RNA干扰抑制小鼠CCR3基因对体内外嗜酸性粒细胞影响的作用研究

发布时间：2018-07-25 06:09

【摘要】：目的： RNAi干扰下调小鼠CCR3基因的表达，在体内和体外观察嗜酸性粒细胞（eosinophil，EOS）的变化情况，为阐明过敏性鼻炎的发生机制提供理论基础。方法：体外培养并纯化小鼠来源的EOS，用细胞染色法进行细胞鉴定。通过磷酸钙沉淀法，将体外合成的特异性CCR3shRNA重组慢病毒载体转染到EOS中。用RT-PCR和Western Blot蛋白印迹法检测慢病毒载体颗粒的抑制效果， AnnexinV-FITC凋亡检测法（(Annexin V-FITC Apoptosis Detection）进行流式细胞计数检测EOS凋亡情况。变应性鼻炎小鼠鼻腔局部分组注入慢病毒载体处理后，RT-PCR和Western Blot蛋白印迹法检测各组小鼠的骨髓、外周血及鼻腔黏膜中EOS的CCR3基因mRNA及蛋白的表达变化情况。流式细胞表面抗原直接标记法检测CD34+细胞的变化情况。结果：（1）RT-PCR和Western Blot表明CCR3shRNA重组慢病毒处理的EOS的CCR3基因在mRNA和蛋白水平表达降低，差异有统计学意义（p 0.05）。（2）流式细胞凋亡术表明CCR3shRNA能够抑制EOS生长并促进细胞凋亡，，差异有统计学意义（p 0.05）。（3）在小鼠骨髓、外周血、鼻腔黏膜中，CCR3shRNA处理组的CCR3基因的mRNA及蛋白表达低于PBS治疗对照及shRNA治疗对照组，差异有统计学意义（p 0.05）。（4）CCR3shRNA处理组的CD34+细胞数与PBS对照及shRNA治疗组相比较差异无统计学意义(p＞0.05)。结论： RNAi干扰下调基因CCR3的表达，在细胞培养和变应性鼻炎动物模型中能有效的影响CCR3基因mRNA和蛋白水平的表达，同时能促进EOS凋亡。小鼠体内注入CCR3shRNA重组慢病毒能有效抑制EOS在局部组织的迁移及浸润。
[Abstract]:Objective:
RNAi interference reduces the expression of CCR3 gene in mice and observe the changes of eosinophil (eosinophil, EOS) in vivo and in vitro, which provides a theoretical basis for elucidating the mechanism of allergic rhinitis.
Method:
EOS was cultured and purified in vitro, and cell identification was carried out by cell staining. The specific CCR3shRNA recombinant lentivirus vector synthesized in vitro was transfected into EOS by calcium phosphate precipitation method. The inhibition effect of lentivirus carrier particles was detected by RT-PCR and Western Blot blot, and AnnexinV-FITC apoptosis assay (Annexin V-F) ITC Apoptosis Detection) flow cytometry was used to detect the apoptosis of EOS. After injection of lentivirus vector in the nasal partial group of allergic rhinitis mice, the expression of CCR3 gene mRNA and protein of EOS in peripheral blood and nasal mucosa was detected by RT-PCR and Western Blot Western blot. The changes of CD34 + cells were detected by direct labeling with surface antigen.
Result:
(1) RT-PCR and Western Blot showed that the expression of CCR3 gene in EOS treated with CCR3 shRNA recombinant lentiviruses was decreased at mRNA and protein levels, and the difference was statistically significant (p 0.05).
(2) Flow cytometry showed that CCR3shRNA could inhibit EOS growth and promote apoptosis, the difference was statistically significant (p 0.05).
(3) in the mice bone marrow, peripheral blood and nasal mucosa, the mRNA and protein expression of CCR3 gene in CCR3shRNA treatment group were lower than that of PBS treatment control and shRNA treatment control group, the difference was statistically significant (P 0.05).
(4) There was no significant difference in the number of CD34 + cells between CCR3shRNA treatment group and PBS control group or shRNA treatment group (p > 0.05).
Conclusion:
The expression of RNAi interference down gene CCR3 can effectively affect the expression of mRNA and protein level of CCR3 gene in the cell culture and allergic rhinitis animal model, and can promote the apoptosis of EOS. The injection of CCR3shRNA recombinant lentivirus in mice can effectively inhibit the migration and infiltration of EOS in the local tissues.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R765.21

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