人单个中性粒细胞IL-8Rβ mRNA丰度的相对半定量研究

INVESTIGATION OF IL-8RΒ mRNA EXPRESSION PROFILE IN SINGLE HUMAN NEUTROPHIL

目的:探讨单个中性粒细胞中白介素-8β型受体(IL-8Rβ)mRNA的表达丰度。方法:采用倍比稀释相对定量单细胞RT-PCR方法分离纯化人外周血中性粒细胞并提取总RNA,针对IL-8RβmRNA设计引物,进行RT-PCR确认IL-8Rβ在中性粒细胞中的基因表达并优化反应条件。收集单个中性粒细胞胞内容物或完整细胞,以相同引物进行RT反应,再将后者所得的cDNA倍比稀释后,与前者共同经过两轮PCR扩增,并将最终产物以BamHI内切酶酶切鉴定产物的特异性。结果:IL-8RβmRNA在人中性粒细胞中有大量表达,单个中性粒细胞中亦能扩增出特异性的IL-8Rβ目的条带,并且将单个细胞内mRNA稀释104倍后仍可扩增出目的条带,BamHI内切酶酶切反应证实为目的产物。结论:人中性粒细胞表达IL-8RβmRNA,mRNA倍比稀释单细胞RT-PCR方法确认其丰度远高于一般mRNA含量,且此方法可在单细胞水平比较细胞内mRNA的丰度。

Aim： To validate the abundance of Interleukin 8 receptor beta（IL-8Rβ） mRNA in single human neutrophil. Methods： Human neutrophils were isolated and purified from volunteers, total RNA was extracted and a regular RT-PCR aiming at IL-8Rβ mRNA was performed to ascertain its expression profile in human neutrophils and optimize the reaction conditions for the following single-cell RT- PeR procedures. Subsequently, single neutrophil or the cellular content was harvested to conduct reverse transcription and two-round PeR with the same primer pairs used before. Serial dilution of single neutrophil cDNA pool was carried out at the .same time with the exact two-round PCR followed. The specificity of this single-cell RT-PCR procedure was verified by the BamHl restriction endonuclease digestion on the final cDNA products. Results： Regular RT-PCR indicated IL-8Rβ mRNA expression in human neutrophils. While single-cell RT-PCR was sensitive enough to detect trace IL-8Rβ mRNA as predicted cDNA product could be amplified from a 10 000 times diluted intracellular specimen from single neutrophil, which indicated an abundant expression of this mRNA in human neutrophil. Moreover, BamHl digestion on the final cDNA product clarified the specificity of this single-cell RT-PeR procedure. Conclusion： This simplified semi-quantitative single-cell RT-PCR procedure specifically confirmed that IL-8Rβ mRNA was highly expressed in human neutrophil, which also provided the possibility of comparing mRNA abundance at single cell level.