【摘要】：目的提取大鼠牙髓干細胞(Dental Pulp Stem Cells,DPSCs)與骨髓間充質(zhì)干細胞(Bone Marrow Mesenchymal Stem Cells, BMSCs),傳代培養(yǎng)觀察二者形態(tài)學,以及其生長差異。免疫熒光染色觀察大鼠牙髓干細胞標志物表達。通過礦化誘導,觀察大鼠牙髓干細胞與骨髓間充質(zhì)干細胞向成骨樣細胞分化能力,并通過免疫熒光對其進行表面成骨標志物檢測,觀察其鈣化結(jié)節(jié)形成能力,對比討論大鼠牙髓干細胞的成骨樣細胞分化能力與特點。 方法通過離心法獲得大鼠骨髓細胞,全骨髓體外貼壁培養(yǎng)擴增,并通過倒置顯微鏡觀察其形態(tài)；通過酶消化法處理大鼠牙髓組織獲得牙髓干細胞,體外培養(yǎng)擴增,通過倒置顯微鏡觀察形態(tài)。通過流式細胞儀檢測所獲得細胞表面CD45,CD90分子表達；通過免疫熒光染色檢測細胞表面STRO-1表達；MTT法分別測得大鼠骨髓間充質(zhì)干細胞與牙髓干細胞的生長曲線,對照分析,比較二者在生長擴增上的差異；分別對大鼠牙髓干細胞與骨髓間充質(zhì)干細胞進行礦化誘導培養(yǎng),2周后對其進行骨鈣素(OCN),牙本質(zhì)泌涎蛋白(DSP)免疫熒光染色；繼續(xù)培養(yǎng)四周,對經(jīng)誘導的大鼠牙髓干細胞進行茜素紅染色觀察鈣結(jié)節(jié)形成情況。 結(jié)果通過離心法獲得的大鼠骨髓細胞經(jīng)過貼壁培養(yǎng),換液逐漸將造血系細胞清除,得到相對純度較高的骨髓間充質(zhì)干細胞貼壁生長；通過酶消化法獲得的大鼠牙髓干細胞第二天可完全貼壁生長；所獲得的細胞經(jīng)流式細胞儀檢測CD45表達陰性,CD90表達陽性,符合間充質(zhì)干細胞特性；免疫熒光染色STRO-1表達陽性,符合干細胞表達特性；MTT法繪制生長曲線可見大鼠牙髓干細胞與骨髓間充質(zhì)干細胞第4天左右進入生長對數(shù)期,牙髓干細胞生長增殖速度快于骨髓間充質(zhì)干細胞；經(jīng)過礦化誘導2周后骨髓間充質(zhì)干細胞與牙髓細胞表達成骨細胞表面標志物OCN陽性,牙髓干細胞表達牙本質(zhì)細胞標志物DSP陽性；經(jīng)4周礦化誘導培養(yǎng),大鼠牙髓干細胞茜素紅染色證實有礦化結(jié)節(jié)生成。 結(jié)論通過離心獲得的骨髓細胞經(jīng)全骨髓貼壁培養(yǎng)可隨著貼壁換液的進行逐漸將造血系細胞清除獲得純度較高的骨髓間充質(zhì)干細胞,其表面標志物檢測證實純度可達80%以上；酶消化法獲得的大鼠牙髓干細胞表面標志物表達符合間充質(zhì)干細胞特點,且生長擴增能力強；通過礦化誘導培養(yǎng),牙髓干細胞可向成骨樣細胞分化,能力與骨髓間充質(zhì)干細胞相當,并可形成礦化結(jié)節(jié)。牙髓干細胞具有較強的增殖能力,并具有成骨樣細胞分化能力,可以作為干細胞治療骨組織缺損修復的理想種子細胞。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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