人嗜中性粒细胞弹性蛋白酶通过肿瘤坏死因子-α转化酶诱导慢性鼻-鼻窦炎中杯状细胞增生的研究
发布时间：2018-12-29 08:33
【摘要】：目的：通过检测中性粒细胞弹性蛋白酶（HNE）、肿瘤坏死因子-α转化酶（TACE）与黏蛋白MUC5AC在CRS患者中的表达情况，以及研究体外细胞培养试验中HNE通过TACE诱导MUC5AC表达的作用，提供HNE通过TACE诱导CRS中杯状细胞增生的证据，为进一步了解CRS的发病机制提供帮助。 方法：取伴或不伴息肉CRS患者鼻窦黏膜各20例，另外取10例正常鼻腔鼻窦黏膜作为对照，用HE染色和PAS染色观察各样本病理改变,用免疫组化的方法分别检测HNE、TACE和MUC5AC在粘膜中的表达情况，并且用荧光定量PCR的方法分别检测组织中HNE、TACE和MUC5AC mRNA的表达情况。建立体外原代鼻黏膜上皮细胞培养模型，并且用细胞免疫化学的方法进行鉴定，稳定传代后给予HNE刺激，并且以肿瘤坏死因子转换酶抑制剂-1(TAPI-1)为干预因素，用荧光定量PCR的方法检测处理后细胞中MUC5ACmRNA的表达情况。 结果：HE染色结果显示，在伴或不伴息肉CRS患者鼻窦黏膜中主要的病理特征是杯状细胞，炎性细胞及黏膜下腺体增生。两组CRS患者鼻窦粘膜上皮层PAS染色均可见强阳性表达，而正常对照组鼻窦粘膜层为弱阳性表达。免疫组化结果显示MUC5AC，TACE和HNE在两组CRS患者的鼻窦黏膜中的表达均高于对照组,且差别均有显著统计学意义(P0.05)，其中，MUC5AC和TACE主要表达于黏膜上皮杯状细胞中,HNE在黏膜上皮和黏膜下腺体中均有表达；两组CRS患者间各指标的表达无差异（P0.05)。荧光定量PCR结果表明伴息肉与不伴息肉CRS患者的鼻窦组织中MUC5AC，TACE和HNE mRNA的表达均增加，且与正常对照组相比有统计学差异(P0.05)，而两组之间并无明显差异(P0.05)。体外培养的鼻粘膜上皮细胞，，经细胞角蛋白AE1抗体染色后证实其为上皮源性，荧光定量PCR检测表明给予HNE刺激后，其MUC5ACmRNA表达水平高于未刺激组，且差异有统计学意义(P0.05);而用肿瘤坏死因子转换酶抑制剂TAPI-1预处理组及单纯使用TAPI-1组，其MUC5ACmRNA表达水平明显下调,并且与对照组及单纯HNE刺激组相比差异均有统计学意义(P0.01)。 结论：在伴与不伴息肉CRS中MUC5AC，TACE和HNE的表达均上调，且HNE通过TACE可诱导鼻黏膜上皮细胞中MUC5AC的增多，表明HNE-TACE信号通路在CRS中杯状细胞增生的过程中具有重要的作用。
[Abstract]:Aim: to investigate the expression of neutrophil elastase (HNE), tumor necrosis factor- 伪 converting enzyme (TACE) and mucin MUC5AC (MUC5AC) in patients with CRS and to study the role of HNE in TACE induced MUC5AC expression in vitro. To provide evidence of TACE induced goblet cell proliferation in CRS by HNE, and to further understand the pathogenesis of CRS. Methods: 20 cases of nasal sinus mucosa with or without polyps, 20 cases of nasal sinus mucosa and 10 cases of normal nasal cavity and sinus mucosa were taken as control. HE staining and PAS staining were used to observe the pathological changes of each sample, and HNE, was detected by immunohistochemical method. The expression of TACE and MUC5AC in mucous membrane and the expression of HNE,TACE and MUC5AC mRNA in tissues were detected by fluorescence quantitative PCR. The primary nasal epithelial cell culture model was established in vitro, and identified by cell immunocytochemistry. After stable passage, HNE was stimulated, and tumor necrosis factor converting enzyme inhibitor 1 (TAPI-1) was used as the intervention factor. The expression of MUC5ACmRNA was detected by fluorescence quantitative PCR. Results: HE staining showed that the main pathological features of nasal sinus mucosa with or without polyp CRS were goblet cells, inflammatory cells and hyperplasia of submucosal glands. Strong positive expression was found in the epithelial layer of nasal sinuses in both groups of CRS patients, while weak positive expression was found in the mucosal layer of nasal sinuses in the normal control group. The results of immunohistochemistry showed that the expression of MUC5AC,TACE and HNE in nasal sinus mucosa of CRS patients was higher than that of control group, and the difference was statistically significant (P0.05). Among them, MUC5AC and TACE were mainly expressed in mucosal goblet cells. HNE was expressed in mucosal epithelium and submucosal gland. There was no difference in the expression of each index between the two groups of CRS patients (P0.05). The results of fluorescence quantitative PCR showed that the expression of MUC5AC,TACE and HNE mRNA in paranasal sinus tissues of patients with polyps and without polyps CRS increased significantly compared with the normal control group (P0.05), but there was no significant difference between the two groups (P0.05). The epithelial cells of nasal mucosa cultured in vitro were confirmed to be epithelial-derived by cytokeratin AE1 antibody staining. Fluorescence quantitative PCR assay showed that the expression of MUC5ACmRNA was higher than that of unstimulated cells after HNE stimulation. The difference was statistically significant (P0.05). The expression of MUC5ACmRNA in TAPI-1 pretreated with TNF- converting enzyme inhibitor and TAPI-1 alone was significantly decreased compared with control group and HNE stimulation group (P0.01). Conclusion: the expression of MUC5AC,TACE and HNE is up-regulated in CRS with and without polyps, and HNE can induce the increase of MUC5AC in nasal epithelial cells through TACE, indicating that HNE-TACE signaling pathway plays an important role in the proliferation of goblet cells in CRS.
【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】：R765.41
本文编号：2394589
[Abstract]:Aim: to investigate the expression of neutrophil elastase (HNE), tumor necrosis factor- 伪 converting enzyme (TACE) and mucin MUC5AC (MUC5AC) in patients with CRS and to study the role of HNE in TACE induced MUC5AC expression in vitro. To provide evidence of TACE induced goblet cell proliferation in CRS by HNE, and to further understand the pathogenesis of CRS. Methods: 20 cases of nasal sinus mucosa with or without polyps, 20 cases of nasal sinus mucosa and 10 cases of normal nasal cavity and sinus mucosa were taken as control. HE staining and PAS staining were used to observe the pathological changes of each sample, and HNE, was detected by immunohistochemical method. The expression of TACE and MUC5AC in mucous membrane and the expression of HNE,TACE and MUC5AC mRNA in tissues were detected by fluorescence quantitative PCR. The primary nasal epithelial cell culture model was established in vitro, and identified by cell immunocytochemistry. After stable passage, HNE was stimulated, and tumor necrosis factor converting enzyme inhibitor 1 (TAPI-1) was used as the intervention factor. The expression of MUC5ACmRNA was detected by fluorescence quantitative PCR. Results: HE staining showed that the main pathological features of nasal sinus mucosa with or without polyp CRS were goblet cells, inflammatory cells and hyperplasia of submucosal glands. Strong positive expression was found in the epithelial layer of nasal sinuses in both groups of CRS patients, while weak positive expression was found in the mucosal layer of nasal sinuses in the normal control group. The results of immunohistochemistry showed that the expression of MUC5AC,TACE and HNE in nasal sinus mucosa of CRS patients was higher than that of control group, and the difference was statistically significant (P0.05). Among them, MUC5AC and TACE were mainly expressed in mucosal goblet cells. HNE was expressed in mucosal epithelium and submucosal gland. There was no difference in the expression of each index between the two groups of CRS patients (P0.05). The results of fluorescence quantitative PCR showed that the expression of MUC5AC,TACE and HNE mRNA in paranasal sinus tissues of patients with polyps and without polyps CRS increased significantly compared with the normal control group (P0.05), but there was no significant difference between the two groups (P0.05). The epithelial cells of nasal mucosa cultured in vitro were confirmed to be epithelial-derived by cytokeratin AE1 antibody staining. Fluorescence quantitative PCR assay showed that the expression of MUC5ACmRNA was higher than that of unstimulated cells after HNE stimulation. The difference was statistically significant (P0.05). The expression of MUC5ACmRNA in TAPI-1 pretreated with TNF- converting enzyme inhibitor and TAPI-1 alone was significantly decreased compared with control group and HNE stimulation group (P0.01). Conclusion: the expression of MUC5AC,TACE and HNE is up-regulated in CRS with and without polyps, and HNE can induce the increase of MUC5AC in nasal epithelial cells through TACE, indicating that HNE-TACE signaling pathway plays an important role in the proliferation of goblet cells in CRS.
【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】：R765.41
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