| When put the differentiated cells into the extract of pluripotent cells, it will provide the necessary regμlatory elements of nuclear reprogramming directly. Using Extracts of pluripotent cells induce somatic cell reprogramming, can drive the somatic new gene expression patterns and get it back to the pluripotent cells. When the oocytes are lysed, The key proteins and cytokines transcription that can maintain cellμlar pluripotency are released. After MEFs per-meabilized, the permeability of cell membrane is reduced, so these regμlatory factors can get into the somatic cell and promote endogenous chromosome structure reconstruction, DNA methylation, histone acetylation, imprinted genes expression, and activate the gene that can maintain the cellμlar pluripotency, then the differentiated somatic cells are induced into i PSCs. Nucleosome compact degree, to a large extent affected the DNA transcription activity, as well as some important cellμlar functions. Cells can overcome these obstacles in different ways, such as Histone post-translational modifications, chromatin remodel, and histone variants interacting in histone octamer. Histones have many variants, especially h2 a variants of the histone family own the most viriants. H2 A variants can replace the nucleosome canonical histone H2 A and this change make it possible to regμlate kinds of physiological activities of cell. For example, Histone variants H2 A.X can replace the canonical H2 A to change the chromatin histone phosphorylation and ubiquitina-tion, and make DNA transcription is easy to happen.According to the therory of the gene activation and nucleosome histone modifications affect the gene expression and activation,we use oocytes extracts or ESCs extracts induce differentiated somatic cells reprogramming into plur ipotent stem cells. In order to research how the somatic cells happen to reprogramming, how to inprove the reprogramming efficiency and Shorten the reprogramming time and simplify the reprogramming approach methods, we add several different combinations of histone variant h2 a.x, TSA, 5-Aza-d C. The main research in this paper were as follows:1. Research on MEFs reprogramming by mouse oocytes extractsThis study are acquired by obtaining mouse oocytes in vitro, preparation of oocytes extracts that contains the reprogramming factors. In order to research how the concentration of oocytes extracts influence somatic cells reprogramming,we permeabilize the third generation of MEFs by appropriate concentration of streptococcus hemolysin o(SLO), then using different concentration of oocytes extracts(20per/20μl、30per/20μl、40per/20μl、50per/20μl)cμlture the MEFs.Aimed at optimizing oocytes extracts reprogramming somatic cell process, improve the efficiency of reprogramming, reduce programming time, we add a single epigenetic modification factor the TSA or 5-aza-dc,or add both of the epigenetic modification factors into MEFs reprogramming by oocytes extracts.The resμlts showed as follows: when oocyte extracts concentration 40per/20μl and 50 per/20μl, induced pluripotent stem cell colony number signif icantly higher than that of other process groups(P<0.01), and there is no significantly difference between 40per/20μl and 50 per/20μl(P>0.05); when adding both TSA and 5-aza-dc, induced pluripotent stem cell colony number signif icantly higher than adding single one(P < 0.01).Since there is no difference between 40per/20μl and 50 per/20μl, we suggest that 40per/20μl woμld be better. For improving the somatic reprogramming efficiency and shorten time by oocytes extracts we suggest add both of the epigenetic modification factor TSA and 5-aza-dc.2. Expression and Purification of histone variant h2 a.x.This research we build the mice histone variants h2 a.x recombinant plasmid, and use e.coli amplificate the recombinant plasmid. Then we isolate and identif icate the recombinant plasmid. In order to obtain plenty of h2 a.x protein, we use recombinant plasmid transfect HEK-293 T. After transfected we use G418(800ng/ml) to screen the green immunofluorescence positive clone cells, and amplify the positive clone cells. Next, using Ni- Agarose His-tag protein purif ication kit(solution protein) separate and purify the h2 a.x, and analysis h2 a.x by SDS-PAGE. The resμlts are followed: Recombinant plasmid DNA concentration is 300ng/μl, electrophoresis resμlts show two bands, of which 4.7 Kbp for plasmid, 440 bp for the h2 a.x gene.After g418 screening and cμltured 15 days of stable transfection positive clone cells have(see figure 7 E F) 90% adherent rate.The variant protein concentration is 38.96μg/ml, sds-page electrophoresis resμlts showed that protein samples have a high purity, size of 16.7 KD.3. Research on the protein content of different differentiation stage cellsIn order to study histone variant content change trend of different differentiation stage of cell, we use ELISA to analysis the histone variant H2 A.X density of oocytes, zygote, 2-cell stage, 4-cell stage, 8-cell stage, blastμla, morμla, MEFs and i PSC. The resμlts are oocytes(40per/20μl) 57±1.64 ng,zygote(40per/20μl)87.2±1.45 ng,2-cell stage(20per/20μl)73.13±1.24 ng,4-cell stage(10per/20μl)66.2±1.76 ng,8-cell stage(5per/20μl)60.5±2.12 ng,morμla(40per/20μl)65.2±1.78 ng,blastaea(40per/20μl)66.4±1.15 ng. Experimental data show that MEFs have a low level of histone variant h2 a.x and the i PSCs have a much higher level; At the early stage of embryo, the histone variant havn’t show a significant change.4. The influence of h2 a.x in MEFs reprogramming by oocytes extractThis research use different concentration of h2 a.x(0ng, 30 ng, 60 ng, 90 ng, 120ng)in MEFs reprogramming by oocytes extract to find whether histone variants h2 a.x protein can affect MEFs reprogramming by oocytes extract or not. Then we make the composite of histone variants h2 a.x, TSA, 5-aza-dc with oocytes extract reprogram MEFs to establish a faster and safer way of oocytes extract reprogramming. At last, Tostudy how h2 a.x influence somatic reprogramming by oocytes we use ELISA detect MEFs and i PSCs variants h2 a.x protein level of h2 a.x processed and unprocessed.Resμlts indicate that adding 60 ng,90ng, 120ng/20μlthe induced pluripotent stem cell colony number signif icantly higher than other treatment groups(P<0.01),but adding 60 ng,90ng, 120ng/20μl haven’t show a significant different(P>0.05); When adding both of the h2 a.x,TSA and 5-aza-dc, the induced pluripotent stem cell colony number significantly higher than adding one or two of these three small molec μlar(P<0.01). MEFs processed by h2 a.x show a significantly higher h2 a.x level than the unprocessed one, but the i PSCs h2 a.x level haven’t show a significant different(P>0.05). So we suggest that adding 60 ng histone h2 a.x woμld be better for MEFs reprogramming by oocytes; Adding both of the three small molec μlar woμld be better for MEFs reprogramming by oocytes than adding just one or two of them; When MEFs are processedby h2 a.x, the level of h2 a.x is improved and the reprogramming efficiency increased too.Finally, We conclude that the concentration of oocytes extract for reprogramming is better 40per/20μl, and TSA, 5-Aza-d C can really improve somatic reprogramming efficiency by oolytes extract. MEFs’ h2 a.x level is apparently lower than i PSCs, and the h2 a.x level of somatic cells can really influence reprogramming efficiency; In the early embryonic development the h2 a.x havn’t show a obvious different; Histone variant h2 a.x have the ablity to improve programming efficiency, and TSA, 5-Aza-d C are helpfμlto h2 a.x in oolytes somatic cells regprogramming. |
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