| Induced pluripotent stem cells(i PSCs) derived from the reprogramming of somatic cellshave more advantages than mbryonic stem cells(ESCs) due to the avoidance of ethical debate and no destroying of the embryo. However, so far, there are still quite a few problems need to be solved for the studies on i PSCs: 1), poor safety; 2) low efficiency; 3) time-consuming. Therefore, in this study, we introduced the magnetic nano technology withaims to building a new method to obtain the i PSCs more quickly, more efficiently and more effectively. Firstly, we constructed four kinds of recombinant plasmids,by the connection of the four humanized transcription factor genes(Oct4, Sox2, Klf4, and c-Myc) with four p EGFP-N1 carriers, then, we coupled the magnetic nanoparticles(Poly-MAG) modified by polyethyleneimine(PEI) with the four recombinant plasmids andtransferred the coupled complex into 293 T cells to make the recombinant proteins expressed. The positive cells with the expression of recombinant proteins were screened by G418 and then extracted and purified. The recombinant proteins were then again connected with the magnetic nanoparticles(Poly-MAG) modified by polyethyleneimine(PEI) to improve the efficiency of the recombinant proteins into the ear f ibroblasts of Guangxi Bama Miniature P igs and reprogramming them into i PSCs. Using this method can completely avoid the tumorigenic risks which caused by viru ses transfection and introduction of exogenous gene. In this study, compared with simply using recombinant proteins connected with the cell penetrating peptides(CPPs), our method made the process of somatic cell reprogramming more effective and efficient. The main research results are as follows:Part 1: Research on the optimization of the isolation methods and culture system for skin fibroblasts of Guangxi Bama Miniature PigsIn order to choose the skin fibroblasts of Guangxi Bama Miniature Pigs which at vigorous growth period as target cells for the reprogramming tests, we used five different methods(4 kinds of enzyme digestive methods and one method of tissue pieces culture) to isolate the ear skin fibroblasts of Guangxi Bama Miniature P igs. The number of total cells and cells mortality rates were calculated to investigate the best isolation method, and then we investigated the effect of d different positions(ears, legs, abdomen, back and tail) of skin fibroblasts of the miniature pigs on the isolation, and results show that: 1) The number of the fibroblasts isolated by 0.25% trypsin treated for 30 min firstly and then 0.2% collagenase for 30 min was significantly higher than other treat ments groups(p<0.05), no signif icant difference for the cells mortality rate with the group treated by 0.125% of trypsin for 60 min(p>0.05), but significantly lower than other groups(p<0.05); 2) Using the tissue culture method, some fibroblasts climbed out from the edge of tissue blocks after 4 daysr, and in the next 5 days, the convergence rate of cells could reached 80%, this method need to spend much more time compared with the enzymic digestion(the convergence rate of cells reached 80% and need 2 days only); 3) Among the five different parts of the skin, the most number of cells with the best adherence was obtained from the ear part; 4) the ear fibroblasts can passage more than 10 generations and the third and fourth generation had the strongest vitality; 5) the cell growth curve showed that, at the 0~3 days after inoculation, cells in a silent growth period, but at 3~8 days after inoculation, cells in a logarithmic growth period, after 8 days cells into smooth growth period, after 10 days, the adherent ability of cells were declined, and presents a negative growth. So, we should choosing 0.25% trypsin+0.04% EDTA digesting 30 min at first and plus 0.2% collagenase digesting 30 min at second to separating the pig ear skin fibroblasts, and using the fibroblasts which at passage 3 or passage 4 and after 3~8 days of training for programming test.Part 2: Research on the optimization of the three kinds of transfection reagents for their mediation of recombinant plasmid DNA into 293 T cells.This experiment aimed to investigate the different transduction efficiency with thr ee kinds of transfection reagents(Lipofectamine ? 2000, PEI and Poly-MAG) into 293 T cells after they connnected with the p EGFP-N1 which containing with plasmid DNA. In the test, we used micro-spectrophotometer and agarose gel electrophoresis respectively for the concentration determination and purity of plasmid DNAs at first, and then, used different concentrations of the above three kinds of transfection reagents coupled with different concentrations of p EGFP-N1 which contain Oct4 plasmid DNA, transfected 293 T cells,the Green Fluorescent Protein(GFP) marked gene was included in the p EGFP-N1 vector, after the transfection, by calculating the transfection efficiency and survival rates of 293 T cells with the various concentrations, we investigated which is best transfection reagent and its optimal transfection concentrations. Finally, for screening the positive clones, we used of G418 screening method. Positive cells with recombinant proteins expressed were extracted and purified, and the micro-spectrophotometer and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was used for the determination of the concentration and purity of recombinant proteins, respectively. The method of Prussian blue staining was used for the qualitative detection of magnetic iron oxide nanoparticles in 293 T cells. The results showed that:(1) The concentration of four recombinant plasmid of transcription factor genes(Oct4, Sox2, Klf4, c-Myc) were:(299ng/μL, 492ng/μL, 500ng/μL and 481ng/μL), respectively, the electrophoretic bands were all matched with Genebank, and their purity is highly.(2) The optimal concentration of three kinds of transfection reagents: Poly-MAG(10μg/m L, incubate 20 min on magnetic plate, the transfection efficiency is 64.09±0.53%), Lipofectamine ? 2000(5μg/m L, the transfection efficiency is 63.82±2.53%), PEI(40μg/m L, the transfection efficiency is 30.20±2.28%), respectively. In terms cells survival rates, the group of Poly-MAG(10μg/m L, incubate 20 min on magnetic plate, the cells survival rate is 91.75±0.62%) was signif icantly higher than the group of PEI(40μg/m L, the cells survival rate is 60.40±3.66%) and the group of Lipofectamine ? 2000(5μg/m L, the cells survival rate is 31.10±5.58%),(p<0.01).(3) In screening of G418 tests, when the concentration of G418 is 600μg/m L or 700μg/m L, the positive rate of GFP+ clones is 86.54±2.35% and 90.00±1.49%, respectively, there have no signif icant difference between them, but both of them were significantly higher than other treatment groups(p < 0.05).(4) After purified, the concentration of four kinds of recombinant proteins(Oct4, Sox2, Klf4, c-Myc) were:(461μg/m L, 434μg/m L, 615μg/m L and 787μg/m L), respectively, by SDS-PAGE detection, the electrophoretic bands were all matched with Genebank, and their purity is high..(5) In the prussian blue stain test, more than 80% of 293 T cells of the group of Poly-MAG(10μg/m L), could dyed blue.In conclusion, we should choose Poly-MAG(10μg/m L, incubate 20 minutes on magnetic plate) for the transfection of 293 T cells. More than 80% of 293 T cells of the group of Poly-MAG(10μg/m L), could dyed blue, suggesting the existence of magnetic iron oxide nanoparticles in this cells. Using G418 with the concentration of 600μg/m L or 700μg/m L to screen 293 T cells, we could obtain the highest rate(86.54±2.35% vs 90.00±1.49%) of GFP+ clones from the living cells. After extract and purify of recombinant proteins, by the concentration and purification testing, all of them are according with the requirement of the subsequent tests.Part 3: Research on the feasibility of using magnetic nano technology combined with the recombinant proteins of humanized transcription factors to reprogramming ear skin fibroblasts of Guangxi Bama Miniature Pigs into i PSCs.This experiment aimed to investigate whether the magnetic nanoparticles coupling with recombinant proteins of transcription factorscould shorten the time for reprogramming somatic cells into i PSCs by recombinant proteins. In the process of this reprogramming, we used magnetic nanoparticles to mediate the recombinant proteins of four transcription factors, and deliveried into the ear skin fibroblasts of Guangxi Bama Miniature Pigs for its reprogramming. The four recombinant proteins of four transcription factors(including 9R poly-arginine, a kind of CPPs) dealed with pig skin fibroblasts was used as control. In order to provide continuously reprogramming "energy" to fibroblasts, in our experiment, we used circulation processing method for the recombinant proteins. To identify the pluripotency of i PSCs-like clones, in this experiment, we observed the morphology, alkaline phosphatase(AKP) staining and Oct4 immunohistochemical staining method. Results showed that:Using of magnetic nanoparticles coupling with recombinant proteins of four transcription factors to reprogramming somatic cells into i PSCs, the number of i PSCs clones of 86 obtained was signif icantly higher than simply using four recombinant proteins to reprogramming the fibroblasts into i PSCs of 54,(P<0.05); The i PSCs-like clones closely adhered to the mouse embryonic fibroblast cells(MEFs) feeder, with a similar appearance of small "is lands", and there was a clear boundary between i PSCs-like c lones and the feeder cells, w The big nuclear and less cytoplasmof the i PSCs-like clones was clearly observed at the high magnification.. By the AKP staining and Oct4 immunohistochemical staining, i PSCs-like clones dyed reddish brown, suggesting that this clones werer i PSCs. In addition, if we simply used ofour recombinant proteins to reprogramming the fibroblasts, it took more than 7 weeks to produce i PSCs, but, if we used magnetic nanoparticles binding with the same four recombinant proteins,it took only 5 weeks to produce i PSCs., suggesting that using magnetic nanoparticles connected with the four recombinant proteins could not only improve the reprogramming efficiency of recombinant proteins mediated, but also could short the time of the process of somatic cells reprogramming.Part 4: Positioning enrichment tests of i PSCs(including magnetic iron oxide nanoparticles).This experiment aimed to investigate that: 1) whether there were magnetic iron oxide nanoparticles in the i PSCs clones; 2) whether magnetic iron oxide nanoparticles could "impeled" i PSCs to enrich at the same position in the magnetic field. In this study, we used prussian blue staining for i PSCs clones, and observed whether its color changed; and added a strong magnetic field at the bottom of the cell culture flask to attract the i PSCs, and follow-up observing the distribution of i PSCs in this flask. The results showed that: i PSCs dyed blue by prussian blue staining, and they could enriched together in additional magnetic field, suggesting the exsistence of the magnetic iron oxide nanoparticles in this i PSCs.Conclusion: We should using of 0.25% trypsin+0.04% EDTA digesting 30 min at first and 0.2% collagenase digesting 30 min at second to separating the pig ear skin fibroblasts; by a magnetic field, using of the surface modified magnetic nanoparticles could not only impr ove the efficiency of DNA binding and reduce the toxicity of 293 T cells transfection, but also improve the reprogramming efficiency and shorten the process time by connecting with recombinant proteins of transcription factors(Oct4, Sox2, Klf4, and c-Myc) to be deliver ied into the somatic cells. The i PSCs produced by the reprogramming of somatic cells mediated by the magnetic nanoparticles binding with recombinant proteins could enrich together in the magnetic field. The present study using of nanotechnology mediated recombinant proteins of humanized transcription factors to reprogramming ear fibroblasts of Guangxi Bama Miniature Pigs into the i PSCs could lay a foundation for clinical disease diagnosis, treatment and regenerative medicine field. |
PDF Full Text Request