The Effects Of Transferrin Receptor And Ferritin Light Chain's Gen And Protein On Stem Cells Labeling With SPIO

BackgroundIn 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(only 10-20ml 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.Since Zuk et al reported that the stem cells in the fat was discovered at first time in 2001,the Adipose tissue-Derived Mesenchymal Stem Cells (ADSCs) 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 tranplanted stem cells in vivo,so that we can evaluate transplant curative effect, optimize transplant method and select appropriate transplant window period. However,it's always a problem for investigator to observe transform, survival status of tranplanted stem cells in vivo.The traditional research method is to do pathological examination after the animals are sacrificed and obtained the organization,which can't satisfy clinical application.And after the animals were sacrificed and observed in vitro,the results can not reflect the survival status in vivo truly and accurately.Observe the nanoparticles of Supraparamagnetic iron oxide (SPIO) labeled stem cells in vivo with MRI could be a noninvasive method,and has the greatest promise.Arbab et al confirmed that the concentration of intracellular iron in labeled stem cells would increased ten or a hundred times than unlabeled ones. The maintenance of cellular iron homeostasis plays a very important role in cellular activity.If cellular iron was overloaded,it woud induce the formation of oxygen radical,and lead to the cell damage. Therefore it is necessary to understand how the cellular iron homeostasis could be maintained,when labeled with SPIO. 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 combine with 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? Pawelczyk et al labeled of stem cells with SPIO using tansfection agent protamine sulfate,and they found that the expression level of TfR's mRNA and protein was reduced temporarily,while the expression level of Fn's mRNA and protein was remained unchanged. Schafer et al detected CD71 (Transferrin Receptor) with Flow Cytometry,they considered that labeled of stem cells with SPIO using tansfection agent had no effect on expression level of gen in stem cells,but they didn't do the quantitative research on TfR's mRNA and protein. The expression level of TfR would be upregulation,when labeled of stem cells with SPIO using no tansfection agent.When labeled with SPIO, the dynamic changes of gen and protein in IRPs/IREs needs further study. Herein,we labeled of ADSCs with SPIO using tansfection agent PLL to preliminarily investigate the effects of intracellular magnetic labeling of ADSCs with SPIO on the expression of TfR and Fn's gen and protein.Purpose1. To establish an optimal method for in vitro separate,cutivate and identify the primitive ADSCs from the SD rat's adipose tissue.2. To investigate the feasibility of intracellular magnetic labeling of ADSCs with SPIO using transfection agent PLL,and to analyze the effect of the viability and proliferation of the labeled stem cells.3. To investigate the effects of this approach on the TfR and Fn's gen and protein of the SPIO labeled ADSCs.Materals and Methods1. Separate,cultivate and identify the primitive ADSCs from SD rat's adipose tissue in vitro.The adipose tissue was obtained from the inguinal area of 3-4 weeks Sague-Dawley rats. Macroscopic small blood vessels and fascia were resected,and the adipose tissue was cut adequately into several pieces.0.25% Ilcollagenase solution was added to about twice the volume, digested in 37℃for 30-45min.And then right amount of low sugar DMEM+10%fetal calf serum were mixed to neutralize enzyme activity. Centrifuged at 1500rpm for 10min,and then threw away liquid supernatant and undigested organization. Low sugar DMEM+10%fetal calf serum were used to resuspend cells,and then cultured in the culture flask.Observe the process of growth of stem cells dynamically with inverted microscope, When the cells were approximately 80 to 90% confluenced, they could be dissociated with Trypsin-EDTA and passaged. Draw the cell growth curve from 1d-7d with P3 cells.The surface makers of stem cells such as CD29,CD31,CD34 and CD44 were identified by Flow Cytometer.2. Labeled the ADSCs with SPIO and transfection agent PLL,and analyzed the effect of the viability and proliferation of the labeled ADSCs,compared with unlabeled ones.The P3 cells of the ADSCs were used, and the reagent of SPIO was Resovist (Schering, Berlin, Germany),with primitive concentration of 28mg/ml. The methods to label the ADSCs with SPIO and transfection agent PLL:the cells were mixtured with the SPIO-PLL complex (SPIO 25μg/ml and PLL 0.75μg/ml) in the serum free media,and shaked up about 30 min at 30r/min in the room temperature. The cultures with the cells adherenced to bottom in 90% were kept over night at 37℃in a couveuse contained 5% CO2. The cells were washed 3 times with PBS to remove excess SPIO-PLL.Prussian blue staining was used to evaluate intracelluar iron uptake with optical microscope. The cells and PLL were cultivated with SPIO(0,12.5,25,50,75μg/ml) in six holes training board for 12h,and the cell viability was assessed with typan blue testing. The cells and PLL were cultivated with SPIO(0,50μg/ml) in six holes training board forld,3d,5d,7d,9d,and the cell proliferation was assessed with MTT testing.3.Analyze the effect of the expression level of TfR and Fn's gen and protein of the labeled ADSCs with SPIO and transfection agent PLL. The ADSCs were labeled with SPIO and transfection agent PLL as above.The total RNA were extracted by the method of Trizol before the cells were labeled(0h),and after the cells were labeled 2h,4h,8h,16h,24h,4d,1w,2w,3w,4w.Reverse transcription liquid were preparated, and the reaction conditions was 37℃15min,85℃5sec,and got the cDNA. The concentration of cDNA was measured,and the PCR reactive fluid was preparated according to the instruction manual. Real-time quantitative PCR was done by two steps,and the expression level of TfR and Fn's mRNA at the above time point were calculated by CT value.Lysis Buffer and PMSF weres used to extract total protein at 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-PAGE was 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.Results1. The rat's ADSCs could be successfully seprated and cultured in vitro by means of 0.25% II collagenase solution.The primary cells were pleomorphic,but they could get more uniform along with the increase of cuture and passage time.The cell growth curve showed the cells proliferated actively.The P4 cells were identified by Flow Cytometer,which found that 98% of the cells expressed CD29,5% expressed CD34,97% expressed CD44.These results were correspond to the characteristic of mesenchymal stem cells.2. It was easy and efficient for intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles,and the viability and proliferation of the labeled ADSCs were not effected within a certain range of concentration.Plussian blue dye stainning of the PLL-SPIO labeled ADSCs revealed that mostly all ADSCs were covered by blue iron particles. When the concentration of the SPIO were 12,25,50,75μg/ml, the time that label rate achieved to 100% were 48h,36h,24h,12h. It was thus the evidence that the intracellular concentration of the iron particle was increased as cultivation time and concentration of the SPIO increased. Typan blue exclusion testing revealed no significant difference of the cell vibility between the labled group and unlabeled group (F=1.512, P=0.271) for the concentration of the SPIO were 13,25,50,75μg/ml, compared with unlabeld cells. MTT testing revealed the proliferation of the labeled ADSCs were lower than the unlabeled ones on 1d (t=2.798, P =0.049),3d (t=3.164, P=0.034),but there was no significant diffrence on 5d (t=1.867, P=0.135),7d(t=2.671, P=0.056),9d (t=0.979, P=0.383) between the two groups.3. The expression of Fn-L's gen and protein were ascended temporarily,while the expression of TfR's gen were reduced temporarily and the expression of TfR's protein maitained unchanged,after intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. Our study found that the expression of Fn-L mRNA between labeled and unlabeled group were shown in Table1. The expression of TfR mRNA between labeled and unlabeled group were shown in Table2.After that,we did the Western Blot for the quantitative examination of the expression of Fn-L and TfR's protein.And our study found that the expression of Fn-L's protein between labeled and unlabeled group were shown in Table 3. The expression of FfR's protein between labeled and unlabeled group had no statistical difference (F=3.555, P=0.132) * F value and P value of the Main effect;# F value and P value of the interaction effect;Two mean comparison was analysed by Independent-Samples T Test;Multiple mean comparison within the groups was analysed by Repeated Measures ANOVA Test.* F value and P value of the Main effect;# F value and P value of the interaction effect;Two mean comparison was analysed by Independent-Samples T Test;Multiple mean comparison within the groups was analysed by Repeated Measures ANOVA Test. ConclusionOur initial results showed that the rat's ADSCs could be successfully seprated and cultured in vitro by means of collagenase solution,and the cellular phenotype were relatively homogeneous,and the cells were proliferated actively. It was easy and efficient for intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles,and the viability and proliferation of the labeled ADSCs were not effected within a certain range of concentration. The influence of expression of Fn-L's gen and protein and and TfR's gen were just temporary,and had no effect on expression of TfR's protein, 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 and ferritin light chain's gen and protein on stem cells labeling with SPIO, within a certain range of concentration.