摘要
目的研究P53特异性抑制剂对骨髓间充质干细胞向心肌样细胞分化的影响。方法分离SD大鼠骨髓间充质干细胞,取第4代骨髓间充质干细胞分为对照组(CON)和P53特异性抑制剂(PFT-α)组。观察骨髓间充质干细胞诱导前后细胞形态变化,流式细胞仪鉴定骨髓间充质干细胞表面抗原及诱导后心肌样细胞分化率,免疫荧光法检测诱导后cTnI、CX-43表达,Western Blot法测定诱导后cTnI、P53、P21蛋白表达情况。结果原代培养的骨髓间充质干细胞2周形成集落,传代诱导后细胞体积变大,呈长梭形,排列趋一致。流式细胞仪测定骨髓间充质干细胞表面抗原CD44阳性表达率为(89.98±1.29)%,CD45阳性表达率为(2.14±0.22)%。心肌样细胞鉴定结果显示:诱导后1周,空白对照组未见cTnI和CX-43免疫荧光表达,PFT-α组少量表达cTnI和CX-43;诱导后4周时,可见空白对照组少量表达cTnI和CX-43,PFT-α组强表达cTnI和CX-43。WesternBlot检测诱导1周时PFT-α组cTnI表达与对照组比较,有统计学差异(P<0.01)。诱导后4周,PFT-α组cTnI表达与对照组比较,有统计学差异(P<0.01)。两组cTnI表达量4周与1周比较有统计学差异(P<0.05)。诱导1周时,PFT-α组几乎不表达P53蛋白,PFT-α组与对照组比较有统计学差异(P<0.05)。诱导4周时,PFT-α组与对照组比较有统计学差异(P<0.05)。PFT-α组诱导4周时P53、P21表达量与1周时比较均有统计学差异(P<0.05)。心肌细胞分化率结果显示,诱导4周时,PFT-α组与空白对照组比较有统计学差异(P<0.01)。结论通过P53特异性抑制剂PFT-α阻断P53-P21蛋白通路,能诱导骨髓间充质干细胞向心肌样细胞分化。
Objective Recently p53 is shown to play an important role in the proliferation and differentiation of bone marrow mesenchymal stem cells(BMMSCs).Methods BMMSCs were isolated from bone marrow of SD rats by density gradient centrifugation.The fourth passage cells were divided into two groups:control group and PFT-α group.The purified BMMSCs were identified by surface antigens and the differentiation rate of BMMSCs was examined by flow cytometry analysis.The expressions of cTnI and CX-43 in BMMSCs after inductions were detected by immunofluorescence microcscopy and those of cTnI,p53 and p21 were detected by Western blot Assay.Results BMSCs of primary culture form colonies at 2 w,passaged cells became larger,elongated spindle,arranged the same trend.At 4 w after induction,BMMSCs represented as closely packed elongated cells with uniform morphology.The results of flow cytometry showed CD44 expression of BMSCs was(89.98±1.29)%,CD45 positive expression rate was(2.14±0.22)%.Immunofluorescence staining showed that at 1 w after induction,no expression of cTnI and CX-43 was observed in CON group,but a little expression of cTnI and CX-43 were observed in the PFT-α group.4 w after induction,CON group only had little expression of cTnI and CX-43,while the PFT-α group demonstrated strong expression of cTnI and CX-43.Western blotting analysis showed that at 1 w after induction,cTnI expression in the PFT-α group,but no cTnI expression was observed in CON group.At 4 w,only a little expression of cTnI was observed in CON group,but in the PFT-α group cTnI expression were significantly increased and much higher than CON group.cTnI expression in every group at 4 w was higher than that of at 1 w.Western blotting analysis showed that at 1 w the expression of p53 and p21 in CON group were stronger compare to PFT-α group(P<0.05),but no expression was observed in PFT-α group.At 4 w,the expression of p53 and p21 in CON group were still strong(P<0.05).The expression of p53 and p21 in PFT-α group and CON group at 4 w were much higher than that of at 1 w(P<0.05).Flow cytometry determined the differentiation rate of cardiomyocyte-like cells and the results showed that differentiation rate of PFT-α group was significant higher than CON group(P<0.01).Conclusions These data open up new possibility of modulating p53-p21 pathway for directed differentiation of BMMSCs into cardiomyocytes,which will be valuable for cardiovascular regenerative medicine.
出处
《中华临床医师杂志（电子版）》
CAS
2011年第10期2878-2885,共8页
Chinese Journal of Clinicians(Electronic Edition)
