气道Orai1通道干预对小鼠实验性变应性鼻炎的影响

发布时间：2018-06-15 19:07

本文选题：Orai1 + 小鼠　；参考：《复旦大学》2012年博士论文

【摘要】：目的：构建针对小鼠ORAI1基因进行RNA干扰从而下调细胞内Orai1蛋白表达的重组慢病毒Lenti-ORAI1-shRNA,并测定重组慢病毒的感染效率与干扰效率。建立Lenti-ORAI1-shRNA感染小鼠鼻腔的体内模型,并测定其对小鼠鼻黏膜中ORAI1基因的转录抑制效率的动态变化；建立Lenti-ORAI1-shRNA气道内干预变应性鼻炎小鼠模型并观察其对鼻炎症状、小鼠鼻腔局部与全身炎症相关免疫细胞功能的影响并初步探寻其机制。材料与方法：针对小鼠ORAI1基因序列设计RNA干扰靶序列,合成靶序列的寡聚DNA,与经ClaI及MluI双酶切的pLVTHM慢病毒载体连接,转化大肠杆菌感受态细胞,产生重组RNA干扰慢病毒表达载体,将pRSV-REV质粒、pMDlg-pRRE粒、pMD2.G与pLVTHM/ORAI1-shDNA重组质粒混合感染293T细胞包装慢病毒并测定病毒滴度。用包装好的Lenti-ORAI1-shRNA感染PANC-1细胞,流式细胞分析测定其感染效率,荧光定量PCR检测其对ORAI1基因的转录抑制效率。将BALB/c小鼠随机分为7组,一组为正常对照组,另外六组为Lenti-ORAI1-shRNA干预组。将干预组小鼠麻醉后,鼻腔滴入8μl0.3%溶血卵磷脂,1小时后鼻腔滴入20μl滴度为1×109tu/ml的Lenti-ORAI1-shRNA进行感染。分别于感染1,3,7,14,21,28天后处死小鼠,测定其鼻黏膜ORAI1mRNA水平。将小鼠随机分为5组：正常对照组(normal),正常小鼠Lenti-ORAI1-shRNA干预组(NLI),变应性鼻炎组(AR),变应性鼻炎小鼠Lenti-GFP干预组(AR-GFP),变应性鼻炎小鼠Lenti-ORAI1-shRNA干预组(AR-LI)。后三组小鼠以).5mg/ml OVA/20mg/ml Al(OH)3溶液腹腔注射3次致敏,40mg/ml OVA鼻腔滴入8次激发建立变应性鼻炎小鼠模型,对NLI组与AR-LI组小鼠于首次鼻腔激发前3天鼻腔滴入20gl滴度为1×109tu／ml的Lenti-ORAI1-shRNA进行干预,对AR-GFP小鼠滴入相同剂量及滴度的Lenti-GFP进行干预。计数各组小鼠末次鼻腔激发后10分钟内喷嚏数及挠鼻数,Real-time RT PCR法测定各组小鼠鼻黏膜及脾脏ORAI1、LTC4S、EAR3、 germline Cε与IL-4mRNA水平,Western Blotting法测定各组小鼠鼻黏膜Orai1、IL-33、IL-17E与TSLP及脾脏Orai1蛋白表达水平,免疫组化法观察各组小鼠鼻黏膜、鼻黏膜相关淋巴与脾脏中Orai1表达分布情况。ELISA法测定各组小鼠鼻腔灌洗液与血清中LTC4、ECP、OVA-IgE与IL-4的浓度。结果：PANC-1细胞经Lenti-ORAI1-shRNA感染后可表达慢病毒所携带的GFP基因而发出绿色荧光,当感染复数为10时,Lenti-ORAI1-shRNA感染PANC-1细胞48小时后感染效率为98.28%,对细胞ORAI1基因的转录抑制效率为90.5%；而用于对照的空载慢病毒在相同条件下感染效率为98.75%,对ORAI1基因的转录抑制效率为-3%。小鼠鼻腔感染Lenti-ORAI1-shRNA后,鼻黏膜ORAI1mRNA水平随感染时间逐渐降低,至第3天时达最低,随后逐渐升高,感染后一月左右Orai1mRNA水平仍低于正常对照组水平。AR-LI组小鼠鼻黏膜与脾脏ORAI1mRNA水平均较AR组下降,其鼻黏膜Orai1蛋白表达水平也低于AR组小鼠,末次鼻腔激发后挠鼻数与喷嚏数较AR组小鼠也都明显减少；AR-GFP组小鼠鼻黏膜与脾脏ORAI1mRNA水平,Orai1表达水平及末次激发后喷嚏数与挠鼻数与AR组小鼠相比均无明显差异。免疫组化结果提示AR-LI组小鼠鼻黏膜上皮层与固有层、鼻黏膜相关淋巴组织、脾脏生发中心等部位Orai1表达均较AR组小鼠减少。AR-LI组小鼠鼻黏膜与脾脏中LTC4S、 EAR3、germline Cε与IL-4的mRNA水平均明显低于AR组小鼠,其鼻腔灌洗液中LTC4、OVA-IgE与IL-4的浓度以及血清中LTC4、ECP、OVA-IgE与IL-4的浓度也均低于AR组小鼠,AR-GFP组小鼠鼻黏膜与脾脏中LTC4S、 EAR3、germline Cε与IL-4的mRNA水平与AR组小鼠无明显差异,其鼻腔灌洗液与血清中LTC4、ECP、OVA-IgE与IL-4的浓度与AR组小鼠也无明显差异。AR组小鼠鼻黏膜中IL-33、IL-17E与TSLP的表达水平均明显高于正常对照组小鼠,AR-LI组小鼠鼻黏膜中IL-33与TSLP的表达水平较AR组有所下降,但IL-17E的表达水平与AR组相比无明显差异。AR-GFP组小鼠鼻黏膜中IL-33、IL-17E与TSLP的表达水平与AR组小鼠相比均无明显差异。结论：成功构建针对ORAI1基因进行RNA干扰的重组慢病毒Lenti-ORAI1-shRNA; Lenti-ORAI1-shRNA能够成功感染PANC-1细胞并有效抑制ORAI1基因的转录水平。Lenti-ORAI1-shRNA鼻腔滴入可成功感染小鼠鼻黏膜细胞,感染后第3天对ORAI1基因的转录抑制效率达到最高。变应性鼻炎小鼠Lenti-ORAI1-shRNA鼻腔干预后可有效下调其鼻黏膜与脾脏中Orai1表达水平,并减轻其鼻炎症状。下调鼻黏膜中Orai1表达不仅可降低变应性鼻炎小鼠鼻黏膜中肥大细胞、嗜酸性粒细胞、B细胞与Th2细胞中相关炎性介质的转录与释放,也可降低全身如脾脏与血清中上述免疫细胞炎性介质的转录与释放。Lenti-ORAI1-shRNA鼻腔干预对小鼠变应性鼻炎炎症的抑制作用可能与其阻碍了鼻黏膜上皮细胞中IL-33与TSLP蛋白的合成和表达有关
[Abstract]:Objective: to construct a recombinant lentivirus Lenti-ORAI1-shRNA that interferes with the RNA interference of the mouse ORAI1 gene and reduce the expression of Orai1 protein in the cell, and determine the infection efficiency and interference efficiency of the recombinant lentivirus. The model of the nasal cavity in the mice infected with Lenti-ORAI1-shRNA is established and the transcriptional inhibition of the ORAI1 gene in the nasal mucosa of the mice is determined. The dynamic changes of efficiency; establish the Lenti-ORAI1-shRNA model of allergic rhinitis in the airway of the mice and observe the effects on the symptoms of rhinitis, the local and systemic inflammation related immune cell function in the nasal cavity of mice and explore its mechanism.
Materials and methods: to design RNA interference target sequence of mouse ORAI1 gene sequence, synthesize oligomeric DNA of target sequence, connect with pLVTHM lentivirus by ClaI and MluI double enzyme, transform Escherichia coli receptive cells, produce recombinant RNA to interfere with lentivirus expression vector, and reorganize pRSV-REV plasmids, pMDlg-pRRE particles, pMD2.G and pLVTHM/ORAI1-shDNA. The plasmid mixed infection 293T cells packed the lentivirus and measured the virus titer. The infected PANC-1 cells were infected with the packed Lenti-ORAI1-shRNA, the flow cytometry was used to determine the infection efficiency. The fluorescence quantitative PCR was used to detect the transcriptional inhibition efficiency of the ORAI1 gene. The BALB/c mice were randomly divided into 7 groups, one group was the normal control group and the other six groups were Lenti-ORAI1. -shRNA intervention group. After the intervention group was anesthetized, the nasal cavity was dripped into the 8 u l0.3% hemolytic lecithin, and the nasal drip was 1 * 109tu/ml Lenti-ORAI1-shRNA after 1 hours. The mice were killed after 1,3,7,14,21,28 infection and the ORAI1mRNA level of the nasal mucosa was measured. The rats were randomly divided into 5 groups: normal control group (normal), positive. The normal mice Lenti-ORAI1-shRNA intervention group (NLI), allergic rhinitis group (AR), allergic rhinitis mice Lenti-GFP intervention group (AR-GFP), allergic rhinitis mice Lenti-ORAI1-shRNA intervention group (AR-LI). The latter three groups of mice with.5mg/ml OVA/20mg/ml Al (OH) 3 solution intraperitoneal injection 3 times, 40mg/ml 8 times to stimulate the establishment of allergic rhinitis The mouse model was used to intervene in the NLI and AR-LI mice at the 20gl drop of 1 x 109tu / ml 3 days before the first nasal excitation, and the AR-GFP mice were treated with the same dose and titer Lenti-GFP. The number of sneezes and the number of nose in 10 minutes after the last nasal stimulation of the mice in each group and the Real-time RT PCR method were measured. The nasal mucosa and spleen of each group were ORAI1, LTC4S, EAR3, germline C epsilon and IL-4mRNA level. Western Blotting method was used to determine the expression level of Orai1, IL-33, IL-17E, TSLP and spleen. The concentrations of LTC4, ECP, OVA-IgE and IL-4 in nasal lavage fluid and serum of mice were determined.
Results: PANC-1 cells can express the GFP gene carried by lentivirus after Lenti-ORAI1-shRNA infection and send out green fluorescence. When the complex number is 10, the infection efficiency of Lenti-ORAI1-shRNA infected PANC-1 cells after 48 hours is 98.28% and the transcriptional inhibition efficiency of ORAI1 gene is 90.5%. The infection efficiency was 98.75%, and the transcriptional inhibition efficiency of ORAI1 gene was -3%. mouse nasal infection Lenti-ORAI1-shRNA, the ORAI1mRNA level of nasal mucosa gradually decreased with the infection time, and reached the lowest at third days, and then gradually increased. The level of Orai1mRNA was still lower than that of normal control group.AR-LI mice after one month. The level of ORAI1mRNA in spleen was lower than that in group AR, and the expression level of Orai1 protein in nasal mucosa was also lower than that in group AR. The number of nose and sneeze in the last nasal cavity were also significantly lower than that of the AR group. The level of ORAI1mRNA in the nasal mucosa and spleen in the AR-GFP group, the level of Orai1 expression, the number of sneezes after the last excitation and the number of the nose were compared with the mice in the AR group. The results of immunohistochemistry showed that the expression of Orai1 in nasal mucosa epithelium and lamina propria, nasal mucosa associated lymphoid tissue and spleen germinal center of AR-LI mice was lower than that of group AR mice and the mRNA levels of LTC4S, EAR3, germline C epsilon and IL-4 in the nasal mucosa and spleen of group.AR-LI mice were significantly lower than those in AR group, and their nasal lavage was more than that of the mice. The concentration of LTC4, OVA-IgE and IL-4 in the liquid and the concentration of LTC4, ECP, OVA-IgE and IL-4 in the serum were also lower than that of the AR mice. There was no significant difference between the nasal mucosa and the spleen in the AR-GFP group. The expression level of IL-33, IL-17E and TSLP in the nasal mucosa of.AR mice was significantly higher than that in the normal control group. The expression level of IL-33 and TSLP in the nasal mucosa of the AR-LI group was lower than that in the AR group, but the expression level of IL-17E was not significantly different from that in the AR group. There was no significant difference between the AR group and the mice.
Conclusion: the recombinant lentivirus Lenti-ORAI1-shRNA was successfully constructed for the RNA interference of the ORAI1 gene, and Lenti-ORAI1-shRNA could successfully infect PANC-1 cells and effectively inhibit the transcriptional level of ORAI1 gene,.Lenti-ORAI1-shRNA instillation could successfully infect the nasal mucosa cells of mice, and the transcriptional inhibition efficiency of ORAI1 gene was reached third days after infection. The expression of Orai1 in nasal mucosa and spleen can be reduced effectively after Lenti-ORAI1-shRNA nasal intervention in allergic rhinitis mice. The expression of Orai1 in nasal mucosa can not only reduce the inflammatory mediators of mast cells, eosinophil cells, B cells and Th2 cells in nasal mucosa of allergic rhinitis mice. Transcription and release can also reduce the transcriptional and release of the systemic inflammatory mediators of the spleen and serum, such as the spleen and serum, and the inhibitory effect of.Lenti-ORAI1-shRNA nasal intervention on inflammation of allergic rhinitis in mice may be related to the inhibition of the synthesis and expression of IL-33 and TSLP protein in the epithelial cells of the nasal mucosa.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R765.21

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