胎鼠肺泡Ⅱ型上皮细胞生长状况及ABCA3表达变化的初步研究
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国家自然科学基金(81270725)


Preliminary study of the fetal rat alveolar type Ⅱ epithelial cell growth and ABCA3 expression changes
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    摘要:

    目的:观察原代培养胎鼠肺泡Ⅱ型上皮细胞(type Ⅱ alveolar epithelial cell,AECⅡ) 的生长状况及AECⅡ特异性蛋白ATP结合盒转运蛋白A3(ATP binding cassette transporter A3,ABCA3)的表达变化,为有关新生儿肺发育及肺部疾病的研究奠定一定基础-方法:采用胰酶联合胶原酶消化肺组织,差速离心及差速贴壁法纯化细胞;电子显微镜鉴定AECⅡ细胞;倒置相差显微镜观察不同时间点细胞生长状态;MTT法绘制细胞生长曲线;免疫荧光技术观察 ABCA3动态表达变化-结果:电镜下观察到AECⅡ特征性板层小体,大小及数量不等,胞膜外缘分布有刺状微绒毛;MTT法测得铺板后第24-36-48-72-96-120 h的吸光度值分别为0.177 ± 0.009,0.193 ± 0.011,0.212 ± 0.019,0.253 ± 0.019,0.243 ± 0.012,0.192 ± 0.011-光学显微镜下细胞形态逐渐拉长,变扁平,细胞内颗粒逐渐减少-免疫荧光示ABCA3表达逐渐下降-结论:原代培养的胎鼠AECⅡ在培养早期生长状况最佳,ABCA3表达最丰富,故在早期(即培养72 h以内)进行肺发育疾病相关机制的研究较为理想-

    Abstract:

    Objective:To observe the growth of type Ⅱ alveolar epithelial cell(AECⅡ) and dynamic expression of AECⅡ specific protein ATP binding cassette transporter A3(ABCA3) in vitro. Methods:Lung tissues of 19-day fetal rats were digested with trypsin and collagenase,then screened for AECⅡ with different centrifugal force and repeated attachment. AECⅡ was identified by electron microscopy. Growth status and shape of attached cells in different time were observed with inverted phase contrast microscope,and the cell growh was assayed by MTT assay. The dynamic expression of ABCA3 in AECⅡ was detected by immunofluorescence. Results:The characteristic lamellar bodies of ACEⅡ with varying size and amount were observed under electron microscopy. The obsorbance value of AECⅡ cells by MTT at 24 h,36 h,48 h,72 h,96 h,120 h was 0.177 ± 0.009,0.193 ± 0.011,0.212 ± 0.019, 0.253 ± 0.019,0.243 ± 0.012,0.192 ± 0.011 respectively. Cell morphology under microscope gradually became elongating and flattening,and the intracellular particles gradually reduced. Simultaneously,the immunofluorescence assay result showed that ABCA3 expression in AECⅡ cells gradually decreased with the culture time prolonged. Conclusion:Primary cultured AECⅡ has the best growth status and the most abundant ABCA3 expression in the early culture time,therefore this period (primary culture for 72 hours or less) is optimum time to research mechanism of diseases associated with lung development.

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张 盼,张晓群,邱 洁,杨 洋,周晓玉,周晓光.胎鼠肺泡Ⅱ型上皮细胞生长状况及ABCA3表达变化的初步研究[J].南京医科大学学报(自然科学版),2012,(11):1488-1492

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  • 收稿日期:2012-07-05
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