本文選題：CPT-11 + 遲發(fā)性腹瀉��； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:伊立替康(Irinotecan,CPT-11)是臨床大腸癌常用的化療藥,其劑量限制性毒性反應(yīng)為遲發(fā)性腹瀉,嚴(yán)重影響了患者的生活質(zhì)量和對(duì)化療的耐受。本研究目的:1.建立CPT-11所致小鼠遲發(fā)性腹瀉模型;2.探討預(yù)先灌胃小柴胡湯對(duì)CPT-11所致小鼠遲發(fā)性腹瀉是否有預(yù)防作用;3.通過超高效液相色譜串聯(lián)三重四級(jí)桿質(zhì)譜法(Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry,UPLC-MS/MS)測(cè)定CPT-11及其代謝產(chǎn)物以及小柴胡湯中有效成分含量,探尋小柴胡湯作用于遲發(fā)性腹瀉的物質(zhì)基礎(chǔ),為機(jī)制研究奠定基礎(chǔ)。方法:1.建立小鼠遲發(fā)性腹瀉模型:健康雄性C57BL6小鼠20只,適應(yīng)性飼喂高脂飼料三周后,隨機(jī)分為模型組(n=10)和正常組(n=10),連續(xù)腹腔注射CPT-11 7天,每天一次,劑量為50 mg·kg-1,正常組腹腔注射等量的右旋糖苷40葡萄糖注射液,每天觀察小鼠的肛門,拍照記錄,收集糞便,用尿糞便隱血試劑盒測(cè)定血便情況。2.小柴胡湯干預(yù)研究:健康雄性C57BL6小鼠40只,適應(yīng)性飼喂高脂飼料三周后,隨機(jī)分為4組,每組10只:空白對(duì)照組(等容生理鹽水),遲發(fā)性腹瀉模型組(等容生理鹽水),小柴胡湯組(1500 mg·kg-1·d-1),洛哌丁胺組(0.5333 mg·kg-1·d-1),連續(xù)灌胃17天,每天1次。于實(shí)驗(yàn)第4~10天,除空白對(duì)照組外,其余各組小鼠均腹腔注射CPT-11 7天誘導(dǎo)遲發(fā)性腹瀉。在注射CPT-11的第0、2、4、6、8、10天取小鼠尾尖血,置預(yù)涂肝素的微量離心管(eppendorf tube,EP管)中,取血時(shí)間為注射后2 h。觀察小鼠體重變化,收集糞便用尿糞便隱血試劑盒測(cè)定血便,實(shí)驗(yàn)第18天所有小鼠乙醚麻醉后取心臟血,置預(yù)涂肝素的EP管中,為尋找CPT-11代謝產(chǎn)物,測(cè)定小柴胡湯有效成分含量,然后處死小鼠取的肝臟、腎臟、大腸、小腸組織,分為兩部分保存,一部分凍存于-80℃冰箱,用于測(cè)量組織中藥物含量;另一部分存于4%甲醛溶液,用于制作病理切片,了解組織損傷。3.采用UPLC-MS/MS方法,測(cè)定組織及血液樣品中CPT-11及其代謝產(chǎn)物和小柴胡湯中有效成分的含量。結(jié)果:1.遲發(fā)性腹瀉模型建立:(1)與正常組相比,模型組小鼠皮毛污穢、潮濕、結(jié)塊,身體發(fā)抖,抓拿時(shí)不掙扎,肛門烏黑紅腫;(2)正常組小鼠體重上升了0.57±0.41g,模型組小鼠下降了5.98±2.08g;(3)正常組小鼠血便率為0,模型組于連續(xù)腹腔注射cpt-11后第7天血便率達(dá)100%(p0.01)。2.小柴胡湯干預(yù)研究:(1)體重比較:第1天和第4天各組小鼠體重?zé)o明顯差異(p0.05),第10天空白對(duì)照組小鼠體重高于其他組小鼠(p0.05),說明遲發(fā)性腹瀉模型能導(dǎo)致小鼠體重顯著降低;第17天空白對(duì)照組小鼠體重仍高于其他組(p0.05),但小柴胡湯組小鼠體重高于模型對(duì)照組(p0.05),說明小柴胡湯能一定程度地減輕小鼠體重降低的癥狀;(2)血便率:正常對(duì)照組、模型對(duì)照組、小柴胡湯組、洛哌丁胺組血便率分別為0%、100%、50%、60%;實(shí)驗(yàn)第8~17天,小柴胡湯組和洛哌丁胺組小鼠血便率均顯著低于模型對(duì)照組(p0.01),且小柴胡湯組小鼠血便率低于洛哌丁胺組(p0.05);(3)病理組織學(xué)觀察:空白對(duì)照組小鼠大腸和小腸結(jié)構(gòu)完整,模型對(duì)照組小鼠大腸和小腸黏膜上皮細(xì)胞伴明顯彌漫性壞死脫落和炎癥細(xì)胞浸潤(rùn),小柴胡湯組和洛哌丁胺組小鼠腸黏膜組織均趨于正常;連續(xù)注射cpt-117天對(duì)肝臟和腎臟無明顯損傷。3.藥物濃度含量測(cè)定:(1)cpt-11及其代謝產(chǎn)物:小柴胡湯組小鼠注射cpt-11第6天時(shí)cpt-11血藥濃度最高,與模型對(duì)照組第4天的最高濃度沒有顯著性差異(p0.05);模型對(duì)照組sn-38最高濃度是實(shí)驗(yàn)組的7倍;模型對(duì)照組注射cpt-11第4天時(shí)sn-38g血藥濃度最高,而小柴胡湯組是第6天,顯著高于模型對(duì)照組(p0.05),除了第4天實(shí)驗(yàn)組血藥濃度比對(duì)照組低外,其他天數(shù)實(shí)驗(yàn)組血藥濃度均高于對(duì)照組;模型對(duì)照組cpt-11濃度在各組織中排序?yàn)樾∧c肝腎大腸,sn-38和sn-38g濃度在各組織中排序?yàn)樾∧c腎大腸肝,模型對(duì)照組小鼠cpt-11、sn-38、sn-38g均在小腸中分布最多(p0.05),小柴胡湯組cpt-11濃度在各組織中排序?yàn)楦文I小腸大腸,sn-38和sn-38g濃度在各組織中排序?yàn)楦涡∧c腎大腸,小柴胡湯組小鼠cpt-11、sn-38、sn-38g這三種物質(zhì)均在肝中分布最多(p0.05),小柴胡湯組各成分在各組織的含量均顯著小于模型對(duì)照組;(2)小柴胡湯中17種有效成分:注射cpt-11后0、2、4、6天血液中只能測(cè)到14種成分,黃芩苷、槲皮素和黃芩苷元這三種成分在檢測(cè)限以下,而各組織中均可以測(cè)到這3種成分,提示這3種成分具有器官靶向性;甘草酸的血藥濃度遠(yuǎn)遠(yuǎn)高于其他成分,在各組織中只少量存在,可能是甘草酸主要在血液中代謝。在肝中漢黃芩素的濃度最高(21700.7430ng/ml),而柴胡皂苷a的濃度最低(12.4930 ng/mL);在腎臟中人參皂苷Rd的濃度最高(21563.0884ng/mL),柴胡皂苷a的含量最低(20.7965 ng/mL);在大腸中漢黃芩素的濃度最高(26370.0697 ng/mL),柴胡皂苷a的含量最低(3.7625 ng/mL);在小腸中人參皂苷Rd的濃度最高(22622.3167 ng/mL),人參皂苷Rg1的含量最低(45.0834 ng/mL)。結(jié)論:1.首次使用C57BL6小鼠,適應(yīng)性飼喂高脂飼料三周后腹腔注射CPT-11 50 mg/kg,連續(xù)7天,每天一次,能成功建立CPT-11所致遲發(fā)性腹瀉小鼠模型;2.小柴胡湯能預(yù)防CPT-11所致小鼠血便,使遲發(fā)性腹瀉小鼠體重正常增長(zhǎng)、降低血便率、改善腸組織損傷;3.小柴胡湯中含有17種活性成分,主要干預(yù)SN-38的代謝,能抑制CPT-11、SN-38、SN-38G的膽汁外排并加速這三種物質(zhì)的消除;CPT-11的腸肝循環(huán)能引起遲發(fā)性腹瀉,小柴胡湯預(yù)防CPT-11所致遲發(fā)性腹瀉是多個(gè)靶器官共同作用的,可能與抑制腸肝循環(huán)有關(guān),可為后續(xù)作用機(jī)制研究提供參考。
[Abstract]:Objective: Irinotecan (CPT-11) is a commonly used chemotherapeutic agent in clinical colorectal cancer. The dose restrictive toxicity is delayed diarrhea, which seriously affects the patient's quality of life and tolerance to chemotherapy. 1. the purpose of this study was to establish a model of delayed diarrhea caused by CPT-11 in mice, and 2. to explore the effect of small Bupleurum Decoction on CPT-11 induced mice in advance. Does delayed diarrhea have a preventive effect; 3. the content of CPT-11, its metabolites and the effective components in small Bupleurum soup are determined by super high performance liquid chromatography tandem mass spectrometry (Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry, UPLC-MS/MS), and the substance base of small Bupleurum Decoction on delayed diarrhea. Foundation. Methods: 1. a model of delayed diarrhea in mice was established: 20 healthy male C57BL6 mice were randomly divided into model group (n=10) and normal group (n=10) after adaptive feeding of high fat diet for three weeks. A continuous intraperitoneal injection of CPT-11 for 7 days, once a day, was 50 mg. Kg-1, and the normal group was intraperitoneally injected with the equal dextran 40 glucoside. Glucose injection, daily observation of the mice's anus, photographing records, collecting feces, using urine fecal occult kit to determine the blood stool.2. small Bupleurum Decoction intervention study: 40 healthy male C57BL6 mice, adaptive feeding high fat feed for three weeks, randomly divided into 4 groups, each group of 10: blank control group (equal volume of saline), delayed diarrhea model group (equal volume saline), small Chaihu Decoction group (1500 mg. Kg-1. D-1), luperidine group (0.5333 mg. Kg-1. D-1), continuously gavage for 17 days, 1 times a day. On day 4~10 of the experiment, except for the blank control group, the rest of the mice were intraperitoneally injected with CPT-11 for 7 days to induce delayed diarrhea. In the micro centrifuge tube (Eppendorf tube, EP tube), the time of taking blood was 2 h. after injection to observe the body weight change of mice. The excrement was collected by urine fecal occult blood reagent box for blood stool. All mice were taken heart blood after eighteenth days of ether anaesthesia, and the EP tube precoated with heparin was used to find the metabolite of CPT-11 and the content of the effective component of Xiao Chai Hu soup was determined. Then the liver, kidney, large intestine and small intestine tissue were put to death in mice, and two parts were stored in two parts. Some were frozen in the refrigerator to measure the drug content in the tissue. The other was stored in 4% Formaldehyde Solution to make pathological sections. The tissue damage.3. was used to determine CPT-11 and its metabolism in tissue and blood samples. Results: the content of the effective components in the material and small Bupleurum soup. Results: 1. the model of delayed diarrhea was established: (1) compared with the normal group, the mice in the model group were filthy, wet, caking, trembling, holding and holding no struggles, and anus black and red swelling; (2) the weight of the normal group increased by 0.57 + 0.41g, and the model mice decreased by 5.98 + 2.08g; (3) normal group mice blood The rate of stool was 0. The rate of blood stool in the model group was 100% (P0.01).2. small Chaihu Decoction after seventh days of continuous intraperitoneal injection of CPT-11. (1) weight comparison: there was no significant difference in weight between the first days and fourth days (P0.05), and the weight of the blank control group was higher than that of the other group (P0.05) on the tenth day, indicating that the delayed diarrhea model could lead to the weight of the mice. The weight of the mice in the seventeenth day blank control group was still higher than that of the other groups (P0.05), but the weight of the mice in the small Chaihu soup group was higher than that of the model control group (P0.05), indicating that the small Bupleurum soup could reduce the symptoms of the weight loss of the mice to a certain extent; (2) the blood stool rate: the normal control group, the model control group, the small Bupleurum soup group and the luperidine group were 0%, 10 respectively. 0%, 50%, 60%; on day 8~17, the blood stool rate of mice in the small Chaihu soup group and the loperbuamine group were significantly lower than that in the model control group (P0.01), and the blood stool rate of the mice in the small Chaihu Decoction group was lower than that of the loperbuchine group (P0.05); (3) the histopathological observation: the large intestine and small intestine structure of the blank control group was complete, and the mice in the model control group of large intestine and small intestinal mucosa epithelium were in the control group. The intestinal mucosal tissue in the small Chaihu soup group and the roperamine group tended to be normal, and the concentration of.3. in the liver and kidney was not significantly damaged by the continuous injection of cpt-117 days. (1) CPT-11 and its metabolites: the mice of the small Chaihu soup group were injected with CPT-11 at sixth days after the injection of CPT-11. The highest concentration was no significant difference between the model control group and the control group at fourth days (P0.05); the highest concentration of SN-38 in the model control group was 7 times of the experimental group; the model control group had the highest concentration of sn-38g blood at fourth days of CPT-11 injection, and the small Bupleurum soup group was sixth days, significantly higher than the model control group (P0.05), except for the blood concentration ratio of the fourth day experimental group. The concentration of blood drug in the experimental group was higher than that of the control group. The concentration of CPT-11 in the model control group was arranged in the small intestine liver and kidney large intestine, and the concentration of SN-38 and sn-38g was arranged in the small intestine and kidney large intestine in the tissues, and the mice in the model control group, CPT-11, SN-38, and sn-38g were all distributed most in the small intestine (P0.05), and the small Bupleurum soup group was CPT-11 concentrated. The degree of SN-38 and sn-38g concentration in the tissues of the liver and kidney of the small intestine, the concentration of SN-38 and sn-38g in the tissues of the liver and kidney large intestine, CPT-11, SN-38 and sn-38g in the small Bupleurum soup group were most distributed in the liver (P0.05), and the content of each component in the small Chaihu soup group was significantly smaller than that in the model control group; (2) 17 species in the small Bupleurum soup were found in the small Bupleurum soup. Effective components: 14 components can be detected only in the blood 0,2,4,6 days after CPT-11, baicalin, quercetin and baicalin, and the three components are below the detection limit, and all of the 3 components can be detected in each tissue, suggesting that these 3 components have organ targeting, and the concentration of glycyrrhizic acid is far higher than that of other components, and only a small amount in each tissue is stored. In the liver, the concentration of Radix scutellarin is the highest (21700.7430ng/ml), and the concentration of Bupleurum saponin A is the lowest (12.4930 ng/mL); the concentration of ginsenoside Rd in the kidney is the highest (21563.0884ng/mL), and the content of Bupleurum saponins A is the lowest (20.7965 ng/mL), and the concentration of baicalein in the large intestine is the highest (26370.06 97 ng/mL), the content of saponins a was the lowest (3.7625 ng/mL); the concentration of ginsenoside Rd in the small intestine was the highest (22622.3167 ng/mL) and the content of ginsenoside Rg1 was the lowest (45.0834 ng/mL). Conclusion: 1., C57BL6 mice were first used, and three weeks after adaptive feeding of high fat feed, the peritoneal injection of CPT-11 50 mg/kg, for 7 days, once a day, could successfully establish CPT. The mice model of delayed diarrhea caused by -11, 2. small Chaihu Decoction can prevent the blood stool of mice caused by CPT-11, make the mice of delayed diarrhea normal weight increase, reduce the rate of blood stool and improve the injury of intestinal tissue. 3. small Bupleurum soup contains 17 kinds of active ingredients, which mainly interfere with the metabolism of SN-38, and can inhibit the bile excretion of CPT-11, SN-38, SN-38G and accelerate these three substances. CPT-11's intestinal hepatic circulation can cause delayed diarrhea. The prevention of delayed diarrhea caused by the small Bupleurum soup is the common effect of multiple target organs, which may be related to the inhibition of the intestinal liver circulation, and can provide reference for the follow-up mechanism.

【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R965

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本文編號(hào)：1873271