针刺俞募穴改善FC小鼠胃肠传输功能的GDNF-PI3K-AKT信号转导机制研究

发布时间：2018-06-20 11:19

本文选题：针刺 + 俞募穴　；参考：《成都中医药大学》2016年博士论文

【摘要】：目的：.本研究在针刺大肠俞募穴治疗功能性便秘疗效肯定的基础上,探讨俞募穴治疗功能性便秘中EGC细胞及GDNF-P13K-Akt信号转导通路的作用,旨在探索针刺大肠俞募穴治疗该病的效应机制,以期为针刺治疗本病的临床选穴及应用提供科学依据。方法：1、实验一选昆明小鼠80只,随机分为以下5组：空白对照组、模型组、针刺组、药物组、空白针刺组,每组各16只。采用复方地芬诺酯灌胃法复制功能性便秘模型,选取双侧天枢、大肠俞交替针刺,每次电针治疗30min,1次/d,5次为一个疗程,疗程之间休息2d,共治疗2个疗程。分别于第1、2疗程结束后取胃、小肠和远端结肠,检测胃排空率、小肠推进率、结肠平滑肌张力变化幅度。2、实验二、三选昆明小鼠80只,随机分为以下5组：对照组、模型组、针刺组、EGC对照组、EGC针刺组,每组各16只。模型制作及电针操作同实验一。各组分别于第1、2疗程结束后取远端结肠,运用数字扫描显微镜、电镜、细胞凋亡技术观察结肠组织形态、平滑肌细胞超微结构、平滑肌细胞凋亡情况；利用免疫组化、原位杂交、PCR及Western Blot法检GDNF-PI3K-Akt信号通路及其下游mTOR蛋白的表达水平。结果：1、胃肠传输功能检测：(1)胃排空率模型组小鼠胃排空明显延迟(P0.01),针刺和药物均可显著加快便秘小鼠的胃排空速度(P0.01,P0.01)；空白对照组与空白针刺组比较,两者胃排空率的差异无统计学意义(P0.05)。第1、2疗程间进行比较,各组胃排空率差异无统计学意义(P0.05)。(2)小肠推进率模型组小鼠小肠推进率明显减慢(P0.01),针刺和药物均可显著加快便秘小鼠的小肠推进速度(P0.01,P0.01)；空白对照组与空白针刺组比较,两者小肠推进率的差异无统计学意义(P0.05)。第1、2疗程间进行比较,各组小肠推进率差异无统计学意义(P0.05)。(3)结肠平滑肌平均张力变化百分数模型组小鼠结肠平滑肌张力变化幅度呈降低趋势,但与对照组比较,差异无统计学意义(P0.05)；与模型组比较,针刺可增强便秘小鼠的结肠平滑肌张力变化幅度(P0.05)；空白对照组与空白针刺组比较,两者结肠平滑肌张力变化幅度百分数的差异无统计学意义(P0.05)。第1、2疗程间比较,各组内结肠平滑肌张力变化不明显(P0.05)。2、结肠组织形态学观察对照组结肠神经丛数量较多,内可见神经节细胞排列整齐均匀。模型组结肠神经丛数量减少,神经节细胞排列紊乱,内见空泡样变,细胞数量减少。针刺组结肠形态学与空白对照组比较无显著性改变。EGC对照组和EGC针刺组结肠组织形态改变与模型组相似,两者形态改变差别不明显。3、平滑肌细胞超微结构观察对照组平滑肌细胞染色质分布均匀,线粒体,粗面内质网,核糖体等结构清晰。模型组平滑肌细胞细胞核染色质聚集,线粒体明显肿胀,粗面内质网扩张。针刺组平滑肌细胞染色质分布均匀,胞浆中线粒体,粗面内质网,核糖体等结构清晰。EGC对照组和EGC针刺组比较,两者平滑肌细胞超微结构的变化不明显。4、平滑肌细胞凋亡检测与对照组比较,模型组凋亡细胞阳性表达率增强(P0.01)；与模型组比较,针刺组凋亡细胞阳性表达率下降(P0.01)；抑制EGC细胞后,针刺对平滑肌细胞凋亡的改善作用不明显(P0.05)。第1、2疗程组内比较发现,各组随疗程变化均不明显(P0.05)。5、GDNF-PI3K-Akt信号转导通路检测(1)GDNF蛋白的表达与对照组比较,模型组结肠内GDNF蛋白的表达水平显著降低(P0.01)；与模型组比较,针刺组GDNF蛋白的含量升高(P0.01)；抑制EGC后,针刺对GDNF蛋白表达水平的调节作用不明显(P0.05)。第1、2疗程间进行比较发现,EGC针刺组2个疗程时GDNF蛋白的表达水平与1个疗程比较显著升高(P0.05),其余组变化均不明显(P0.05)。(2)Rai蛋白及其mRNA的表达与对照组比较,模型组结肠内Rai蛋白及其mRNA的表达水平降低(P0.01,P0.05)；与模型组比较,针刺组Rai蛋白及其mRNA的表达水平呈增强趋势,但差异无统计学意义(P0.05)；与EGC对照组比较,EGC针刺组结肠内Rai蛋白及其mRNA的表达增强(P0.05,P0.05)。第1、2疗程组内进行比较,对照组2个疗程时Rai蛋白的表达水平与1个疗程比较显著升高(P0.01),其余组两者表达水平随疗程变化不明显(P0.05,P0.05)。(3)RET蛋白及其mRNA的表达与对照组比较,模型组结肠内RET蛋白及其mRNA的含量显著降低(P0.01,P0.01)；与模型组比较,针刺组两者的表达水平增强(P0.01)：与EGC对照组比较,EGC针刺组结肠内RET蛋白及其mRNA的表达水平变化不明显(P0.05,P0.05)。第1、2疗程组内比较发现,各组内两者的表达水平变化均不明显(P0.05,P0.05)。(4) PI3K、Akt表达水平与对照组比较,模型组结肠内PI3K、Akt的含量升高(P0.01)；与模型组比较,针刺组Akt的表达水平降低(P0.05),PI3K的表达水平呈下降趋势,但差异无统计学意义(P0.05)。EGC对照组与EGC针刺组比较,两组PI3K、Akt的表达水平变化均不明显(P0.05,P0.05)。第1、2疗程组内比较发现,各组PI3K、Akt的表达水平变化均不明显(P0.05,P0.05)。(5)mTOR蛋白的表达与对照组比较,模型组结肠内mTOR蛋白的含量升高(P0.01)；针刺可抑制结肠内mTOR的表达,但与模型组比较差异无统计学意义(P0.05)；EGC对照组与EGC针刺组比较,mTOR表达水平的变化不明显(P0.05)。第1、2疗程组内比较发现,各组mTOR的表达水平变化均不明显(P0.05)。结论：1、在健康状态下,针刺大肠俞募穴对胃肠传输功能的改善作用不明显。2、在疾病状态下,针刺大肠俞募穴可能通过促使EGC分泌产生GDNF,调控PI3K-Akt信号通路,下调mTOR的表达,抑制细胞凋亡,进而改善结肠组织形态及胃肠传输功能,治疗功能性便秘。3、抑制EGC后,针刺大肠俞募穴对结肠组织形态、GDNF-PI3K-Akt信号通路表达水平的调控作用不明显。从反面证实了EGC细胞在针刺大肠俞募穴治疗功能性便秘中的重要作用。
[Abstract]:Objective: To explore the role of EGC cells and GDNF-P13K-Akt signal transduction pathway in the treatment of functional constipation by Yu Muxue in the treatment of functional constipation by needling large intestine Shu Points in the treatment of functional constipation. The purpose of this study is to explore the effect mechanism of acupuncture on the treatment of the disease by needling large intestine Yu Muxue in order to provide the clinical selection and application of acupuncture in the treatment of this disease. Scientific basis. Methods: 1, 80 Kunming mice were selected and randomly divided into 5 groups: blank control group, model group, acupuncture group, drug group, blank acupuncture group, 16 rats in each group. The model of functional constipation was replicated by Fufang pinactone gavage method, bilateral Tianshu, 30min, 1 times /d and 5 times were selected. After 2 courses of treatment, 2 courses were treated. After the end of the course of treatment, the gastric emptying, small intestine and distal colon were taken to detect gastric emptying rate, small intestine propulsion rate,.2 of colonic smooth muscle tension, two and three Kunming mice were selected, randomly divided into 5 groups: control group, model group, acupuncture group, EGC control group, EGC acupuncture group, each group. Group 16 each. Model making and electroacupuncture operation and experimental one. Each group took the distal colon after the end of the 1,2 course. The morphology of colon tissue, the ultrastructure of smooth muscle cells and the apoptosis of smooth muscle cells were observed with the technique of digital scanning microscope, electron microscope, and cell apoptosis. Immunization, in situ hybridization, PCR and Western Blot were used to examine GDN. The expression level of F-PI3K-Akt signaling pathway and its downstream mTOR protein. Results: 1, gastrointestinal transmission function test: (1) gastric emptying in mice with gastric emptying rate was significantly delayed (P0.01), acupuncture and drugs could significantly accelerate the gastric emptying speed of constipated mice (P0.01, P0.01); blank control group was compared with blank acupuncture group, and the gastric emptying rate of the two groups was compared with that of blank acupuncture group. The difference was not statistically significant (P0.05). There was no significant difference in gastric emptying rate in each group (P0.05). (2) small intestinal propulsion rate of small intestine propelling model group was significantly slower (P0.01), acupuncture and drugs could significantly accelerate the speed of small intestine push in constipation mice (P0.01, P0.01); blank control group was compared with blank acupuncture group. There was no significant difference in the small intestinal propulsion rate (P0.05). There was no significant difference in the small intestinal propulsive rate between the two groups (P0.05). (3) the variation of colonic smooth muscle tension in the model group was lower than that in the model group (P0.05), but there was no significant difference compared with the control group (P0.05 Compared with the model group, acupuncture could enhance the amplitude of colonic smooth muscle tension in the constipation mice (P0.05). Compared with the blank control group, there was no significant difference between the blank control group and the blank acupuncture group (P0.05). The changes of colonic smooth muscle tension in each group were not significantly changed (P0.05).2 in the group 1,2 course. The number of colon nerve plexus in the control group was more, and the number of ganglion cells arranged neatly and even. The number of colonic plexus in the model group was reduced, the ganglion cells were arranged in disorder, the number of vacuoles changed and the number of cells decreased. The colonic morphology of the acupuncture group had no significant changes in the.EGC control group and the EGC acupuncture group. The morphological changes of the colonic tissue were similar to those in the model group. The morphological changes of the two groups were not obvious.3. The ultrastructure of the smooth muscle cells was observed in the control group. The chromatin of the smooth muscle cells in the control group was evenly distributed, the mitochondria, the rough endoplasmic reticulum, the ribosome and other structures were clear. The chromatin of the cell nuclei of the smooth muscle cells in the model group was clustered, the mitochondria were swollen and the rough endoplasmic reticulum The chromatin of smooth muscle cells in the acupuncture group was evenly distributed, the mitochondria in the cytoplasm, the rough endoplasmic reticulum and the ribosome were clear in the.EGC control group and the EGC acupuncture group. The changes of the ultrastructure of the smooth muscle cells were not obvious.4, the apoptosis detection of smooth muscle cells was compared with the control group, and the positive expression rate of the apoptotic cells in the model group was enhanced (P0.01). Compared with the model group, the positive expression rate of apoptotic cells in the acupuncture group decreased (P0.01). After the inhibition of EGC cells, the effect of acupuncture on the apoptosis of smooth muscle cells was not obvious (P0.05). In the 1,2 course group, it was found that the changes of each group were not obvious (P0.05).5, GDNF-PI3K-Akt signal transduction pathway was detected (1) the expression of GDNF protein and the control group Compared with the model group, the expression level of GDNF protein in the colon was significantly decreased (P0.01). Compared with the model group, the content of GDNF protein in the acupuncture group increased (P0.01). After the inhibition of EGC, the regulation effect of acupuncture on the expression of GDNF protein was not obvious (P0.05). The expression level of GDNF protein in the 2 course of the EGC acupuncture group was compared with that of the EGC acupuncture group, and the expression level of the GDNF protein was 1. Compared with the control group, the expression level of Rai protein and mRNA in the colon of the model group decreased (P0.01, P0.05). Compared with the model group, the expression level of Rai protein and mRNA in the acupuncture group was enhanced, but the difference was not statistically significant compared with the model group, but the difference was not statistically significant. (2) the expression of Rai protein and its mRNA in the model group decreased (P0.01, P0.05) in the model group. (P0.05); compared with the EGC control group, the expression of Rai protein and mRNA in the colon of the EGC acupuncture group was enhanced (P0.05, P0.05). The expression level of the Rai protein in the control group was compared in the 2 course of the control group. The level of the expression of the Rai protein in the control group was significantly higher than that in the 1 course (P0.01). The levels of the other groups were not obvious (P0.05, P0.05). (3) protein and The expression of mRNA in the colon of the model group was significantly lower than that in the control group (P0.01, P0.01). Compared with the model group, the expression level of the two groups was enhanced (P0.01). Compared with the EGC control group, the expression level of RET protein and mRNA in the colon of the EGC acupuncture group was not significantly changed (P0.05, P0.05). It was found that the expression level of the two groups was not obvious (P0.05, P0.05). (4) PI3K, the expression level of Akt in the model group was higher than that of the control group (P0.01). Compared with the model group, the expression level of Akt in the acupuncture group decreased (P0.05) and the expression level of PI3K decreased, but the difference was not statistically significant (P0.05). The expression level of PI3K and Akt in the two groups was not significantly changed (P0.05, P0.05) in the two groups and the EGC acupuncture group. In the 1,2 course, the expression level of PI3K and Akt was not significantly changed (P0.05, P0.05). (5) the expression of the mTOR protein was higher than that of the control group. There was no significant difference in the expression of mTOR in the intestinal tract (P0.05). The changes of mTOR expression level in the EGC control group and the EGC group were not obvious (P0.05). In the 1,2 course, the changes in the expression level of mTOR were not obvious (P0.05). 1, in the healthy state, the acupuncture of the needling large intestine Shu Points to the gastrointestinal transmission The effect of functional improvement is not obvious.2, in the condition of disease, the acupuncture of the acupuncture point of the acupuncture of large intestine may be stimulated by EGC secretion to produce GDNF, regulate the PI3K-Akt signal pathway, reduce the expression of mTOR, inhibit the cell apoptosis, and then improve the morphology of the colon and the gastrointestinal transmission function, and treat the functional constipation.3 and the inhibition of EGC, acupuncture on the colon Shu point to colonic group The regulatory role of the expression level of GDNF-PI3K-Akt signal pathway is not obvious. The important role of EGC cells in the treatment of functional constipation is confirmed by the reverse side.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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