| Objective:(1).To identify the effect of IL-? on the degeneration and apoptosis of NP-MSCs,then to construct the in vitro IDD model which can simulate the in-vivo IDD pathological processes.(2).To identify the Huc-MSCs' protective effect on NP-MSCs by using transwell coculture system,and to observe the mechanisms of the protective effect.Method:(1).Primary Huc-MSCs and NP-MSCs were cultured from human umbilical cord and rat tail,then identified as stem cells by cell morphology,stem cell surface markers,and other methods.(2).CCK8 assay was used to find the appropriate concentration of IL-1? to induce NP-MSCs into degeneration and apoptosis in vitro.Cell apoptosis was detected by flow cytometry with Annexin V/PI staining.Caspase3,caspase8 and caspase9 were detected by spectrophotometry,and the expression of COL2A1A1?AGC was detected by RT-PCR and West-blot.(3)Huc-MSCs and NP-MSCs induced by IL-1? was co-cultured in Transwell cocultured system,Cell apoptosis was detected by flow cytometry with Annexin V/PI staining.Caspase3,caspase8 and caspase9 were detected by spectrophotometry,the expression of extracellular matrix AGC,COL2A1A1 and extracellular matrix hydrolysisrelated enzymes MMP3 and ADAMTS4 were detected by RT-PCR and Western-blot,to detect the protective mechanisms of Huc-MSCs.(4)Selective signaling pathway inhibitor LY294002 was used to inhibit the PI3K/Akt signaling pathway selectively,to identify the effect of PI3K/Akt signaling pathway on the protective mechanism of Huc-MSCs.Results:(1).Anchorage-dependent spindle cells were isolated and cultured from the intervertebral disc of SD rats and neonatal umbilical cord perivascular mesenchymal tissue.The cells were cultured for 6-7 generations without significant changes in cell morphology.The cells cultured from rat nail showed high expression of surface markers of mesenchymal stem cells such as CD44,CD29 and CD90,but no expression of surface markers of hematopoietic stem cells such as CD45 and CD34.While cells from human umbilical was detected high expression of human mesenchymal stem cell surface marker CD29,but no expression of CD45 hematopoietic stem cell surface markers.(2).The inhibition effect of IL-1?on NP-MSCs was enhanced with the increase of the concentration and the duration of exposure.10 ng/ml IL-1?for 24 hours could observably induced degeneration of NP-MSCs cells when the effect turn to apoptosis or necrosis.(3).Cell experiments were divided into groups according to different treatment methods: Group A(control group),Group B(IL-1?treatment group)and Group C(coculture group).The cell apoptosis rates of Group A,Group B and Group C were 50.533±1.301,69.467±1.069,and 57.333±0.451.The apoptosis rate of Group B and Group C was higher than that of Group A(p<0.05).P <0.05),the apoptosis rate of Group C was suppressed than that of Group B after co-culture with Huc-MSCs(p<0.05).The activity of caspase-3,caspase-8 and caspase-9 in Group B were promoted than those in Group A(p<0.05),suppressed in Group C(p=0.000<0.05).P = 0.000 < 0.05;P =0.000<0.05).(4).We import group D(LY294002+ Huc-MSCs co-culture group),the mRNA and protein expression of Bcl2,AGC and COL2A1A1 in Group B were suppressed by IL-1?,the suppression was partly blocked in Group C and Group D,and the expression of Bcl2 in Group D was lower than that in Group C(P < 0.05).The expression of extracellular matrix hydrolase MMP3 and ADMATS4 in Group B was promoted.The expression of MMP3 and ADMATS4 in group C and group D was suppressed compared with Group B and Group A(P < 0.05).while in Group D has more expression than that in Group C(P < 0.05).Conclusions:(1).Spindle cells could be isolated and cultured to passages from Rat nail intervertebral discs and neonatal umbilical cord perivascular mesenchymal tissue.These cells have high expression of mesenchymal stem cell phenotype and do not express hematopoietic stem cell phenotype,and can be identified as NP-MSCs and Huc-MSCs.(2).10ng/ml IL-1?action 24 h can significantly inhibit the proliferation of NP-MSCs,the extracellular matrix secrete,and induce the apoptosis of NP-MSCs,which can be used to create IDD in vitro model.(3).Non-contact co-culture of Huc-MSCs and NP-MSCs can reduce the effect of IL-1?to NP-MSCs.which reflect in reduce cell apoptosis,increase the secretion extracellular matrix,and reduce the expression of MMP3,ADMATS4 and other inflammatory factors of hydrolytic extracellular matrix.PI3K/Akt signaling pathway plays an important role in this protective effect. |
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