【摘要】：目的：一、研究脈沖電磁場(puslsed electromagnetic fields,PEMFs)對成骨細(xì)胞(Osteoblast, OB)的作用,探究PEMFs治療骨質(zhì)疏松的的作用機(jī)理。二、研究PEMFs對造血干細(xì)胞龕中的主要組成成分——ALCAM+Sca-1-成骨細(xì)胞的作用,探究PEMFs對造血干細(xì)胞歸巢的作用。 方法：一、取新生SD大鼠頭蓋骨,通過骨組織塊培養(yǎng)法分離、培養(yǎng)成骨細(xì)胞。取傳代第三代細(xì)胞分為5組：1-70組、2-70組、3-70組、4-70組、對照組,其中前4組為作用組,分別接受1mT,70Hz；2mT,70Hz；3mT,70Hz；4mT,70Hz的PEMFs作用,對照組不接受PEMFs作用,同PEMFs組一致置于相同環(huán)境下、磁場作用距離外。按照分組劑量進(jìn)行離體PEMFs作用(0.5h/d,連續(xù)作用4d),作用結(jié)束后,對各組分別進(jìn)行細(xì)胞堿性磷酸酶(alkaline phosphatase, ALP)活性、核結(jié)合因子α1(core-binding factorα1, cbfα1/RUNX2)表達(dá)、體外成骨能力指標(biāo)——礦化結(jié)節(jié)(mineralized nodules)形成能力檢測。 二、24只C57/6小鼠隨機(jī)分為兩組：PEMF組和對照組,每組12只小鼠。PEMF組小鼠接受PEMFs作用,劑量為2mT,70Hz,1h/次,1次/d,對照組不接受PEMFs作用,同PEMFs組一致置于相同環(huán)境下、磁場作用距離外,1h次,1次/d。連續(xù)作用4w。作用結(jié)束后,兩組分別取股骨、脛骨,沖出骨髓(bone marrow, BM)后,研磨至碎片,經(jīng)膠原酶消化,收集細(xì)胞經(jīng)淋巴細(xì)胞分離液分離,磁珠系統(tǒng)分選出CD31-CD45-TER119-細(xì)胞,再經(jīng)流式細(xì)胞儀分選ALCAM+Sca-1-成骨細(xì)胞。分選出的ALCAM+Sca-1-成骨細(xì)胞經(jīng)TRIzol法提取Total RNA,逆轉(zhuǎn)錄,基因芯片雜交,洗脫,檢測,進(jìn)行數(shù)據(jù)分析。 結(jié)果：一、經(jīng)PEMFs作用后,與對照組相比,成骨細(xì)胞ALP活性降低,cbfα1表達(dá)增多,礦化結(jié)節(jié)數(shù)量顯著增多。 二、經(jīng)PEMFs作用后,PEMF組與對照組相比,ALCAM+Sca-1-.成骨細(xì)胞的基因上調(diào)表達(dá)成骨細(xì)胞功能相關(guān)基因,與造血干細(xì)胞歸巢相關(guān)的細(xì)胞粘附分子、趨化分子基因,調(diào)節(jié)造血干細(xì)胞的細(xì)胞因子基因。結(jié)論：一、PEMFs作用成骨細(xì)胞可促進(jìn)成骨細(xì)胞功能分化,增強(qiáng)成骨細(xì)胞的細(xì)胞外 基質(zhì)礦化。 二、PEMFs作用促使造血干細(xì)胞龕中的主要組成成分——ALCAM+Sca-1-成骨細(xì)胞上調(diào)表達(dá)造血干細(xì)胞歸巢相關(guān)基因,在基因水平上推斷PEMFs有助于造血干細(xì)胞歸巢重建造血功能。
[Abstract]:Objective: to investigate the effect of pulsed electromagnetic field (puslsed electromagnetic fields,PEMFs) on (Osteoblast, OB) of osteoblasts and explore the mechanism of PEMFs in the treatment of osteoporosis. Secondly, to study the effect of PEMFs on ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, and to explore the effect of PEMFs on homing of hematopoietic stem cell. Methods: firstly, osteoblasts were isolated from the skull of newborn SD rats by bone mass culture. The third passage cells were divided into 5 groups: 1-70 group, 2-70 group, 3-70 group, 4-70 group, and control group. The first four groups were treated with 1mTTn70Hzn2mT70Hzn2mT70HzHzO3mTZN 4mTzTn70 Hz PEMFs, the control group was not treated with PEMFs, and was placed in the same environment as PEMFs group, the former group was treated in the same environment, and the control group was treated in the same environment as the PEMFs group. The magnetic field acts outside the range. PEMFs was treated in vitro (0.5 h / d for 4 days) according to the group dose. The alkaline phosphatase (alkaline phosphatase, ALP) activity and the expression of core-binding factor 偽 1 (cbf 偽 1/RUNX2) were observed in each group after the treatment. The ability of mineralized nodules to form (mineralized nodules) was measured as an index of osteogenic ability in vitro. Second, 24 C57 / 6 mice were randomly divided into two groups: PEMF group and control group, 12 mice in each group. The mice in PEMF group were treated with PEMFs at a dose of 2mTTn70Hz1, once a day. The control group was not treated with PEMFs, and was placed in the same environment as PEMFs group. Outside the magnetic field, 1 time, 1 time per day. Continuous action for 4 weeks. After the treatment, femur, tibia, bone marrow (bone marrow, BM) were washed out, then ground to pieces, digested by collagenase, collected cells were separated by lymphocytes, and CD31-CD45-TER119- cells were separated by magnetic beads system. ALCAM Sca-1- osteoblasts were separated by flow cytometry. The selected ALCAM Sca-1- osteoblasts were extracted Total RNA, reverse transcription, gene chip hybridization, elution, detection and data analysis by TRIzol. Results: first, compared with the control group, the ALP activity of osteoblasts decreased, the expression of cbf 偽 1 increased, and the number of mineralized nodules increased significantly after PEMFs treatment. Second, after treated with PEMFs, ALCAM Sca-1-. in PEMF group was higher than that in control group. The genes of osteoblasts up-regulate the expression of osteoblast function-related genes, the cell adhesion molecules and chemotactic molecules associated with homing of hematopoietic stem cells, and the cytokine genes of hematopoietic stem cells. Conclusion: firstly, PEMFs can promote the differentiation of osteoblasts and enhance the extracellular matrix mineralization of osteoblasts. Secondly, ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, was induced by PEMFs to up-regulate the expression of homing related genes of hematopoietic stem cells. It was concluded that PEMFs could help hematopoietic stem cells to homing and reconstructing hematopoietic function at the gene level.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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本文編號(hào)：2290661