本文選題：傣藥抗腫瘤驗方 + 子宮肌瘤��； 參考：《云南中醫(yī)學院》2017年碩士論文

【摘要】：目的:(1)從民族藥中尋找新藥是世界公認的重要途徑,傣藥中蘊藏著豐富的防治疑難疾病亟待研發(fā)的寶藏,傣藥抗腫瘤驗方就是其中之一。本項目用現(xiàn)代藥理實驗的方法,去驗證民間長久盛傳的抗腫瘤驗方,為尋找新藥,開發(fā)新藥打下基礎(chǔ)。(2)子宮肌瘤是由細胞突變、基因刺激導致的平滑肌纖維異常增生而成的良性腫瘤。育齡期婦女子宮肌瘤發(fā)病率達70%以上,嚴重影響女性的身體健康及生育能力。傣藥抗腫瘤驗方是民間治療腫瘤、包塊的有效傣醫(yī)驗方,其在臨床上用于治療子宮肌瘤,發(fā)現(xiàn)具有良好的療效,通過查閱國內(nèi)外相關(guān)文獻,沒有發(fā)現(xiàn)關(guān)于該驗方藥效學研究的文獻報道。因此,本研究采用子宮肌瘤模型小鼠的動物模型、以小鼠子宮病理形態(tài)、腫瘤壞死因子-α、白細胞介素-2、雌、孕激素及其受體水平、一氧化氮合酶的活性表達為檢測指標,對傣藥抗腫瘤驗方的抗子宮肌瘤作用進行研究。方法:1.供試品的制備采用乙醇提取、純水提取、分別濃縮后合并濃縮液的方法制備實驗用藥。2.子宮肌瘤小鼠模型的篩選與復制雌激素誘導單模法造模。3.民族藥經(jīng)驗方對小鼠實驗性子宮肌瘤模型的影響造模的同時分別相應(yīng)的灌胃給藥,檢測病理形態(tài)和激素水平變化。4.傣藥抗腫瘤驗方對小鼠實驗性子宮肌瘤模型子宮病理改變的影響采用he染色觀察小鼠實驗性子宮肌瘤模型子宮病理改變,并測量子宮肌壁的厚度。5.傣藥抗腫瘤驗方對小鼠實驗性子宮肌瘤模型血清中e2、p、tnf-α、il-2的影響采用競爭elisa法檢測小鼠血清中e2、p的水平,采用雙抗體夾心elisa法檢測小鼠血清中il-2、tnf-α的含量。6.傣藥抗腫瘤驗方對小鼠實驗性子宮肌瘤模型子宮組織nos活性的影響采用化學比色法檢測小鼠子宮組織nos活性。7.傣藥抗腫瘤驗方對小鼠實驗性子宮肌瘤模型局部組織er、pr表達的影響采用免疫化學組織染色方法觀察小鼠子宮組織er、pr的陽性表達,采用motic數(shù)碼醫(yī)學圖像分析系統(tǒng),測定陽性目標總面積。結(jié)果:1.供試品的制備本實驗中共有2個來源于傣族民間的驗方,分別是傣藥抗腫瘤驗方(以下簡稱原方組)和抗腫瘤蟲方(以下簡稱蟲方組)。其中傣藥抗腫瘤驗方中有海藻和甘草違背了中藥配伍“十八反”的規(guī)定,所以又加了2個拆方實驗組,即傣藥抗腫瘤驗方原方保留海藻,去掉甘草的一組(以下簡稱去甘草組)傣藥抗腫瘤驗方原方保留甘草,去掉海藻的一組(以下簡稱去海藻組),共計4個驗方組。制得原方組混懸液、去甘草組混懸液、去海藻組混懸液、蟲方組混懸液的提取濃度均為1.26g生藥/ml,劑量均為12.64g生藥/kg。篩方實驗后,又重新制得有效原方組混懸液(高劑量25.45g生藥/kg;中劑量12.64g生藥/kg;低劑量4.21g生藥/kg)。2.子宮肌瘤小鼠模型的篩選與復制成功復制子宮肌瘤小鼠模型。正常組小鼠子宮呈y字型,質(zhì)地光滑均勻,色澤淡紅,宮體纖細,無結(jié)節(jié)和腫脹現(xiàn)象。模型組小鼠子宮色澤比較暗沉,質(zhì)地不均勻,有的小鼠子宮出現(xiàn)畸形和水腫現(xiàn)象。病理切片顯示皺紋狀排列的平滑肌層呈局灶性增生,肌細胞呈橢圓形,細胞核染色深,位于細胞的中央,子宮內(nèi)膜間質(zhì)彌漫大量炎性細胞,外膜層平滑肌組織厚度明顯增加。與正常組相比,模型組小鼠子宮系數(shù)、子宮濕重均增加(p0.01,p0.01)。3.民族藥經(jīng)驗方對小鼠子宮病理模型的影響原方組能改善子宮肌瘤小鼠的病理組織形態(tài),同時可以有效的降低子宮肌瘤小鼠體內(nèi)e2、p的水平,比較有顯著性意義(p0.01,p0.01),蟲方組和原方組對子宮肌瘤小鼠模型抑制作用結(jié)果相近,但蟲方組對于子宮肌瘤模型肉眼可見的水腫改善程度小。去甘草組和去海藻組對小鼠子宮肌瘤模型抑制作用微弱。4.原方組對小鼠實驗性子宮肌瘤模型子宮病理改變的影響原方組各劑量均能改善子宮肌瘤小鼠的病理組織形態(tài),光鏡下可見用藥組子宮平滑肌局灶性增生區(qū)域明顯減少,大多數(shù)細胞核呈梭形、桿狀,肌細胞排列較整齊,能顯著降低子宮臟器系數(shù)、子宮濕重和子宮內(nèi)膜厚度,比較有顯著性意義(p0.01)。5.原方組對小鼠實驗性子宮肌瘤模型血清中e2、p、tnf-α、il-2的影響⑴各組血清中e2水平的檢測結(jié)果顯示,模型組血清e2水平明顯高于正常對照組(p0.01);陽性藥組和用藥組的e2水平低于模型組,兩組間差異具有統(tǒng)計學意義(p0.01)。⑵各組血清p水平的檢測結(jié)果顯示,模型組血清p水平明顯高于正常對照組(p0.01);陽性藥組和用藥中、低劑量組的p水平低于模型組,兩組間具有統(tǒng)計學意義(p0.01),用藥高劑量組的p水平也顯著降低(p0.05)。⑶各組血清TNF-α水平檢測結(jié)果表明,模型組TNF-α水平顯著升高(P0.05),各給藥組TNF-α水平有降低趨勢,高劑量組和中劑量組有顯著性差異(P0.05)陽性藥和低劑量組有極顯著性差異(P0.01)。⑷各組血清IL-2水平檢測結(jié)果表明,模型組IL-2水平顯著降低,除高劑量組外,其余給藥組IL-2水平顯著升高(P0.05),高劑量組IL-2水平與模型組比較無顯著性差異。6.原方組對小鼠實驗性子宮肌瘤模型子宮組織NOS的影響各組小鼠子宮組織NOS活性測試結(jié)果表明,模型組NOS活性顯著升高(P0.01),各給藥組NOS活性顯著降低(P0.01)。7.原方組對小鼠實驗性子宮肌瘤模型局部組織ER、PR表達的影響原方組可以降低子宮肌瘤模型ER、PR的表達。結(jié)論:本研究成功復制了小鼠實驗性子宮肌瘤模型,對傣藥抗腫瘤驗方等四個方子進行篩選,結(jié)果顯示原方組與其他驗方組相比,有很好的治療優(yōu)勢。并對原方組進行深入的研究,發(fā)現(xiàn)傣藥抗腫瘤驗方原方組對子宮肌瘤具有明顯的抑制作用,其機制與降低血清激素水平及相應(yīng)受體的表達、降低子宮肌瘤小鼠體內(nèi)的腫瘤壞死因子(TNF-α)、升高IL-2的水平及降低NOS的活性表達有關(guān)。
[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.
【學位授予單位】：云南中醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R29

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