大鼠牙髓干细胞与骨髓间充质干细胞分化成骨样细胞能力对比研究
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摘要
目的提取大鼠牙髓干细胞(Dental Pulp Stem Cells,DPSCs)与骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells, BMSCs),传代培养观察二者形态学,以及其生长差异。免疫荧光染色观察大鼠牙髓干细胞标志物表达。通过矿化诱导,观察大鼠牙髓干细胞与骨髓间充质干细胞向成骨样细胞分化能力,并通过免疫荧光对其进行表面成骨标志物检测,观察其钙化结节形成能力,对比讨论大鼠牙髓干细胞的成骨样细胞分化能力与特点。
方法通过离心法获得大鼠骨髓细胞,全骨髓体外贴壁培养扩增,并通过倒置显微镜观察其形态；通过酶消化法处理大鼠牙髓组织获得牙髓干细胞,体外培养扩增,通过倒置显微镜观察形态。通过流式细胞仪检测所获得细胞表面CD45,CD90分子表达；通过免疫荧光染色检测细胞表面STRO-1表达；MTT法分别测得大鼠骨髓间充质干细胞与牙髓干细胞的生长曲线,对照分析,比较二者在生长扩增上的差异；分别对大鼠牙髓干细胞与骨髓间充质干细胞进行矿化诱导培养,2周后对其进行骨钙素(OCN),牙本质泌涎蛋白(DSP)免疫荧光染色；继续培养四周,对经诱导的大鼠牙髓干细胞进行茜素红染色观察钙结节形成情况。
结果通过离心法获得的大鼠骨髓细胞经过贴壁培养,换液逐渐将造血系细胞清除,得到相对纯度较高的骨髓间充质干细胞贴壁生长；通过酶消化法获得的大鼠牙髓干细胞第二天可完全贴壁生长；所获得的细胞经流式细胞仪检测CD45表达阴性,CD90表达阳性,符合间充质干细胞特性；免疫荧光染色STRO-1表达阳性,符合干细胞表达特性；MTT法绘制生长曲线可见大鼠牙髓干细胞与骨髓间充质干细胞第4天左右进入生长对数期,牙髓干细胞生长增殖速度快于骨髓间充质干细胞；经过矿化诱导2周后骨髓间充质干细胞与牙髓细胞表达成骨细胞表面标志物OCN阳性,牙髓干细胞表达牙本质细胞标志物DSP阳性；经4周矿化诱导培养,大鼠牙髓干细胞茜素红染色证实有矿化结节生成。
结论通过离心获得的骨髓细胞经全骨髓贴壁培养可随着贴壁换液的进行逐渐将造血系细胞清除获得纯度较高的骨髓间充质干细胞,其表面标志物检测证实纯度可达80%以上；酶消化法获得的大鼠牙髓干细胞表面标志物表达符合间充质干细胞特点,且生长扩增能力强；通过矿化诱导培养,牙髓干细胞可向成骨样细胞分化,能力与骨髓间充质干细胞相当,并可形成矿化结节。牙髓干细胞具有较强的增殖能力,并具有成骨样细胞分化能力,可以作为干细胞治疗骨组织缺损修复的理想种子细胞。
Comparative study on the capacity of rat dental pulp stem cells and bone marrow mesenchymal stem cells in differentiating to osteoblast-like cells
Objective research on extraction of rat dental pulp stem cells (DPSCs) and bone marrow mesenchymal stem cells (BMSCs). To take observation of the differences in their morphology and growth curve. Staining to distinguish the stem cell's marker expression.Inducing DPSCs and BMSCs in differentiating to osteoblast-like cells. Taking observation of the expression of markers on osteoblast after inducing and the ability of bone-formation. Taking discussion of characteristics of rat dental pulp stem cells on differentiating to osteoblast-like cells.
Methods Rat bone marrow mesenchymal stem cells were obtained by centrifugation, the whole one marrow was cultivated in DMEM medium, taking morphologic observation by inverted microscope. Dental pulp stem cells were obtained by enzymatic digestion, takeing morphologic observation after proliferation in vitro. Scaning CD45 and CD90 expression on cells by flow cytometry (FCM), detecting the expression of STRO-1 by immunofluorescence growth curves of BMSCs and DPSCs were measured by MTT, taking comparison of cell growth. Both of DPSCs and BMSCs were induced to osteoblast-like cells The expression of OCN and DSP on cells were detected by immunofluorescence after 2-weeks-induction. calcific nodules which were secreted by DPSCs were stained by alizarin red after 4-weeks-induction
Results BMSCs obtained by centrifugation was purified after adhered cultivation, hematopoietic cells were removed gradually. DPSCs obtained by enzymatic digestion can be completely adherent in the second day and proliferated vigorously. Cells exhibited a negative expression of CD45 and a positive expression of CD90 which consisted with the characteristics of mesenchymal stem cells. The positive STRO-1 expression in inmmunofluorescence fitted with stem cells'feature. Growth curve shows that both DPSCs and BMSCs went into logarithmic phase in the fourth day. DPSCs had a higher proliferation than BMSCs. After 2-weeks-induction, both DPSCs and BMSCs express a positive OCN molecule, and the DSP expression was positive on DPSCs. The formation of calcific nodules could be proved by alizarin red staining at the bottle of DPSCs medium after 4-weeks-induction.
Conclusions BMSCs obtained by centrifugation could be purified by the whole bone marrow cells adhered cultivation, hematopoietic cells were removed by medium changes. The purity of BMSCs which was detected by FCM could be higher than 80% in P2. DPSCs obtained by enzymatic digestion consisted with the characteristics of mesenchymal stem cells and perform a higher proliferation ability. After mineralized induction, DPSCs differentiate into osteoblast-like cells just like BMSCs in the molecule expression, there were also calcific nodules at the bottle of DPSCs'medium. It means that both the proliferation ability and differentiation ability into osteoblast-like cells of DPSCs were brilliant, which offers a ideal cells for repairation of bone defect.
方法通过离心法获得大鼠骨髓细胞,全骨髓体外贴壁培养扩增,并通过倒置显微镜观察其形态；通过酶消化法处理大鼠牙髓组织获得牙髓干细胞,体外培养扩增,通过倒置显微镜观察形态。通过流式细胞仪检测所获得细胞表面CD45,CD90分子表达；通过免疫荧光染色检测细胞表面STRO-1表达；MTT法分别测得大鼠骨髓间充质干细胞与牙髓干细胞的生长曲线,对照分析,比较二者在生长扩增上的差异；分别对大鼠牙髓干细胞与骨髓间充质干细胞进行矿化诱导培养,2周后对其进行骨钙素(OCN),牙本质泌涎蛋白(DSP)免疫荧光染色；继续培养四周,对经诱导的大鼠牙髓干细胞进行茜素红染色观察钙结节形成情况。
结果通过离心法获得的大鼠骨髓细胞经过贴壁培养,换液逐渐将造血系细胞清除,得到相对纯度较高的骨髓间充质干细胞贴壁生长；通过酶消化法获得的大鼠牙髓干细胞第二天可完全贴壁生长；所获得的细胞经流式细胞仪检测CD45表达阴性,CD90表达阳性,符合间充质干细胞特性；免疫荧光染色STRO-1表达阳性,符合干细胞表达特性；MTT法绘制生长曲线可见大鼠牙髓干细胞与骨髓间充质干细胞第4天左右进入生长对数期,牙髓干细胞生长增殖速度快于骨髓间充质干细胞；经过矿化诱导2周后骨髓间充质干细胞与牙髓细胞表达成骨细胞表面标志物OCN阳性,牙髓干细胞表达牙本质细胞标志物DSP阳性；经4周矿化诱导培养,大鼠牙髓干细胞茜素红染色证实有矿化结节生成。
结论通过离心获得的骨髓细胞经全骨髓贴壁培养可随着贴壁换液的进行逐渐将造血系细胞清除获得纯度较高的骨髓间充质干细胞,其表面标志物检测证实纯度可达80%以上；酶消化法获得的大鼠牙髓干细胞表面标志物表达符合间充质干细胞特点,且生长扩增能力强；通过矿化诱导培养,牙髓干细胞可向成骨样细胞分化,能力与骨髓间充质干细胞相当,并可形成矿化结节。牙髓干细胞具有较强的增殖能力,并具有成骨样细胞分化能力,可以作为干细胞治疗骨组织缺损修复的理想种子细胞。
Comparative study on the capacity of rat dental pulp stem cells and bone marrow mesenchymal stem cells in differentiating to osteoblast-like cells
Objective research on extraction of rat dental pulp stem cells (DPSCs) and bone marrow mesenchymal stem cells (BMSCs). To take observation of the differences in their morphology and growth curve. Staining to distinguish the stem cell's marker expression.Inducing DPSCs and BMSCs in differentiating to osteoblast-like cells. Taking observation of the expression of markers on osteoblast after inducing and the ability of bone-formation. Taking discussion of characteristics of rat dental pulp stem cells on differentiating to osteoblast-like cells.
Methods Rat bone marrow mesenchymal stem cells were obtained by centrifugation, the whole one marrow was cultivated in DMEM medium, taking morphologic observation by inverted microscope. Dental pulp stem cells were obtained by enzymatic digestion, takeing morphologic observation after proliferation in vitro. Scaning CD45 and CD90 expression on cells by flow cytometry (FCM), detecting the expression of STRO-1 by immunofluorescence growth curves of BMSCs and DPSCs were measured by MTT, taking comparison of cell growth. Both of DPSCs and BMSCs were induced to osteoblast-like cells The expression of OCN and DSP on cells were detected by immunofluorescence after 2-weeks-induction. calcific nodules which were secreted by DPSCs were stained by alizarin red after 4-weeks-induction
Results BMSCs obtained by centrifugation was purified after adhered cultivation, hematopoietic cells were removed gradually. DPSCs obtained by enzymatic digestion can be completely adherent in the second day and proliferated vigorously. Cells exhibited a negative expression of CD45 and a positive expression of CD90 which consisted with the characteristics of mesenchymal stem cells. The positive STRO-1 expression in inmmunofluorescence fitted with stem cells'feature. Growth curve shows that both DPSCs and BMSCs went into logarithmic phase in the fourth day. DPSCs had a higher proliferation than BMSCs. After 2-weeks-induction, both DPSCs and BMSCs express a positive OCN molecule, and the DSP expression was positive on DPSCs. The formation of calcific nodules could be proved by alizarin red staining at the bottle of DPSCs medium after 4-weeks-induction.
Conclusions BMSCs obtained by centrifugation could be purified by the whole bone marrow cells adhered cultivation, hematopoietic cells were removed by medium changes. The purity of BMSCs which was detected by FCM could be higher than 80% in P2. DPSCs obtained by enzymatic digestion consisted with the characteristics of mesenchymal stem cells and perform a higher proliferation ability. After mineralized induction, DPSCs differentiate into osteoblast-like cells just like BMSCs in the molecule expression, there were also calcific nodules at the bottle of DPSCs'medium. It means that both the proliferation ability and differentiation ability into osteoblast-like cells of DPSCs were brilliant, which offers a ideal cells for repairation of bone defect.
引文
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