胃蛋白酶在喉咽上皮细胞炎症恶性转化中的作用

Laryngopharyngeal reflux is believed to be caused by retrograde flow of gastric contents which impairs pharyngeal and laryngeal mucosa by direct contact, however, the association between the refluxate and laryngeal carcinoma remains unclear. Recent study supports a role for refluxed pepsin in the promotion of epithelial proliferation and tumorigenesis of the larynx and pharynx. Pepsin significantly altered the expression of 27 genes implicated in carcinogenesis, and also affected the expression of 22 microRNAs known to be implicated in human head and neck cancers. Our previous study indicatd that acid and weakly acid reflux events were detected in vocal cord nudes, vocal cord leukoplakia and laryngeal carcinoma patients. Laryngopharyngeal reflux was a risk factor for laryngeal cancer, and there was no correlation between reflux and tobacco and alcohol. The aim of this study was to evaluate a role for refluxed pepsin in the promotion of inflammation to malignant transformation in laryngeal and pharyngeal epithelial cells. We intend to detect pepsin by ELISA in patients’ saliva and by IHC in tissue samples to examine the relationship between the refluxate and precancerous lesions and laryngeal carcinoma. In virto, the model of pepsin-indued inflammation malignant transformation of epithelial cell in the pharynx and larynx will be established. Based on this model, the changes of NF-kB, STAT3, inflammatory cytokines, EMT markers and other appropriate channels will be detected. These studies will demonstrate a significant role for pepsin in the promotion of inflammation to malignant transformation.

胃内容物反流到食道外导致咽喉粘膜损伤，导致一系列咽喉反流性疾病，但是否参与咽喉粘膜癌变存在争论。有研究发现，胃蛋白酶促进咽喉上皮细胞增殖，改变了与癌病相关的27种基因表达变化，也影响与头颈鳞癌相关的22个microRNAs的表达。我们的临床研究显示喉癌及癌前病变患者存在酸性及弱酸反流，咽喉反流是声带粘膜癌变的危险因素之一，且与抽烟、饮酒无相关性。本研究拟探讨胃蛋白酶是否参与喉咽上皮细胞的炎症恶性转化过程。通过ELISA及免疫组化法检测正常人、声带息肉、癌前及咽喉癌患者唾液和组织中胃蛋白酶的表达，证实反流事件与咽喉慢性炎症及恶性癌变的相关性；体外建立胃蛋白酶诱导喉咽上皮细胞炎症恶性转化模型，检测转录调控因子NF-kB、STAT3、炎性细胞因子、EMT等相应通路改变，为咽喉反流参与咽喉癌变提供理论证据。

胃内容物反流到食道外导致咽喉粘膜损伤，导致一系列咽喉反流性疾病，但是否参与咽喉粘膜癌变存在很大争论。有研究发现，胃蛋白酶促进咽喉上皮细胞增殖，改变了与癌病相关的27种基因表达变化，也影响与头颈鳞癌相关的22个microRNAs的表达。我们前期临床研究显示喉癌及癌前病变患者存在酸性及弱酸反流，咽喉反流是声带粘膜癌变的危险因素之一。本项目中，我们通过体内外实验检测pepsin在正常、声带息肉、喉癌前病变、下咽癌、喉癌患者唾液、组织标本中表达情况及不同浓度pepsin对喉咽上皮、喉癌Hep-2细胞生物学特性的影响，探讨pepsin在喉癌发生发展中的作用；通过BD 流式液相多重蛋白定量技术（CBA）、荧光定量PCR、Western 、免疫荧光检测和信号通路检测等，对胃蛋白酶参与喉咽上皮细胞炎症恶性化的可能机制进行探讨。该项目按照研究计划和研究进度，进展顺利，取得的主要成果包括：（1）在组织标本中，pepsin在健康对照组、声带息肉组、癌前病变组、喉癌组表达依次高，与24 h MII-pH监测的酸反流次数、酸反流时间、酸反流时间百分比、平均酸清除时间有显著正相关。（2）在细胞实验中，我们成功培养出喉上皮原代细胞，CK免疫荧光检测证明该细胞来自上皮细胞。(3)通过不同浓度pepsin刺激喉癌Hep-2细胞的MTT、Transwell及细胞划痕试验证实Pepsin可促进喉癌细胞体外增殖、迁移。（3）在细胞试验中，我们观察到pepsin可使细胞形态从原来的类圆形变成长梭形， PCR、Western发现证实随着Pepsin浓度增高，上皮标志物E-cadherin下调而间质标志物Vimentin、β-catenin及其下游转录因子snail、slug表达上调，同时我们在喉癌组织标本中也验证了Pepsin表达与E-cadherin表达呈负相关，与Vimcntin、β-catenin表达呈正相关。说明pepsin可诱导喉癌发生上皮-间质转化，使其恶性程度增加。（4）细胞试验中CBA结果提示随着Pepsin刺激浓度的增加，IL-8分泌显著增多，喉癌组织中IL-8的表达也与Pepsin呈正相关。对于pepsin刺激下的Hep-2细胞加入IL-8抑制剂阻滞IL-8受体CXCR2后，上皮间质转化诱导作用明显减弱，细胞增殖及迁移能力显著减少，同时snail的表达下调。提示Pepsin可能是通过激活肿