy of current therapies is little, the diseased (or damaged) nerve cells and nerve fiber can neither regenerate nor recover functions. Stem cells have capabilities of self-renewing and differentiating to a variety of somatic cells. Stem cell transplantation in the treatment of neurological diseases and injuries, most likely to cure such disorders from the pathology and basic pathogenesis, bring patients with new hope of rehabilitation and recovery.Adult stem cells which commonly used in research on stem cell transplantation therapy are neural stem cells and mesenchymal stem cells. Considering cells source and related clinical value issues such as security and ethics, adult mesenchymal stem cells, particularly adipose-derived mesenchymal stem cells and perinatal mesenchymal stem cells have great potential for future development.There are a variety of neurons and glial cells that have different subtypes and functions,which constitute a complex network in nervous system.As far as a certain neurological diseases, in most cases only one particular type of neural cells occurred lesions, transplantation therapy to replace the targeted diseased cells is needed. In vitro under specific conditions, MSCs can be induced to differentiate into neural cells (neurons and glia). With the rise of gene therapy, in order to import therapeutic gene into MSCs for transplantation, targeted modification of one particular type of neural cells is needed. Thus,precisely orientation for a particular type of cell is very necessary among mesenchymal stem cell transplantation and gene therapy for neurological disease. Non-tissue(cell)-specific viral promoters (E.G. human cytomegalovirus immediate early promoter, CMV promoter) driving expression of target gene in all types of cells will produce great side effects,and gene expression will gradual decay or even disappear. Neural cell-specific expressed proteins commonly include synapsin ? (SYN1, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), nestin , neuron-specific enolase (NSE) and so on. In short, in order to monitor neural differentiation status of MSCs and avoid side effects of gene therapy, it is need to use tissue (cell)-specific promoters to modulate target gene expression in specific cell types.Ferritin is an intracellular iron-storage protein having ferroxidase activity,which is developed as for MRI endogenous imaging molecule with high detection sensitivity. Ferritin is an inherent and endogenous protein in human body without toxic and immunogenic, overexpression has no effect on cell growth. Ferritin increases cellular iron uptake and intracellular iron content makes redistribution of intracellular iron and extracellular iron, leads to inhomogeneity of magnetic field intensity inside and outside the cells, brings about shortened T2 relaxation time of tissue, and the reduction extent of T2 values is positive linear correlation with ferritin expression, T2 relaxation time is positively correlated with the brightness of MRI images. MRI is a noninvasive imaging technology; MRI imaging techniques combined with contrast function of ferritin can be used to continuous, real-time dynamic tracking of stem cells transplanted into the body.As far as donor and recipient are concerned, there are individual differences of MSCs. In order to achieve reliable and satisfactory therapeutic effect,it is necessary to accurately monitor and track MSCs transplanted into recipient body. MSCs have important clinical significance in the repair and regeneration of nervous system, but the status of distribution, migration, growth and differentiation of MSCs after transplantation in vivo is not clear, which limited MSCs clinical application, and the molecular mechanisms of MSCs to repair lesion and damage of nervous system also needs further research. There are quite a few literatures in the past reported that ferritin as endogenous MRI imaging molecule was used to track migration of stem cells in vivo, but the related research that using neural cell-specific promoter regulated the expression of ferritin to monitor the status of growth and differentiation of MSCs in the state have not been reported.The purpose of the research is to monitor hADMSCs neural differentiation status in vitro by marking hADMSCs using combined technology of the neural cell-specific promoters and ferritin as MRI endogenous marker and detecting of cell type specific ferritin expression regulated by neural cell-specific promoter and is aimed to provide a theoretical basis for further experiment of monitoring biological security and differentiation status of hADMSCs in vivo.Methods1 Obtain target gene template:Total RNA and genomic DNA as templates for coding sequence of human ferritin heavy chain (hFTH1) and GFAP, MBP promoter sequence were extracted from Cultured K562 cells and U251 cells respectively, primers were designed to amplify these sequences by PCR. Purified PCR products were ligated to T vector,transformed into E. coli strain DH5a, screening cultured through ampicillin resistance, extracted plasmids through miniprep, initial screening through colony PCR, sequenced. The sequencing results were verified by aligning with sequence published in NCBI through BLAST.2 Construct recombinant expression vectors:2.1 Amplify target gene sequence to introduce the relevant restriction sites:Using T vector plasmids with correct sequencing results as template, amplified hFTHl sequence by PCR method,introduced Mlu ? and Cla ? restriction sites at 5'end and 3' end respectively; Amplified hGFAP promoter sequence and mutated the 33rd Nucleotide A in the 3' end to Nucleotide T by PCR method, introduced EcoR ?and Mlu ? restriction sites at 5' end and 3' end respectively; Amplified hMBP promoter sequence by PCR method, introduced EcoR ? and Mlu ? restriction sites at 5' end and 3' end respectively. Using the plasmids containing the CMV or hSYN1 promoter sequence as templates, amplified CMV and hSYNl promoter sequence respectively, introduced EcoR ? and Mlu ? restriction sites at 5' end and 3' end respectively.2.2 Construct recombinant expression vectors pLVTHM-H1-hFTH1 (semi-finished product):pLVTHM vector and hFTH1 PCR products were double digested with the restriction enzymes Mlu ? and Cla ?, so pLVTHM vector plasmid was linearized.Large fragment digestion products of the former and digestion products of the latter were ligated to gain pLVTHM-H1-hFTH1 vector.2.3 Construct recombinant expression vectors (finished product):pLVTHM-H1-hFTH1 vector was double digested with the restriction enzymes EcoR ? and Mlu ? and the large fragment digestion products was retrieved.As restriction site described in paragraph 2.1,the PCR products of 4 promoter sequences were double digested with the corresponding restriction enzymes respectively. Large fragment digestion products of the former and digestion products of the latter were ligated, i.e. H1 promoter sequence was substituted for 4 promoter sequence aforementioned respectively, to gain 4 recombinant expression vectors,one is constitutively (no tissue cell-specific) ferritin expression vector, named pLVTHM-CMV-hFTH1; the other three are neural cell-specific ferritin expression vector, named pLVTHM-hS YN1 -hFTH1,pLVTHM-GFAP-hFTH1,pLVTHM-MBP-hFTH1 respectively, transformed into E. coli strain DH5a,screening cultured through ampicillin resistance, extracted plasmids through miniprep, initial screening through EcoR ? and Mlu ? double digestion, sequenced.The sequencing results were verified by aligning with sequence published in NCBI through BLAST.3 extract plasmids through midiprep:pLVTHM vectors (empty vector control), 4 recombinant expression vectors plasmids with correct sequencing results, psPAX2 (packaging plasmid) and pMD2.G(envelope plasmid), 7 plasmids (strains) in total, were cultured to amplify, extract plasmids through midiprep.4 Package lentivirus by transfecting HEK-293T cells through DNA-calcium phosphate co-precipitation methodpLVTHM and 4 recombinant expression vectors aforementioned respectively along with psPAX2 and pMD2.G vector in accordance with an appropriate proportion transiently co-transfected HEK-293T cells to package lentivirus,concentrated by ultrafiltration, determined the lentivirus titer by infecting HEK-293T cells with serial gradient dilutions of lentivirus and observing percent of GFP positive cells, obtained 5 lentiviral, which is named as same as the corresponding recombinant expression vector.5 Construct lentivirus stable infected hADMSCs cell lineshADMSCs was infected with 5 lentivirus at suitable MOI respectively,pr otamine sulfate as co-transfection agent, observed the percent of GFP positive cells,make sure that the infection rate not less than 70%,if necessary, adju sted and optimized the infection method or screened GFP-positive cells by G FP-tagged fluorescence-activated cell sorting technology. Names and number o f each group hADMSCs was described as below: 1 hADMSCs; 2 hADMSCs-pLVTHM; 3 hADMSCs-pLVTHM-CMVpromoter-hFTHl; 4 hADMSCs- pLVTHM-hSYN1promoter-hFTH1; 5 hADMSCs-pLVTHM-hGFAPpromoter-hFTHl; 6 hADMS Cs-pLVTHM-hMBPpromoter-hFTHl; 7 hADMSCs-pLVTHM-hSYNlpromoter-h-FTH1-induced to neuron; 8 hADMSCs-pLVTHM-hGFAPpromoter-hFTH1-induced to ast rocyte; 9 hADMSCs-pLVTHM-hMBPpromoter-hFTHl-induced to oligodendrocyte.hADMSCs of group 4, 5, 6 were infected with lentivirus expressing ferritin under the regulation of neurons, astrocytes, oligodendrocytes specific promoter respectively, and then were induced to the corresponding cell types, so hAD MSCs of group 7, 8, 9 were obtained. 72h prior to the detection of each gro up hADMSCs ferritin expression, intracellular iron content and MRI imaging in vitro, ferric ammonium citrate was added in the medium to the final conc entration of 200?M, to provide hADMSCs with iron source to synthesize ferri tin.6 Effect of lentivirus infection on hADMSCs proliferation detected by CCK-8 methodThe control group hADMSCs and 5 lentivirus stable infected groups hADMSCs (i.e. the first 6 groups as described in the paragraph methods 5) were seeded with appropriate density on 96-well plate and cultured, OD450 of each hADMSCs group were determined by CCK-8 assay at the time points of 24h, 48h,72h and 96h after vaccination, wells for each hADMSCs group at each time point were in triplicates.7 Induce HADMSCs differentiated to neurons, astrocytes and oligodendrocytes7.1 Induce HADMSCs differentiated to neuronshADMSCs was pre-induced with Neurobasal medium containing 1.0mM?-mercaptoethanol (?-ME), 10ng/ml basic fibroblast growth factor (bFGF) for 24h;then induced with Neurobasal medium containing 3mM ?-ME, 10% FBS for 48h.7.2 Induce HADMSCs differentiated to astrocytehADMSCs was pre-induced with DMEM (high glucose) medium containing 20ng/ml epidermal growth factor (EGF), 20ng/ml bFGF, 100U/ml penicillin,100?g/ml streptomycin, 2mM L-glutamine, 1% N2 supplement for 3d; then induced with DMEM (high glucose) containing 1mM N6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (dbcAMP), 0.5mM 3-Isobutyl-l-methylxanthine (IBMX), 5ng/ml human platelet derived growth factor(hPDGF), 50ng/ml human neuregulin 1-?1 (human neuregulin 1-betal/HRG1-betal EGF Domain), 20ng/ml hbFGF, 100U/ml penicillin, 100?g/ml streptomycin, 2mM L-glutamine for 3d.7.3 Induce HADMSCs differentiated to oligodendrocyteshADMSCs was pre-induced with Neurobasal A medium containing 20ng/ml bFGF, 20ng/ml EGF, 1% N2 supplement, 2mM L-glutamine for 48h; then induced with Neurobasal A medium containing 500ng/ml IGF, 50ng/ml NT-3, 5ng/ml PDGF,1% N2 supplement for 3d.7.4 detect neural cell specific markers by ImmunofluorescenceInduced hADMSCs Were detected expression of neural cell specific markers by immunofluorescence, (nestin, NSE for neuron; GFAP for astrocytes; MBP for oligodendrocytes), to determine whether the hADMSCs was successfully induced differentiation towards neural cells.8 detect ferritin expression of each group hADMSCs by immunofluorescence9 detect hFTH1 mRNA expression of each group hADMSCs by qRT-PCR10 detect ferritin expression of each group hADMSCs by western blot11 Qualitatively observe intracellular iron content and distribution of each group hADMSCs by Prussian blue staining12 quantitative detection of intracellular iron content of each group hADMSCs by ICP-MSEach group hADMSCs (approximately 1×106 cells per group) was digested to prepared sample solution, and the iron content was detected and then divided by the amount of cells to obtain the iron content within single cell of each group hADMSCs.13 Each group hADMSCs MRI imaging in vitroEach group hADMSCs (1×106 cells per group) were embedded in 1%agarose-PBS gel, to detect T2 of each group hADMSCs by field strength 3.0T MRI imaging.14 statistical analysisQuantitative experimental results was analyzed using SPSS 13.0 statistical software, the mean and standard in each group (each condition) of the measurement data was calculated, expressed as (x±s). Result of method 6 was analyzed by factorial analysis of variance (two-factor, group and incubation time), the main and two-factor interactions effects was analyzed, mean differences between the various levels of the same factors were compared by one way analysis of variance (one way ANOVA), if variances homogeneity, F value was adopted, if variances heterogeneity,Welch value was adopted; Multiple comparison test, if variances homogeneity, using LSD method, if variances heterogeneity, using Dunnett's T3 method. Mean differences between groups of the results of method 9, 10, 12, 13 were compared using one-way ANOVA. The correlation between the four experiments results were analyzed by bivariate correlation analysis (Spearman correlation coefficient)separately. Significant levels are all P<0.05, represents that mean difference between groups compared was statistically significant.Results1 Construct recombinant expression vectorsThe BLAST results of sequencing results of 4 constructed recombinant expression vectors aligning with sequences published in NCBI and actual designed was verified correct.2. Package lentivirus5 lentivirus were packaged and concentrated, titers is in the range of 2×108-6×108.3 Construct lentivirus stable infected hADMSCs cell lines5 lentivirus stable infected hADMSCs cell lines was constructed, the percent of GFP positive cells were in the range of 60% -70%.4 Effect of lentivirus infection on hADMSCs proliferation detected by CCK-8 methodOD450 mean differences between groups (the control group hADMSCs and 5 lentivirus stable infected groups hADMSCs, i.e. the first 6 groups) at the same time point assayed by CCK-8 were not statistically significant (main effect F=2.622,P=0.036; 24h F=2.062, P=0.041; 48h F=2.184, P=0.124; 72h F = 0.158, P=0.973;96h F=0.607, P=0.697); OD450 mean differences of between time points in each group hADMSCs were statistically significant, OD450 gradually increased with the passage of culture time (main effect F=226.620, P<0.001, group 1, F=554.972,P<0.001; group 2, F=2049.169,P<0.001; group 3, F=585.452,P<0.001; group 4,F=633.111, P<0.001; group 5, F=524.362, P<0.001; group 6, F=1525.607,P<0.001). There was no interaction effect between the two factors of group and culture time (F=1.054, P<0.421), the growth curve showed typical logarithmic growth phase and plateau phase, the growth curve showed typical logarithmic growth phase and plateau .phase, indicated lentivirus infections had no effect on hADMSCs proliferation.5 Induce HADMSCs differentiated to neurons, astrocytes and oligodendrocytesnestin red fluorescence varied from strong positive to weak positive, NSE red fluorescence varied from the weak positive to strong positive, indicated that hADMSCs was successfully induced differentiation to neuron; GFAP red fluorescence varied from weak positive to strong positive, indicated that hADMSCs was successfully induced differentiation to astrocytes; MBP red fluorescence varied from weak positive to strong positive,indicated that hADMSCs was successfully induced differentiation to oligodendrocyte;6 detect ferritin expression of each group hADMSCs by immunofluorescenceFerritin red fluorescence were observed in each group hADMSCs, indicated that ferritin expressed in all hADMSCs groups, groups 1 and 2 weakly expressed,expression increased in group 3 compared with the previous two groups; expression increased in group 4, 5, 6 compared with group 1, but were weaker than group 3;expression significantly increased in group 7, 8, 9 compared with group 4, 5, 6 group respectively.7 detect hFTH1 mRNA expression of each group hADMSCs by qRT-PCRhFTHl mRNA 2-??Ct mean of each group hADMSCs were 1.000±0.000,1.076±0.026, 1.549±0.314, 1.452±0.316, 1.280±0.164, 1.199±0.104, 5.997±0.445,7.130±0.683,5.744±0.369 respectively,Mean differences between groups were statistically significant (F= 69.029, P<0.001). Expression significantly increased in groups 7, 8, 9 compared with groups 4, 5, 6 respectively,indicated that the process of inducted differentiation activated and enhanced the functions of corresponding neural cell-specific promoters, so hFTH1 gene transcription levels were significantly increased.8 detect ferritin expression of each group hADMSCs by western blotFerritin expression semi-quantitative levels mean of each group hADMSCs were 0.400±0.003,0.460±0.011, 0.676±0.012,0.541±0.022,0.503±0.007,0.606±0.010, 1.224±0.002,1.250±0.018,1.032±0.014 respectively, Mean differences between groups were statistically significant (F=2150.319, P<0.001).Expression significantly increased in groups 7, 8, 9 compared with groups 4, 5, 6 respectively, indicated that the process of inducted differentiation activated and enhanced the functions of corresponding neural cell-specific promoters, so that ferritin expression level were significantly increased.9 Qualitatively observe intracellular iron content and distribution of each group hADMSCs by Prussian blue stainingBlue granules were seen in all hADMSCs groups, indicated that ferritin expressed, uptaked and bound extracellular iron. A few scattered intracellular blue granules was seen in groups 1,2; the amount of blue granules in group 3 increased significantly compared with the previous two groups; the amount of blue granules in groups 4,5,6 increased compared with group 1, but was less than group 3; the amount of blue granules in groups 7,8,9 increased significantly compared with groups 4,5,6, gathered into a piece distribution.10 quantitative detection of intracellular iron content of each group hADMSCs by ICP-MSIntracellular iron content (pg/cell) mean of each group hADMSCs were 48.861±0.297,58.583±0.789,126.264±0.653,145.771±0.634,58.267±0.702,67.556±0.416, 468.441±0.572, 668.652 ± 0.739, 444.308±0.527 respectively,mean differences between groups were statistically significant (F=433247.6, P<0.001).Intracellular iron content significantly increased in groups 7, 8, 9 compared with groups 4, 5, 6 respectively, indicated that the process of inducted differentiation activated and enhanced the functions of corresponding neural cell-specific promoters, hFTH1 gene transcription levels were significantly increased, thus made the translation levels and synthesis of ferritin significantly increased,uptaked and bound a large amount of extracellular iron,so that the Intracellular iron content greatly increased.11 determine T2 value of each group hADMSCs by MRI in vitroT2 values (ms) mean of each group hADMSCs were 212.75±33.00,173.25±10.81,133.75±30.14,123.25±10.72,101.25±14.66,174.00±23.24,35.25±3.30,27.50±4.12,28.25±11.41 respectively,mean differences between groups were statistically significant (F=55.147,P<0.001). T2 values significantly decreased in groups 7, 8, 9 compared with groups 4, 5, 6 respectively, indicated that the process of inducted differentiation activated and enhanced the functions of corresponding neural cell-specific promoters, thus made the translation levels and synthesis of ferritin significantly increased, the intracellular iron content greatly increased, resulted in inhomogeneity of magnetic field strength between extracellular and intracellular, so that T2 significantly reduced.12 bivariate correlation analysisSpearman correlation coefficient between the results of hADMSCs hFTHl mRNA expression and ferritin expression, intracellular iron content, T2 were 0.950(P<0.001), 0.933 (P<0.001), -0.900 (P=0.001) respectively; Spearman correlation coefficient between the results of ferritin expression and intracellular iron content,were 0.933 (P<0.001), -0.783 (P=0.013) respectively; Spearman correlation coefficient between the results of intracellular iron content and T2 were -0.800(P=0.010), indicated that statistically significant correlation between the 4 results is present (T2 results showed a negative correlation with other three results), the 4 experimental results can be cross-validated with each other.Conclusions and innovation:Neural cells-specific promoter can regulate the relatively specific expression of ferritin as endogenous MRI marker molecule in hADMSCs, which will link the neural cell differentiation status of hADMSCs in vitro to MRI; So MRI can be used to monitor the status of neural cell differentiation status of hADMSCs. |
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