【摘要】：目的:基于“炎癥誘導(dǎo)血管新生”假說從NF-κB及VEGF信號(hào)通路研究腸瑞灌腸劑治療放射性直腸炎的分子機(jī)制,為放射性直腸炎的臨床治療提供新的藥物作用靶點(diǎn)和新的研究思路方法。方法:以腸瑞灌腸劑7.2g/Kg、3.6g/Kg、1.8g/Kg高、中、低三個(gè)劑量灌腸6MV-X線直線加速器單次劑量27Gy照射致放射性直腸炎C57/BL小鼠,分別設(shè)地塞米松慶聯(lián)合慶大霉素、八味錫類散為對(duì)照,灌腸給藥,每日一次,連續(xù)2周。觀察小鼠一般狀況、糞便,剖取直腸,HE染色觀察其病理改變,免疫組化分析放射性直腸炎小鼠模型直腸組織中NF-κB、VEGF的表達(dá)、PCR檢測直腸組織NF-κB、VEGF表達(dá)以及ELISA檢測小鼠血清中PKA的含量,探討三者之間的關(guān)系。結(jié)果:1.腸瑞灌腸劑對(duì)放射性直腸炎C57/BL小鼠直腸組織病理學(xué)影響(1)光鏡下直腸粘膜病變分級(jí)比較,照射后各組粘膜均有不同程度改變,與空白組正常粘膜相比(P㩳0.05)均有統(tǒng)計(jì)學(xué)差異,提示造模成功。(2)模型組與中藥對(duì)照組、腸瑞低劑組比較(P0.05),差別無統(tǒng)計(jì)學(xué)意義;而與治療高、中劑量組及西藥對(duì)照組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義,即可說明腸瑞高中劑量以及對(duì)照西藥均能改善直腸粘膜組織炎癥。(3)治療高劑組與中、西藥陽性對(duì)照組比較(P㩳0.05),差別均有統(tǒng)計(jì)學(xué)意義,說明高劑量腸瑞灌腸劑療效優(yōu)于對(duì)照西藥地塞米松加慶大霉素以及對(duì)照中藥八味錫類散。而與低劑組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義,說明治療放射性直腸炎高劑量腸瑞灌腸劑療效優(yōu)于低劑量藥物。2.免疫組化分析腸瑞灌腸劑對(duì)放射性直腸炎C57/BL小鼠直腸組織中NF-κB、VEGF的表達(dá)的影響(1)空白對(duì)照組亦見少量NF-κB、VEGF表達(dá),考慮與灌腸器械刺激有關(guān),可以忽略。照射后各組均明顯出現(xiàn)了NF-κB、VEGF表達(dá),但與空白組比較比較(P㩳0.05)差別有統(tǒng)計(jì)學(xué)意義。說明NF-κB、VEGF表達(dá)是直腸粘膜受放射線照射刺激后產(chǎn)生的。(2)治療組與模型組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義,說明腸瑞組、地塞米松合慶大組以及八味錫類散組均能夠抑制NF-κB以及VEGF的表達(dá);腸瑞高劑組與西藥陽性對(duì)照組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義;腸瑞高、中劑量組與中藥陽性對(duì)照組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義。說明高劑量腸瑞灌腸劑較陽性對(duì)照組能夠顯著下調(diào)放射性直腸炎直腸組織NF-κB、VEGF表達(dá)水平,尚不能說明腸瑞中劑量組療效優(yōu)于西藥陽性對(duì)照組。3.PCR檢測腸瑞灌腸劑對(duì)放射性直腸炎C57/BL小鼠直腸組織中NF-κB、VEGF的表達(dá)的影響(1)統(tǒng)計(jì)結(jié)果顯示,空白對(duì)照組可見極少量NF-κB、VEGF表達(dá)(考慮與灌腸器械刺激有關(guān),可以忽略),其它各組均有不同程度的NF-κB、VEGF表達(dá),組間比較,各組與空白組比較,差別有統(tǒng)計(jì)學(xué)意義(P㩳0.05),提示正常鼠直腸粘膜極少量或不表達(dá)NF-κB、VEGF,直腸粘膜受照射刺激后發(fā)生了NF-κB、VEGF基因表達(dá)的改變。(2)模型組NF-κB、VEGF表達(dá)明顯增高,其它各組與之相比較,差別有統(tǒng)計(jì)學(xué)意義(P㩳0.05),說明經(jīng)過治療后出現(xiàn)了二者表達(dá)量的變化。(3)治療高、中劑量組NF-κB、VEGF表達(dá)顯著減少,與西藥對(duì)照組、中藥對(duì)照組比較,差別有統(tǒng)計(jì)學(xué)意義(P㩳0.05),說明腸瑞灌腸劑高、中劑量組能夠顯著下調(diào)放射性直腸炎直腸NF-κB、VEGF表達(dá),因而能夠控制炎癥,抑制血管新生。4.ELISA法測定腸瑞灌腸劑對(duì)放射性直腸炎C57/BL小鼠血清中蛋白激酶A(PKA)的含量的影響(1)空白對(duì)照組及各治療組與模型組相比,差別有統(tǒng)計(jì)學(xué)意義(p0.05),提示造模成功。(2)模型組、西藥、中藥陽性對(duì)照組以及腸瑞中、低劑量組與空白組比較,(p0.05)差別有統(tǒng)計(jì)學(xué)意義,腸瑞高劑量組與空白組比較(p0.05),差別無統(tǒng)計(jì)學(xué)意義。(3)腸瑞高劑量組分別與西藥、中藥陽性對(duì)照組比較(P㩳0.05),差別有統(tǒng)計(jì)學(xué)意義;而腸瑞中、低劑量組分別與陽性對(duì)照組比較,差別無統(tǒng)計(jì)學(xué)意義。說明腸瑞高劑量組能夠明顯抑制蛋白激酶A(PKA)的含量。結(jié)論:腸瑞灌腸劑能夠迅速改善放射性直腸炎小鼠的一般狀況,及時(shí)恢復(fù)進(jìn)食與體重,及時(shí)保護(hù)和修復(fù)腸粘膜,促進(jìn)潰瘍愈合,能顯著改善光鏡下病理變化及結(jié)構(gòu)異常。以炎癥因子為靶點(diǎn),從腸瑞灌腸劑通過降低蛋白激酶A(PKA)的含量進(jìn)而下調(diào)IL-1β、NF-ΚB信號(hào)表達(dá),從而明顯抑制VEGF表達(dá),達(dá)到控制炎癥反應(yīng)、止血,促進(jìn)腸粘膜修復(fù)的目的,證實(shí)了“炎癥誘導(dǎo)血管新生”的假說。高劑量腸瑞灌腸劑治療小鼠放射性直腸炎方面療效顯著。
[Abstract]:Objective: Based on the "inflammatory induced angiogenesis" hypothesis from NF- kappa B and VEGF signaling pathway to study the molecular mechanism of enteral enema in the treatment of radionuclitis, to provide new drug targets and new ways of thinking for the clinical treatment of radionitis. Methods: three, 7.2g/Kg, 3.6g/Kg, 1.8g/Kg, middle and low. Dose enema 6MV- X-ray linear accelerator single dose 27Gy irradiation of radionuclitis C57/BL mice, respectively, dexamethasone combined with gentamicin, eight flavors of tin as the control, enema administration, once a day for 2 weeks, observation of the general state of the mice, feces, rectum, HE staining to observe the pathological changes, immunohistochemical analysis of radioactivity The expression of NF- kappa B, VEGF in rectal tissue of proctitis mice, PCR detection of NF- kappa B in rectal tissue, VEGF expression and the content of PKA in serum of mice detected by ELISA, and to explore the relationship between the three groups. Results: 1. the effect of enteral enema on the rectal histopathology of C57/BL mice of radionuclitis (1) comparison of the classification of rectal mucosal lesions under light microscope, illumination. The mucosa of each group changed in varying degrees, and compared with the normal mucosa of the blank group (P? 0.05), there were statistical differences, suggesting that the model was successful. (2) there was no statistical difference between the model group and the traditional Chinese medicine control group and the intestinal Rui lower agent group (P0.05), but the difference was statistically significant compared with the treatment group, medium dose group and Western medicine control group (P? 0.05). It showed that the dosage of Chang Rui high school and the control western medicine could improve the inflammation of rectal mucosa tissue. (3) the difference of the high dose group and the western medicine positive control group (P? 0.05) was statistically significant, indicating that the effect of high dose enteric enema was better than that of the control group of dexamethasone plus gentamicin and the eight flavour of the traditional Chinese medicine. Comparison (P? 0.05), the difference was statistically significant, indicating that the therapeutic effect of high dose enteric enema on radionitis is better than low dose.2. immunohistochemical analysis of the effect of intestinal rayon enema on the expression of NF- kappa B and VEGF in rectal tissue of C57/BL mice with radionuclitis (1) a small amount of NF- kappa B, VEGF expression, and the consideration of enema in the blank control group NF- kappa B, VEGF expression appeared in all groups after irradiation, but compared with the blank group (P? 0.05), the difference was statistically significant. It indicated that NF- kappa B, VEGF expression was produced by radiation exposure of the rectal mucosa. (2) the treatment group was compared with the model group (P? 0.05), the difference was statistically significant, indicating the intestinal Rui group, The expression of NF- kappa B and VEGF in the group of dexamethasone Heqing and the group of eight stannic groups could inhibit the expression of kappa kappa B and VEGF. The difference was statistically significant between the intestinal reagents group and the western medicine positive control group (P? 0.05), and the middle dose group was compared with the positive control group of traditional Chinese medicine (P? 0.05), and the difference was statistically significant. The group can significantly reduce the NF- kappa B and VEGF expression in rectal tissue of radionuclitis, and the effect of the intestinal Rui middle dose group is better than that of the western medicine positive control group. The effect of.3.PCR detection on the expression of NF- kappa B and VEGF in rectal tissue of C57/BL mice of radioactive proctitis (1) shows that a small amount of NF in the blank control group shows a very small amount of NF. - kappa B, VEGF expression (considering the irritation of the enema device, neglecting), the other groups had different degrees of NF- kappa B, VEGF expression, compared with the blank group, the difference was statistically significant (P? 0.05), suggesting that the normal rat rectum mucosa was very small or non expression of NF- kappa B, VEGF, and the rectal mucosa was irradiated with NF- kappa B, VEGF base occurred. (2) the expression of NF- kappa B and VEGF in the model group was significantly higher than that in other groups (P? 0.05), indicating the changes in the expression of two. (3) the treatment was high, the medium dose group NF- kappa B, the VEGF expression decreased significantly, and the difference was statistically significant compared with the western medicine control group and the traditional Chinese medicine control group (P). 0.05), indicating that the enteric enema is high, the medium dose group can significantly down regulate the expression of NF- kappa B and VEGF in rectum of radionuclitis, and thus can control the inflammation and inhibit the effect of the neovascularization.4.ELISA method on the content of protein kinase A (PKA) in the serum of C57/BL mice of radionuclitis (1) blank control group and each treatment group and model The difference was statistically significant (P0.05). (2) the model group, the western medicine, the Chinese medicine positive control group and the Chang Ruizhong, the low dose group and the blank group were compared with the blank group, and the difference was statistically significant. The difference between the high dose group and the blank group (P0.05) was not statistically significant. (3) the intestinal RRH dose group was positive for the western medicine and the Chinese medicine positive. Compared with the control group (P? 0.05), the difference was statistically significant, but the low dose group had no statistically significant difference compared with the positive control group. It was indicated that the content of protein kinase A (PKA) could be significantly inhibited by the intestinal RI dose group. Conclusion: the intestinal reagents can quickly improve the general condition of the radionuclitis mice and restore the food in time. And weight, timely protection and repair of intestinal mucosa, promote ulcer healing, can significantly improve the pathological changes and structural abnormalities under the light microscope. Targeting the inflammatory factors, the expression of IL-1 beta and NF- B signal is down regulated by the intestinal reagents by reducing the protein kinase A (PKA) content, thus obviously inhibiting the expression of VEGF, controlling the inflammatory reaction, stopping bleeding and promoting the effect. The purpose of intestinal mucosal repair is to confirm the hypothesis of "inflammation inducing angiogenesis". High dose enema has a significant effect on the treatment of radiation proctitis in mice.
【學(xué)位授予單位】：山西省中醫(yī)藥研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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