呼吸道合胞病毒对大鼠原代气道上皮细胞表达胸腺基质淋巴细胞生成素的影响

Effects of respiratory syncytial virus on the expression of thymic stromal lymphopoietin in primary rat airway epithelial cells

目的:探讨分离和培养大鼠原代气道上皮细胞(primary rat airway epithelial cell,PRAEC)的方法,研究呼吸道合胞病毒(respiratory syncytial virus,RSV)对PRAEC表达胸腺基质淋巴细胞生成素(thymicstromal lymphopoietin,TSLP)的影响。方法:采用改良的酶消化法分离PRAEC,并在专用的支气管上皮培养基中培养。在培养成功的原代细胞中加入RSV以感染PRAEC。应用Real-time PCR检测正常的和受到RSV感染的PRAEC中TSLP基因表达。结果:获得纯度较高(约为97%)的PRAEC,细胞成活率为90%,获得细胞量为(1.02±0.33)×106个细胞/鼠,且存活时间较长。正常PRAEC表达少量的TSLP,但是在受到RSV感染后其TSLP表达出现增高,且TSLP的表达在一定范围内与RSV的滴度成正相关。结论:本实验成功地分离并培养了PRAEC,在此基础上进行的实验证实了RSV感染确实能够促进气道上皮细胞表达TSLP,为进一步研究RSV感染在哮喘发病中的作用机制奠定了基础。

Objective To explore the methods for isolating and culturing primary rat airway epithelial cell （PRAEC）, and to investigate the effects of respiratory syncytial virus （RSV） on the expression of thymic stromal lymphopoietin （TSLP） in PRAECs. Methods PRAECs,which were isolated by digestion with prognase and cultured in airway epithelial cell medium （AECM）, were exposed to different titers of RSV（MOI = 2.5, 5,10） for different periods （6 h, 12 h, 18 h, 24 h）. The production of TSLP mRNA was measured by real-time quantitative PCR. Results The average yield of PRAECs was （1.02±0.33）×10^6cells/trachea. Cell viability determined by trypan blue exclusion was 90%, and the purity was 97% as determined by pan-eytokeratin. Exposure of PRAECs to RSV （MOI = 10） induced a rapid （6 h）,high （12 h）, and persistent （18 h） increase in TSLP mRNA compared to non-treated PRAECs （P 〈 0.05）. Conclusion We have successfully isolated and cultured PRAECs. And in vitro experiments confirmed that RSV infection could indeed promote the expression of TSLP in PRAECs,which may contribute to the intiation and development of asthma.