RNA干擾抑制小鼠CCR3基因?qū)w內(nèi)外嗜酸性粒細胞影響的作用研究
發(fā)布時間:2018-07-25 06:09
【摘要】:目的: RNAi干擾下調(diào)小鼠CCR3基因的表達,在體內(nèi)和體外觀察嗜酸性粒細胞(eosinophil,EOS)的變化情況,為闡明過敏性鼻炎的發(fā)生機制提供理論基礎。 方法: 體外培養(yǎng)并純化小鼠來源的EOS,用細胞染色法進行細胞鑒定。通過磷酸鈣沉淀法,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蛋白印跡法檢測慢病毒載體顆粒的抑制效果, AnnexinV-FITC凋亡檢測法((Annexin V-FITC Apoptosis Detection)進行流式細胞計數(shù)檢測EOS凋亡情況。變應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及蛋白的表達變化情況。流式細胞表面抗原直接標記法檢測CD34+細胞的變化情況。 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和蛋白水平表達降低,差異有統(tǒng)計學意義(p 0.05)。 (2)流式細胞凋亡術表明CCR3shRNA能夠抑制EOS生長并促進細胞凋亡,,差異有統(tǒng)計學意義(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及蛋白表達低于PBS治療對照及shRNA治療對照組,差異有統(tǒng)計學意義(p 0.05)。 (4)CCR3shRNA處理組的CD34+細胞數(shù)與PBS對照及shRNA治療組相比較差異無統(tǒng)計學意義(p>0.05)。 結(jié)論: RNAi干擾下調(diào)基因CCR3的表達,在細胞培養(yǎng)和變應性鼻炎動物模型中能有效的影響CCR3基因mRNA和蛋白水平的表達,同時能促進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。
[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.
【學位授予單位】:南昌大學
【學位級別】:碩士
【學位授予年份】:2014
【分類號】:R765.21
本文編號:2142861
[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.
【學位授予單位】:南昌大學
【學位級別】:碩士
【學位授予年份】:2014
【分類號】:R765.21
【參考文獻】
相關期刊論文 前4條
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2 申跡;柯霞;洪蘇玲;曾慶;梁傳余;李同英;唐安洲;;Epidemiological Features of Allergic Rhinitis in Four Major Cities in Western China[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2011年04期
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本文編號:2142861
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