本文選題：芫花根水提取物 + 炎性因子��； 參考：《南京中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:通過芫花根水提物與空白對照及黃芩油膏對比,外用于大鼠肛瘺術后及肛緣水腫換藥,觀察創(chuàng)面水腫、炎癥反應的變化情況及創(chuàng)面的愈合情況。以此驗證芫花根治療痔瘺疾病的有效性與其抗炎、消腫的藥理作用相關,并且為芫花根的進一步探索研究提供動物實驗研究基礎。方法:實驗一:在大鼠背上制備肛瘺術后創(chuàng)面模型,將造模成功的大鼠分成A組(生理鹽水組)、B組(芫花根組)、C組(黃芩油膏組),分別使用生理鹽水、芫花根水提物、黃芩油膏浸泡的紗布給大鼠創(chuàng)面換藥。在用藥3、7、14天時,取各組的創(chuàng)面組織進行HE染色和MASSON染色,觀察肉芽組織變化、炎細胞浸潤情況和纖維化變化,并應用PCR定量檢測各組大鼠血清中IL-6、IL-12的表達及使用流式細胞術檢測各組大鼠外周血中Treg/Th17比例。實驗二:建立大鼠肛門巴豆油腫脹模型,造模成功后,將大鼠分成A組(正常組)、B組(生理鹽水組)、C組(芫花根組)、D組(黃芩油膏組),A組不做任何處理,B、C、D組每隔4、24、48小時分別將生理鹽水、芫花根水提物、黃芩油膏涂于腫脹的肛門處,48.5小時后,取各組大鼠肛周腫脹部位組織進行HE染色和MASSON染色,觀察大鼠肛緣腫脹程度、血管生成情況、炎細胞浸潤情況和纖維化變化,并行免疫組化檢測SM22 α蛋白的表達,免疫熒光染色定位SM22 α蛋白,以及應用PCR和ELISA方法檢測各組大鼠血清中IL-17和TNF-α的表達情況。數(shù)據(jù)均采用SPSS18.0分析。結果:實驗一中,PCR定量檢測結果顯示:用藥3天后,A、B、C組IL-12的表達量用X±S表示分別為1.02±0.28、2.52±1.01、2.78±1.83,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05(P=0.011),而B、C組對比,統(tǒng)計學上無明顯差異,P0.05(P=0.648)。三組中IL-6表達量用 X±S 表示分別為 1.04±0.46、0.84±0.29、0.69±0.33,P0.05(P=0.157),無統(tǒng)計學差異。用藥7天后,三組IL-12的表達量分別為0.80±0.24、2.75± 1.13、3.02±2.21,經(jīng)統(tǒng)計分析存在顯著性差,P0.05(P=0.006),而B、C組對比,無明顯差異,P0.05(P=0.700)。三組中IL-6表達量分別為1.86±0.56、0.62±0.21、0.53±0.23,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05,而B、C組對比,則無明顯差異。P0.05(P=0.589)。用藥14天后,三組IL-12的表達量分別為0.49±0.21、3.48±1.45、4.19±1.48,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05,而B、C組對比,則無明顯差異,P0.05(P=0.237)。三組中IL-6表達量分別為2.84±0.99、0.49±0.23、0.31±0.14,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05,而B、C組對比,則無明顯差異P0.05(P=0.516)。HE染色顯示:用藥3天后A、B、C三組均有纖維脂肪組織中見大量急慢性炎細胞浸潤,局部肌肉組織變性壞死,血管增生,管壁增厚,A組中還見間質水腫和血管閉塞,說明A組炎癥表現(xiàn)更重,B、C較A組稍輕,兩組炎癥程度相當。用藥7天后三組纖維脂肪組織中仍有大量急慢性炎細胞浸潤,A、C組局部肌肉組織變性壞死、鈣化,B、C組局部有肉芽組織形成,說明B、C組創(chuàng)面開始修復。用藥14天后B、C組組織中炎細胞浸潤較A組明顯減少,且有玻璃樣改變,說明創(chuàng)面開始有瘢痕組織形成。Masson染色顯示:用藥3天后創(chuàng)面組織中見大量粘液及有膠原纖維生成,并且B、C組中多于A組。用藥7天后創(chuàng)面組織中膠原纖維增多,且B、C組比A組多。用藥14天后A組創(chuàng)面組織中膠原纖維增減少B、C組中膠原纖維增多。流式細胞術檢測外周血Treg/Th17比例的結果用X±S表示為:用藥3天后,A、B、C組分別是0.663±0.042、0.770±0.009、0.926±0.002,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05(P=0.004)。用藥7天后,分別是0.565±0.001、1.077±0.033、1.372±0.065,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05(P=0.001)。用藥14天后,分別是0.295±0.011、1.722 ±0.074、2.362±0.187,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05(P=0.001)。實驗二中,PCR定量檢測TNF-α及IL-17指標的結果顯示:A、B、C、D四組中TNF-α 的表達量以 X±S 表示分別為 0.88±0.21、5.03±1.31、2.63±0.96、1.57±0.27,P0.05,存在顯著差異,而C、D組對比,統(tǒng)計學上無明顯差異,P0.05(P=0.073)。四組中IL-17表達量為0.92±0.26、3.37±0.85、2.15±0.45、1.48±0.39,經(jīng)統(tǒng)計分析存在顯著性差異,P0.05,而C、D組對比,統(tǒng)計學上無明顯差異,P0.05(P=0.072)。ELISA定量檢測TNF-α及IL-17指標的結果與PCR檢測結果相符,P值均小于0.05,存在顯著性差異。HE染色示:A組見正常的肛周粘膜、肌肉等組織。B、C、D組的肛周粘膜、肌肉組織中均見急慢性炎細胞浸潤,C、D組較A組少,C組較D炎細胞浸潤更少,且C組中未見肌肉組織壞死,并伴有小血管增生,說明C、D組用藥后較A組炎癥性水腫消退,且C組炎癥、水腫程度最輕。MASSON染色觀察肛緣纖維化程度的結果為:A組中見正常肛緣組織,B組見大量粘液及膠原纖維生成,C組和D組經(jīng)藥物治療后見少量粘液及膠原纖維生成。免疫組化檢測SM22α蛋白的表達用IOD值表示結果,A、B、C、D分別為8841.39±49841.88、4339.75±24700.33、7211.28±29230.33、7702.81±39702.19,四組經(jīng)統(tǒng)計學分析,有顯著性差異,P0.05,但是C、D兩組比較,統(tǒng)計學上無明顯差異,P0.05(P=0.166)。免疫熒光染色顯示:SM22α蛋白及STIM1蛋白位于細胞質內(nèi),紅色熒光位于細胞核中,所以Orail蛋白位于細胞質中。結論:芫花根水提物具有減輕痔瘺術后炎癥反應、術后水腫的作用。在創(chuàng)面愈合的早中期減少炎細胞滲出,增加膠原纖維產(chǎn)生,提高SM22 α蛋白的表達,進而促進痔瘺術后創(chuàng)面的愈合。
[Abstract]:Objective: To compare the water extract of the root of the Daphne Daphne with the blank control and the Scutellaria baicalensis ointment, and to observe the edema, the changes of the inflammatory reaction and the healing of the wounds in the rats after the operation of anal fistula and the edema of the anal border, in order to verify the effectiveness of the root canal for the treatment of hemorrhoids and the pharmacological effects of the anti inflammation and swelling of the disease, and to be the root of the Daphne Daphne. Further exploration and research provide the basis of animal experiment. Experiment 1: to prepare the wound model after anal fistula operation on the back of the rat, the rats were divided into A group (physiological saline group), group B (Daphne root group), group C (Scutellaria baicalensis ointment group), using physiological saline, water extract of Daphne Daphne root, and gauze soaked in Scutellaria ointment to the wound of rats. After 3,7,14 days, the wound tissue of each group was stained with HE and MASSON, and the changes of granulation tissue, infiltration of inflammatory cells and fibrosis were observed. The expression of IL-6 and IL-12 in the serum of each group was detected by PCR and the proportion of Treg/Th17 in the peripheral blood of each group was detected by flow cytometry. Experiment two: establishment of experiment two: After the model was successful, the rats were divided into A group (normal group), group B (normal saline group), group C (Daphne root group), group D (Scutellaria baicalensis ointment group), A group did not do any treatment, B, C, D group every 4,24,48 hour respectively, the saline, Daphne root water extract, Scutellaria baicalin was coated on the swollen anus, after 48.5 hours, take each group big HE staining and MASSON staining were performed in the tissue of swollen perianal area. The swelling of the anal margin, angiogenesis, infiltration of inflammatory cells and fibrosis were observed in rats. The expression of SM22 alpha protein was detected by immunohistochemistry, SM22 alpha protein was detected by immunofluorescence staining, and IL-17 and TNF in serum of rats were detected by PCR and ELISA methods. The data were analyzed by SPSS18.0. Results: in the first experiment, PCR quantitative detection results showed that the expression of IL-12 in A, B and C group was 1.02 + 0.28,2.52 + 1.01,2.78 + 1.83 respectively after 3 days of drug use, and there were significant differences in statistical analysis, P0.05 (P= 0.011). 8) in the three group, the expression of IL-6 in the three group was 1.04 + 0.46,0.84 + 0.29,0.69 + 0.33, P0.05 (P=0.157), and there was no statistical difference. The expression of IL-12 in the three groups was 0.80 + 0.24,2.75 + 1.13,3.02 + 2.21 respectively after 7 days of drug use, and there was a significant difference in statistical analysis and P0.05 (P= 0.006). The expression of IL-6 was 1.86 + 0.56,0.62 + 0.21,0.53 + 0.23 respectively. There were significant differences in statistical analysis, P0.05, but there was no significant difference in.P0.05 (P=0.589) in B and C groups. The expression of IL-12 in the three groups was 0.49 + 0.21,3.48 + 1.48, respectively. The expression of IL-6 was P0.05 (P=0.237). The expression of IL-6 in the three groups was 2.84 + 0.99,0.49 + 0.23,0.31 + 0.14, respectively, and there was a significant difference by statistical analysis, P0.05, while B, C group contrast, there was no significant difference between P0.05 (P=0.516).HE staining: 3 days of A, three groups of fibrous adipose tissue were found to see a large number of acute and chronic inflammatory cells infiltration, local muscle tissue. Degeneration and necrosis, vascular hyperplasia and thickening of the wall of the tube, A group also found interstitial edema and vascular occlusion, which showed that A group was more severe in inflammation, B, C was slightly lighter than that in A group, and the two groups of inflammation were similar. There were still a large number of acute and chronic inflammatory cells in three groups of fibrous adipose tissue after 7 days, A, C group muscle tissue degeneration and necrosis, calcification, B, and C group local granulation tissue The formation of B, C group wound began to repair. After 14 days, B, the infiltration of inflammatory cells in group C was significantly lower than that in the A group, and there was a glass like change. It showed that the wound began to have scar tissue to form.Masson staining showed that a large number of mucus and collagen fibrinolysis were found in the wound tissue after 3 days of drug use, and B and C group were more than the A group. 7 days after the drug use, the wound group was used. The collagen fibers in the fabric increased, and the B, C group were more than the A group. The collagen fibers in the wound tissue of group A decreased B, and the collagen fibers increased in group C 14 days later. The result of flow cytometry for detecting the proportion of Treg/Th17 in peripheral blood was expressed as X + S: 3 days after medication, A, B, and C groups were 0.663 + + 0.002, respectively. The statistical analysis was significant. Difference, P0.05 (P=0.004). 7 days after medication, 0.565 + 0.001,1.077 + 0.033,1.372 + 0.065 respectively, statistically significant differences, P0.05 (P=0.001). 14 days after drug use, 0.295 + 0.011,1.722 + 0.074,2.362 + 0.187 respectively, statistically significant differences, P0.05 (P=0.001). The results showed that the expression of TNF- alpha in A, B, C, D four groups was 0.88 + 0.21,5.03 + 1.31,2.63 + 0.96,1.57 + 0.27 and P0.05, and there were significant differences. There was no significant difference between P0.05, C and D group. The results of P0.05 (P=0.072).ELISA quantitative detection of TNF- A and IL-17 were consistent with the results of PCR detection, P values were less than 0.05, and there was a significant difference in.HE staining. Chronic inflammatory cell infiltration, C, D group less than the A group, C group less infiltration than D inflammatory cells, and C group no muscle tissue necrosis, accompanied by small vascular hyperplasia, indicating C, D group after the use of A group inflammatory edema subsided, C group inflammation, edema degree of the least.MASSON staining observation of the degree of anal fibrosis in the case of the normal anal margin in the A group A large number of mucus and collagen fibers were produced. A small amount of mucus and collagen fibers were found in group C and group D after treatment. The expression of SM22 alpha protein was expressed with IOD values by immunohistochemistry. A, B, C, D were 8841.39 + 49841.884339.75 + 24700.337211.28 + 39702.19, respectively. The four groups were statistically analyzed with significant differences. 05, but C, D two groups compared, statistically no significant difference, P0.05 (P=0.166). Immunofluorescence staining showed that the SM22 alpha protein and STIM1 protein in the cytoplasm, red fluorescence in the nucleus, so the Orail protein in the cytoplasm. Conclusion: Daphne root water extract has the effect of alleviated inflammatory reaction after hemorrhoids operation, postoperative edema. In the wound. In the early and middle stages of healing, the inflammatory cells exudate, the production of collagen fibers is increased, the expression of SM22 alpha protein is increased, and the wound healing after hemorrhoid fistula operation is promoted.
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5

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