肠道病毒EV71相关受体在人二倍体细胞KMB17表面的构成分析

Analysis of enterovirus 71 associated receptors expression on the membrane of human diploid cell line KMB17 membrane

目的研究人二倍体细胞KMB17表面与肠道病毒EV71感染相关的受体Toll样受体4(TLR4)、血小板衍生生长因子受体α多肽(pDGFR-αR1)的表达情况,以及细胞周期、细胞代次、EV71感染对这两种受体表达的影响。方法使用荧光标记的特异抗体:PE-TLR4、FITC-PDGFR-αR1标记不同细胞代次、处于不同细胞周期时段的KMB17细胞,应用流式细胞仪检测表达这两种受体蛋白的阳性细胞率,以及EV71感染前后,阳性细胞率的变化。使用NCBI primer blast设计TLR4、PDGFR-αR1特异的引物,提取不同细胞代次、细胞周期时段KMB17细胞的RNA,使用real-timePCR检测这些受体蛋白特异mRNA的量。结果 KMB17细胞在使用血清饥饿法进行G0/G1同步化后约22h进入S期,在36h进入G2期,在48h左右完成分裂。PDGFR-αR1、TLR4平均阳性细胞的比例分别为4.47%和11.82%;通过流式细胞仪检测,在G0/G1期,PDGFR-αR1、TLR4的平均阳性细胞比例分别为4%和8.9%;在S期,分别为2.6%和9%;在G2/M期,分别为4.4%和13.5%;EV71病毒感染后,PDGFR-αR1阳性的细胞比率由2.6%上升至4.0%,TLR4阳性细胞比率分别为11.6%和12.8%。结论人二倍体细胞KMB17表面表达肠道病毒相关的受体TLR4和PDGFR-αR1,表达PDGFR-αR1的阳性细胞较TLR4少,不同代次的KMB17细胞这2种受体阳性细胞比例的差异不大,PDGFR-αR1阳性细胞在G0期较多,TLR4阳性细胞在G2/M期较多;EV71病毒感染后,PDGFR-αR1、TLR4阳性细胞的比率变化不大,表明这两种受体分子在KMB17细胞表面表达较稳定,不受EV71感染的影响。

Objective To detect the expression level of enterovirus associated receptors： Toll like receptor 4 （TLR4） and platelet derived growth factor receptor ct polypeptide （PDGFR-αR1） on the membrane of human diploid cell line KMB17, and also to determine whether different cell cycle phases, cell passages and EV71 infection may affect these receptors expression. Methods KMB17 cells in different passages and cell cycle phases were labeled with fluorescent conjugated PE-TLR4 or FITC-PDGFR-ctR1 monoclonal antibodies. Cellular expression level of these receptors was detected and compared by FACS before and after EV71 infection. TLR4 and PDGFR-αR1 gene-specific primers were designed using NCBI primer blast. Total RNA was extracted from KMB17 cells in different passages and cell cycle phases, and TLR4 and PDGFR-αR1 mRNAs were detected by real time PCR. Results KMB 17 cells entered S phase and G2 phase about 22 hours and 36 hours respectively, and completed one cell cycle around 48 hours, after synchronization in G0/G1 phases by serum starvation. The mean ratio of PDGFR-αR1 and TLR4 positive cells detected by FACS was 4.47% and 11.82% respectively. In G0/G1 phase, the ratio was 4% and 8.9%; in S phase, the ratio was 2.6% and 9%; and in G2/M phase, the ratio was 4.4% and 13.5% respectively. The ratio of PDGFR-αR1 positive cells changed from 2.6% to 4.0% andTLR4 positive cells changed from 11.6% to 12.8% post EV71 infection. Concluslons TLR4 and PDGFR-aR1 were expressed on the membrane of human diploid cell KMB17. PDGFR-aR1 positive cells were less than that of TLR4. No obvious fluctuations of the positive ratios were observed in the different passages of KMB17 cells. In different cell phases, more PDGFR-αRls were expressed in GO phase while more TLR4s expressed in G2/M phase. EV71 infection has little effect on the PDGFR-αR1 or TLR4 expression rate.