本文關(guān)鍵詞：潰瘍油干預(yù)糖尿病潰瘍愈合的實(shí)驗(yàn)及臨床研究　出處：《北京中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蛋白酪氨酸磷酸酶1B 潰瘍油 生長因子 糖尿病潰瘍 晚期糖基化終末產(chǎn)物

【摘要】：研究目的：糖尿病潰瘍中醫(yī)歸屬于脫疽范圍,是糖尿病的嚴(yán)重的慢性并發(fā)癥之一,由代謝紊亂導(dǎo)致的血管病變、神經(jīng)病變,加之糖尿病免疫功能受損繼發(fā)感染而協(xié)同發(fā)病,是臨床一種常見的慢性難愈性創(chuàng)面。難愈性糖尿病潰瘍?nèi)匀皇悄壳瓣P(guān)注的重點(diǎn)和難點(diǎn),糖尿病潰瘍發(fā)生發(fā)展的機(jī)制主要與糖尿病發(fā)生發(fā)展的相關(guān)機(jī)制及糖尿病潰瘍難愈機(jī)制相關(guān),慢性創(chuàng)面愈合過程需要多種細(xì)胞、生長因子及細(xì)胞外基質(zhì)的協(xié)同參與,構(gòu)成復(fù)雜的生物網(wǎng)絡(luò)共同促進(jìn)潰瘍愈合,細(xì)胞因子缺失或者與靶細(xì)胞受體信號(hào)轉(zhuǎn)導(dǎo)“失耦聯(lián)”都會(huì)導(dǎo)致創(chuàng)面不愈合或延期愈合,中藥外治糖尿病潰瘍能夠明顯促進(jìn)創(chuàng)面愈合,但是中藥外治如何調(diào)控相應(yīng)靶細(xì)胞仍需要研究,中藥復(fù)方潰瘍油在長期的臨床實(shí)踐中歸納總結(jié)而成,臨床中用于濕熱蘊(yùn)結(jié)型糖尿病潰瘍患者,本課題通過研究潰瘍油干預(yù)糖尿病潰瘍愈合的臨床和實(shí)驗(yàn)研究來進(jìn)一步闡明創(chuàng)面愈合的機(jī)制,研究潰瘍油促進(jìn)糖尿病潰瘍愈合過程中對于相關(guān)通路的影響作用及其在臨床中的療效評(píng)定。研究方法：1.實(shí)驗(yàn)研究：選購80-100g SPF級(jí)SD雄性大鼠,按隨機(jī)數(shù)字表法隨機(jī)分為空白對照組、模型組、西藥組、中藥組,其中模型組、西藥組、中藥組按體重50mg/kg一次性快速腹腔注射STZ溶液造糖尿病模型,空白對照組一次性腹腔注射等體積不含STZ的O.1mmol/L檸檬酸-檸檬酸鈉緩沖液,分別于3天、7天麻醉成功后測量尾靜脈血糖,以連續(xù)兩次不禁食血糖16.7mmol/L的大鼠為成模糖尿病大鼠,成模后,在無菌條件下分別在大鼠背部剪取造模區(qū)皮膚,并創(chuàng)面外敷冰醋酸一周形成缺損性皮膚潰瘍大鼠模型。糖尿病潰瘍大鼠造模成功后,四組每天以碘伏消毒創(chuàng)面換藥,中藥組外敷潰瘍油,西藥組外敷乳酸依沙吖啶溶液,四層醫(yī)用無菌紗布包扎固定。分別于用藥后3、7、14天,取適量皮下組織進(jìn)行固定、石蠟包埋、切片后HE染色進(jìn)行形態(tài)學(xué)觀察,免疫組化染色檢測組織中AGEs、 PDGF、 VEGF、 PTP1B的含量,并應(yīng)用Image Pro Plus分析積分光密度值;于3、7、14天取適量皮下組織經(jīng)液氮速凍后經(jīng)Western blot測定AGEs、 PDGF、 VEGF、 PTP1B蛋白表達(dá),掃描膠片并分析凈光密度值。2.臨床研究：臨床中收集60例糖尿病潰瘍濕熱蘊(yùn)結(jié)型患者,隨機(jī)分組為對照組、治療組,、分別在兩組患者入院第一日,收集基本資料,兩組每天予以碘伏消毒創(chuàng)面換藥后,治療組外敷潰瘍油,對照組外敷乳酸依沙吖啶溶液,四層醫(yī)用無菌紗布包扎固定。于治療0、14天治療組和對照組分別評(píng)價(jià)中醫(yī)證候療效,檢測WBC、NE%、ESR、CRP、TGF-β、VEGF、PDGF等各項(xiàng)指標(biāo),最后經(jīng)統(tǒng)計(jì)學(xué)分析結(jié)果。研究結(jié)果：1.實(shí)驗(yàn)研究：糖尿病潰瘍大鼠造模成功后,各組創(chuàng)面有少量滲血滲液,周圍組織水腫,空白對照組大鼠整體狀態(tài)良好,進(jìn)食進(jìn)水正常,毛色光亮,活動(dòng)靈敏,體重明顯增長,模型組、潰瘍油組、乳酸依沙吖啶組大鼠整體狀態(tài)較差,進(jìn)食少,多飲水,毛色暗無光澤,毛發(fā)潮濕,蜷縮怕冷,活動(dòng)不靈敏,體重增長不明顯,多尿,味腥臭。四組大鼠體重在造模前及造模成功后第三天基本屬于同一基線,而在造模第三天到十四天,空白對照組大鼠體重增長明顯,模型組、潰瘍油組及乳酸依沙吖啶組體重增長緩慢。四組大鼠在造模前血糖均處于正常水平,空白對照組在造模第三天、第七天、第十四天血糖處于正常水平,模型組、潰瘍油組及乳酸依沙吖啶組第三天血糖高于正常水平,均達(dá)到糖尿病模型的標(biāo)準(zhǔn),模型組、潰瘍油組及乳酸依沙吖啶組第七天血糖有所下降,均達(dá)到糖尿病模型的標(biāo)準(zhǔn),第七天到第十四天血糖值趨于穩(wěn)定保持在成模模型的水平。免疫組化分析在各組中的表達(dá),AGEs蛋白表達(dá)量在第三天時(shí)潰瘍油組顯著低于空白對照組、模型組、乳酸依沙吖啶組,P0.05,第七天時(shí)潰瘍油組顯著高于模型組,P0.05,潰瘍油組與空白對照組、乳酸依沙吖啶組相比未見明顯差異,P0.05,第十四天時(shí),潰瘍油組顯著低于空白對照組,P0.05,與其他兩組相比未見明顯差異,P0.05。PDGF蛋白表達(dá)量在第三天時(shí)潰瘍油組明顯高于空白對照組、模型組、乳酸依沙吖啶組,P0.05,在第七天、第十四天均未見明顯差異。VEGF蛋白表達(dá)量在第三天潰瘍油組較乳酸依沙吖啶組相比明顯升高,P0.05,第七天、第十四天時(shí)潰瘍油組均明顯高于乳酸依沙吖啶組,P0.01。PTPIB蛋白表達(dá)量在第三天潰瘍油組明顯低于模型組,P0.01,在第七天潰瘍油組明顯高于空白對照組、模型組、乳酸依沙吖啶組,P0.01,第十四天潰瘍油組明顯低于模型組,P0.01,且潰瘍油組高于乳酸依沙吖啶組,P0.01。蛋白免疫印跡結(jié)果顯示：AGEs在四組中的蛋白表達(dá)含量分別為第三天時(shí)潰瘍油組均低于空白對照組、模型組、乳酸依沙吖啶組,其中潰瘍油組顯著低于模型組,P0.05,第七天時(shí)潰瘍油組與模型組未見明顯差異,潰瘍油組顯著高于乳酸依沙吖啶組,P0.01,第十四天未見明顯差異。PDGF在第三天時(shí)潰瘍油組高于空白對照組、模型組、乳酸依沙吖啶組,且潰瘍油組顯著高于模型組,P0.05,第七天、第十四天潰瘍油組高于空白對照組、模型組、乳酸依沙吖啶組,但未見顯著性差異。VEGF在第三天潰瘍油組高于空白對照組、模型組、乳酸依沙吖啶組,且潰瘍油組顯著高于空白對照組,P0.05,第七天、第十四天未見明顯差異。PTPIB在第三天時(shí)潰瘍油組低于模型組、乳酸依沙吖啶組,在第七天、第十四天潰瘍油組與其他組間未見明顯差異。2.臨床研究：通過觀察糖尿病,潰瘍患者應(yīng)用潰瘍油治療前后的中醫(yī)證候療效,主癥積分可見對照組和治療組在0天時(shí)未見明顯差異,P0.05,在治療14天后,治療組的主癥積分經(jīng)治療后明顯低于對照組,p0.01。次證積分在治療0天和14天時(shí)均未見明顯的差異,P0.05�？偘Y證候積分在治療0天時(shí)乳酸依沙吖啶組和潰瘍油組未見明顯差異,P0.05,在治療14天后,潰瘍油組的總癥證候積分明顯低于乳酸依沙吖啶組,p0.01。中醫(yī)證候療效對比,對照組的總有效率為86.67%,治療組的總有效率為96.67%,治療組的總有效率優(yōu)于對照組。炎癥相關(guān)指標(biāo),治療前兩組白細(xì)胞計(jì)數(shù)、中性粒細(xì)胞百分比、CRP均無明顯差異,P0.05,血沉在治療前對照組明顯低于治療組,P0.01。治療14天后,對照組和治療組相比較,白細(xì)胞計(jì)數(shù)、中性粒細(xì)胞百分比、CRP、ESR均治療組明顯低于對照組,P0.01。生長因子在兩組治療前后的變化,PDGF、TGF-β在治療前對照組和治療組未見明顯差異,P0.05,在治療14天后,治療組均顯著高于對照組,p0.05。 VEGF在0天時(shí)對照組和治療組在存在非常明顯差異,P0.01,在14天,治療組與對照組未見顯著性差異,p0.05。結(jié)論：潰瘍油干預(yù)糖尿病潰瘍大鼠能夠促進(jìn)局部肉芽組織生長并促進(jìn)微血管形成,明顯加速創(chuàng)面的愈合,其機(jī)制可能與潰瘍油能夠有效的抑制AGEs和PTPIB,并能夠促進(jìn)PDGF、 VEGF生長相關(guān)。潰瘍油外敷治療濕熱蘊(yùn)結(jié)型糖尿病潰瘍患者取得較好的臨床療效,能夠明顯提高創(chuàng)面總有效率,有效的控制炎癥反應(yīng),在創(chuàng)面修復(fù)過程中能夠明顯提高促愈因子PDGF、 TGF-β的濃度,加速創(chuàng)面愈合,對于糖尿病潰瘍慢性創(chuàng)面的愈合和組織再生有較好的促進(jìn)作用。
[Abstract]:Objective: diabetic ulcers TCM belongs to the scope of experience, is one of the serious chronic complications of diabetes, metabolic disorder caused by vascular disease, neuropathy, diabetes and impaired immune function in secondary infection and co morbidity, is a common clinical chronic wounds. Refractory diabetic ulcer is still a key and difficult problem concerned the mechanism of pathogenesis of diabetic ulcer mainly with the occurrence and development of diabetes and diabetic ulcer healing, chronic wound healing process requires multiple cells involved in growth factors and extracellular matrix, constitute a complex biological network jointly promote ulcer healing, cytokine and receptor deficiency or signal transduction "decoupling" will lead to wound healing or delayed healing, traditional Chinese Medicine external treatment of diabetic ulcer can obviously promote wound Healing, but traditional Chinese medicine how to control the corresponding target cells still need to study traditional Chinese medicine compound ulcer oil in the long-term clinical practice summarized into patients shireyunjiexing for clinical diabetic ulcers, ulcer oil intervention through the study of clinical and experimental research of diabetic ulcer healing to further elucidate the mechanism of wound healing, research ulcer oil promote diabetic ulcer healing effect on the pathway and its role in the clinical evaluation of therapeutic effect. Methods: 1. experimental study: buy 80-100g SPF SD male rats were randomly divided into control group, model group, western medicine group, Chinese medicine group, the model group, western medicine group, Chinese medicine group established the diabetic model according to the weight of 50mg/kg rapid one-time intraperitoneal injection of STZ solution, the blank control group were injected equal volume without STZ O.1mmol/L. Citric acid sodium citrate buffer, respectively, in 3 days, 7 days after the success of anesthesia to measure blood glucose of tail vein for two times and not fasting blood glucose 16.7mmol/L rats as diabetic rats, after model, under aseptic conditions respectively in rat models of skin wound and cut back. A week deposited glacial acetic acid formation model of defect of skin ulcers in rats. The diabetic ulcer rats after modeling successfully, four groups every day to change the traditional Chinese medicine group with iodine to disinfect the wound, ulcer oil, western medicine group with Ethacridine Lactate Solution, four layers of medical sterile gauze bandage fixed. In use after 3,7,14 days, take appropriate amount of subcutaneous tissue was fixed after slicing, paraffin embedding, HE staining was used to observe the morphology, immunohistochemical staining of AGEs, VEGF, PDGF expression, PTP1B content, and the application of Image Pro Plus analysis of integral optical density at 3,7,14 days; take appropriate amount of subcutaneous tissue by liquid nitrogen Frozen after Western blot PDGF, VEGF, AGEs assay, PTP1B protein expression, scanning the film and analysis of net optical density value of the.2. clinical research: collected 60 cases of diabetic ulcer of damp heat accumulation type of the clinical patients, were randomly divided into control group and treatment group, respectively, in two groups of patients into the hospital on the first day, the collection of basic data. Two groups every day to iodophor disinfection wound dressing, treatment group and control group were treated with Topical Ulcer oil, Ethacridine Lactate Solution, four layers of medical sterile gauze bandage fixed. In treatment 0,14 days of treatment group and control group respectively. The evaluation of TCM syndrome curative effect, detection of WBC, NE%, ESR, CRP, VEGF, TGF- beta, PDGF indexes etc. Finally, the statistical analysis results. Results: 1. experimental study: diabetic ulcer rats after successful wound with a small amount of blood oozing exudate, edema, blank control group rats were in good overall condition, water consumption is Often, a sleek, sensitive action, body weight increased significantly, the model group, the ulcer oil group, ethacridine lactate group rats whole state is poor, eat less, drink more water, hair color is dark and dull, wet hair curled up, cold, not sensitive, weight growth is not obvious, polyuria, weight taste stench. Four groups of rats before modeling and after modeling third days basically belong to the same baseline, and on the third day to the fourteen day, the rats weight in blank control group increased significantly, the model group, the ulcer oil group and ethacridine lactate group weight increased slowly. The four groups of rats before modeling blood glucose are all in normal level, the blank control group made in third days, seventh days, fourteenth days at the normal level of blood glucose, model group, ulcer oil group and ethacridine lactate group third days blood glucose higher than the normal level, reached the standard of diabetes model, model group, ulcer oil group and ethacridine lactate Group seventh days blood glucose decreased, reached the diabetes model standard, seventh to fourteenth days of blood glucose value tends to remain stable at the level of expression into model. Immunohistochemical analysis in each group, the expression of AGEs protein in the third day oil ulcer group was significantly lower than that of the control group, model group, lactate acridine group, P0.05, day seventh ulcer oil group was significantly higher than that of model group, P0.05, ulcer oil group and blank control group, there were no obvious differences, ethacridine lactate group P0.05, fourteenth day, ulcer oil group was significantly lower than the control group, P0.05, compared with the other two groups had no significant difference, the expression of P0.05.PDGF protein in third days of ulcer oil group was significantly higher than that of the blank control group, model group, ethacridine lactate group, P0.05, in seventh days, fourteenth days, there were no significant differences between the expression of.VEGF protein in third days ulcer oil group than lactic acid Ethacridine group is higher than P0.05, seventh days, fourteenth days ulcer oil group were significantly higher than that of ethacridine lactate group, the expression of P0.01.PTPIB protein in third days ulcer oil group was significantly lower than the model group, P0.01, on the seventh day of ulcer oil group was significantly higher than that of the blank control group, model group, ethacridine lactate group, P0.01 fourteenth days, ulcer oil group was significantly lower than the model group, P0.01, and ulcer oil group was higher than that of ethacridine lactate group, P0.01. protein Western blot analysis showed that AGEs protein expression in the four groups were third days when ulcer oil group were lower than the blank control group, model group, ethacridine lactate group, the ulcer oil group was significantly lower than that of model group, P0.05, day seventh ulcer oil group and model group showed no significant difference, ulcer oil group was significantly higher than that of ethacridine lactate group, P0.01 fourteenth days, there was no significant difference between the.PDGF on the third day break Ulcer oil group was higher than that of the blank control group, model group, ethacridine lactate group and ulcer oil group was significantly higher than that of model group, P0.05, seventh days, fourteenth days ulcer oil group was higher than that of the blank control group, model group, ethacridine lactate group, but no significant difference in the third day of.VEGF group was higher than that of blank control group ulcer oil, model group, ethacridine lactate group and ulcer oil group was significantly higher than that of control group, P0.05, seventh days, fourteenth days and there is no significant difference between.PTPIB ulcer in third days of oil group was lower than that of model group, ethacridine lactate group, on the seventh day, fourteenth day clinical study of ulcer oil group and the other groups had no significant difference.2.: To observe the curative effect of TCM Syndromes of diabetes, ulcer patients before and after the application of ulcer oil treatment, the main symptoms integral were control group and treatment group on day 0 showed no significant difference, P0.05, after 14 days of treatment, the main symptoms of the treatment group After treatment was significantly lower than the control group, p0.01. syndrome in the treatment of 0 days and 14 days were no obvious differences in the total syndrome scores of P0.05. lactic acid in the treatment of 0 days of ethacridine group and ulcer oil group had no obvious difference, P0.05, after 14 days of treatment, the symptom syndrome integral ulcer oil group the group was significantly lower than that of ethacridine lactate, p0.01. comparison of TCM syndrome curative effect, the total efficiency of the control group was 86.67%, the total efficiency of the treatment group was 96.67%, the total efficiency of the treatment group than the control group. The related indicators of inflammation, before treatment in two groups of white blood cell count, neutrophil percentage, CRP had no obvious the difference, P0.05, erythrocyte sedimentation rate in the control group before treatment was significantly lower than the treatment group, 14 days after P0.01. treatment, the treatment group and the control group compared to the white blood cell count, neutrophil percentage, CRP, ESR in treatment group were significantly lower than the control group, the P0.01. growth factor in the two groups before treatment After the change, PDGF, TGF- beta control group and treatment group had no significant difference before treatment, P0.05, after 14 days of treatment, the treatment group were significantly higher than control group, p0.05. VEGF on the 0 day of the treatment group and the control group in the presence of a very significant difference, P0.01, on the 14 day, there were no significant differences. Conclusion: treatment group and control p0.05. ulcer oil intervention in diabetic ulcer rats can promote granulation tissue growth and promote angiogenesis, accelerate wound healing, and its mechanism may be related to ulcer oil can inhibit AGEs and PTPIB effectively, and can promote the growth of PDGF, VEGF. Get better clinical effect in patients with ulcer oil shireyunjiexing treatment of diabetic ulcers, wounds can significantly improve the total efficiency, effective control of inflammation, can significantly improve the healing factor PDGF in wound healing process, the concentration of TGF- beta, accelerate wound healing, to It has a good promoting effect on the healing and tissue regeneration of the chronic wound of diabetic ulcer.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259

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