国际呼吸杂志
國際呼吸雜誌
국제호흡잡지
INTERNATIONAL JOURNAL OF RESPIRATION
2012年
8期
569-573
,共5页
姜迪譞%胡瑞成%戴爱国%谭双香
薑迪譞%鬍瑞成%戴愛國%譚雙香
강적현%호서성%대애국%담쌍향
Ⅱ型肺泡上皮细胞%原代培养%传代培养%表型%分化
Ⅱ型肺泡上皮細胞%原代培養%傳代培養%錶型%分化
Ⅱ형폐포상피세포%원대배양%전대배양%표형%분화
Alveolar type Ⅱ epithelial cells%Primary culture%Subculture%Phenotype%Differentiation
目的 通过对离体培养的原代细胞和第3代Ⅱ型肺泡上皮细胞(alveolar typeⅡepithelial cells,AECⅡ)进行分化表型鉴定,探索AECⅡ离体传代培养的可行性.方法 联合应用肺循环灌注、支气管肺泡灌洗、双酶联合螯合剂灌注消化、物理过滤、差速离心、贴壁纯化、免疫吸附等方法分离和纯化大鼠AECⅡ.纯化的AECⅡ接种于基质胶包被的培养皿,在含10μg/L角质细胞生长因子、5％胎牛血清的DMEM/F12培养基中进行离体培养.收集原代和第3培养细胞,透射电镜观察细胞超微结构,免疫细胞化学染色检测肺泡表面活性蛋白A(surfactant protein A,SP-A)表达情况,并进行碱性磷酸酶染色鉴定细胞分化表型.结果 AECⅡ产量为(21.3±5.8)×106/鼠,活细胞比例为(96.5±0.8)％.细胞培养至第3代,其形态、生长特征与原代培养细胞无明显差异.透射电镜观察证实原代细胞及第3代细胞均具备AECⅡ的特征性结构,SP-A免疫细胞化学染色显示原代细胞及第3代细胞阳性比例均超过95％,碱性磷酸酶染色显示原代细胞及第3代细胞阳性比例分别为(85.2±3.7)％和(90.4±4.2)％.结论 在适宜的培养条件下,离体培养的第3代AECⅡ能够维持相对稳定的分化表型,可以用于离体实验.
目的 通過對離體培養的原代細胞和第3代Ⅱ型肺泡上皮細胞(alveolar typeⅡepithelial cells,AECⅡ)進行分化錶型鑒定,探索AECⅡ離體傳代培養的可行性.方法 聯閤應用肺循環灌註、支氣管肺泡灌洗、雙酶聯閤螯閤劑灌註消化、物理過濾、差速離心、貼壁純化、免疫吸附等方法分離和純化大鼠AECⅡ.純化的AECⅡ接種于基質膠包被的培養皿,在含10μg/L角質細胞生長因子、5％胎牛血清的DMEM/F12培養基中進行離體培養.收集原代和第3培養細胞,透射電鏡觀察細胞超微結構,免疫細胞化學染色檢測肺泡錶麵活性蛋白A(surfactant protein A,SP-A)錶達情況,併進行堿性燐痠酶染色鑒定細胞分化錶型.結果 AECⅡ產量為(21.3±5.8)×106/鼠,活細胞比例為(96.5±0.8)％.細胞培養至第3代,其形態、生長特徵與原代培養細胞無明顯差異.透射電鏡觀察證實原代細胞及第3代細胞均具備AECⅡ的特徵性結構,SP-A免疫細胞化學染色顯示原代細胞及第3代細胞暘性比例均超過95％,堿性燐痠酶染色顯示原代細胞及第3代細胞暘性比例分彆為(85.2±3.7)％和(90.4±4.2)％.結論 在適宜的培養條件下,離體培養的第3代AECⅡ能夠維持相對穩定的分化錶型,可以用于離體實驗.
목적 통과대리체배양적원대세포화제3대Ⅱ형폐포상피세포(alveolar typeⅡepithelial cells,AECⅡ)진행분화표형감정,탐색AECⅡ리체전대배양적가행성.방법 연합응용폐순배관주、지기관폐포관세、쌍매연합오합제관주소화、물리과려、차속리심、첩벽순화、면역흡부등방법분리화순화대서AECⅡ.순화적AECⅡ접충우기질효포피적배양명,재함10μg/L각질세포생장인자、5％태우혈청적DMEM/F12배양기중진행리체배양.수집원대화제3배양세포,투사전경관찰세포초미결구,면역세포화학염색검측폐포표면활성단백A(surfactant protein A,SP-A)표체정황,병진행감성린산매염색감정세포분화표형.결과 AECⅡ산량위(21.3±5.8)×106/서,활세포비례위(96.5±0.8)％.세포배양지제3대,기형태、생장특정여원대배양세포무명현차이.투사전경관찰증실원대세포급제3대세포균구비AECⅡ적특정성결구,SP-A면역세포화학염색현시원대세포급제3대세포양성비례균초과95％,감성린산매염색현시원대세포급제3대세포양성비례분별위(85.2±3.7)％화(90.4±4.2)％.결론 재괄의적배양조건하,리체배양적제3대AECⅡ능구유지상대은정적분화표형,가이용우리체실험.
Objective To exploration the feasibility of alveolar type Ⅱ epithelial cells (AEC Ⅱ ) in vitro subculture by identification the differentiation phenotype of primary and 3rd generation cells.Methods Rat AEC Ⅱ were isolated and purified by sequent processes composed with lung circulation perfusion,bronchoalveolar lavage,dual-enzyme and chelator infusion digestion,physical filtration,differential centrifugation,adherent purification,immunoadsorption. Purified AEC Ⅱ then were inoculated into Matrigel-coated culture dishes,and in vitro cultured by DMEM/F12 medium containing 10 μg/Lkeratinocyte growth factor and 5％ fetal bovine serum.The primary and the 3rd generation cells were collected for differentiation phenotype identification by transmission electron microscope observe cell ultrastructure,immunocytochemical detection surfactant protein A (SP-A) expression and the alkaline phosphatase staining appraisement.Results The total yields of AEC Ⅱ were (21.3±5.8))× 106 cells per rat,and the proportion of living cells reached to (96.5 ± 0.8 ) ％.The morphology and growth characteristics of the 3rd generation cells were not obvioursly different from the primary cultured cells.Transmission electron microscope confirmed that the 3rd generation cells possess AEC Ⅱ characteristic ultrastructure as well as the primary cells.SP-A immunocytochemical staining demonstrated that the proportions of positive cells for the primary and the 3rd generation exceeded 95％,alkaline phosphatase staining positive rates of primary cells and 3rd generation cell were (85.2±3.7) ％ and (90.4±4.2) ％respectively.Conclusions As supportted with appropriate in vitro culture conditions,the 3rd generation AEC Ⅱ is capable to maintain relatively stable diferentiation phenotype,and could be be used in in vitro experiments.
