傣药抗肿瘤验方对小鼠实验性子宫肌瘤抑制作用的研究

发布时间：2018-07-06 18:29

本文选题：傣药抗肿瘤验方 + 子宫肌瘤　；参考：《云南中医学院》2017年硕士论文

【摘要】：目的:(1)从民族药中寻找新药是世界公认的重要途径,傣药中蕴藏着丰富的防治疑难疾病亟待研发的宝藏,傣药抗肿瘤验方就是其中之一。本项目用现代药理实验的方法,去验证民间长久盛传的抗肿瘤验方,为寻找新药,开发新药打下基础。(2)子宫肌瘤是由细胞突变、基因刺激导致的平滑肌纤维异常增生而成的良性肿瘤。育龄期妇女子宫肌瘤发病率达70%以上,严重影响女性的身体健康及生育能力。傣药抗肿瘤验方是民间治疗肿瘤、包块的有效傣医验方,其在临床上用于治疗子宫肌瘤,发现具有良好的疗效,通过查阅国内外相关文献,没有发现关于该验方药效学研究的文献报道。因此,本研究采用子宫肌瘤模型小鼠的动物模型、以小鼠子宫病理形态、肿瘤坏死因子-α、白细胞介素-2、雌、孕激素及其受体水平、一氧化氮合酶的活性表达为检测指标,对傣药抗肿瘤验方的抗子宫肌瘤作用进行研究。方法:1.供试品的制备采用乙醇提取、纯水提取、分别浓缩后合并浓缩液的方法制备实验用药。2.子宫肌瘤小鼠模型的筛选与复制雌激素诱导单模法造模。3.民族药经验方对小鼠实验性子宫肌瘤模型的影响造模的同时分别相应的灌胃给药,检测病理形态和激素水平变化。4.傣药抗肿瘤验方对小鼠实验性子宫肌瘤模型子宫病理改变的影响采用he染色观察小鼠实验性子宫肌瘤模型子宫病理改变,并测量子宫肌壁的厚度。5.傣药抗肿瘤验方对小鼠实验性子宫肌瘤模型血清中e2、p、tnf-α、il-2的影响采用竞争elisa法检测小鼠血清中e2、p的水平,采用双抗体夹心elisa法检测小鼠血清中il-2、tnf-α的含量。6.傣药抗肿瘤验方对小鼠实验性子宫肌瘤模型子宫组织nos活性的影响采用化学比色法检测小鼠子宫组织nos活性。7.傣药抗肿瘤验方对小鼠实验性子宫肌瘤模型局部组织er、pr表达的影响采用免疫化学组织染色方法观察小鼠子宫组织er、pr的阳性表达,采用motic数码医学图像分析系统,测定阳性目标总面积。结果:1.供试品的制备本实验中共有2个来源于傣族民间的验方,分别是傣药抗肿瘤验方(以下简称原方组)和抗肿瘤虫方(以下简称虫方组)。其中傣药抗肿瘤验方中有海藻和甘草违背了中药配伍“十八反”的规定,所以又加了2个拆方实验组,即傣药抗肿瘤验方原方保留海藻,去掉甘草的一组(以下简称去甘草组)傣药抗肿瘤验方原方保留甘草,去掉海藻的一组(以下简称去海藻组),共计4个验方组。制得原方组混悬液、去甘草组混悬液、去海藻组混悬液、虫方组混悬液的提取浓度均为1.26g生药/ml,剂量均为12.64g生药/kg。筛方实验后,又重新制得有效原方组混悬液(高剂量25.45g生药/kg;中剂量12.64g生药/kg;低剂量4.21g生药/kg)。2.子宫肌瘤小鼠模型的筛选与复制成功复制子宫肌瘤小鼠模型。正常组小鼠子宫呈y字型,质地光滑均匀,色泽淡红,宫体纤细,无结节和肿胀现象。模型组小鼠子宫色泽比较暗沉,质地不均匀,有的小鼠子宫出现畸形和水肿现象。病理切片显示皱纹状排列的平滑肌层呈局灶性增生,肌细胞呈椭圆形,细胞核染色深,位于细胞的中央,子宫内膜间质弥漫大量炎性细胞,外膜层平滑肌组织厚度明显增加。与正常组相比,模型组小鼠子宫系数、子宫湿重均增加(p0.01,p0.01)。3.民族药经验方对小鼠子宫病理模型的影响原方组能改善子宫肌瘤小鼠的病理组织形态,同时可以有效的降低子宫肌瘤小鼠体内e2、p的水平,比较有显著性意义(p0.01,p0.01),虫方组和原方组对子宫肌瘤小鼠模型抑制作用结果相近,但虫方组对于子宫肌瘤模型肉眼可见的水肿改善程度小。去甘草组和去海藻组对小鼠子宫肌瘤模型抑制作用微弱。4.原方组对小鼠实验性子宫肌瘤模型子宫病理改变的影响原方组各剂量均能改善子宫肌瘤小鼠的病理组织形态,光镜下可见用药组子宫平滑肌局灶性增生区域明显减少,大多数细胞核呈梭形、杆状,肌细胞排列较整齐,能显著降低子宫脏器系数、子宫湿重和子宫内膜厚度,比较有显著性意义(p0.01)。5.原方组对小鼠实验性子宫肌瘤模型血清中e2、p、tnf-α、il-2的影响⑴各组血清中e2水平的检测结果显示,模型组血清e2水平明显高于正常对照组(p0.01);阳性药组和用药组的e2水平低于模型组,两组间差异具有统计学意义(p0.01)。⑵各组血清p水平的检测结果显示,模型组血清p水平明显高于正常对照组(p0.01);阳性药组和用药中、低剂量组的p水平低于模型组,两组间具有统计学意义(p0.01),用药高剂量组的p水平也显著降低(p0.05)。⑶各组血清TNF-α水平检测结果表明,模型组TNF-α水平显著升高(P0.05),各给药组TNF-α水平有降低趋势,高剂量组和中剂量组有显著性差异(P0.05)阳性药和低剂量组有极显著性差异(P0.01)。⑷各组血清IL-2水平检测结果表明,模型组IL-2水平显著降低,除高剂量组外,其余给药组IL-2水平显著升高(P0.05),高剂量组IL-2水平与模型组比较无显著性差异。6.原方组对小鼠实验性子宫肌瘤模型子宫组织NOS的影响各组小鼠子宫组织NOS活性测试结果表明,模型组NOS活性显著升高(P0.01),各给药组NOS活性显著降低(P0.01)。7.原方组对小鼠实验性子宫肌瘤模型局部组织ER、PR表达的影响原方组可以降低子宫肌瘤模型ER、PR的表达。结论:本研究成功复制了小鼠实验性子宫肌瘤模型,对傣药抗肿瘤验方等四个方子进行筛选,结果显示原方组与其他验方组相比,有很好的治疗优势。并对原方组进行深入的研究,发现傣药抗肿瘤验方原方组对子宫肌瘤具有明显的抑制作用,其机制与降低血清激素水平及相应受体的表达、降低子宫肌瘤小鼠体内的肿瘤坏死因子(TNF-α)、升高IL-2的水平及降低NOS的活性表达有关。
[Abstract]:Objective: (1) it is an important way to find new drugs from national medicine, and there are abundant treasures in Dai medicine to prevent and cure difficult diseases. The antitumor prescription of Dai medicine is one of them. This project uses modern pharmacological experiments to verify the anti tumor prescription of the people for a long time, so as to find new drugs and develop new drugs. (2) uterine myoma is a benign tumor resulting from abnormal proliferation of smooth muscle fibers caused by cell mutation and gene stimulation. The incidence of uterine myoma in women of childbearing age is over 70%, which seriously affects the health and fertility of women. The treatment of uterine leiomyoma has been found to have a good effect. No literature reports on the pharmacodynamics of this test have been found by consulting the relevant literature at home and abroad. Therefore, this study adopts the animal model of uterine myoma model mice, with the pathological morphology of uterus, tumor necrosis factor - alpha, interleukin -2, female, progesterone and its receptor water. The activity of the activity of nitric oxide synthase is the test index, and the anti uterine fibroid effect of the antitumor prescription of the Dai medicine is studied. Method: 1. the preparation of the test products was prepared by ethanol extraction, pure water extraction, and concentrated solution after concentration, and the method of preparing experimental drug.2. uterine fibroid mouse model and replicating estrogen induced single mode method were prepared. The effect of model.3. on the model of experimental uterine myoma in mice and the corresponding administration of the model of the mice with the model of uterine myoma. The changes of the pathological morphology and hormone levels were measured by.4.. The effect of the anti tumor prescription of the Dai medicine on the uterine pathological changes of the experimental uterine fibroids in mice was observed by HE staining and the experimental uterine fibroid model was observed in mice. The effect of.5. Dai medicine on the serum E2, P, tnf- alpha and IL-2 in the serum of the experimental uterine fibroid model of mice by using the competitive ELISA method to detect the level of E2 and P in the serum of mice, and the detection of IL-2 in the serum of mice by the double antibody sandwich ELISA method, and the content of tnf- alpha in the anti tumor test prescription of the Dai medicine on mice. The effect of NOS activity in uterine tissue of experimental uterine fibroid model, the chemical colorimetric method was used to detect the NOS activity of the uterine tissue in mice. The effect of.7. on the local tissue Er, the expression of PR in the experimental uterine fibroid model of mice was determined by the immunohistochemical staining method to observe the positive expression of ER in the uterus of mice and the positive expression of PR, and the number of Motic was used. The total area of the positive target was determined by the code medical image analysis system. Results: 1. the preparation of the test products was 2 from the Dai Folk test side, respectively, the antitumor prescription of Dai medicine (hereinafter referred to as the original group) and the anti tumor insect prescription (hereinafter referred to as the insect cube group). Among them, there are algae and licorice in the anti tumor test of Dai medicine. For the "Eighteen anti" regulations, we added 2 dismantling experimental groups, that is, the antitumor prescription of the Dai medicine, the original prescription of the Dai medicine, and the group (hereinafter referred to as the Glycyrrhiza uralensis group) for the antitumor prescription, and the group of algae (hereinafter referred to as the algae group), a total of 4 test groups. The concentration of suspensions in the suspension liquid and the suspension of the seaweed group were all 1.26g /ml, and the dosage was all /kg. sieving of 12.64g. The effective original group suspension (high dose 25.45g raw drug /kg, the medium dose 12.64g raw medicine /kg, the low dose 4.21g raw medicine / kg).2. uterine myoma mice model was reproduced and replicated successfully. A mouse model of uterine myoma was made. The uterus of the normal group was Y type, with smooth and uniform texture, pale red color, thin uterine body, no nodules and swelling. The color and texture of the uterus in the model group were dark and uneven, and the uterus appeared abnormal and edema in some mice. The pathological slices showed that the smooth smooth muscle layer arranged in the wrinkled form showed focal hyperplasia. The muscle cells were oval, the nucleus dyed deep, located in the center of the cells, the endometrium permeated a large number of inflammatory cells, and the thickness of the smooth muscle tissue in the outer membrane increased obviously. Compared with the normal group, the uterus coefficient and the wet weight of the uterus in the model group increased (P0.01, P0.01).3. people medicine experience on the mouse uterus pathological model The histopathological morphology of uterine myoma mice could be improved and the levels of E2 and P in the mice were significantly reduced (P0.01, P0.01), and the inhibitory effects on the mice model of uterine myoma were similar, but the improvement of the edema in the myoma model was small. The inhibitory effect of Glycyrrhiza group and seaweed group on the mouse uterine myoma model the effect of weak.4. original group on the pathological changes of uterine fibroid model in mice can improve the pathological morphology of the uterus myoma mice. The nucleus of the cell was spindle, rod like, and the muscle cells were arranged neatly. It could significantly reduce the uterus organ coefficient, the uterus wet weight and the endometrium thickness. The effect of.5. original group on the serum E2, P, tnf-, and IL-2 in the serum of mice experimental uterine fibroid model (1) the test results of the serum E2 level in each group showed that the serum E2 water in the model group was E2. The level of E2 in the positive drug group and the drug group was lower than that of the model group (P0.01), and the difference between the two groups was statistically significant (P0.01). (2) the serum level of P in each group showed that the serum P level of the model group was significantly higher than that of the normal control group (P0.01); the P level of the positive drug group and the low dose group was lower than that of the model group. The two groups had statistical significance (P0.01), and the level of P in the high dose group was also significantly decreased (P0.05). (3) the level of serum TNF- alpha in each group showed that the level of TNF- alpha in the model group was significantly higher (P0.05), the level of TNF- alpha in each group was reduced, and there was a significant difference between the high dose group and the middle dose group (P0.05) and the low dose group. Significant difference (P0.01). 4. The results of serum IL-2 level in each group showed that the level of IL-2 in the model group decreased significantly, except for the high dose group, the IL-2 level in the other group was significantly increased (P0.05). There was no significant difference in the IL-2 level between the high dose group and the model group. The effect of.6. on the NOS of the uterine fibroid model of the rat model was different from that of the group.6.. The test results of NOS activity in mouse uterus showed that the activity of NOS in the model group was significantly increased (P0.01), and the activity of NOS in each group decreased significantly (P0.01).7. original group on the localized tissue of the uterine fibroid model in mice. The expression of PR could reduce the expression of ER and PR of the uterine myoma model. Conclusion: This study successfully replicated the mice. The experimental uterine myoma model was screened for four prescription of the antitumor prescription of the Dai medicine. The results showed that the original group had a good therapeutic advantage compared with the other group, and had a thorough study on the original prescription group. It was found that the antitumor prescription group of the Dai medicine has a clear inhibitory effect on the uterine myoma, and its mechanism and reducing the serum hormone water. The expression of corresponding receptors decreased the level of tumor necrosis factor (TNF- alpha), increased the level of IL-2 and decreased the activity of NOS in uterine fibroids.
【学位授予单位】：云南中医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R29

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