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
发布时间：2019-01-17 11:50
【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。
[Abstract]:Objective to investigate the morphology and growth of rat dental pulp stem cells (Dental Pulp Stem Cells,DPSCs) and bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Stem Cells, BMSCs),). Expression of rat dental pulp stem cell markers was observed by immunofluorescence staining. The differentiation ability of rat dental pulp stem cells and bone marrow mesenchymal stem cells into osteoblast-like cells was observed by mineralization induction, and the osteogenic markers on the surface were detected by immunofluorescence, and the ability of calcified nodule formation was observed. The differentiation ability and characteristics of osteoblast like cells of rat dental pulp stem cells were discussed. Methods Rat bone marrow cells were obtained by centrifugation. The whole bone marrow was amplified by adherent culture in vitro and its morphology was observed by inverted microscope. Dental pulp stem cells were obtained from rat dental pulp tissue by enzyme digestion and were cultured and amplified in vitro. Morphology was observed by inverted microscope. The expression of CD45,CD90 on the cell surface was detected by flow cytometry, and the expression of STRO-1 on the cell surface was detected by immunofluorescence staining. The growth curves of rat bone marrow mesenchymal stem cells and dental pulp stem cells were measured by MTT method. Rat dental pulp stem cells and bone marrow mesenchymal stem cells were mineralized and cultured, and then were stained with osteocalcin (OCN), dentin secreting sialoprotein (DSP) immunofluorescence staining after 2 weeks. After cultured for four weeks, calcium nodules were observed by alizarin red staining on induced rat dental pulp stem cells. Results the bone marrow cells of rats obtained by centrifugation were gradually removed from hematopoietic lineage cells through adherent culture, and bone marrow mesenchymal stem cells (BMSCs) with relatively high purity were obtained by adherent growth. Rat dental pulp stem cells obtained by enzyme digestion could completely adhere to the wall on the second day, and the obtained cells showed negative expression of CD45 and positive expression of CD90 by flow cytometry, which accorded with the characteristics of mesenchymal stem cells. The expression of STRO-1 was positive by immunofluorescence staining, which was consistent with the expression characteristics of stem cells. The growth curve of rat dental pulp stem cells and bone marrow mesenchymal stem cells entered the logarithmic phase on the 4th day, and the growth rate of dental pulp stem cells was faster than that of bone marrow mesenchymal stem cells. After 2 weeks of mineralization induction, bone marrow mesenchymal stem cells and dental pulp cells expressed positive expression of osteoblast surface marker OCN and dental pulp stem cells expressed dentin cell marker DSP. After 4 weeks of mineralization induced culture, the formation of mineralized nodules was confirmed by alizarin red staining of rat dental pulp stem cells. Conclusion Bone marrow mesenchymal stem cells with high purity can be obtained by centrifugation by whole bone marrow adherent culture. The purity of hematopoietic mesenchymal stem cells can be more than 80%. The expression of surface markers of rat dental pulp stem cells obtained by enzyme digestion was consistent with the characteristics of mesenchymal stem cells and the ability of growth and amplification was strong. Through mineralization induction and culture, dental pulp stem cells can differentiate into osteoblast-like cells, and have the same ability as bone marrow mesenchymal stem cells and form mineralized nodules. Dental pulp stem cells have strong proliferative ability and osteoblast-like cell differentiation ability, which can be used as the ideal seed cells for the treatment of bone tissue defects.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2410033
[Abstract]:Objective to investigate the morphology and growth of rat dental pulp stem cells (Dental Pulp Stem Cells,DPSCs) and bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Stem Cells, BMSCs),). Expression of rat dental pulp stem cell markers was observed by immunofluorescence staining. The differentiation ability of rat dental pulp stem cells and bone marrow mesenchymal stem cells into osteoblast-like cells was observed by mineralization induction, and the osteogenic markers on the surface were detected by immunofluorescence, and the ability of calcified nodule formation was observed. The differentiation ability and characteristics of osteoblast like cells of rat dental pulp stem cells were discussed. Methods Rat bone marrow cells were obtained by centrifugation. The whole bone marrow was amplified by adherent culture in vitro and its morphology was observed by inverted microscope. Dental pulp stem cells were obtained from rat dental pulp tissue by enzyme digestion and were cultured and amplified in vitro. Morphology was observed by inverted microscope. The expression of CD45,CD90 on the cell surface was detected by flow cytometry, and the expression of STRO-1 on the cell surface was detected by immunofluorescence staining. The growth curves of rat bone marrow mesenchymal stem cells and dental pulp stem cells were measured by MTT method. Rat dental pulp stem cells and bone marrow mesenchymal stem cells were mineralized and cultured, and then were stained with osteocalcin (OCN), dentin secreting sialoprotein (DSP) immunofluorescence staining after 2 weeks. After cultured for four weeks, calcium nodules were observed by alizarin red staining on induced rat dental pulp stem cells. Results the bone marrow cells of rats obtained by centrifugation were gradually removed from hematopoietic lineage cells through adherent culture, and bone marrow mesenchymal stem cells (BMSCs) with relatively high purity were obtained by adherent growth. Rat dental pulp stem cells obtained by enzyme digestion could completely adhere to the wall on the second day, and the obtained cells showed negative expression of CD45 and positive expression of CD90 by flow cytometry, which accorded with the characteristics of mesenchymal stem cells. The expression of STRO-1 was positive by immunofluorescence staining, which was consistent with the expression characteristics of stem cells. The growth curve of rat dental pulp stem cells and bone marrow mesenchymal stem cells entered the logarithmic phase on the 4th day, and the growth rate of dental pulp stem cells was faster than that of bone marrow mesenchymal stem cells. After 2 weeks of mineralization induction, bone marrow mesenchymal stem cells and dental pulp cells expressed positive expression of osteoblast surface marker OCN and dental pulp stem cells expressed dentin cell marker DSP. After 4 weeks of mineralization induced culture, the formation of mineralized nodules was confirmed by alizarin red staining of rat dental pulp stem cells. Conclusion Bone marrow mesenchymal stem cells with high purity can be obtained by centrifugation by whole bone marrow adherent culture. The purity of hematopoietic mesenchymal stem cells can be more than 80%. The expression of surface markers of rat dental pulp stem cells obtained by enzyme digestion was consistent with the characteristics of mesenchymal stem cells and the ability of growth and amplification was strong. Through mineralization induction and culture, dental pulp stem cells can differentiate into osteoblast-like cells, and have the same ability as bone marrow mesenchymal stem cells and form mineralized nodules. Dental pulp stem cells have strong proliferative ability and osteoblast-like cell differentiation ability, which can be used as the ideal seed cells for the treatment of bone tissue defects.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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