發(fā)布時間：2018-05-19 11:58

  本文選題：分離培養(yǎng)方法 + 破骨細(xì)胞��； 參考：《中國現(xiàn)代醫(yī)學(xué)雜志》2017年18期

【摘要】：目的建立一種簡便高效的體外分離大鼠骨髓單核細(xì)胞的方法,觀察其體外生長特性,并誘導(dǎo)其分化為破骨細(xì)胞。方法無菌條件下分離出SD大鼠的脛骨和股骨,使用環(huán)氧樹脂管(EP管)和移液槍頭快速分離骨髓組織,再用紅細(xì)胞裂解液去除紅細(xì)胞。細(xì)胞培養(yǎng)過夜后收集懸浮細(xì)胞,加入巨噬細(xì)胞集落刺激因子(M-CSF)繼續(xù)培養(yǎng)擴(kuò)增后得到貼壁的骨髓單核細(xì)胞。觀察骨髓單核細(xì)胞生長過程中的形態(tài)學(xué)特征;通過細(xì)胞計數(shù)試劑盒法(CCK-8)測繪細(xì)胞的生長曲線;用流式細(xì)胞儀檢測細(xì)胞表面CD11b的表達(dá);在加入M-CSF和核因子κB受體活化因子配基(RANKL)誘導(dǎo)分化后,用抗酒石酸酸性磷酸酶(TRAP)染色和降鈣素受體(CTR)免疫熒光染色鑒定其是否能分化為成熟破骨細(xì)胞。結(jié)果通過該方法獲得的大鼠骨髓單核細(xì)胞培養(yǎng)1 d后基本呈小圓形,雜細(xì)胞較少。3 d后細(xì)胞數(shù)目稍多,兩端開始出現(xiàn)觸角,5 d后數(shù)目增多,細(xì)胞呈橢圓形,兩端觸角明顯;細(xì)胞的增殖依賴于M-CSF;流式結(jié)果顯示,通過此方法獲得的單核細(xì)胞純度較高;TRAP染色和CTR免疫熒光染色結(jié)果提示,通過該方法獲得的單核細(xì)胞可以誘導(dǎo)為成熟破骨細(xì)胞。結(jié)論通過EP管和移液槍頭裝置可快速分離獲取單核細(xì)胞,獲得的細(xì)胞表型穩(wěn)定,適合用于骨代謝疾病的進(jìn)一步研究。
[Abstract]:Objective to establish a simple and efficient method for isolating rat bone marrow monocytes in vitro and to observe their growth characteristics in vitro and induce them to differentiate into osteoclasts. Methods the tibia and femur of SD rats were isolated under aseptic condition. The bone marrow tissue was separated quickly by using epoxy resin tube and EP tube. Then the erythrocyte was removed by erythrocyte lysis solution. Suspension cells were collected after overnight cell culture, and macrophage colony stimulating factor (M-CSF) was added to obtain adherent bone marrow monocytes. Morphological characteristics of bone marrow monocytes were observed, cell growth curve was measured by cell count kit (CCK-8), CD11b expression on cell surface was detected by flow cytometry. After induction of differentiation by adding M-CSF and nuclear factor- 魏 B receptor activator ligand (RANKL), the differentiation of osteoclasts was identified by tartrate-resistant acid phosphatase staining and calcitonin receptor immunofluorescence staining. Results the monocytes of rat bone marrow obtained by this method were basically small round after 1 day culture, the number of cells was slightly more after less than 3 days, the number of cells increased after 5 days, the number of cells was oval, and the antennae of both ends were obvious. The results of flow cytometry showed that the monocytes obtained by this method were highly purified by trap staining and CTR immunofluorescence staining, which indicated that the monocytes obtained by this method could be induced into mature osteoclasts. Conclusion Mononuclear cells can be rapidly isolated and obtained by EP tube and liquid transfer device. The obtained cells have stable phenotypes and are suitable for further study of bone metabolic diseases.
【作者單位】： 南京中醫(yī)藥大學(xué)骨傷研究所(骨傷修復(fù)與重建新技術(shù)實驗室);南京中醫(yī)藥大學(xué)附屬醫(yī)院骨傷科;
【基金】：國家自然科學(xué)基金面上項目(No:81473692) 江蘇省自然科學(xué)基金青年基金項目(No:BK20151007)
【分類號】：R329.2

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