瘢痕灸预处理对预防原发性肝癌大鼠Wnt信号通路相关基因的影响
发布时间：2018-07-22 13:15
【摘要】：目的:本研究采用二乙基亚硝氨(DEN)诱导致原发性肝癌大鼠模型作为研究对象,采用麦粒状艾炷,分别在大鼠足三里穴、肝俞穴实施瘢痕灸预处理。观察不同穴位瘢痕灸预处理对原发性肝癌大鼠Wnt信号传导通路相关基因表达的影响,从而进一步探讨瘢痕灸对原发性肝癌的作用机制,明确瘢痕灸预防肝癌发生、发展的现代机理,为今后瘢痕灸预防原发性肝癌,控制疾病发展提供分子生物学依据。材料和方法:SPF级Wistar大鼠共48只,雄性,使用随机数字表法分为空白组(12只)、模型组(12只)、预处理肝俞组(12只)和预处理足三里组(12只)。在适应性喂养一周后,对预处理肝俞组和预处理足三里组进行瘢痕灸,将艾绒做成一壮为麦粒大小,质量约为0.0005g/壮,12壮/穴,每壮使其燃尽,空白组和模型组大鼠不做瘢痕灸处理。治疗结束后,除空白组外,均采用腹腔注射DEN的方法建立大鼠原发性肝癌模型。DEN用量按照每千克体重50mg计算,造模持续14周:1-4周,每周注射2次;5-14周,每周注射1次,注射时间均在上午8:30--10:30。造模结束后,大鼠禁食一天,用10%水合氯醛麻醉,开腹行腹主动脉采血,离心取血清备检,取部分大鼠肝脏组织用无菌EP管保存,液氮浸泡后,-80℃冰箱冷冻,其余肝脏组织采用4%多聚甲醛固定,避光保存。检测大鼠血清中的甲胎蛋白(AFP)、γ-谷胺酰转肽酶(γ-GT)、岩藻糖苷酶(AFU)含量;Rt-PCR法检测肝脏组织Wnt信号通路中Wnt3a、Wnt10b的m RNA表达。HE染色法观察大鼠肝脏组织结构病理形态变化。结果:1 HE染色结果显示:与空白组相比,模型组、预处理肝俞组和预处理足三里组大鼠肝组织肝小梁、肝窦呈无序排列,并伴有不同程度的深染;与模型组相比,预处理肝俞组和预处理足三里组癌细胞数量明显减少,其中预处理肝俞组效果更为显著。2血清学检验:与空白组对比,模型组、预处理足三里组和预处理肝俞组大鼠血清中AFP、AFU、γ-GT的含量均出现不同程度增高,差异有统计学意义(p0.05);与模型组比较,预处理足三里组和预处理肝俞组大鼠血清中AFP、AFU、γ-GT含量均明显降低,差异具有统计学意义(p0.05);与预处理足三里组比较,预处理肝俞组AFP、AFU、γ-GT含量均显著升高,差异有统计学意义(p0.05)。3 Rt-PCR结果示:与空白组比较,模型组、预处理肝俞组和预处理足三里组大鼠肝组织中Wnt10b、Wnt3a的m RNA表达量均明显升高,差异有统计学意义(p0.05);与模型组比较,预处理肝俞组和预处理足三里组大鼠Wnt10b、Wnt3a的m RNA表达均明显降低,差异有统计学意义(p0.05);与预处理肝俞组比较,预处理足三里组相关基因表达均显著升高,差异有统计学意义(p0.05)。结论:1瘢痕灸肝俞穴和足三里穴可以调节Wnt信号通路中Wnt10b、Wnt3a的基因表达水平,有效减少癌细胞的增殖。2瘢痕灸肝俞穴和足三里穴对原发性肝癌均有不同程度的预防作用,但分析比较各指标间的差异,肝俞穴的作用效果要优于足三里穴,因此本研究认为瘢痕灸肝俞穴治疗原发性肝癌具有特异性。
[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.
【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245
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