本文關(guān)鍵詞：基于“有故無殞”理論評(píng)價(jià)雷公藤甲素對(duì)荷瘤小鼠的肝毒性反應(yīng)　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 雷公藤甲素 荷瘤小鼠 抗腫瘤 細(xì)胞凋亡 肝毒性 氧化應(yīng)激 毒效關(guān)系

【摘要】：研究目的根據(jù)中醫(yī)理論"有故無殞"思想,以CT26細(xì)胞及荷瘤小鼠為研究對(duì)象,在評(píng)價(jià)雷公藤甲素抗腫瘤作用的同時(shí),比較觀察不同劑量雷公藤甲素對(duì)正常生理狀態(tài)及荷瘤病理狀態(tài)小鼠的肝毒性反應(yīng),為雷公藤的臨床應(yīng)用及深度開發(fā)提供更多的資料。研究方法離體實(shí)驗(yàn):雷公藤甲素(50nM)孵育CT26細(xì)胞12小時(shí)、24小時(shí),經(jīng)MTS法檢測(cè)細(xì)胞的活力、計(jì)算增殖抑制率;用流式細(xì)胞術(shù)檢測(cè)雷公藤甲素(25nM和50nM)誘導(dǎo)CT26細(xì)胞凋亡的作用。整體實(shí)驗(yàn):以雄性BALB/C小鼠為研究對(duì)象,將2×106個(gè)CT26細(xì)胞注入小鼠右前肢下的皮下部位,建立荷瘤小鼠模型。采用隨機(jī)分組的方法將實(shí)驗(yàn)動(dòng)物分為假手術(shù)組、腫瘤模型組、雷公藤甲素治療組(0.4mg/kg),每組10只,腹腔注射連續(xù)給藥21天后,取材,用以觀察雷公藤甲素抗CT26結(jié)腸癌的藥效。觀測(cè)指標(biāo)包括:小鼠體重、腫瘤生長(zhǎng)情況及行為學(xué)變化,測(cè)量腫瘤的長(zhǎng)度和寬度。通過體重及腫瘤監(jiān)測(cè)、腫瘤組織冰凍切片TUNEL染色及免疫組織化學(xué)法檢測(cè)腫瘤組織冰凍切片內(nèi)Caspase-3的表達(dá)情況等方法觀察其抗腫瘤作用。對(duì)正常小鼠及荷瘤小鼠在相同條件下暴露低劑量(0.1mg/kg)、中劑量(0.2mg/kg)、高劑量(0.4mg/kg)的雷公藤甲素,腹腔注射連續(xù)給藥25天后,取材,通過大體觀察、血清生化檢測(cè)(ALT、AST)、肝組織勻漿液檢測(cè)(MDA、SOD、GSH-PX)、組織病理學(xué)觀察、iNOS、Nrf2蛋白表達(dá)分析等方法對(duì)比不同劑量雷公藤甲素對(duì)正常生理狀態(tài)及荷瘤病理狀態(tài)下小鼠的肝毒性反應(yīng)。研究結(jié)果(1)雷公藤甲素可顯著抑制CT26的生長(zhǎng)增殖并誘導(dǎo)其細(xì)胞凋亡。(2)0.4mg/kg雷公藤甲素腹腔注射給藥,雷公藤甲素可顯著抑制荷瘤小鼠皮下腫瘤的生長(zhǎng),小鼠行為學(xué)及體重出現(xiàn)顯著改變,腫瘤組織內(nèi)出現(xiàn)明顯的細(xì)胞凋亡。(3)對(duì)于丙氨酸氨基轉(zhuǎn)移酶(ALT),隨著雷公藤甲素藥物劑量的增加,正常組及腫瘤組的ALT均增加,在高劑量時(shí),正常組ALT高于腫瘤組;對(duì)于天門冬氨酸氨基轉(zhuǎn)移酶(AST),隨著雷公藤甲素藥物劑量的增加,正常組及腫瘤組的AST變化不明顯,腫瘤組的AST值明顯高于正常組。(4)肝組織勻漿檢測(cè)結(jié)果表明:對(duì)于丙二醛(MDA),隨著雷公藤甲素藥物劑量的增加,正常組及腫瘤組的MDA含量均增加,在低劑量及中劑量時(shí),正常組要高于腫瘤組;對(duì)于超氧化物歧化酶(SOD),隨著雷公藤甲素藥物劑量的增加,正常組及腫瘤組的SOD活力均降低,正常組與腫瘤組間比較無統(tǒng)計(jì)學(xué)差異;對(duì)于谷胱甘肽過氧化物酶(GSH-PX),隨著雷公藤甲素藥物劑量的增加,正常組及腫瘤組的GSH-PX活力均降低,在低劑量及高劑量時(shí),腫瘤組要高于正常組。(5)肝組織HE染色結(jié)果表明:隨著雷公藤甲素藥物劑量的增加,肝臟組織細(xì)胞病變?cè)矫黠@,正常組的肝臟組織損傷程度明顯重于腫瘤組。(6)iNOS免疫組化顯色結(jié)果表明:隨著雷公藤甲素藥物劑量的增加,iNOS在肝臟組織中的表達(dá)隨之增加,在低劑量及中劑量時(shí),正常組與腫瘤組之間iNOS的表達(dá)沒有顯著性差異;在高劑量時(shí),正常組的iNOS的陽(yáng)性表達(dá)要明顯高于腫瘤組。(7)Nrf 2免疫組化及Western Blot結(jié)果表明:隨著雷公藤甲素藥物劑量的增加,Nrf 2蛋白在肝臟組織中的表達(dá)隨之增加,在低劑量時(shí),正常組與腫瘤組之間Nrf2的表達(dá)量沒有顯著性差異,在中劑量及高劑量時(shí),正常組Nrf2的表達(dá)量要明顯高于腫瘤組。研究結(jié)論1.雷公藤甲素具有顯著的抗腫瘤作用,其初步機(jī)制與誘導(dǎo)細(xì)胞凋亡有關(guān);2.雷公藤甲素在病理狀態(tài)下小鼠的肝毒性反應(yīng)比正常生理狀態(tài)下的肝毒性反應(yīng)要輕;3.雷公藤甲素的肝毒性發(fā)生機(jī)制與誘導(dǎo)氧化應(yīng)激有關(guān);4.高劑量的雷公藤甲素對(duì)的正常組及腫瘤組小鼠均可產(chǎn)生明顯的肝毒性反應(yīng),提示雷公藤甲素用量超出一定范圍后,無論機(jī)體處于生理狀態(tài)還是病理狀態(tài),其肝毒性反應(yīng)程度均很嚴(yán)重。
[Abstract]:The purpose of the study according to the theory of TCM "so no artist" thought, using CT26 cells and tumor bearing mice as the research object, in the evaluation of triptolide antitumor effect at the same time, comparative observation of liver toxicity of different dose of triptolide on normal physiological state and pathological state of tumor bearing mice, provide more information for clinical application and the depth of the development of Tripterygium. Research methods in vitro: triptolide (50nM) CT26 cells were incubated for 12 hours, 24 hours, were detected by MTS activity, calculation of inhibition rate; flow cytometry was used to detect the effects of triptolide (25nM and 50nM) induced CT26 cell apoptosis. The whole experiment in male BALB/C mice as the research object, the 2 x 106 CT26 cells were injected into the subcutaneous site of mice under the right forelimb, establishing tumor bearing mice model. Using the method of randomized experimental animal tumor divided into sham operation group, model group, Lei The triptolide treatment group (0.4mg/kg), 10 rats in each group, intraperitoneal injection after 21 days of medication, were used to observe the effect of triptolide against CT26 colon cancer. The observation index included: mice weight, tumor growth and behavior changes, measure the length and width. The tumor weight and tumor monitoring the frozen tumor tissue, detection of tumor tissue sections with TUNEL staining and immunohistochemistry of frozen sections in the expression of Caspase-3 was observed. The anti-tumor effect of normal mice and tumor bearing mice in the same conditions exposed to low dose (0.1mg/kg), middle dose (0.2mg/kg), high dosage (0.4mg/kg) triptolide. Intraperitoneally administered continuously for 25 days, from the general observation of serum biochemical detection (ALT, AST), the detection of liver tissue homogenate (MDA, SOD, GSH-PX, iNOS), histopathological observation, the expression of Nrf2 protein in the methods of comparative analysis of different agents The amount of liver toxicity of triptolide on normal physiological and pathological conditions in tumor bearing mice. Results (1) the proliferation of triptolide could significantly inhibit CT26 and induce cell apoptosis. (2) 0.4mg/kg triptolide intraperitoneal injection, triptolide can significantly inhibit subcutaneous tumor of mice bearing the growth of mice behavior and body weight had significant change, significant apoptosis occurred in the tumor tissue. (3) to alanine aminotransferase (ALT), with the increase of triptolide dose, normal group and tumor group ALT were increased at high doses, ALT higher than the normal group in tumor group for; aspartate aminotransferase (AST), with the increase of triptolide dose, AST changes the normal group and tumor group was not obvious, the AST value of tumor group was significantly higher than the normal group. (4) the detection results of liver homogenate showed that for C Two aldehyde (MDA), with the increase of triptolide dose, the content of MDA in normal group and tumor group were increased in the low dose and middle dose, normal group is higher than that of tumor group; for superoxide dismutase (SOD), with the increase of triptolide dose, normal group and the tumor group SOD activity decreased, no significant difference between normal group and tumor group; for glutathione peroxidase (GSH-PX), with the increase of triptolide dose, normal group and tumor group GSH-PX activity decreased in the low dose and high dose group, the tumor is higher than that of the normal group. (5) liver tissue HE staining results showed that with the increase of triptolide dose, tissue cells of liver is more obvious, the degree of liver tissue injury in normal group was significantly heavier than tumor group. (6) iNOS immunohistochemical coloration. The results show that with triptolide drug dose The increased expression of iNOS in liver tissues increased in low dose and middle dose, there was no significant difference in expression of iNOS between normal group and tumor group; at high doses, the positive expression of iNOS in the normal group was higher than that of tumor group. (7) Nrf 2 immunohistochemistry and Western Blot the results showed that with the increase of triptolide dose, the expression of Nrf 2 protein in liver tissues increased at low doses, there is no significant difference between the expression of Nrf2 between normal group and tumor group, the middle dose and high dose, the expression of Nrf2 in the normal group was significantly higher than the tumor group. Conclusion 1. triptolide has significant anti-tumor effects, the mechanisms involved in the induction of apoptosis; liver toxicity of triptolide in 2. pathological conditions of mice are lighter than liver toxicity in normal condition; 3. Triptolide Liver toxicity mechanism and oxidative stress induced by about 4.; high dose of triptolide on the normal group and tumor group of mice liver can produce significant toxicity, suggesting that triptolide content beyond a certain range, regardless of the body in a physiological state or pathological conditions, the degree of liver toxicity was very serious.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張?zhí)鞁?韓森;張瑋;趙福建;李健;;雷公藤甲素通過誘導(dǎo)細(xì)胞自噬促進(jìn)結(jié)腸癌CT26細(xì)胞死亡[J];解剖學(xué)報(bào);2016年06期

2 閔群燕;;用雷公藤多甙治療慢性腎小球腎炎的效果研究[J];當(dāng)代醫(yī)藥論叢;2016年03期

3 王寧;馬慧萍;漆欣筑;蒙萍;賈正平;;Nrf2-ARE信號(hào)通路在機(jī)體氧化應(yīng)激損傷防護(hù)中的研究進(jìn)展[J];解放軍醫(yī)藥雜志;2015年12期

4 南麗紅;鄭燕芳;徐偉;李煌;褚克丹;毛澤玲;楊瀾;何一博;黃枚;;不同炮制方法對(duì)雷公藤的急性毒性和抗炎作用的影響[J];時(shí)珍國(guó)醫(yī)國(guó)藥;2015年08期

5 張晶;李曉芳;劉昱;吳明澤;;腫瘤流行病學(xué)現(xiàn)狀及抗腫瘤中藥的研究進(jìn)展[J];山東化工;2015年12期

6 劉澤洲;許可嘉;張?zhí)鞁?易小烈;趙福建;楊美娟;牛建昭;李健;;雷公藤甲素誘導(dǎo)小鼠急性肝損傷的形態(tài)學(xué)研究[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2015年17期

7 劉建群;張國(guó)華;高俊博;;烘箱煨制雷公藤藥效毒性及成分變化研究[J];亞太傳統(tǒng)醫(yī)藥;2015年10期

8 龍曉芝;耿耘;郭晴晴;;近年中藥抗腫瘤有效成分及作用機(jī)制研究進(jìn)展[J];中華中醫(yī)藥學(xué)刊;2015年04期

9 王寶娟;付濱;張童燕;李彥;;雷公藤甲素免疫調(diào)節(jié)機(jī)制研究進(jìn)展[J];河北中醫(yī);2015年03期

10 張世應(yīng);;雷公藤的毒性研究[J];湖北中醫(yī)雜志;2015年03期

，

本文編號(hào)：1383675