摘要
支气管哮喘病因极其复杂,炎症、气道重塑和气道神经控制的变化是引起哮喘反复恶化及持久气流阻塞的根本原因。免疫异常及变态反应是哮喘发病中的关键因素。IL-23是新近发现的一种维持慢性炎症和抵抗胞内细菌感染的重要的促炎因子,研究证实,IL-23可刺激CD4+T细胞的一个新型亚群-Th17亚群,特征性产生前炎症细胞因子IL-17。IL-17在支气管哮喘中促炎作用得到认同,IL-23作为上游调控因子是否也参与支气管哮喘的发病呢?本实验通过pSRZSi-IL-23p19表达质粒进行体外、体内转染,采用RT-PCR、Western blot、酶联免疫反应、组织形态学观察等方法,于观察pSRZSi-IL-23p19对哮喘模型相关炎症因子的影响,探讨IL-23与哮喘模型的关系。本研究证实IL-23参与了支气管哮喘的发病,并通过IL-23/IL-17途径起作用;特异性抑制IL-23异常表达,可减轻肺组织的炎症、中性粒细胞募集及嗜酸性粒细胞浸润;提出应用RNA干扰技术,通过阳离子脂质体介导可有效行呼吸道转染;证实呼吸道转染pSRZSi-IL-23p19可减轻哮喘肺部炎症。该研究为支气管哮喘发病机制的研究提供了新的思路,并可能为今后哮喘的治疗开辟新的途径。
Objective
The etiopathogenisis of bronchial asthma is complex. Asthma is chronic airway inflammation with the characteristics of intermittent and reversible airway obstruction, bronchial hyperresponsiveness, lymphocytes, eosinophil infiltration of airway submucosal. Asthma is also a kind of abnormal immune allergic diseases with Th1/Th2 imbalance and Th2 cells in advantages. However, a study shows that Th1/Th2 imbalance reverse can not to reduce asthma attack, so this will be a challenge to Th2 advantage theory.
IL-23 discovered in 2000 was a new type of cytokine. IL-23 becomes one of the hot spots research in recent years because of its complex biological characteristics and important role in the biological field. IL-23 was researched deeply in autoimmune disease, tumor, infection and so on. IL-23 can enhance the role of Th1 immune response, and the role of promotion pro-inflammatory cytokine secretion, such as IL-17.So there are contradictions for IL-23 in the pathogenesis of asthma.
RNA interference is a kind of highly conserved gene silencing technology. Elbashir Studied and founded that 21nt double-stranded RNA fragment can reduce the expression of targeting gene in different mammalian cell lines such as T lymphocytes, epithelial cells, liver cells and so on. RNAi technology is the same as gene knockout and gene silencing in functions, and more convenient than Gene knockout technology.
We established animal model of asthma, and detected the expression of IL-23 in the peripheral blood and lung tissue of asthmatic mice. pSRZsi-IL-23p19 expression plasmid was established With RNAi technology, cationic lipid-mediated transfected T lymphocytes and respiratory tract of mice, then we observed the expression changes of IL-23 and the impact of the airway inflammation and cytokine secretion in vitro and in vivo to explain the role of IL-23 in the pathogenesis of asthma and the impact of airway inflammation in asthma using IL-23siRNA technology.
Method
1、pSRZsi-IL-23p19 expression plasmid was constructed, amplificated and identificated.
2、Mouse model of asthma were established per literature improvement, observation changes of ethology、leves of IL-4/IFN-γ、Count of eosinophil and neu neutrophil in BALF and Structural changes in lung tissue of mouse.
3、pSRZsi-IL-23p19 expression plasmid and Lipofectamine 2000cationic transfected T lymphocytes of asthma murine in different proportions to determination IL-23p19/IL-17 mRNA expression level with RT-PCR semi-quantitative method and IL-23 protein content changes with western blot in T lymphocytes, IL-4、IFN-γchanges of culture supernatants with ELISA.
4、The experimental anima randomly divided into control group, asthma model group, hormone therapy group, the empty vector intervention group, and recombinant plasmid intervention. Asthma model were constructed using intraperitoneal injection of 100μl OVA suspension in the days of 3、9、16 , and excited by air compressed pump in the days of 17、19、21、23、25 by 30 minutes each time and five days. Control group were constructed using intraperitoneal injection of 100μl PBS containing 0.2 mg aluminum hydroxide in the days of 3、6、9, stimulation was the same as model group. Allergy and excitation of recombinant plasmid intervention was the same as asthma model. Recombinant plasmid was transfected through nasal cavity in the day of 16、18、20、22、24,and the mousse was killed in a specified time after the last excitation. IL-23, IL-17, IL-4 and IFN-γlevels in serum were detected in ELISA; IL-23p19/IL-17mRNA levels were detected in RT-PCR; Expression features of IL-23 in lung tissue were detected in immunohistochemistry; Structural changes in lung tissue were observed by HE; Count of eosinophil and neu neutrophil in BALF.
Resurt
1、pSRZsi-IL-23p19 expression plasmid was requirement for our contrive via develop pSRZsi-IL-23p19 and appraisement amplification production with .
2、Eosinophil cells in BALF was increased. Massive infiltration of inflamma-tory cells , goblet cell hyperplasia, mucosal wall damage, mucus suppository form were saw in pulmonary histologically, so the model successfully established.
3、Effect of pSRZsi-IL-23p19 expression plasmid transfection in vitro on T ymphocyte cells of asthma of mouse:
①After lipofectamine 2000 cationic liposome-mediated pSRZsi-IL- 23p19 expression plasmid was transfected to T lymphocyte, we found that the highest transfection efficiency is in 1:2 proportion.
②IL-23p19/IL-17mRA and IL-23 protein content of T lymphocyte cells in asthma group was higher than control group, and interference group has lower than asthma group.There was no significant difference between intervention group and control group.
③IFN-γlevel of T lymphocyte supernatant in asthma group was lower than co-ntrol group, no significant difference between intervention group and asthma group. IL-4 level of T lymphocyte supernatant in asthma group was higher than control group, after n the treatment of intervention was lower.
4、Effect of pSRZsi-IL-23p19 expression plasmid transfection in vivo on asthma model:
①Lipofectamine 2000 cationic liposome-mediated pSRZsi-IL-23p19 expression plasmid transfection in vivo was succssfull because we can see the green fluorescent protein around bronchial under fluorescence microscopy.
②The levels of IL-23p19/IL-17 mRNA of lung tissue in asthma group was hig her than control group, and lower after hormone therapy. After 24-hour of transfection was lower,after 48-hour of transfection was lower than after hormone therapy.There was no significant difference between vacancy intervention group and asthma group .The level of IL-23/IL-17 in serum and the result of immunohis-tochemistry are the same as expression levels in lung tissue. IL-23 and IL-17 was positively correlated.
③The levels of IL-4 in serum in asthma group was higher than control group,and higher than in the treatment of hormone,there was no significant difference between vacancy intervention group and asthma group.In the treatment of inter-vention the leves of IL-4 in serum was lower,but extent was inferior to in group of hormone-treated group.
④The levels of IFN-γin serum in asthma group was lower than control group,and in the treatment of hormone higher than asthma group. Treatment of inter-vention no significant effect on IFN-γ. there was no significant difference bet-ween vacancy intervention group and asthma group.
Conclusion
1、IL-23 participates in and contributes to the occurrence of asthma, and Associa-ted with IL-17,this pathway is different from Th1/Th2 Disequilibriump pathway.
2、pSRZsi-IL-23p19 expression plasmid transfection in vitro can reduced IL-23 expression in T lymphocyte cells, thus abaissement IL-17 expression in T lymphocyte cells.
3、pSRZsi-IL-23p19 expression plasmid transfection in vivo via nose ,this may abaissement IL-23/IL-17 expression in pulmorary tissue and reduced IL-23/IL-17 leves in blood serum.
4、Via inhibit IL-23 gene expression,can abaissement Neu count in pulmorary tissue, depress leve of IgE in blood serum , abatement inflammation condition ,these effccts is implement may via abaissement IL-17 expression and transform humoral immunity.
5、RNA interfere technique is application in athma, may implement target gene silencing.
引文
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