The Association Study Of Binding Activity Of IRPs/IREs And Iron Concentration On Rat ADSCs Labeling With SPIO

BackgroundWith the development of the experimental medicine, stem cell transplantation technology is also undergoing rapid changes. In recent years, stem cell transplantation is the hot spot in medical science research, because of the attractive prospect in repairing injuryed tissues such as myocardial infarct, myocardial infarct and diabetes.Firstly, we should gain sufficient sources of stem cells in order to do stem cell transplantation. Candidates for such strategies include bone marrow mesenchyme stem cells(BMSCs) and embryonic stem cells(ESCs) in clinical application. There are still some limitations to their practical use, including lack of source(only10-20ml bone marrow per person) and a invasive way for BMSCs, immunologicrejection and ethical issues for ESCs. So the type of stem cells that is abundant and can be easily accepted by patient is badly in need. So, we should gain sufficient sources of stem cells in order to do stem cell transplantation.Since Zuk et al reported that the stem cells in the fat was discovered at first time in2001, the Adipose tissue-Derived Mesenchymal Stem Cells (ADMSCs) became an new important source of stem cells, which has brought to people’s attention.The evaluation of stem cell therapy needs to reflect the situation of migrate, homing, proliferation and differentiation of transplanted stem cells in vivo, so that we can evaluate transplant curative effect, optimize transplant method and select appropriate transplant window period. Recently, many researches are focused on tracking of cells following transplantation, and magnetic resonance imaging (MRI) is considered to be one of the best techniques to determine the bio-distribution and migration of transplanted stem cells. Many researches found that cells labeled with superparemagnetic iron oxide(SPIO) could be intravital, dynamical, sensitive and non-invasive tracked by magnetic resonance imaging (MRI).The direct effect of SPIO labeling on cells is iron overload. Initial studies showed that iron in labeled cells would be increased10to more than100times than that in unlabeled cells. In this case, the primary problem need to clarify is whether cellular iron homeostasis can be maintained. Iron, is a very important trace elements of the cells. When labeled with SPIO, the dynamic changes of iron concentration in the cells needs further study.The main purpose of the stem cell transplant treatment, is transplanted stem cells into specific target organs or tissues, to make it differentiated into specific organization cells, perform specific organs or tissues of the corresponding function. Therefore, the SPIO should be stay enough time within cells, then, MRI could detect the labeling cells. MRI can accurately and comprehensively judge the transplanted stem cells in the body of all kinds of situation, and curative effect is accurate assessment for transplantation. Initial study reported that SPIO labeled cells could be visualized by MR more than21days. So, it is necessary to clarify the iron concentration after SPIO labeling of stem cells, whether the magnetic resonance imaging (MRI) could detect the labeling cell, it is also a key of stent cell transplantation.Iron Regulatory Proteins/Iron Responsive Elements System(IRPs/IREs) is the most important system to maintain cellular iron homeostasis, which is consist of Transferrin Receptor(TfR), Ferritin(Fn) and Iron Regulatory Proteins(IRPs). The expression of both TfR and Fn are regulated by IRPs in the post-transcriptional level. IRPs achieve regulatory function to combinewith highly conserved Untranslated Region(UTR) in TfR and Fn mRNA.The binding site between UTR in TfR, Fn mRNA and IRPs is called Iron Response Elements(IREs). As intracellular iron of SPIO labeled stem cells will be overload, whether cellular iron homeostasis can be maintained? When labeled with SPIO, the dynamic changes of protein in IRPs/IREs and the binding activity of IRPs/IREs needs further study.Purpose1. To investigate the effects of iron content on rat ADSCs labeling with SPIO.2. To preliminarily investigate the feasibility of MRI to detect the rat ADSCslabeling with SPIO.3. To investigate the effects of Fn-H, Fn-L and TfR protein expression, and thebinding activity of IRPs/IREs on rat ADSCs labeling with SPIO.Materials and methods1. Isolation and culture of rat ADSCsAdipose tissue was obtained from the inguen of3-4weeks old male Sprague-Dawley(SD) rats. After washing with phosphate-buffered saline (PBS)3to4times, the adipose tissue was minced with eye scissors into less than lmm3per piece and dissociated in0.25%collagenase type II in PBS solution for35-45minutes at37℃with gentle shaking, followed by centrifugation at1500g for10minutes. Cells pellets were re-suspended in Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG), containing10%fetal bovine serum, and cultured at37℃in humid air with5%CO2atmosphere. After being seeded for24hours, unattached cells and debris were removed, and adherent cells were continue to culture. Fresh medium was changed every3days. The cells with70%-80%confluence were released with0.25%trypsin-EDTA and subsequently cultured for two passages prior to labeling.2. Identification of rat ADSCs (Detection of the surface antigen on rat ADSCs)The surface makers of stem cells such as CD29, CD31, CD44, and CD45were identified by Flow Cytometer.3. ICP-MS detect the iron concentration on rat ADSCs labeling with SPIO.The reagent of SPIO was Resovist (Schering, Berlin, Germany) with primitive concentration of28mg/ml. SPIO and PLL were put into a tube containing serum-free medium. Then, the solution containing SPIO and PLL was allowed to mix in a rotator for30minutes. After that, fetal bovine serum at final concentration of10%was added into the solution. Second passage rat ADSCs with80%-90%confluence of the surface area of culture plate were used to label. The SPIO was at final concentrations of50μg/ml (the ratio of SPIO and PLL was1:0.03). Incubating at37℃in humid air with5%CO2atmosphere.After incubating for0hour(without labeled),2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells.At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.4. SPIO labeling rat ADSCs, MRI detect the iron concentration in the cell4.1Magnetic resonance imaging (MRI) scan parameters3.0T GE superconducting mri scanner, head coil, T2*WI scanning, and get the R2*mapping figure. FAST GRE T2*WI:line of coronary scanning, TR200ms, TE2.2ms, double Angle Flip Angle60°, thick layer2mm, layer spacing0mm, Matrix:256x256, NEX:10, FOV15cm.4.2Standard concentration solution-MRI scanThe SPIO standard of Fe concentration in the solution:50ug/ml,40ug/ml,30ug/ml,25ug/ml,20ug/ml,15ug/ml,10ug/ml,5ug/ml,2.5ug/ml,1ug/ml. More than1ml solution concentration, partial shipments at EP tube (capacityof.2ml)Put the standard concentration SPIO solution EP tube in a special container, filled with copper sulfate solution (10tendency/1), MRI scans, obtain T2*value and R2*value. R2*value and the concentration of fitting straight line, get linear equations.4.3SPIO cell labeling solution-MRI scanAfter incubating for2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1x106cells/ml.After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells. At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml of gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1×106cells/ml.Put the EP tube of different time after SPIO labeling in a special container that filled with copper sulfate solution (10mmol/1). MRI scans. Obtain T2*value and R2*value, put R2*value into linear equations, the corresponding density are obtained.5Analyze the effect of the expression level of TfR and Fn’s protein of the labeled ADSCs with SPIO and transfection agent PLLThe ADSCs were labeled with SPIO and transfection agent PLL as above. Lysis Buffer and PMSF weres used to extract total proteinat the time point of Oh,16h,24h,4d,lw,2w,3w,4w,and the protein concentration was measured by means of BCA. Western Blot:①the protein was degenerated and reducted,②SDS-PAGEwas preparated,③Sample was loaded,④Electrophoresis,⑤Transmembrane,⑥Ponceau staining,⑦Immunoglobulin binding sites at PVDF were closed,⑧Wash membrane, primary antibody incubation, wash membrane, secondary antibody inucubation, wash membrane,⑨Displayed by ECL,and exposured with film.The results were scaned by scanners, and the expression level of TfR and Fn’s protein at the above time point were quantitative analysised by Molecular Analysis image analysis software.6SPIO labeling rat ADSCs, RNA Binding Protein Immunoprecipitation(RIP) detect IREs/IRPs binding activitySPIO labeling rat ADSCs, at corresponding time points (labeled before,1day,2days,4days,7days,14days,21days and28days), according to the following RIP experiment, the steps are as follows:6.1Using antibody to capture the endogenous RNA binding protein in the cytoplasm 6.2To prevent nonspecific combination of RNA6.3Through precipitation along to separate RNA binding protein and its combination of RNA6.4The combination of RNA sequence identification:by microarray (RIP-Chip), quantitative RT-PCR method7Statistical methodsAll calculation using SPSS20.0statistical analysis system, unified hypothesis testing with double side inspection, test statistics and their corresponding P values are given. Inspection level is0.05, or P<0.05think the difference was statistically significant. This experiment all the data for the measurement data, using the mean±standard deviation.7.1ICP-MS measurement of different points in time the comparison of stem cells within the iron concentration using a single set of repetitive measure analysis of variance, the comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance.7.2MRI measurements:iron ion concentration and R2*value using linear regression analysis. MRI measurement of intracellular iron concentration of the true value of the predicted values and the icp-ms measurement comparison between using matching t test.7.3Compare rat ADSCs before and marking of different time points after Fn-H, Fn-L and TfR protein expression level, and the combination of IRPs and IREs activity level, the comparison of different time using a single set of repetitive measure analysis of variance, comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance. Results1. Rat ADSCs could be successfully isolated and cultured in vitro by means of0.25%Ⅱ collagenase solution. After culturing for24hours, some pleomorphic and adherent cells were present. Following by changing of medium and transferring of culture, cells were become more uniform, showing a typical fibroblast-like appearance with the long spindle morphology. After primarily culturing for6-7days, cells were needed to transfer. And then, cells were transferred every3-4days.2The P4cells were identified by Flow Cytometer, which found that96.5%of the cells expressed CD44,98.4%of the cells expressed CD29, while only8.4%of the cells expressed CD31, CD45cells express is only2.5%. The results show that the separation of cell phenotype is relatively uniform. These results were correspondto the characteristic of mesenchymal stem cells.3. ICP-MS detection SPIO labeling rat ADSCs iron content in the cell:not tag group of cells, iron concentration0.393+/-0.027pg/cell. Measurement,2hours after SPIO labeling higher iron content that is visible in the cell, concentration of iron2.627+/-0.207pg/cell, continue to rise gradually, after the second day after mark, the mean of iron ion concentration reached a maximum,35.315+/-2.041pg/cell, reduce gradually, after28days was1.350+/-0.260pg/cell.By a single set of repetitive measure analysis of variance, iron concentration in different time have significant difference (F=761.129, P<0.001). Based on a single set of repetitive measure analysis of variance of multiple comparison (LSD) method were analyzed, and found2h and3w,12h,8h and4d there was no statistically significant difference with1w (P>0.05), the comparison of differences between other groups have statistical significance (P<0.05). 4MRI quantitative analysis of stem cells after SPIO labeling4.1MRI scan standard iron concentration solutionStandard concentration iron solution by MRI scan, measured T2*value, translates into R2*value. Iron concentration as a dependent variable, R2*value as independent variables, the iron concentration (Y) and R2*value (X) linear regression analysis, the final fitting the linear regression equation is:the model test results for, was statistically significant (F=4579.44, P<0.001), R=0.992, R=0.983, square iron concentration and R2*value exists strong linear relationship. Upon examination, the regression equation of the R2*value coefficient was statistically significant (t=67.672, P<0.001). MRI scan standard concentration SPIO solution, concentration of iron and the R2*value linear fitting results are as follows:,y=-5.672+0.296x4.2MRI scan the solution of cells labeling by SPIO, calculate iron concentration in the cellMRI scans SPIO labeling cells in different time (with icp-ms measurement for the same batch of cells, different processing methods), measuring the T2*value and R2*value. According to R2*value SPIO labeling cells into linear equations, the predictive value of iron ion concentration. ICP ir-MS on concentration and MRI measurement of iron concentration no statistical differences between the two groups (F=0.291, P=0.291). For each time point matching t test found that2days,4days,7days and14days of the comparison between the two groups had no statistical significance (P>0.05), other time points were statistically significant (P<0.05).5SPIO labeling of ADSCs, Fn-L, Fn-H and TfR protein expressionOur study found that the expression of Fn-L’s, Fn-H’s and TfR’s protein were shown in Table1. Table1Effects of labeling of rADSCs with SPIO on the expression of Fn-H, Fn-L and TfR protein 6.1Binding activity of Fn-L mRNA and IRP12days after SPIO labeled, the binding activity of IRP1and Fn-L mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05).6.2Binding activity of TfR mRNA and IRP14days and7days after SPIO labeled, the binding activity of IRP1and TfR mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05)ConclusionsIt can be successfully isolated ADSCs from rat adipose tissue. Rat ADSCs are one type of mesenchymal stem cells. They are not only easy to isolate, but also have active proliferation capability and stable biological characteristics. It is easy and efficient to label cells with SPIO by using PLL.SPIO labeling stem cells, ICP can accurately detect the iron concentration of stem cells. MRI can accurately predict iron concentration in the cells. MRI has a high consistency with ICP. The influenceof expression of Fn-L’s, TfR’s and Fn-H’s protein were just temporary, after intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. These indicated that it would not induce irreversible influence on the expression of transferrin receptor, ferritin light chain’s and high chain’s protein on stem cells labeling with SPIO, within a certain range of concentration. SPIO-labeled stem cells, is safe and reliable.