TRPC6在Barrett食管发生中的作用研究

发布时间：2018-08-13 19:44

【摘要】：目的:Barrett食管(Barrett’s esophagus,BE)被定义为食管复层鳞状上皮被单层柱状上皮所取代的病理现象。BE是胃食管反流病(Gastroesophageal reflex disease,GERD)的并发症,与食管腺癌的发生密切相关。它已经被公认为是食管腺癌最重要的癌前病变,其发展为食管腺癌的危险性高达正常人的30~50倍[1]。目前抑酸治疗对反流性食管炎治疗有效,从而减少BE的发生。这提示酸性刺激在BE的发生过程中起到重要作用,但其具体发生机制目前尚不清楚,因此本课题拟通过研究酸性刺激如何促进BE发生的作用机制,为BE的临床治疗靶点提供理论支持。方法:1.钙离子成像技术检测不同pH值及阻断剂对SHEE细胞内钙离子的变化。2.免疫组织化学方法检测正常人食管下段粘膜和Barrett食管下段病变粘膜中CDX2、TRPC6的表达情况。3.免疫印迹法检测pH处理24小时以后HEEC细胞株中CDX2、TRPC6的蛋白表达情况。结果:1.钙离子成像技术结果显示:(1)与对照组(pH 7.4)比较,在pH值为7.0、6.5、6.0时,SHEE细胞内的钙离子水平无明显差异(P0.05),在pH值为5.5、5.0时,SHEE细胞内的钙离子水平明显升高,差异有统计学意义(P0.05);(2)加入细胞内钙螯合剂BAPTA-AM和TRPC6阻断剂SKF96365后,SHEE细胞内钙离子浓度增高被显著抑制(P0.05)。2.免疫组织化学法结果显示:CDX2、TRPC6在正常食管粘膜组织和BE食管粘膜组织中均有表达,与正常食管粘膜组(NE)比较,Barrett食管粘膜组(BE)中的表达水平均明显升高,差异有统计学意义(P0.05),且CDX2与TRPC6在BE中的表达具有相关性。3.免疫印迹结果显示:pH值为5.5处理HEEC细胞24小时后与对照组(pH 7.2)相比TRPC6和CDX2蛋白表达上调(P0.05),加入阻断剂BAPTA-AM和SKF96365后,CDX2与TRPC6蛋白增加被显著抑制(P0.05)。结论:酸暴露激活TRPC6通道,使细胞内钙离子水平升高,进而上调CDX2的表达,促进Barrett食管的发生。
[Abstract]:Objective to define Barrett's Barrett's esophagus (Barrett's esophagus be) as a pathological phenomenon in which esophageal squamous epithelium is replaced by monolayer columnar epithelium. Be is a complication of (Gastroesophageal reflex disease of gastroesophageal reflux disease and is closely related to the occurrence of esophageal adenocarcinoma. It has been recognized as the most important precancerous lesion of esophageal adenocarcinoma. Acid suppression therapy is effective for reflux esophagitis, thus reducing the incidence of be. This suggests that acid stimulation plays an important role in the development of be, but its specific mechanism is not clear, so this paper intends to study the mechanism of acid stimulation to promote the occurrence of be. To provide theoretical support for the clinical target of be. Method 1: 1. Calcium ion imaging technique was used to detect the changes of calcium ions in SHEE cells with different pH values and blockers. Immunohistochemical method was used to detect the expression of CDX2 / TRPC6 in the lower esophageal mucosa of normal subjects and the lesion mucosa of Barrett's lower esophagus. The protein expression of CDX2 and TRPC6 in HEEC cells was detected by Western blotting after 24 hours of pH treatment. The result is 1: 1. The results of calcium imaging showed that: (1) compared with the control group (pH 7.4), there was no significant difference in the intracellular calcium level in SHEE cells at pH 7.0 ~ 6.5 ~ 6.0 (P0.05), but the intracellular calcium level in SHEE cells was significantly increased at pH 5.55 ~ 5.0. The difference was statistically significant (P0.05); (2) after the addition of intracellular calcium chelator BAPTA-AM and TRPC6 blocker SKF96365, the increase of intracellular calcium ion concentration was significantly inhibited (P0.05). 2. The results of immunohistochemistry showed that the expression of (BE) in the normal esophageal mucosa and be esophageal mucosa was significantly higher than that in the normal esophageal mucosa group and in the be esophageal mucosal tissue, compared with the normal esophageal mucosa group, the expression of (BE) in the normal esophageal mucosa group was significantly higher than that in the normal esophageal mucosa group. The difference was statistically significant (P0.05), and the expression of CDX2 and TRPC6 in be was related. 3. The results of Western blot showed that the expression of TRPC6 and CDX2 protein was up-regulated 24 hours after the treatment of HEEC cells with a pH value of 5.5 (P0.05), and the increase of CDX2 and TRPC6 protein was significantly inhibited by the addition of BAPTA-AM and SKF96365 (P0.05). Conclusion: acid exposure activates TRPC6 channels, elevates intracellular calcium level, upregulate the expression of CDX2 and promote the development of Barrett esophagus.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R571

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