【摘要】：目的： RNAi干擾下調(diào)小鼠CCR3基因的表達(dá)，在體內(nèi)和體外觀察嗜酸性粒細(xì)胞（eosinophil，EOS）的變化情況，為闡明過(guò)敏性鼻炎的發(fā)生機(jī)制提供理論基礎(chǔ)。 方法： 體外培養(yǎng)并純化小鼠來(lái)源的EOS，用細(xì)胞染色法進(jìn)行細(xì)胞鑒定。通過(guò)磷酸鈣沉淀法，將體外合成的特異性CCR3shRNA重組慢病毒載體轉(zhuǎn)染到EOS中。用RT-PCR和Western Blot蛋白印跡法檢測(cè)慢病毒載體顆粒的抑制效果， AnnexinV-FITC凋亡檢測(cè)法（(Annexin V-FITC Apoptosis Detection）進(jìn)行流式細(xì)胞計(jì)數(shù)檢測(cè)EOS凋亡情況。變應(yīng)性鼻炎小鼠鼻腔局部分組注入慢病毒載體處理后，RT-PCR和Western Blot蛋白印跡法檢測(cè)各組小鼠的骨髓、外周血及鼻腔黏膜中EOS的CCR3基因mRNA及蛋白的表達(dá)變化情況。流式細(xì)胞表面抗原直接標(biāo)記法檢測(cè)CD34+細(xì)胞的變化情況。 結(jié)果： （1）RT-PCR和Western Blot表明CCR3shRNA重組慢病毒處理的EOS的CCR3基因在mRNA和蛋白水平表達(dá)降低，差異有統(tǒng)計(jì)學(xué)意義（p 0.05）。 （2）流式細(xì)胞凋亡術(shù)表明CCR3shRNA能夠抑制EOS生長(zhǎng)并促進(jìn)細(xì)胞凋亡，，差異有統(tǒng)計(jì)學(xué)意義（p 0.05）。 （3）在小鼠骨髓、外周血、鼻腔黏膜中，CCR3shRNA處理組的CCR3基因的mRNA及蛋白表達(dá)低于PBS治療對(duì)照及shRNA治療對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（p 0.05）。 （4）CCR3shRNA處理組的CD34+細(xì)胞數(shù)與PBS對(duì)照及shRNA治療組相比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p＞0.05)。 結(jié)論： RNAi干擾下調(diào)基因CCR3的表達(dá)，在細(xì)胞培養(yǎng)和變應(yīng)性鼻炎動(dòng)物模型中能有效的影響CCR3基因mRNA和蛋白水平的表達(dá)，同時(shí)能促進(jìn)EOS凋亡。小鼠體內(nèi)注入CCR3shRNA重組慢病毒能有效抑制EOS在局部組織的遷移及浸潤(rùn)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R765.21

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