瘢痕灸預處理對預防原發(fā)性肝癌大鼠Wnt信號通路相關(guān)基因的影響
發(fā)布時間：2018-07-22 13:15
【摘要】：目的:本研究采用二乙基亞硝氨(DEN)誘導致原發(fā)性肝癌大鼠模型作為研究對象,采用麥粒狀艾炷,分別在大鼠足三里穴、肝俞穴實施瘢痕灸預處理。觀察不同穴位瘢痕灸預處理對原發(fā)性肝癌大鼠Wnt信號傳導通路相關(guān)基因表達的影響,從而進一步探討瘢痕灸對原發(fā)性肝癌的作用機制,明確瘢痕灸預防肝癌發(fā)生、發(fā)展的現(xiàn)代機理,為今后瘢痕灸預防原發(fā)性肝癌,控制疾病發(fā)展提供分子生物學依據(jù)。材料和方法:SPF級Wistar大鼠共48只,雄性,使用隨機數(shù)字表法分為空白組(12只)、模型組(12只)、預處理肝俞組(12只)和預處理足三里組(12只)。在適應(yīng)性喂養(yǎng)一周后,對預處理肝俞組和預處理足三里組進行瘢痕灸,將艾絨做成一壯為麥粒大小,質(zhì)量約為0.0005g/壯,12壯/穴,每壯使其燃盡,空白組和模型組大鼠不做瘢痕灸處理。治療結(jié)束后,除空白組外,均采用腹腔注射DEN的方法建立大鼠原發(fā)性肝癌模型。DEN用量按照每千克體重50mg計算,造模持續(xù)14周:1-4周,每周注射2次;5-14周,每周注射1次,注射時間均在上午8:30--10:30。造模結(jié)束后,大鼠禁食一天,用10%水合氯醛麻醉,開腹行腹主動脈采血,離心取血清備檢,取部分大鼠肝臟組織用無菌EP管保存,液氮浸泡后,-80℃冰箱冷凍,其余肝臟組織采用4%多聚甲醛固定,避光保存。檢測大鼠血清中的甲胎蛋白(AFP)、γ-谷胺酰轉(zhuǎn)肽酶(γ-GT)、巖藻糖苷酶(AFU)含量;Rt-PCR法檢測肝臟組織Wnt信號通路中Wnt3a、Wnt10b的m RNA表達。HE染色法觀察大鼠肝臟組織結(jié)構(gòu)病理形態(tài)變化。結(jié)果:1 HE染色結(jié)果顯示:與空白組相比,模型組、預處理肝俞組和預處理足三里組大鼠肝組織肝小梁、肝竇呈無序排列,并伴有不同程度的深染;與模型組相比,預處理肝俞組和預處理足三里組癌細胞數(shù)量明顯減少,其中預處理肝俞組效果更為顯著。2血清學檢驗:與空白組對比,模型組、預處理足三里組和預處理肝俞組大鼠血清中AFP、AFU、γ-GT的含量均出現(xiàn)不同程度增高,差異有統(tǒng)計學意義(p0.05);與模型組比較,預處理足三里組和預處理肝俞組大鼠血清中AFP、AFU、γ-GT含量均明顯降低,差異具有統(tǒng)計學意義(p0.05);與預處理足三里組比較,預處理肝俞組AFP、AFU、γ-GT含量均顯著升高,差異有統(tǒng)計學意義(p0.05)。3 Rt-PCR結(jié)果示:與空白組比較,模型組、預處理肝俞組和預處理足三里組大鼠肝組織中Wnt10b、Wnt3a的m RNA表達量均明顯升高,差異有統(tǒng)計學意義(p0.05);與模型組比較,預處理肝俞組和預處理足三里組大鼠Wnt10b、Wnt3a的m RNA表達均明顯降低,差異有統(tǒng)計學意義(p0.05);與預處理肝俞組比較,預處理足三里組相關(guān)基因表達均顯著升高,差異有統(tǒng)計學意義(p0.05)。結(jié)論:1瘢痕灸肝俞穴和足三里穴可以調(diào)節(jié)Wnt信號通路中Wnt10b、Wnt3a的基因表達水平,有效減少癌細胞的增殖。2瘢痕灸肝俞穴和足三里穴對原發(fā)性肝癌均有不同程度的預防作用,但分析比較各指標間的差異,肝俞穴的作用效果要優(yōu)于足三里穴,因此本研究認為瘢痕灸肝俞穴治療原發(fā)性肝癌具有特異性。
[Abstract]:Objective: in this study, two ethyl nitrosamine (DEN) induced primary liver cancer rat model was used as the research object. The wheat grain moxa cone was used to pretreat the scar moxibustion at the Zusanli point and Gan Shu point in rats. The effect of Different Acupoint Moxibustion on the expression of Wnt signal transduction pathway related gene expression in primary liver cancer rats was observed. To further explore the mechanism of scar moxibustion on primary liver cancer, to clarify the modern mechanism of scar moxibustion to prevent the occurrence of liver cancer and to provide molecular basis for the prevention of primary liver cancer by scar moxibustion, and to control the development of the disease. Materials and methods: 48 Wistar rats were divided into blank group (12 rats) by random number table method. Model group (12), pretreated liver Yu Group (12) and pretreated Zusanli group (12). After one week of adaptive feeding, cicatricial moxibustion was carried out to the pretreated liver and pretreated Zusanli group. The quality of the cicatre was made into a strong grain size of 0.0005g/ Zhuang, 12 Zhuang / acupoint, every Zhuang, and the blank group and model group did not do scar moxibustion. After the treatment, the.DEN dosage of rat primary liver cancer was established by intraperitoneal injection of DEN in addition to the blank group. The model lasted for 14 weeks, 1-4 weeks, 2 times a week and 1 times a week for 5-14 weeks. The injection time was at the end of the 8: 30--10:30. model in the morning, and the rats were fasted one day with 10% chlorinated chlorine. Aldehyde anesthesia, abdominal aorta blood extraction, centrifuge serum preparation, extract some rat liver tissue preservation with aseptic EP tube, liquid nitrogen soaked, -80 centigrade refrigerators frozen, the rest of the liver tissue was fixed with 4% polyformaldehyde, to avoid light preservation. Test the rat serum of alpha fetoprotein (AFP), gamma glutamyl transpeptidase (gamma -GT), fucosidase (AFU) content; Rt-PCR method was used to detect the Wnt signal pathway of liver tissue, Wnt3a, Wnt10b m RNA expression.HE staining method to observe the pathological changes of liver tissue structure of rats. Results: 1 HE staining results showed that compared with the blank group, the model group, pretreated liver Shu group and pretreated Zusanli group rat liver trabecula, the hepatic sinusoids were arranged disorderly and accompanied by different courses. Compared with the model group, the number of cancer cells in the pretreated liver Yu group and the pretreated Zusanli group decreased significantly, and the effect of pretreated liver Yu group was more significant.2 serological test: compared with the blank group, the content of AFP, AFU and gamma -GT in the model group, the pretreated Zusanli group and the pretreated liver Shu group were all increased in varying degrees. The difference was statistically significant (P0.05). Compared with the model group, the content of AFP, AFU and gamma -GT in the serum of pretreated Zusanli group and pretreated liver Yu group decreased significantly (P0.05). Compared with the pre treated Zusanli group, the content of AFP, AFU, and gamma -GT in the pre treated liver Shu group increased significantly, the difference was statistically significant (P0.05).3 Rt-. PCR results showed that compared with the blank group, the expression of M RNA in the liver tissue of the pretreated liver Yu group and the pretreated Zusanli group was significantly higher than that of the model group, and the difference was statistically significant (P0.05). Compared with the model group, the expression of Wnt10b and Wnt3a in the pretreated liver and pretreated Zusanli group was significantly lower than that in the pretreated Zusanli group. There was a significant difference in the expression of the m RNA of Wnt3a. There was a significant difference in the expression of M RNA in the pre treated and pretreated Zusanli group. Statistical significance (P0.05); compared with the pretreated liver Shu group, the expression of the related genes in the pretreated Zusanli group increased significantly, and the difference was statistically significant (P0.05). Conclusion: 1 the scar moxibustion Gan Shu point and Zusanli point can regulate the gene expression level of Wnt10b, Wnt3a in the Wnt signaling pathway, and effectively reduce the proliferation of.2 scar moxibustion at the liver and foot points of the scar moxibustion. Three li points have different preventive effects on primary liver cancer, but the analysis and comparison of the difference between the various indexes, the effect of the liver Shu point is better than the Zusanli point. Therefore, this study holds that the scar moxibustion Gan Shu point is specific for the treatment of primary liver cancer.
【學位授予單位】：遼寧中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R245
[Abstract]:Objective: in this study, two ethyl nitrosamine (DEN) induced primary liver cancer rat model was used as the research object. The wheat grain moxa cone was used to pretreat the scar moxibustion at the Zusanli point and Gan Shu point in rats. The effect of Different Acupoint Moxibustion on the expression of Wnt signal transduction pathway related gene expression in primary liver cancer rats was observed. To further explore the mechanism of scar moxibustion on primary liver cancer, to clarify the modern mechanism of scar moxibustion to prevent the occurrence of liver cancer and to provide molecular basis for the prevention of primary liver cancer by scar moxibustion, and to control the development of the disease. Materials and methods: 48 Wistar rats were divided into blank group (12 rats) by random number table method. Model group (12), pretreated liver Yu Group (12) and pretreated Zusanli group (12). After one week of adaptive feeding, cicatricial moxibustion was carried out to the pretreated liver and pretreated Zusanli group. The quality of the cicatre was made into a strong grain size of 0.0005g/ Zhuang, 12 Zhuang / acupoint, every Zhuang, and the blank group and model group did not do scar moxibustion. After the treatment, the.DEN dosage of rat primary liver cancer was established by intraperitoneal injection of DEN in addition to the blank group. The model lasted for 14 weeks, 1-4 weeks, 2 times a week and 1 times a week for 5-14 weeks. The injection time was at the end of the 8: 30--10:30. model in the morning, and the rats were fasted one day with 10% chlorinated chlorine. Aldehyde anesthesia, abdominal aorta blood extraction, centrifuge serum preparation, extract some rat liver tissue preservation with aseptic EP tube, liquid nitrogen soaked, -80 centigrade refrigerators frozen, the rest of the liver tissue was fixed with 4% polyformaldehyde, to avoid light preservation. Test the rat serum of alpha fetoprotein (AFP), gamma glutamyl transpeptidase (gamma -GT), fucosidase (AFU) content; Rt-PCR method was used to detect the Wnt signal pathway of liver tissue, Wnt3a, Wnt10b m RNA expression.HE staining method to observe the pathological changes of liver tissue structure of rats. Results: 1 HE staining results showed that compared with the blank group, the model group, pretreated liver Shu group and pretreated Zusanli group rat liver trabecula, the hepatic sinusoids were arranged disorderly and accompanied by different courses. Compared with the model group, the number of cancer cells in the pretreated liver Yu group and the pretreated Zusanli group decreased significantly, and the effect of pretreated liver Yu group was more significant.2 serological test: compared with the blank group, the content of AFP, AFU and gamma -GT in the model group, the pretreated Zusanli group and the pretreated liver Shu group were all increased in varying degrees. The difference was statistically significant (P0.05). Compared with the model group, the content of AFP, AFU and gamma -GT in the serum of pretreated Zusanli group and pretreated liver Yu group decreased significantly (P0.05). Compared with the pre treated Zusanli group, the content of AFP, AFU, and gamma -GT in the pre treated liver Shu group increased significantly, the difference was statistically significant (P0.05).3 Rt-. PCR results showed that compared with the blank group, the expression of M RNA in the liver tissue of the pretreated liver Yu group and the pretreated Zusanli group was significantly higher than that of the model group, and the difference was statistically significant (P0.05). Compared with the model group, the expression of Wnt10b and Wnt3a in the pretreated liver and pretreated Zusanli group was significantly lower than that in the pretreated Zusanli group. There was a significant difference in the expression of the m RNA of Wnt3a. There was a significant difference in the expression of M RNA in the pre treated and pretreated Zusanli group. Statistical significance (P0.05); compared with the pretreated liver Shu group, the expression of the related genes in the pretreated Zusanli group increased significantly, and the difference was statistically significant (P0.05). Conclusion: 1 the scar moxibustion Gan Shu point and Zusanli point can regulate the gene expression level of Wnt10b, Wnt3a in the Wnt signaling pathway, and effectively reduce the proliferation of.2 scar moxibustion at the liver and foot points of the scar moxibustion. Three li points have different preventive effects on primary liver cancer, but the analysis and comparison of the difference between the various indexes, the effect of the liver Shu point is better than the Zusanli point. Therefore, this study holds that the scar moxibustion Gan Shu point is specific for the treatment of primary liver cancer.
【學位授予單位】：遼寧中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R245
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