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生姜瀉心湯預(yù)防伊立替康所致遲發(fā)性腹瀉的機(jī)理研究

發(fā)布時間:2018-05-08 01:13

  本文選題:生姜瀉心湯 + 伊立替康 ; 參考:《北京中醫(yī)藥大學(xué)》2017年博士論文


【摘要】:目的1.在伊立替康遲發(fā)性腹瀉大鼠模型上,研究探討中藥生姜瀉心湯對腸粘膜細(xì)胞凋亡、細(xì)胞增殖和干細(xì)胞標(biāo)志物表達(dá)、肝臟UGT1A1、腸道β-葡萄糖醛酸酶活性的影響,揭示該方在預(yù)防伊立替康腸毒性的作用靶點(diǎn)和作用機(jī)制,為中藥預(yù)防化療性腹瀉提供科學(xué)依據(jù)。2.為臨床應(yīng)用生姜瀉心湯預(yù)防伊立替康腸毒性提供實(shí)驗依據(jù)。方法中藥的作用具有多靶點(diǎn)的特點(diǎn),探索中藥生姜瀉心湯在不同靶點(diǎn)抑制化療性腸毒性的作用,是闡明對腸毒性預(yù)防機(jī)理的關(guān)鍵。根據(jù)伊立替康的代謝及其腸毒性的發(fā)生機(jī)制,利用伊立替康腹瀉大鼠模型進(jìn)行體內(nèi)試驗,從調(diào)節(jié)肝臟UGT1A1酶、腸道β-葡萄糖醛酸酶以及促進(jìn)腸粘膜自身修復(fù)機(jī)能三個環(huán)節(jié)進(jìn)行研究。1.建立伊立替康腹瀉模型大鼠;2.通過觀察實(shí)驗過程中各組大鼠體重、進(jìn)食量的變化,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠一般狀況的影響;并根據(jù)文獻(xiàn)記載腹瀉等級和評分標(biāo)準(zhǔn),對各組大鼠在伊立替康注射后進(jìn)行腹瀉評估,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腹瀉癥狀的影響;3.通過HE染色技術(shù),觀察伊立替康對腹瀉模型大鼠空腸、回腸、盲腸、結(jié)腸腸粘膜的損傷作用,并對比各組大鼠間不同腸段腸粘膜在鏡下病理上的差異,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腸粘膜損傷的影響;4.通過TUNEL染色和caspase-3酶活性檢測技術(shù),分別定性和定量的檢測各組大鼠空腸腸粘膜細(xì)胞的凋亡狀況,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腸粘膜細(xì)胞凋亡的影響;5.通過免疫組化染色技術(shù),采用增殖細(xì)胞核抗原(proliferating cell nuclear antigen,PCNA)和細(xì)胞黏附分子(homing cell adhesion molecule,CD44)抗體分別染色標(biāo)記空腸中增殖的腸粘膜細(xì)胞和腸隱窩處的腸干細(xì)胞,觀察各組大鼠空腸粘膜上皮細(xì)胞和腸干細(xì)胞在鏡下的染色情況,并通過Image-Pro Plus(IPP)軟件測定所拍攝的圖像中染色部分的整體光密度(Integrated option density,IOD)值,并計數(shù)每個視野下PCNA染色陽性細(xì)胞數(shù)目,定量地分析各組大鼠空腸粘膜上皮細(xì)胞和腸干細(xì)胞表達(dá)的差異性,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腸粘膜細(xì)胞增殖的影響;6.通過實(shí)時定量PCR技術(shù),檢測各組大鼠空腸組織腸干細(xì)胞標(biāo)志物L(fēng)gr5 mRNA表達(dá)情況,從mRNA水平驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腸干細(xì)胞表達(dá)的影響;7.通過酶活性檢測技術(shù),測定各組大鼠腸道糞便中β-葡萄糖醛酸酶活性,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠腸道內(nèi)環(huán)境的影響;8.通過實(shí)時定量PCR技術(shù),檢測各組大鼠肝臟組織UGT1A1 mRNA表達(dá)情況,驗證生姜瀉心湯對伊立替康腹瀉模型大鼠肝臟代謝酶的調(diào)節(jié)作用。結(jié)果1.通過對伊立替康腹瀉模型大鼠一般情況、腹瀉癥狀的觀察,及對其腸粘膜病理、免疫組化的檢測,均證實(shí)伊立替康腹瀉模型大鼠造模成功;2.對各組大鼠一般情況的觀察結(jié)果顯示,從實(shí)驗第7天(末次注射伊立替康后48h)起,中、高劑量中藥組大鼠的累積體重增加值顯著高于模型對照組(p0.05,p0.01),但不同劑量中藥組間的差異不具有統(tǒng)計學(xué)意義,直到第9天,與低劑量中藥組相比,高劑量中藥組大鼠才顯示出更高的累積體重增加值(p0.05)。從實(shí)驗第4天起,與空白對照組相比,模型對照組和低、中、高劑量中藥組大鼠進(jìn)食量顯著減少(p0.01),而各劑量中藥組大鼠進(jìn)食量恢復(fù)明顯快于模型對照組;至實(shí)驗第9天,模型對照組大鼠的進(jìn)食量仍顯著低于空白對照組(p0.01),各劑量中藥組大鼠的進(jìn)食量則顯著高于模型對照組(p0.01),而與空白對照組無差異,但不同劑量中藥組間的差異不具有統(tǒng)計學(xué)意義。對各組大鼠腹瀉癥狀的觀察結(jié)果顯示,末次伊立替康注射24h后,模型對照組和各劑量中藥組大鼠的腹瀉評分均值開始升高,其中模型對照組大鼠腹瀉評分升高幅度明顯高于各劑量中藥組,在末次用藥60h和70h后,與模型對照組相比,中劑量和高劑量中藥組大鼠的腹瀉等級分布均具有明顯差異(p0.05,p0.01)3.腸粘膜病理鏡下觀察結(jié)果顯示,模型對照組大鼠腸粘膜可觀察到伊立替康引起的損傷,包括:腸上皮大體結(jié)構(gòu)不完整、上皮細(xì)胞排列紊亂、腸隱窩和腸絨毛減少、變短等,其中,以空腸、盲腸黏膜的損傷最為嚴(yán)重;而低、中、高劑量中藥組大鼠腸黏膜結(jié)構(gòu)更加完整,腸隱窩和腸絨毛得到了更好的保存。4.TUNEL染色和caspase-3酶活性檢測結(jié)果顯示,中藥組鏡下空腸黏膜陽性凋亡細(xì)胞明顯少于模型對照組,且低、中、高劑量中藥組腸道caspase-3酶活性(U)分別為0.66±0.10、0.48±0.11、0.68±0.19,均明顯低于模型對照組(vs1.00±0.17,p0.01);5.PCNA和CD44染色標(biāo)記免疫組化檢測結(jié)果顯示,高劑量中藥組PCNA陽性細(xì)胞數(shù)明顯高于模型對照組(531.20±101.64 vs 312.40±43.23,p0.01);高劑量中藥組PCNA 的 IOD 值明顯高于模型對照組(2494.93±615.00 vs 1026.26±339.00,p0.05);中、高劑量中藥組CD44的IOD值分別為511.35±83.18、613.04±102.42,均明顯高于模型對照組(vs 360.99± 156.87,p0.05,p0.01);6.實(shí)時定量PCR技術(shù)結(jié)果顯示,與模型對照組相比,各劑量中藥組大鼠腸道Lgr5 mRNA表達(dá)均有所增多,但只有高劑量中藥組Lgr5 mRNA表達(dá)顯著增多,具有統(tǒng)計學(xué)意義(1.75±0.53 vs 0.56±0.54,p0.01);7.β 葡萄糖醛酸酶活性檢測結(jié)果顯示,在伊立替康注射前,與空白對照組、模型對照組相比,低、中劑量中藥組大鼠腸道糞便β-葡萄糖醛酸酶活性無明顯差異;而高劑量中藥組大鼠腸道糞便β-葡萄糖醛酸酶活性明顯低于模型對照組(3.88±1.78 vs 5.92±1.47,p0.05);在注射伊立替康后,低、中、高劑量中藥組β-葡萄糖醛酸酶活性分別為12.56±1.81、13.55±1.17、11.62±1.78,均明顯低于模型對照組(vs 16.09± 1.40,p0.05,p0.01),而各劑量中藥組間的差異并不顯著;8.實(shí)時定量PCR技術(shù)結(jié)果顯示,與空白對照組相比,模型對照組和低、中、高劑量中藥組大鼠肝臟UGT1A1酶mRNA表達(dá)顯著降低;而與模型對照組相比,各劑量中藥組大鼠肝臟UGT1A1酶mRNA的表達(dá)均未表現(xiàn)出明顯差異。結(jié)論1.生姜瀉心湯可有效減輕伊立替康所引起的體重下降,促進(jìn)注射伊立替康后大鼠的進(jìn)食;2.生姜瀉心湯可以降低伊立替康所引起的腹瀉級別和評分;3.生姜瀉心湯可以減輕伊立替康引起的腸粘膜病理性損傷;4.生姜瀉心湯可減少伊立替康所導(dǎo)致的腸道細(xì)胞凋亡;5.生姜瀉心湯可促進(jìn)腸黏膜細(xì)胞的增殖;6.生姜瀉心湯可促進(jìn)腸干細(xì)胞的表達(dá);7.生姜瀉心湯可降低大鼠注射伊立替康后腸道糞便中β-葡萄糖醛酸酶活性;8.生姜瀉心湯對肝臟UGT1A1酶mRNA的表達(dá)無明顯影響。
[Abstract]:Objective 1. to investigate the effect of ginger Xiexin Decoction on intestinal mucosal cell apoptosis, cell proliferation and expression of stem cell markers, liver UGT1A1 and intestinal beta glucuronoate activity in the rat model of irinotecan delayed onset diarrhea, and to reveal the target and mechanism of this prescription in preventing the toxicity of irinotecan. Providing scientific basis for the treatment of diarrhoea,.2. provides experimental basis for the clinical application of ginger Xiexin Decoction in the prevention of irinotecan enterotoxicity. Methods the effect of Chinese medicine has the characteristics of multiple targets. The effect of ginger Xiexin Decoction on the inhibition of chemotherapeutic intestinal toxicity at different targets is explored, and the key to the mechanism of intestinal toxicity prevention is clarified. According to erinoteca's generation A rat model of irinotecan diarrhea rat model was carried out in vivo by using the three links of regulating the liver UGT1A1 enzyme, intestinal beta glucuronoenzyme and promoting the self repair function of the intestinal mucosa to establish the rat model of erineecan diarrhea model by.1., and 2. by observing the weight of each group of rats during the experiment. To verify the effect of ginger Xiexin Decoction on the general condition of the rat model of irinotecan diarrhea model, and to evaluate the diarrhoea in the rats after injection of irinotecan according to the rank of diarrhoea and scoring in the literature, and to verify the effect of ginger Xiexin Decoction on diarrhea in the rat model of irinotecan diarrhea model; 3. The effects of irinotecan on jejunum, ileum, cecum and colon mucosa of rats with diarrhea model, and the pathological changes of intestinal mucosa in different intestinal segments in each group were compared, and the effect of ginger Xiexin Decoction on intestinal mucosal injury in the rat model of irinotecan diarrhea model was verified. 4. by TUNEL staining and caspase-3 enzyme activity detection techniques, respectively The effect of ginger Xiexin Decoction on the apoptosis of intestinal mucosa in the rat model of irinotecan diarrhea model was tested by qualitative and quantitative analysis of the apoptosis of intestinal intestinal mucosa cells in each group. 5. by immunohistochemical staining, proliferating cell nuclear antigen (PCNA) and cell adhesion molecules (homing cell adhesi) were used. On molecule, CD44) antibodies were stained respectively to mark the intestinal stem cells of intestinal mucosa cells and intestinal crypts in the jejunum, and observe the staining of the intestinal mucosal epithelial cells and intestinal stem cells under the microscope in each group, and determine the overall light density (Integrated option de) of the stained parts in the images taken by the Image-Pro Plus (IPP) software. Nsity, IOD) value, and count the number of PCNA staining positive cells in each field of vision, quantitative analysis of the difference in the expression of intestinal mucosal epithelial cells and intestinal stem cells in each group, and verify the effect of ginger Xiexin soup on the proliferation of intestinal mucosa cells in the rat model of irinotecan diarrhea model; 6. through real-time quantitative PCR technique to detect the jejunum tissue in each group The expression of Lgr5 mRNA in the intestinal stem cell marker, the effect of ginger Xiexin Decoction on the expression of intestinal stem cells in the rat model of irinotecan diarrhea model from mRNA level; 7. the activity of beta glucuronuronase in intestinal feces of rats was measured by enzyme activity detection technique, and the internal environment of the rat model of yinecan diarrhea model was verified by the ginger Xiexin soup. 8. through real-time quantitative PCR technique, the expression of UGT1A1 mRNA in rat liver tissues was detected, and the regulation of ginger Xiexin Decoction on liver metabolic enzymes in the rat model of irinotecan diarrhea model was verified. Results 1. through the observation of the general condition of the rat model of the irinotecan diarrhea model, the observation of the diarrhea symptoms, and the immunohistochemistry of the intestinal mucosa and immunohistochemistry. The experimental results of irinotecan diarrhea model rats were confirmed successfully. 2. the general situation of the rats in each group showed that the cumulative weight increase value of the rats in the high dose group of Chinese medicine group was significantly higher than that of the model control group (P0.05, P0.01), but the difference between the different doses of Chinese medicine group was no longer than that of the model control group (48h after the last injection of irinotecan). It was statistically significant, until ninth days, compared with the low dose Chinese medicine group, the high dose group of Chinese medicine group showed higher cumulative weight gain (P0.05). From the fourth day of the experiment, the intake of the rats in the model control group and the low, middle and high dose group of Chinese medicine group decreased significantly (P0.01), and the intake of the rats in each dose group was compared with the blank control group. The recovery of the model control group was significantly faster than that of the model control group. After ninth days of the experiment, the intake of the rats in the model control group was still significantly lower than that in the blank control group (P0.01). The intake of the rats in each dose group was significantly higher than that of the model control group (P0.01), but there was no difference between the control group and the blank control group, but the difference between the different doses of the Chinese medicine group was not statistically significant. The observation of diarrhea symptoms in rats showed that after the injection of 24h, the mean value of diarrhoea score in the model control group and the Chinese medicine group began to rise, and the increase of the diarrhea score in the model control group was significantly higher than that of the traditional Chinese medicine group. The middle dose and the high dose of the model control group were compared with the model control group after the last dose of 60H and 70h. The diarrhea grade distribution in the dose group of the Chinese medicine group had obvious difference (P0.05, P0.01). The observation of 3. intestinal mucosa pathological mirror showed that the intestinal mucosa of the model control group could observe the injury caused by irinotecan, including the incomplete general structure of the intestinal epithelium, the disorder of the epithelial cells, the decrease of the intestinal recess and the intestinal villi, and so on. The mucosal structure of the jejunum and the cecum was the most serious, but the intestinal mucosal structure of the rats in the low, middle and high doses of the Chinese medicine group was more complete. The intestinal recess and intestinal villi were better preserved by.4.TUNEL staining and the detection of Caspase-3 enzyme activity. The positive apoptotic cells of the jejunum mucosa under the traditional Chinese medicine group were significantly lower than those in the model control group, and the middle and high doses were low in the middle and high doses. The intestinal caspase-3 enzyme activity (U) in the drug group was 0.66 + 0.10,0.48 + 0.11,0.68 + 0.19, respectively, which were significantly lower than that in the model control group (vs1.00 0.17, P0.01). The results of 5.PCNA and CD44 staining markers showed that the number of PCNA positive cells in the high dose Chinese medicine group was significantly higher than that in the model control group (531.20 + 101.64 vs 312.40 + 43.23, P0.01). The IOD value of PCNA in the drug group was significantly higher than that in the model control group (2494.93 + 615 vs 1026.26 + 339, P0.05), and the IOD value of CD44 in the high dose Chinese medicine group was 511.35 + 83.18613.04 + 102.42, respectively higher than that of the model control group (vs 360.99 + 156.87, P0.05, P0.01), and 6. real-time quantitative PCR technique results showed that compared with the model control group, each dose was compared with the model control group. The expression of Lgr5 mRNA in the intestinal tract of the rats in the traditional Chinese medicine group increased, but the expression of Lgr5 mRNA in the high dose group was significantly increased, with statistical significance (1.75 + 0.53 vs 0.56 + 0.54, P0.01); 7. beta glucuronide activity detection results showed that before the injection of irinotecan, compared with the empty white control group, the low, medium dose group of Chinese medicine group was compared with the model control group. The activity of beta glucuronate in intestinal feces of rats had no significant difference, but the activity of beta glucuronate in intestinal feces of rats in high dose group was significantly lower than that of model control group (3.88 + 1.78 vs 5.92 + 1.47, P0.05). After injection of irinotecan, the activity of beta glucuronase in low, medium and high dose group was 12.56 + 1.81,13.55 + 1.17,1, respectively 1.62 + 1.78, significantly lower than the model control group (vs 16.09 + 1.40, P0.05, P0.01), but the difference between different doses of traditional Chinese medicine group was not significant; 8. real-time quantitative PCR technique results showed that compared with the blank control group, the expression of UGT1A1 enzyme mRNA in the liver of the model control group and the low, middle and high dose group of Chinese medicine group decreased significantly; and compared with the model control group, each group was compared with the model control group. There was no obvious difference in the expression of UGT1A1 enzyme mRNA in the liver of the rats in the dose group. Conclusion 1. ginger Xiexin Decoction can effectively reduce the weight loss caused by erinotecan and promote the feeding of rats after injection of irinotecan; 2. ginger Xiexin soup can reduce the grade and score of diarrhea caused by erinotecan; 3. ginger diarrhea heart soup can be reduced. 4. ginger Xiexin Decoction can reduce the apoptosis of intestinal cells caused by erinotecan, 5. ginger Xiexin soup can promote the proliferation of intestinal mucosa cells, 6. ginger Xiexin soup can promote the expression of intestinal stem cells, and 7. ginger Xiexin Decoction can reduce the beta glucosaldehyde in intestinal feces after injection of irinotecan in rats The activity of UGT1A1 enzyme mRNA was not significantly affected by ginger 8. Xiexin Decoction.

【學(xué)位授予單位】:北京中醫(yī)藥大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2017
【分類號】:R730.5

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