| Backgroud and Objective: Stem cell-based restorative treatment has bring light to the damaged myocardium after myocardial infraction.The cardiac stem cells(CSCs)because of they have a heart lineage cells similar genetic background,organization specificity,multiple differentiation potential,low immunogenicity and specificity features,has become the best candidate to repair the damaged myocardium.However,the low survival and proliferation ability of the transplanted stem cells in the infracted myocardium has obviously weakened the ability to repair the damaged heart because of the bad local microenvironment after stem cell therapy.Therefore,it is time to develop novel strategies to boost stem cell survival and proliferation.Recent years,the paracrine effect of stem cells on myocardium repair has gradually be taken seriously.And exosomes,as the main product of stem cell paracrine effect,has been widely used to pretreatment stem cells and promote the biological functions because of its safety and efficacy.Some studies have shown that bone marrow mesenchymal stem cells derived exosomes(BMSC-exosomes)can significantly rEDUce the myocardial infarction area,promote the restoration of cardiac function after myocardial infraction.In addition,recent study reports that BMSC-exosomes has ablity to promote C-kitpos CSCs proliferation under oxygen condition.However,the regulatory effect under oxidative stress have not been reported yet.Recent years,hypoxia preconditioning has become a effective means to promote the survival ability in the oxidative stress environment,and studies have shown that hypoxia preconditioning BMSC-exosomes have a better effect in promoting endothelial cells proliferation.So,whether hypoxic preconditioning BMSC-exosomes can repair damaged myocardium after myocardial infractionpromot by improving stem cells proliferation in oxidatie stress environment,and which related mechanism could be involved? Therefore,the study aimed to investigate hypoxic preconditioning BMSC-exosomes could regulate C-kitposCSCs proliferation in oxidative stress environment and then analyze the underlying mechanisms.Methods:Part one1.BMSC was cultured and selected in SD rats with whole bone marrow cells adherent method which we have mastered before.Use the flow cytometry methods to identificate the purity for subsequent experiment.C-kitpos CSCs was cultured and selected in SD rats with enzyme digestion and magnetic bead which we have mastered before.Use the immunofluorescence and flow cytometry methods to identificate the purity for subsequent experiment.2.Exosomes was isolated from P3 bone marrow mesenchymal stem cells by polyethylene glycol combine with ultracentrifugation and ExoQuick-TC Kit.Eectron microscope(ETM)showed that the sizes were inhomogeneous,average diameter of30-100 nm,and the appearance was round or oval,saccate,and light in the low density area.Identification of western blotting showed that exosomes positively expressed CD63,CD9,Hsp70 protein.3.Processed C-kitpos CSCs with different concentration of H2O2(200,and 100,and 50 ?Mol/L)for 2h to simulate hypoxia micro-environment of different degrees.The control group was established with normal cells without H2O2.Detecting the activity of C-kitpos CSCs with CCK-8,further more detect survival rate,early apoptosis rate and necrosis rate with flow cytometry.Finally,make sure that the best reasonable concentration of H2O2 induced C-kitpos CSCs early apoptosis to simulate the hypoxia micro-environment.4.Processed C-kitposCSCs with different concentration of BMSC-exosomes(100,200,300,400,500ug/mL)for different time(6,12,18,24h)to explore the optimal concentration and time to treat cells by CCK-8.Further more,C-kitpos CSCs was co-culture with Di I labled exosomes 24 h to Prove that exosomes can be internalized by C-kitposCSCs.5.To verify the influence of normoxia and hypoxic preconditioning BMSC-exosomes to C-kitpos CSCs Under Oxidative Stress Conditions,the experiment was divided into 4 groups:(1)Control Group: C-kitpos CSCs under normoxia conditions.(2)PBS Group: C-kitpos CSCs Under Oxidative Stress Conditions was treated with PBS.(3)Exo-n Group :C-kitpos CSCs Under Oxidative Stress Conditions was treated with normoxia BMSC-exosomes.(4)Exo-h Group :C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes.Part two6.To prove the effect of hypoxia preconditioning BMSC-exosomes are mainly throug high expression of miR-150 and then activate SRC/CLC3 signaling pathways to promote C-kitposCSCs proliferation Under Oxidative Stress Conditions.We transferred miR-150 mimics to C-kitpos CSCs with life2000 TM as positive control group,the experiment was divided into 7 groups:(1)Control Group: C-kitpos CSCs under normoxia conditions.(2)PBS Group: C-kitpos CSCs Under Oxidative Stress Conditions was treated with PBS.(3)Mimics nagetive control(MNC): cells were transfected with miR-150 mimics negative transfected.(4)miR-150 mimics Group: cells were transfected with miR-150 mimics.(5)Exo-h Group :C-kitpos CSCs Under Oxidative Stress Conditions was treated with normoxia BMSC-exosomes.(6)Exoh+simiR-150 Group :C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes and miR-150 inhibitor.(7)Exoh+Scr(-)Group :C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes and miR-150 negtive inhibitor.we adopted RT-PCR to detect the expression of miR-150 m RNA in normoxia and hypoxic preconditioning BMSC-exosomes,and detect the expression of miR-150 and the target gene SRCIN1 mRNA in each groups.Western blotting to detect the expression of SRCIN1 protein in each groups.To verify the effection of miR-150 and exosomes on cell differentiation in vitro,we adopted RT-PCR to detect the early stage markers such as Nkx2.5,CD31,?-SMA mRNA of myocardial cells in each group.CCK-8method was used to detecte cell viability,EDU and flow cytometry was used to detect the proliferation ability,RT-PCR and Western blotting were observed to detecte the expression of SRC,CLC3,PCNA,CyclinD1 and P21 in each groups.7.The results of this study above eloquently showed that hypoxic preconditioning BMSC-exosomes could promote the proliferation capaciy of C-kitposCSCs Under Oxidative Stress Conditions at a certain extent,To further investigate whether the SRC/CLC3 signaling pathway involved in process,we divided the experiment into five groups :(1)Control Group: C-kitpos CSCs under normoxia conditions.(2)PBS Group: C-kitposCSCs Under Oxidative Stress Conditions was treated with PBS.(3)Exo-h Group:C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes.(4)Exo-h+PP2 Group:C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes and SRC inhibitor.(5)Exo-h+NPPB Group:C-kitpos CSCs Under Oxidative Stress Conditions was treated with hypoxic preconditioning BMSC-exosomes and CLC3 inhibitor.CCK-8 method was used to detecte cell viability,EDU and flow cytometry was used to detect the proliferation ability,RT-PCR and Western blotting were observed to detecte the expression of SRC,CLC3,PCNA,CyclinD1 and P21 in each groups.Results:Part one1.BMSC Cultured with whole bone marrow cells adherent method after 7 days,cells have basicly coverd dishes,and the integration was more than 85%.The rate of cells proliferation accelerated significantly after passage,and passage was carried out every 3 to 4 days.Cells morphology is uniform,like the spindle and fish.Identification of flow cytometry: the results showed that over 90% of rat BMSC express CD29,CD90;but did not express the hematopoietic progenitor cell surface marker CD34.Primary cultured CSCs were adhered to the wall within 48 hours,cell morphology were round highlights and stretched into polygon or triangles after4-5days,then the cell proliferation rate was gradually accelerated in logarithmic form.It reached the period of plateau around 7-8 days.Immunofluorescence detected that c-kit was positive after magnetic bead separation.Whats more,The signs of C-kitposcells were detected by the glow cytometry,and the results showed that C-kit 97.2%,CD45 2.2%,CD34 1.7%.2.Identificating BMSC-exosomes by TEM showed that: the sizes were30-100 nm,and the appearance was round or oval,saccate,and light in the low density area.Identification of western blotting showed that exosomes positively expressed CD63,CD9 and Hsp70 protein.3.CCK8 detection showed that the viability of C-kitposCSCs was decreased to approximate 50% when treated with 100?M H2O2,which compared with the Control group.FCM was applied to observe C-kitpos CSCs apotosis rates,compared with the Control?100?50?M group,the early apoptosis rates were significantly higher while the cell death rates had no significant change(P>0.05)after treatment H2O2(100?mol/L)for 2 hours.Therefore the oxidative stress microenvironment after myocardial infarction in vitro was simulated by100?mol/L H2O2,for 2h of C-kitposCSCs in the following experiment.4.CCK8 detection showed that the viability of C-kitposCSCs after BMSC-exosomes treated has presented a certain amount of time and dose dependent effect.When the treated time was 24 h,the viability in 400ug/m L grouop was higher than 300ug/mL grouop(P< 0.05),but the 500ug/mL group did not show much difference significantly(P> 0.05).So we ultimately selected 400ug/mL and 24 h as the optimal concentration and time to treat cell.C-kitpos CSCs surface appeared red DiI fluorescent after co-cultureing with DiI labled exosomes 24 h,it proved that exosomes can be internalized by C-kitposCSCs.5.CCK8 results showed that the viability of C-kitpos CSCs was decreased seriously after H2O2 treated compared with the Control group(P < 0.05),and there was a significant increase in cell viability after treated with normoxia and hypoxic preconditioning BMSC-exosomes(Exo-n Group and Exo-h Group)compared with the PBS group(P < 0.05),and the effect was much better in Exo-h Group compared with the Exo-n group(P < 0.05).The cell proliferation ability was detected by EDU.The results showed that the proliferation ability of C-kitposCSCs Under Oxidative Stress Conditions was decreased seriously after PBS treated compared with the Control group(P< 0.05),and there was a significant increase in cell proliferationability after treated with Exo-n and Exo-h compared with the PBS group(P< 0.05),and the effect was much better in Exo-h Group compared with the Exo-n group(P <0.05).The cell cycle was detected by FCM,the results showed that cell cycle was seriously blocked in G0/G1 phase after H2O2 treated compared with the Control group(P< 0.05),and cells in G0/G1 phase ratio is rEDUced,S+G2/M phase ratio increased significantly after treated with Exo-n and Exo-h compared with the PBS group(P< 0.05),and the effect was much better in Exo-h Group compared with the Exo-n group(P < 0.05).RT-PCR and Western blotting showed that the expression of PCNA and CyclinD1 was significantly decreased,and the expression of P21 was increased compared with the Control group(P<0.05).The expression of PCNA and CyclinD1 was increased,and the expression of P21 was decreased after treated with Exo-n and Exo-h compared with the PBS group(P < 0.05).Part two6.RT-PCR showed that the expression level of miR-150 was obviously higher in hypoxic preconditioning BMSC-exosomes compared with the normoxia one(P <0.05).And the expression level of miR-150 was obviously higher after treated with miR-150 mimics and Exo-h compared with the PBS group(P < 0.05),while no significant difference was found in MNC group(P>0.05).The expression level of miR-150 was obviously decreased in Exo-h+simiR-150 group compared with the Exo-h group(P < 0.05),while no significant difference was found in Exo-h+Scr(-)group(P>0.05).And the expression level of SRCIN1 mRNA was no significant difference in each groups(P> 0.05).Western blotting showed that the expression level of SRCIN1 protein was obviously decreased after treated with mi R-150 mimics and Exo-h compared with the PBS group(P< 0.05),and increased in Exo-h+simiR-150 group compared with the Exo-h group(P< 0.05),while no significant difference was found in Exo-h+Scr(-)group(P>0.05).RT-PCR showed that there were no significant differences in expression of CD31,Nkx2.5 and ?-SMA in each group,it meant that there was no influenece on the plasticity of C-kitpos CSCs.We also did not found out any difference in Nkx2.5 and CD31,?-SMA at mRNA levels in these three groups by RT-PCR(p >0.05)CCK8 showed that the viability of C-kitposCSCs was obviously higher after treated with miR-150 mimics and Exo-h compared with the PBS group(P< 0.05),and the viability was obviously lower in Exo-h+simiR-150 group compared with the Exo-h group(P< 0.05).EDU showed that cell proliferation ability was obviously higher after treated with miR-150 mimics and Exo-h comparedwith the PBS group(P<0.05),and the proliferation ability was obviously lower in Exo-h+simiR-150 group compared with the Exo-h group(P< 0.05).FCM showed that cells in G0/G1 phase ratio is rEDUced,S+G2/M phase ratio was increased significantly after treated with miR-150 mimics and Exo-h compared with the PBS group(P< 0.05),and cells in S+G2/M phase ratio was obviously lower in Exo-h+simiR-150 group compared with the Exo-h group(P<0.05).RT-PCR and Western blotting showed that the expression of SRC,CLC3,PCNA and CyclinD1 was increased,and P21 was decreased after treated with miR-150 mimics and Exo-h compared with the PBS group(P< 0.05),The expression of SRC,CLC3,PCNA and CyclinD1 was decreased,and P21 was increased in Exo-h+simiR-150 group compared with the Exo-h group(P < 0.05).7.CCK8 showed that the viability of C-kitposCSCs was obviously decreased after treated with Exo-h+PP2 and Exo-h+NPPB compared with Exo-h group(P<0.05),EDU showed that the cell proliferation ability was obviously decreased after treated with Exo-h+PP2 and Exo-h+NPPB compared with Exo-h group(P<0.05),FCM showed that cells in G0/G1 phase ratio is increased,S+G2/M phase ratio was rEDUced significantly after treated with Exo-h+PP2 and Exo-h+NPPB compared with Exo-h group(P < 0.05).RT-PCR and Western blotting showed that the expression of SRC,CLC3,PCNA and CyclinD1 was decreased,and P21 was increased after treated with Exo-h+PP2 and Exo-h+NPPB compared with Exo-h group(P < 0.05).Conclusion:1.Both normoxia and hypoxic preconditioning BMSC-exosomes have the ablity to promote C-kitpos CSCs proliferation Under Oxidative Stress Conditions,and the hypoxic preconditioning one do much better;2.miR-150 can promote C-kitpos CSCs proliferation Under Oxidative Stress Conditions obviously.The expression level of miR-150 was obviously higher in hypoxic preconditioning BMSC-exosomes compared with the normoxia one,hypoxic preconditioning BMSC-exosomes could be mainly throug passing miR-150 to C-kitposCSCs and then activate SRC/CLC3 signaling pathways to promote cell proliferation under Oxidative Stress Conditions. |
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