本文選題：針刺 + 俞募穴�。� 參考：《成都中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的：.本研究在針刺大腸俞募穴治療功能性便秘療效肯定的基礎(chǔ)上,探討俞募穴治療功能性便秘中EGC細(xì)胞及GDNF-P13K-Akt信號(hào)轉(zhuǎn)導(dǎo)通路的作用,旨在探索針刺大腸俞募穴治療該病的效應(yīng)機(jī)制,以期為針刺治療本病的臨床選穴及應(yīng)用提供科學(xué)依據(jù)。方法：1、實(shí)驗(yàn)一選昆明小鼠80只,隨機(jī)分為以下5組：空白對(duì)照組、模型組、針刺組、藥物組、空白針刺組,每組各16只。采用復(fù)方地芬諾酯灌胃法復(fù)制功能性便秘模型,選取雙側(cè)天樞、大腸俞交替針刺,每次電針治療30min,1次/d,5次為一個(gè)療程,療程之間休息2d,共治療2個(gè)療程。分別于第1、2療程結(jié)束后取胃、小腸和遠(yuǎn)端結(jié)腸,檢測(cè)胃排空率、小腸推進(jìn)率、結(jié)腸平滑肌張力變化幅度。2、實(shí)驗(yàn)二、三選昆明小鼠80只,隨機(jī)分為以下5組：對(duì)照組、模型組、針刺組、EGC對(duì)照組、EGC針刺組,每組各16只。模型制作及電針操作同實(shí)驗(yàn)一。各組分別于第1、2療程結(jié)束后取遠(yuǎn)端結(jié)腸,運(yùn)用數(shù)字掃描顯微鏡、電鏡、細(xì)胞凋亡技術(shù)觀察結(jié)腸組織形態(tài)、平滑肌細(xì)胞超微結(jié)構(gòu)、平滑肌細(xì)胞凋亡情況；利用免疫組化、原位雜交、PCR及Western Blot法檢GDNF-PI3K-Akt信號(hào)通路及其下游mTOR蛋白的表達(dá)水平。結(jié)果：1、胃腸傳輸功能檢測(cè)：(1)胃排空率模型組小鼠胃排空明顯延遲(P0.01),針刺和藥物均可顯著加快便秘小鼠的胃排空速度(P0.01,P0.01)；空白對(duì)照組與空白針刺組比較,兩者胃排空率的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。第1、2療程間進(jìn)行比較,各組胃排空率差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(2)小腸推進(jìn)率模型組小鼠小腸推進(jìn)率明顯減慢(P0.01),針刺和藥物均可顯著加快便秘小鼠的小腸推進(jìn)速度(P0.01,P0.01)；空白對(duì)照組與空白針刺組比較,兩者小腸推進(jìn)率的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。第1、2療程間進(jìn)行比較,各組小腸推進(jìn)率差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)結(jié)腸平滑肌平均張力變化百分?jǐn)?shù)模型組小鼠結(jié)腸平滑肌張力變化幅度呈降低趨勢(shì),但與對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與模型組比較,針刺可增強(qiáng)便秘小鼠的結(jié)腸平滑肌張力變化幅度(P0.05)；空白對(duì)照組與空白針刺組比較,兩者結(jié)腸平滑肌張力變化幅度百分?jǐn)?shù)的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。第1、2療程間比較,各組內(nèi)結(jié)腸平滑肌張力變化不明顯(P0.05)。2、結(jié)腸組織形態(tài)學(xué)觀察對(duì)照組結(jié)腸神經(jīng)叢數(shù)量較多,內(nèi)可見神經(jīng)節(jié)細(xì)胞排列整齊均勻。模型組結(jié)腸神經(jīng)叢數(shù)量減少,神經(jīng)節(jié)細(xì)胞排列紊亂,內(nèi)見空泡樣變,細(xì)胞數(shù)量減少。針刺組結(jié)腸形態(tài)學(xué)與空白對(duì)照組比較無(wú)顯著性改變。EGC對(duì)照組和EGC針刺組結(jié)腸組織形態(tài)改變與模型組相似,兩者形態(tài)改變差別不明顯。3、平滑肌細(xì)胞超微結(jié)構(gòu)觀察對(duì)照組平滑肌細(xì)胞染色質(zhì)分布均勻,線粒體,粗面內(nèi)質(zhì)網(wǎng),核糖體等結(jié)構(gòu)清晰。模型組平滑肌細(xì)胞細(xì)胞核染色質(zhì)聚集,線粒體明顯腫脹,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張。針刺組平滑肌細(xì)胞染色質(zhì)分布均勻,胞漿中線粒體,粗面內(nèi)質(zhì)網(wǎng),核糖體等結(jié)構(gòu)清晰。EGC對(duì)照組和EGC針刺組比較,兩者平滑肌細(xì)胞超微結(jié)構(gòu)的變化不明顯。4、平滑肌細(xì)胞凋亡檢測(cè)與對(duì)照組比較,模型組凋亡細(xì)胞陽(yáng)性表達(dá)率增強(qiáng)(P0.01)；與模型組比較,針刺組凋亡細(xì)胞陽(yáng)性表達(dá)率下降(P0.01)；抑制EGC細(xì)胞后,針刺對(duì)平滑肌細(xì)胞凋亡的改善作用不明顯(P0.05)。第1、2療程組內(nèi)比較發(fā)現(xiàn),各組隨療程變化均不明顯(P0.05)。5、GDNF-PI3K-Akt信號(hào)轉(zhuǎn)導(dǎo)通路檢測(cè)(1)GDNF蛋白的表達(dá)與對(duì)照組比較,模型組結(jié)腸內(nèi)GDNF蛋白的表達(dá)水平顯著降低(P0.01)；與模型組比較,針刺組GDNF蛋白的含量升高(P0.01)；抑制EGC后,針刺對(duì)GDNF蛋白表達(dá)水平的調(diào)節(jié)作用不明顯(P0.05)。第1、2療程間進(jìn)行比較發(fā)現(xiàn),EGC針刺組2個(gè)療程時(shí)GDNF蛋白的表達(dá)水平與1個(gè)療程比較顯著升高(P0.05),其余組變化均不明顯(P0.05)。(2)Rai蛋白及其mRNA的表達(dá)與對(duì)照組比較,模型組結(jié)腸內(nèi)Rai蛋白及其mRNA的表達(dá)水平降低(P0.01,P0.05)；與模型組比較,針刺組Rai蛋白及其mRNA的表達(dá)水平呈增強(qiáng)趨勢(shì),但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；與EGC對(duì)照組比較,EGC針刺組結(jié)腸內(nèi)Rai蛋白及其mRNA的表達(dá)增強(qiáng)(P0.05,P0.05)。第1、2療程組內(nèi)進(jìn)行比較,對(duì)照組2個(gè)療程時(shí)Rai蛋白的表達(dá)水平與1個(gè)療程比較顯著升高(P0.01),其余組兩者表達(dá)水平隨療程變化不明顯(P0.05,P0.05)。(3)RET蛋白及其mRNA的表達(dá)與對(duì)照組比較,模型組結(jié)腸內(nèi)RET蛋白及其mRNA的含量顯著降低(P0.01,P0.01)；與模型組比較,針刺組兩者的表達(dá)水平增強(qiáng)(P0.01)：與EGC對(duì)照組比較,EGC針刺組結(jié)腸內(nèi)RET蛋白及其mRNA的表達(dá)水平變化不明顯(P0.05,P0.05)。第1、2療程組內(nèi)比較發(fā)現(xiàn),各組內(nèi)兩者的表達(dá)水平變化均不明顯(P0.05,P0.05)。(4) PI3K、Akt表達(dá)水平與對(duì)照組比較,模型組結(jié)腸內(nèi)PI3K、Akt的含量升高(P0.01)；與模型組比較,針刺組Akt的表達(dá)水平降低(P0.05),PI3K的表達(dá)水平呈下降趨勢(shì),但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。EGC對(duì)照組與EGC針刺組比較,兩組PI3K、Akt的表達(dá)水平變化均不明顯(P0.05,P0.05)。第1、2療程組內(nèi)比較發(fā)現(xiàn),各組PI3K、Akt的表達(dá)水平變化均不明顯(P0.05,P0.05)。(5)mTOR蛋白的表達(dá)與對(duì)照組比較,模型組結(jié)腸內(nèi)mTOR蛋白的含量升高(P0.01)；針刺可抑制結(jié)腸內(nèi)mTOR的表達(dá),但與模型組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；EGC對(duì)照組與EGC針刺組比較,mTOR表達(dá)水平的變化不明顯(P0.05)。第1、2療程組內(nèi)比較發(fā)現(xiàn),各組mTOR的表達(dá)水平變化均不明顯(P0.05)。結(jié)論：1、在健康狀態(tài)下,針刺大腸俞募穴對(duì)胃腸傳輸功能的改善作用不明顯。2、在疾病狀態(tài)下,針刺大腸俞募穴可能通過(guò)促使EGC分泌產(chǎn)生GDNF,調(diào)控PI3K-Akt信號(hào)通路,下調(diào)mTOR的表達(dá),抑制細(xì)胞凋亡,進(jìn)而改善結(jié)腸組織形態(tài)及胃腸傳輸功能,治療功能性便秘。3、抑制EGC后,針刺大腸俞募穴對(duì)結(jié)腸組織形態(tài)、GDNF-PI3K-Akt信號(hào)通路表達(dá)水平的調(diào)控作用不明顯。從反面證實(shí)了EGC細(xì)胞在針刺大腸俞募穴治療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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245

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