【摘要】：目的:造血干細(xì)胞(Hematopoietic stem cell HSC)目前被廣泛用于移植治療惡性血液系統(tǒng)疾病,采集足夠量的造血干細(xì)胞是移植治療成功的關(guān)鍵前提,近年研究明確,骨髓微環(huán)境中存在著兩種以上的骨髓龕,其中骨內(nèi)膜龕對(duì)維持造血干細(xì)胞的穩(wěn)定靜止起著重要作用,而成骨細(xì)胞是骨內(nèi)膜龕的重要組成部分,大量實(shí)驗(yàn)表明,G-CSF動(dòng)員后成骨細(xì)胞數(shù)量減少,功能減低,破骨細(xì)胞作為與成骨細(xì)胞相反作用的細(xì)胞,在動(dòng)員過(guò)程中發(fā)生什么變化,值得我們?nèi)パ芯?本研究以小鼠為研究對(duì)象,觀察G-CSF動(dòng)員對(duì)小鼠成骨、破骨細(xì)胞的影響,以及通過(guò)PTH誘導(dǎo)激活破骨細(xì)胞,觀察對(duì)造血干細(xì)胞動(dòng)員的影響。方法:利用流式細(xì)胞計(jì)數(shù)檢測(cè)動(dòng)員0、3、5天小鼠外周血干細(xì)胞比例,觀察G-CSF誘導(dǎo)動(dòng)員的情況,通過(guò)實(shí)時(shí)定量-多聚酶鏈?zhǔn)椒磻?yīng)(RQ-PCR)檢測(cè)動(dòng)員前后骨髓細(xì)胞骨鈣素(Osteocalcin OCN)、基質(zhì)細(xì)胞衍生因子-1(Stromal cell-derived factor-1 SDF-1)和基質(zhì)金屬蛋白酶9(Matrix metalloproteinases9 MMP9)的m RNA表達(dá),免疫組織化學(xué)檢測(cè)骨髓OCN(成骨細(xì)胞特異性表達(dá))陽(yáng)性細(xì)胞的數(shù)量來(lái)比較動(dòng)員前后成骨細(xì)胞的數(shù)量和功能上的變化,ELISA法檢測(cè)小鼠周血TRACP-5b的濃度反應(yīng)破骨細(xì)胞活性,抗酒石酸磷酸酶(tartrate-resistant acid phosphatase TRAP)染色檢測(cè)小鼠股骨中破骨細(xì)胞數(shù)量,評(píng)價(jià)G-CSF動(dòng)員對(duì)成骨、破骨細(xì)胞的影響。大劑量PTH(Parathyroid hormone甲狀旁腺素)激活破骨細(xì)胞,觀察激活破骨細(xì)胞后能否產(chǎn)生HSC動(dòng)員效應(yīng)。結(jié)果:1.骨髓SDF-1和OCN表達(dá)減少,提示成骨細(xì)胞功能降低;MMP9表達(dá)增高,提示中性粒細(xì)胞釋放MMP9,骨髓基質(zhì)發(fā)生蛋白水解。2.顯微鏡下觀察動(dòng)員后骨髓成骨細(xì)胞數(shù)量減少,形態(tài)由原來(lái)卵圓形變?yōu)樗笮沃帘馄较А?.小鼠外周血TRACP-5b增多,0天(4.43±0.52IU/L)、5天(10.49±1.75m IU/m L),P0.05,提示破骨細(xì)胞活性增高;TRAP染色提示注射G-CSF后破骨細(xì)胞數(shù)量增多。4.大劑量PTH激活破骨細(xì)胞能夠產(chǎn)生HSC動(dòng)員效應(yīng)。結(jié)論G-CSF動(dòng)員后成骨細(xì)胞數(shù)量及活性降低,導(dǎo)致SDF-1趨化能力減弱,破壞造血干細(xì)胞靜止?fàn)顟B(tài)引發(fā)動(dòng)員;破骨細(xì)胞激活后可能與成骨細(xì)胞相互作用影響動(dòng)員發(fā)生。
[Abstract]:Objective: hematopoietic stem cell (Hematopoietic stem cell HSC) has been widely used in the treatment of malignant hematological diseases. The acquisition of sufficient amount of hematopoietic stem cells is the key prerequisite for the successful treatment of hematopoietic stem cells. There are more than two kinds of bone marrow niches in bone marrow microenvironment, in which endomembrane niches play an important role in maintaining the stability of hematopoietic stem cells, and osteoblasts are important components of osteoblast niches. A large number of experiments showed that the number and function of osteoblasts decreased after G-CSF mobilization. The changes of osteoclasts in the mobilization process of osteoclasts, which are opposite to those of osteoblasts, are worthy of our study. To observe the effect of G-CSF mobilization on osteogenesis and osteoclast in mice, and to observe the effect of PTH on the mobilization of hematopoietic stem cells. Methods: the percentage of peripheral blood stem cells (PBSCs) in mice mobilized for 3 days was detected by flow cytometry, and the mobilization induced by G-CSF was observed. The expression of osteocalcin (Osteocalcin OCN), stromal cell derived factor 1 (Stromal cell-derived factor-1 SDF-1) and matrix metalloproteinase 9 (Matrix metalloproteinases9 MMP9) in bone marrow cells before and after mobilization was detected by real-time quantification-polymerase chain reaction (RQ-PCR). The number of bone marrow OCN (osteoblast specific expression) positive cells was detected by immunohistochemistry to compare the changes in the number and function of osteoblasts before and after mobilization. Elisa was used to detect the concentration of TRACP-5b in peripheral blood of mice, which reflected the osteoclast activity. Anti-tartrate phosphatase (tartrate-resistant acid phosphatase TRAP) staining was used to detect the number of osteoclasts in the femur of mice and to evaluate the effect of G-CSF mobilization on osteoblasts and osteoclasts. High dose PTH (Parathyroid hormone parathyroid hormone (PTH (Parathyroid hormone) activated osteoclasts to observe whether HSC mobilization could be produced after activation of osteoclasts. The result is 1: 1. The decreased expression of SDF-1 and OCN in bone marrow suggested that the function of osteoblasts decreased and the expression of MMP9 increased, suggesting that neutrophils released MMP9, bone marrow stromal proteolysis. 2. Under microscope, the number of bone marrow osteoblasts decreased and the shape of bone marrow osteoblasts changed from oval to fusiform to flat disappearance. 3. The increase of TRACP-5b in peripheral blood of mice was (4.43 鹵0.52IU/L) / 5d (10.49 鹵1.75m IU/m / L) P0.05, suggesting that the increased activity of osteoclasts and trap staining suggested that the number of osteoclasts increased after G-CSF injection. High dose PTH activates osteoclasts and produces HSC mobilization. Conclusion the number and activity of osteoblasts decreased after G-CSF mobilization, which resulted in the decrease of chemotactic ability of SDF-1, which resulted in the mobilization of osteoclasts after the activation of osteoclasts, and the activation of osteoclasts may influence the mobilization of osteoblasts.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

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本文編號(hào)：2224913