牛膝多糖诱导单核细胞HLA-DR_αmRNA表达的实验研究

Expermental Research of Monocytes Stimulated by Achyranthes Biolenatata Polysaccharides That Lead to Expression of HLA-DR_α mRNA

目的:研究单核细胞的分离培养并检测牛膝多糖(ABPS)诱导单核细胞HLA-DRαmRNA的表达变化。 方法:采集健康成人的血液,肝素抗凝,用淋巴细胞分离液密度梯度离心法分离外周血单个核细胞,用贴壁法分离 单核细胞。接种细胞到24孔板,同一时间不同ABPS浓度或同一浓度ABPS不同时间37℃、5%CO2培养。RT PCR检测HLA-DRαmRNA表达水平并且进行PCR产物验证。结果:(1)鉴定单核细胞:在PBMC中,流式细胞术 检测到CD14阳性细胞占12.0%,黏附得到的单核细胞中,流式细胞术检测到CD14阳性细胞占83.1%。(2)同一 时间不同浓度ABPS上调HLA-DRαmRNA表达。(3)不同时间同一浓度ABPS上调HLA-DRαmRNA表达。(4) 引物序列和产物序列分别进行BLAST表明PCR产物为NM_019111。结论:在体外,ABPS上调单核细胞HLA -DRαmRNA,有显著的剂量和时间效应关系。提示ABPS能激活单核细胞并有增强免疫功能。

Objective: To study a isolation and cultivation method of monocytes and investigate the HLA-DR αmRNA expression of monocytes induced by Achyranthes bidentata polysaccharides (ABPS) in vitro. Methods: Heparinied blood were collected immediately from healthy grown-up.The monocytes were separated by the anchoring technique. In 24-well flat-bottom plates, freshly isolated cells were cultured with serially diluted ABPS-RPMI1640 concentration for identical time or with distinct time for same ABPS-RPMI1640 concentration at 37℃ in 5%CO 2. Detection of HLA-DR α mRNA expression with RT-PCR and the PCR product was verified by sequencing. Results: (1) Identification of monocytes: The CD14 + number cell of PBMC is 12.0% with flow cytometry(anti-CD14) detection; The CD14 + number of obtained monocytes by the anchoring technique that were detected with flow cytometry(anti-CD14), is 83.1%. (2) Identical time and different ABPS up-regulation HLA-DR αmRNA expression of monocytes. (3) Different time and identical ABPS also up-regulation HLA-DR αmRNA expression of monocytes. (4) To make a BLAST at NCBI for primer sequence and product sequence show that our product sequence were NM_019111.2.Conclusions: In vitro, ABPS can induce the expression of HLA-DR αmRNA in the monocytes, which the expression of HLA-DR αmRNA has significantly dose and time effect, and have show that ABPS can activate the monocytes and reinforce immunological function.