应用蛋白芯片技术对电针足阳明经（穴）促胃黏膜损伤修复过程中相关蛋白磷酸化的研究
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摘要
目的：通过对电针足阳明经(穴)促胃黏膜损伤修复过程的研究,探讨电针足阳明经(穴)对胃黏膜损伤修复效应的影响。采用蛋白芯片技术观察电针足阳明经(穴)对胃黏膜损伤修复相关信号蛋白磷酸化的变化,全面揭示与上述过程有关的信号分子及通路,从蛋白组学角度阐明电针足阳明经(穴)对胃黏膜损伤的修复机制,从而更好地理解针灸治病机理以及胃经与胃的相关性,为针灸临床应用提供一定的实验及理论依据。方法：40只SD健康大鼠随机分为4组,即空白组、模型组、针刺治疗组、针刺对照组,10只／组；本研究分为三个部分,第一部分：采用无水乙醇灌胃方法制备胃黏膜损伤大鼠模型,GUTH法行胃黏膜损伤指数计数后,随机挑选5只／组行HE染色法观察胃黏膜形态学改变,免疫组化法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA).第二部分：剩余大鼠5只／组,提取胃黏膜细胞,采用720种磷酸化抗体蛋白芯片技术检测电针足阳明经(穴)对胃黏膜损伤修复相关信号蛋白磷酸化的变化,筛选出各组差异性表达的磷酸化信号蛋白。第三部分：采用第一部分和第二部分的实验标本,应用免疫印迹(Western-blot)方法检测大鼠胃黏膜细胞磷酸化raf-1蛋白表达,免疫组化法检测磷酸化细胞外信号调节激酶(extracellular signal-regulated kinase, ERK1/2)蛋白与细胞周期因子D1 (Cell cycle protein D1,cyclin D1)蛋白表达。
结果：
1.针刺治疗组可明显降低胃黏膜损伤指数的计数,胃黏膜损伤指数值显著低于模型组和针刺对照组(P＜0.01),而与空白组比较差异无显著性意义(P＞0.05)；模型组与空白组比较,胃黏膜损伤指数值显著升高,差异具有显著性意义(P<0.01)。
2.针刺足阳明经(穴)可促进胃黏膜细胞增殖,针刺治疗组PCNA-LI与模型组比较差异具有显著性意义(P<0.01)。
3.胃黏膜细胞720抗体蛋白芯片结果显示：与空白组比较,模型组的蛋白磷酸化水平变化大于1.5倍的蛋白中有26种出现上调,61种出现下调；与模型组比较,针刺治疗组的蛋白磷酸化水平变化大于1.5倍的蛋白中有84种出现上调,12种出现下调,针刺对照组则只有12种出现上调,3种出现下调；与针刺对照组比较,针刺治疗组的磷酸化水平差异蛋白在蛋白数量、蛋白功能种类与信号转导通路的归属上均明显优于针刺对照组。
4.针刺足阳明经(穴)可明显激活RAF-1、ERK1/2蛋白磷酸化的水平,也可上调cyclin D1蛋白的水平,上述三个指标针刺治疗组与空白组、模型组、针刺对照组比较,差异均具有显著性或非常显著性意义(P<0.01或P<0.05)。
结论：
1.电针足阳明经(穴)能明显改善受损胃黏膜形态学的改变,促PCNA指数增加。提示电针足阳明经(穴)能明显促胃黏膜损伤修复,且促细胞增殖是电针足阳明经(穴)促胃黏膜损伤修复的重要环节。
2.电针足阳明经可双向调整模型组上下调的蛋白,且调整的蛋白涉及多个功能种类,归属多个信号转导通路。提示多靶点、多途径与多条信号转导通路激活是电针足阳明胃经(穴)促胃黏膜损伤修复的重要特征。
3.电针足阳明经(穴)与对照点比较,能特异性激活MAPKS信号转导通路,提示MAPKS信号转导通路与电针足阳明经(穴)促胃黏膜损伤修复密切相关。
4.电针足阳明经(穴)促胃黏膜损伤修复过程中激活多种功能类型蛋白和多条信号转导通路的激活的特点符合针灸“综合性”调整作用的基本特点。采用蛋白芯片技术探讨中医药及针灸对机体的整体调节作用,对阐释中医整体观的认识具有一定的促进作用。
5.电针对胃黏膜修复作用具有一定的穴位特异性,为经脉(穴)—脏腑相关相对特异性提供一定的实验依据。
Objective:To study the effect of the stimulation of ST with EA on the injury repairing of gastric mucosa. By observing the changes of related signal protein phosphorylation under the stimulation of ST with EA, the related signal molecule and pathway were revealed, and from the proteome analysis, the mechanism of injury repairing of Stomach mucosa under the stimulation of ST with EA was expounded which applied certain experimental and theoretic basis for clinical applying and was helpful to understand the therapeutic mechanism of EA and the relationship between ST and stomach.
Methods:40 SD rats were randomly divided into 4 groups:contrast group, model group, EA treating group and EA contrast group with 10 rats per group. The research was divided into three parts:Part 1:The stomach mucosa injury model was set by gastric administration with ethanol. The injury indexes of stomach mucosa were measured by GUTH. The morphology changes of stomach mucosa in 5 randomly-selected rats were observed by HE. The proliferating cell nuclear antigen (PCNA) was tested by immunohistochemical method. Part 2:The rest SD rats were randomly divided into 4 groups with 5 rats per group. The stomach mucosa was extracted. The changes of related signal protein phosphorylation of stomach mucosa injury repairing with EA at ST were detected by 720 kinds of phosphorylation of antibody protein chip tech. And the differential expressed phosphorylation signal protein in each group was screened. Part 3:the express of raf-1 protein of stomach mucosa in the samples of part 1 and 2 were detected with Western-blot and the expression of ERK 1/2 protein and cyclin D1 protein were detected by SP.
Results:
1. The number of the injury index of stomach mucosa in EA treating group was decreased obviously, and compared with model group and EA contrast group, the injury index was remarkable lower (P<0.01); compared with contrast group, the difference was insignificant (P>0.05); compared with contrast group, the index in model group was increased obviously and the deference was remarkable significant (P＜0.01).
2. EA at ST can promote the proliferation of stomach mucosa cell. Compared with model group, the difference of the level of PCNA-LI in EA treatment group and model group was remarkable significant (P<0.01).
3.720 kinds of phosphorylation of antibody protein chip tech detection shows: compared with contrast group,84 kinds of protein that was up regulated more than 1.5 folds in the phosphorylation change were up-regulated,12 kinds were down-regulated; while in EA contrast group, only 17 kinds were up-regulated and 3 were down-regulated; compared with EA contrast group, the number, function type and signal transduction pathway of phosphorylation signal protein were much better than that of EA contrast group.
4. EA at ST can obviously activate the level of RAF-1 and phosphorylation of protein and up regulated the level of cyclin D1 protein. The difference among the groups were all remarkable significant (P＜0.01 or P＜0.05).
Conclusion:
1. EA at ST can obviously improve the morphology changes of stomach mucosa and increase the index of PCNA-LI which indicates that EA at ST can obviously improve the morphology changes of stomach mucosa
2. EA at ST can dual-directionally regulate the proteins of model group which involves various functional types and belongs to multiple signal transduction pathways. It indicates multi-target and multiple-way is the important characteristic of EA at ST to repair the stomach mucosa.
3. Compared with contrast group, EA at ST can activate the MAPKS signal transduction pathway specifically which indicates MAPKS signal transduction pathway is close related with repairing of EA at ST to promote the repairing of stomach mucosa.
4. EA at ST can obviously activate various functional types of proteins and several signal transduction pathway which accord with the basic characteristics of acupuncture such as holism and comprehensibility. Studying the holistic regulating effect of TCM and acupuncture by using protein chip tech can promote the understanding and applying of holism in TCM.
结果：
1.针刺治疗组可明显降低胃黏膜损伤指数的计数,胃黏膜损伤指数值显著低于模型组和针刺对照组(P＜0.01),而与空白组比较差异无显著性意义(P＞0.05)；模型组与空白组比较,胃黏膜损伤指数值显著升高,差异具有显著性意义(P<0.01)。
2.针刺足阳明经(穴)可促进胃黏膜细胞增殖,针刺治疗组PCNA-LI与模型组比较差异具有显著性意义(P<0.01)。
3.胃黏膜细胞720抗体蛋白芯片结果显示：与空白组比较,模型组的蛋白磷酸化水平变化大于1.5倍的蛋白中有26种出现上调,61种出现下调；与模型组比较,针刺治疗组的蛋白磷酸化水平变化大于1.5倍的蛋白中有84种出现上调,12种出现下调,针刺对照组则只有12种出现上调,3种出现下调；与针刺对照组比较,针刺治疗组的磷酸化水平差异蛋白在蛋白数量、蛋白功能种类与信号转导通路的归属上均明显优于针刺对照组。
4.针刺足阳明经(穴)可明显激活RAF-1、ERK1/2蛋白磷酸化的水平,也可上调cyclin D1蛋白的水平,上述三个指标针刺治疗组与空白组、模型组、针刺对照组比较,差异均具有显著性或非常显著性意义(P<0.01或P<0.05)。
结论：
1.电针足阳明经(穴)能明显改善受损胃黏膜形态学的改变,促PCNA指数增加。提示电针足阳明经(穴)能明显促胃黏膜损伤修复,且促细胞增殖是电针足阳明经(穴)促胃黏膜损伤修复的重要环节。
2.电针足阳明经可双向调整模型组上下调的蛋白,且调整的蛋白涉及多个功能种类,归属多个信号转导通路。提示多靶点、多途径与多条信号转导通路激活是电针足阳明胃经(穴)促胃黏膜损伤修复的重要特征。
3.电针足阳明经(穴)与对照点比较,能特异性激活MAPKS信号转导通路,提示MAPKS信号转导通路与电针足阳明经(穴)促胃黏膜损伤修复密切相关。
4.电针足阳明经(穴)促胃黏膜损伤修复过程中激活多种功能类型蛋白和多条信号转导通路的激活的特点符合针灸“综合性”调整作用的基本特点。采用蛋白芯片技术探讨中医药及针灸对机体的整体调节作用,对阐释中医整体观的认识具有一定的促进作用。
5.电针对胃黏膜修复作用具有一定的穴位特异性,为经脉(穴)—脏腑相关相对特异性提供一定的实验依据。
Objective:To study the effect of the stimulation of ST with EA on the injury repairing of gastric mucosa. By observing the changes of related signal protein phosphorylation under the stimulation of ST with EA, the related signal molecule and pathway were revealed, and from the proteome analysis, the mechanism of injury repairing of Stomach mucosa under the stimulation of ST with EA was expounded which applied certain experimental and theoretic basis for clinical applying and was helpful to understand the therapeutic mechanism of EA and the relationship between ST and stomach.
Methods:40 SD rats were randomly divided into 4 groups:contrast group, model group, EA treating group and EA contrast group with 10 rats per group. The research was divided into three parts:Part 1:The stomach mucosa injury model was set by gastric administration with ethanol. The injury indexes of stomach mucosa were measured by GUTH. The morphology changes of stomach mucosa in 5 randomly-selected rats were observed by HE. The proliferating cell nuclear antigen (PCNA) was tested by immunohistochemical method. Part 2:The rest SD rats were randomly divided into 4 groups with 5 rats per group. The stomach mucosa was extracted. The changes of related signal protein phosphorylation of stomach mucosa injury repairing with EA at ST were detected by 720 kinds of phosphorylation of antibody protein chip tech. And the differential expressed phosphorylation signal protein in each group was screened. Part 3:the express of raf-1 protein of stomach mucosa in the samples of part 1 and 2 were detected with Western-blot and the expression of ERK 1/2 protein and cyclin D1 protein were detected by SP.
Results:
1. The number of the injury index of stomach mucosa in EA treating group was decreased obviously, and compared with model group and EA contrast group, the injury index was remarkable lower (P<0.01); compared with contrast group, the difference was insignificant (P>0.05); compared with contrast group, the index in model group was increased obviously and the deference was remarkable significant (P＜0.01).
2. EA at ST can promote the proliferation of stomach mucosa cell. Compared with model group, the difference of the level of PCNA-LI in EA treatment group and model group was remarkable significant (P<0.01).
3.720 kinds of phosphorylation of antibody protein chip tech detection shows: compared with contrast group,84 kinds of protein that was up regulated more than 1.5 folds in the phosphorylation change were up-regulated,12 kinds were down-regulated; while in EA contrast group, only 17 kinds were up-regulated and 3 were down-regulated; compared with EA contrast group, the number, function type and signal transduction pathway of phosphorylation signal protein were much better than that of EA contrast group.
4. EA at ST can obviously activate the level of RAF-1 and phosphorylation of protein and up regulated the level of cyclin D1 protein. The difference among the groups were all remarkable significant (P＜0.01 or P＜0.05).
Conclusion:
1. EA at ST can obviously improve the morphology changes of stomach mucosa and increase the index of PCNA-LI which indicates that EA at ST can obviously improve the morphology changes of stomach mucosa
2. EA at ST can dual-directionally regulate the proteins of model group which involves various functional types and belongs to multiple signal transduction pathways. It indicates multi-target and multiple-way is the important characteristic of EA at ST to repair the stomach mucosa.
3. Compared with contrast group, EA at ST can activate the MAPKS signal transduction pathway specifically which indicates MAPKS signal transduction pathway is close related with repairing of EA at ST to promote the repairing of stomach mucosa.
4. EA at ST can obviously activate various functional types of proteins and several signal transduction pathway which accord with the basic characteristics of acupuncture such as holism and comprehensibility. Studying the holistic regulating effect of TCM and acupuncture by using protein chip tech can promote the understanding and applying of holism in TCM.
引文
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