【摘要】：目的建立穩(wěn)定的成年小鼠骨細(xì)胞分離和鑒定方法,檢測其標(biāo)志性基因和成纖維細(xì)胞生長因子受體(fibroblast growth factor receptor,FGFR)表達(dá)。方法將3月齡C57小鼠的脛骨和股骨分離后剪除干骺端,沖去骨髓,切割成約1 mm碎片,用Ⅰ型膠原酶和EDTA溶液交替消化以獲取骨細(xì)胞。分別通過形態(tài)觀察、ALP染色、OC染色和E11/gp38染色鑒定骨細(xì)胞。qRT-PCR檢測骨細(xì)胞標(biāo)志性基因表達(dá),并與骨細(xì)胞系IDG-SW3和MLO-Y4比較。檢測FGFRs在骨細(xì)胞的表達(dá)。結(jié)果分離出的骨細(xì)胞呈多樹突狀或星狀,并借突觸彼此連接。ALP染色可見成骨細(xì)胞染色陽性,骨細(xì)胞染色陰性。OC染色成骨細(xì)胞和骨細(xì)胞均陽性,成纖維細(xì)胞陰性,而E11/gp38染色僅見骨細(xì)胞染色陽性。實(shí)驗(yàn)所得細(xì)胞表達(dá)骨細(xì)胞特異基因DMP1、SOST、FGF23,表明骨細(xì)胞成功分離,且此原代骨細(xì)胞與IDG-SW3和MLOY4細(xì)胞系表達(dá)有差異。FGFR1~3在骨細(xì)胞中均有表達(dá)。結(jié)論成功分離成年小鼠原代骨細(xì)胞,發(fā)現(xiàn)FGFR1~3在原代骨細(xì)胞中均有表達(dá)。
[Abstract]:Objective to establish a stable method for the isolation and identification of adult mouse bone cells and to detect the expression of its iconic gene and fibroblast growth factor receptor (fibroblast growth factor receptor,FGFR). Methods the tibia and femur of 3-month-old C57 mice were separated and the metaphysis was cut off, then the bone marrow was cut off and cut into about 1 mm fragment. The bone cells were digested alternately with type I collagenase and EDTA solution to obtain bone cells. Bone cells were identified by morphological observation, ALP staining, OC staining and E11/gp38 staining, respectively. QRT-PCR was used to detect the expression of symbolic genes in bone cells and compared with bone cell lines IDG-SW3 and MLO-Y4. The expression of FGFRs in bone cells was detected. Results the isolated osteocytes were dendritic or stellate and connected with each other by synapses. ALP staining showed that osteoblasts were positive, osteoblasts were negative, OC staining was positive for osteoblasts and osteocytes, and fibroblasts were negative. E11/gp38 staining was only positive for bone cells. The expression of osteocyte specific gene DMP1,SOST,FGF23, indicated that osteocytes were isolated successfully, and the expression of FGFR1~3 was different from that of IDG-SW3 and MLOY4 cell lines. FGFR1~3 was expressed in all bone cells. Conclusion the primary bone cells of adult mice were successfully isolated and the expression of FGFR1~3 was found in primary bone cells.
【作者單位】： 第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所:創(chuàng)傷實(shí)驗(yàn)室 骨代謝修復(fù)中心 創(chuàng)傷燒傷與復(fù)合傷國家重點(diǎn)實(shí)驗(yàn)室;蘭州軍區(qū)蘭州總醫(yī)院急診科;第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所康復(fù)科;
【基金】：國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃,2014CB942904) 國家自然科學(xué)基金國際合作與交流項(xiàng)目(81220108020) 國家自然科學(xué)基金重點(diǎn)項(xiàng)目(81030036) 國家自然科學(xué)基金面上項(xiàng)目(81471092)~~
【分類號】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 朱瑩;戚華兵;王權(quán);王曉鳳;黃啟釗;陳林;;構(gòu)建FGFR3基因沉默慢病毒載體及其對ATDC5細(xì)胞增殖和分化的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2013年11期

2 李曉剛;蘇楠;唐玉彬;溫軒;唐浚洲;陳思宇;李燦;杜曉蘭;陳林;;FGF2對破骨細(xì)胞體外分化以及骨吸收功能的直接調(diào)控作用[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2014年07期

【共引文獻(xiàn)】

相關(guān)期刊論文 前5條

1 谷國良;KalervoH. V，

本文編號：2342087