东南大学学报(医学版)
東南大學學報(醫學版)
동남대학학보(의학판)
Journal of Southeast University (Medical Science Edition)
2015年
5期
684-688
,共5页
石任任%王珊%何颖%邹泽红%张可俊%李林梅%肖春梅%关志澳%陶爱林
石任任%王珊%何穎%鄒澤紅%張可俊%李林梅%肖春梅%關誌澳%陶愛林
석임임%왕산%하영%추택홍%장가준%리림매%초춘매%관지오%도애림
肥大细胞%骨髓%组胺检测%诱导培养%小鼠
肥大細胞%骨髓%組胺檢測%誘導培養%小鼠
비대세포%골수%조알검측%유도배양%소서
mast cell%bone marrow%histamine detection%induced culture%mice
目的:探讨体外诱导培养小鼠骨髓细胞来源肥大细胞的方法,为体外组胺激发试验提供试材。方法:采用白细胞介素3(IL-3)和干细胞因子(SCF)联合刺激小鼠骨髓细胞4、6周诱导其分化为肥大细胞,光镜下观察细胞形态,甲苯胺蓝染色观察细胞成熟度,蛋白免疫印迹法分析表面 FcεRⅠ和CD117表达情况, HistaReader 501组胺仪检测细胞脱颗粒能力。结果:诱导培养4、6周后的细胞为大小均一且具折光性的圆形悬浮细胞;甲苯胺蓝染色显示培养4、6周后的细胞胞核染为蓝色,胞质含有丰富的粗大紫红色颗粒,镜下统计成熟肥大细胞比例达85%以上;蛋白免疫印迹法分析发现培养4、6周后的细胞皆表达FcεRⅠ和CD117,其中培养6周的细胞FcεRⅠ表达水平更高;组胺仪检测培养6周的细胞发现,经浓度为100、500、1000μmol· L -1的钙离子载体刺激后,与阴性对照组相比,细胞组胺释放量依次为3.86%、17.04%及23.47%( P<0.05),其脱颗粒具有钙离子载体浓度依赖性。结论:获得大量高纯度的小鼠骨髓来源肥大细胞,可促进后期的组胺激发试验及Ⅰ型变态反应机制研究。
目的:探討體外誘導培養小鼠骨髓細胞來源肥大細胞的方法,為體外組胺激髮試驗提供試材。方法:採用白細胞介素3(IL-3)和榦細胞因子(SCF)聯閤刺激小鼠骨髓細胞4、6週誘導其分化為肥大細胞,光鏡下觀察細胞形態,甲苯胺藍染色觀察細胞成熟度,蛋白免疫印跡法分析錶麵 FcεRⅠ和CD117錶達情況, HistaReader 501組胺儀檢測細胞脫顆粒能力。結果:誘導培養4、6週後的細胞為大小均一且具摺光性的圓形懸浮細胞;甲苯胺藍染色顯示培養4、6週後的細胞胞覈染為藍色,胞質含有豐富的粗大紫紅色顆粒,鏡下統計成熟肥大細胞比例達85%以上;蛋白免疫印跡法分析髮現培養4、6週後的細胞皆錶達FcεRⅠ和CD117,其中培養6週的細胞FcεRⅠ錶達水平更高;組胺儀檢測培養6週的細胞髮現,經濃度為100、500、1000μmol· L -1的鈣離子載體刺激後,與陰性對照組相比,細胞組胺釋放量依次為3.86%、17.04%及23.47%( P<0.05),其脫顆粒具有鈣離子載體濃度依賴性。結論:穫得大量高純度的小鼠骨髓來源肥大細胞,可促進後期的組胺激髮試驗及Ⅰ型變態反應機製研究。
목적:탐토체외유도배양소서골수세포래원비대세포적방법,위체외조알격발시험제공시재。방법:채용백세포개소3(IL-3)화간세포인자(SCF)연합자격소서골수세포4、6주유도기분화위비대세포,광경하관찰세포형태,갑분알람염색관찰세포성숙도,단백면역인적법분석표면 FcεRⅠ화CD117표체정황, HistaReader 501조알의검측세포탈과립능력。결과:유도배양4、6주후적세포위대소균일차구절광성적원형현부세포;갑분알람염색현시배양4、6주후적세포포핵염위람색,포질함유봉부적조대자홍색과립,경하통계성숙비대세포비례체85%이상;단백면역인적법분석발현배양4、6주후적세포개표체FcεRⅠ화CD117,기중배양6주적세포FcεRⅠ표체수평경고;조알의검측배양6주적세포발현,경농도위100、500、1000μmol· L -1적개리자재체자격후,여음성대조조상비,세포조알석방량의차위3.86%、17.04%급23.47%( P<0.05),기탈과립구유개리자재체농도의뢰성。결론:획득대량고순도적소서골수래원비대세포,가촉진후기적조알격발시험급Ⅰ형변태반응궤제연구。
Objective:To investigate induced culture methods of bone marrow-derived mast cells(BMMCs) to provide materials for allergen challenge assay.Methods: BMMCs were differentiated from bone marrow cells cultured for 4 to 6 weeks with interleukin-3(IL-3) and stem cell factor (SCF) supplemented.The cell features were characterized by light microscopy and toluidine blue staining.The maturity of the cells was investigated by analyzing the membrane markers FcεRⅠand CD117 by Western blotting, and potency of BMMCs was calculated through histamine release assay by HistaReader 501 instrument.Results: The induced BMMCs, all in uniformed size with refraction, were observed to be filled with typical purple granules.The mature mast cells accounted for more than 85% of all cells.FcεRⅠand CD117 were positively expressed in BMMCs and FcεRⅠwere more expressive after 6 weeks of culture.The rate of histamine release by BMMCs cultured for 6 weeks were 3.86%, 17.04%and 23.47% ( P <0.05 ) after stimulated with calcium ionophore at concentrations of 100 , 500 and 1 000 μmol· L -1 , compared to control group.Conclusion:A large amount of highly purified BMMCs have been obtained, which will facilitate the subsequent allergen challenge assay and mechanism research on typeⅠallergy.