溃疡油干预糖尿病溃疡愈合的实验及临床研究

发布时间：2018-01-12 19:06

本文关键词：溃疡油干预糖尿病溃疡愈合的实验及临床研究　出处：《北京中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的：糖尿病溃疡中医归属于脱疽范围,是糖尿病的严重的慢性并发症之一,由代谢紊乱导致的血管病变、神经病变,加之糖尿病免疫功能受损继发感染而协同发病,是临床一种常见的慢性难愈性创面。难愈性糖尿病溃疡仍然是目前关注的重点和难点,糖尿病溃疡发生发展的机制主要与糖尿病发生发展的相关机制及糖尿病溃疡难愈机制相关,慢性创面愈合过程需要多种细胞、生长因子及细胞外基质的协同参与,构成复杂的生物网络共同促进溃疡愈合,细胞因子缺失或者与靶细胞受体信号转导“失耦联”都会导致创面不愈合或延期愈合,中药外治糖尿病溃疡能够明显促进创面愈合,但是中药外治如何调控相应靶细胞仍需要研究,中药复方溃疡油在长期的临床实践中归纳总结而成,临床中用于湿热蕴结型糖尿病溃疡患者,本课题通过研究溃疡油干预糖尿病溃疡愈合的临床和实验研究来进一步阐明创面愈合的机制,研究溃疡油促进糖尿病溃疡愈合过程中对于相关通路的影响作用及其在临床中的疗效评定。研究方法：1.实验研究：选购80-100g SPF级SD雄性大鼠,按随机数字表法随机分为空白对照组、模型组、西药组、中药组,其中模型组、西药组、中药组按体重50mg/kg一次性快速腹腔注射STZ溶液造糖尿病模型,空白对照组一次性腹腔注射等体积不含STZ的O.1mmol/L柠檬酸-柠檬酸钠缓冲液,分别于3天、7天麻醉成功后测量尾静脉血糖,以连续两次不禁食血糖16.7mmol/L的大鼠为成模糖尿病大鼠,成模后,在无菌条件下分别在大鼠背部剪取造模区皮肤,并创面外敷冰醋酸一周形成缺损性皮肤溃疡大鼠模型。糖尿病溃疡大鼠造模成功后,四组每天以碘伏消毒创面换药,中药组外敷溃疡油,西药组外敷乳酸依沙吖啶溶液,四层医用无菌纱布包扎固定。分别于用药后3、7、14天,取适量皮下组织进行固定、石蜡包埋、切片后HE染色进行形态学观察,免疫组化染色检测组织中AGEs、 PDGF、 VEGF、 PTP1B的含量,并应用Image Pro Plus分析积分光密度值;于3、7、14天取适量皮下组织经液氮速冻后经Western blot测定AGEs、 PDGF、 VEGF、 PTP1B蛋白表达,扫描胶片并分析净光密度值。2.临床研究：临床中收集60例糖尿病溃疡湿热蕴结型患者,随机分组为对照组、治疗组,、分别在两组患者入院第一日,收集基本资料,两组每天予以碘伏消毒创面换药后,治疗组外敷溃疡油,对照组外敷乳酸依沙吖啶溶液,四层医用无菌纱布包扎固定。于治疗0、14天治疗组和对照组分别评价中医证候疗效,检测WBC、NE%、ESR、CRP、TGF-β、VEGF、PDGF等各项指标,最后经统计学分析结果。研究结果：1.实验研究：糖尿病溃疡大鼠造模成功后,各组创面有少量渗血渗液,周围组织水肿,空白对照组大鼠整体状态良好,进食进水正常,毛色光亮,活动灵敏,体重明显增长,模型组、溃疡油组、乳酸依沙吖啶组大鼠整体状态较差,进食少,多饮水,毛色暗无光泽,毛发潮湿,蜷缩怕冷,活动不灵敏,体重增长不明显,多尿,味腥臭。四组大鼠体重在造模前及造模成功后第三天基本属于同一基线,而在造模第三天到十四天,空白对照组大鼠体重增长明显,模型组、溃疡油组及乳酸依沙吖啶组体重增长缓慢。四组大鼠在造模前血糖均处于正常水平,空白对照组在造模第三天、第七天、第十四天血糖处于正常水平,模型组、溃疡油组及乳酸依沙吖啶组第三天血糖高于正常水平,均达到糖尿病模型的标准,模型组、溃疡油组及乳酸依沙吖啶组第七天血糖有所下降,均达到糖尿病模型的标准,第七天到第十四天血糖值趋于稳定保持在成模模型的水平。免疫组化分析在各组中的表达,AGEs蛋白表达量在第三天时溃疡油组显著低于空白对照组、模型组、乳酸依沙吖啶组,P0.05,第七天时溃疡油组显著高于模型组,P0.05,溃疡油组与空白对照组、乳酸依沙吖啶组相比未见明显差异,P0.05,第十四天时,溃疡油组显著低于空白对照组,P0.05,与其他两组相比未见明显差异,P0.05。PDGF蛋白表达量在第三天时溃疡油组明显高于空白对照组、模型组、乳酸依沙吖啶组,P0.05,在第七天、第十四天均未见明显差异。VEGF蛋白表达量在第三天溃疡油组较乳酸依沙吖啶组相比明显升高,P0.05,第七天、第十四天时溃疡油组均明显高于乳酸依沙吖啶组,P0.01。PTPIB蛋白表达量在第三天溃疡油组明显低于模型组,P0.01,在第七天溃疡油组明显高于空白对照组、模型组、乳酸依沙吖啶组,P0.01,第十四天溃疡油组明显低于模型组,P0.01,且溃疡油组高于乳酸依沙吖啶组,P0.01。蛋白免疫印迹结果显示：AGEs在四组中的蛋白表达含量分别为第三天时溃疡油组均低于空白对照组、模型组、乳酸依沙吖啶组,其中溃疡油组显著低于模型组,P0.05,第七天时溃疡油组与模型组未见明显差异,溃疡油组显著高于乳酸依沙吖啶组,P0.01,第十四天未见明显差异。PDGF在第三天时溃疡油组高于空白对照组、模型组、乳酸依沙吖啶组,且溃疡油组显著高于模型组,P0.05,第七天、第十四天溃疡油组高于空白对照组、模型组、乳酸依沙吖啶组,但未见显著性差异。VEGF在第三天溃疡油组高于空白对照组、模型组、乳酸依沙吖啶组,且溃疡油组显著高于空白对照组,P0.05,第七天、第十四天未见明显差异。PTPIB在第三天时溃疡油组低于模型组、乳酸依沙吖啶组,在第七天、第十四天溃疡油组与其他组间未见明显差异。2.临床研究：通过观察糖尿病,溃疡患者应用溃疡油治疗前后的中医证候疗效,主症积分可见对照组和治疗组在0天时未见明显差异,P0.05,在治疗14天后,治疗组的主症积分经治疗后明显低于对照组,p0.01。次证积分在治疗0天和14天时均未见明显的差异,P0.05。总症证候积分在治疗0天时乳酸依沙吖啶组和溃疡油组未见明显差异,P0.05,在治疗14天后,溃疡油组的总症证候积分明显低于乳酸依沙吖啶组,p0.01。中医证候疗效对比,对照组的总有效率为86.67%,治疗组的总有效率为96.67%,治疗组的总有效率优于对照组。炎症相关指标,治疗前两组白细胞计数、中性粒细胞百分比、CRP均无明显差异,P0.05,血沉在治疗前对照组明显低于治疗组,P0.01。治疗14天后,对照组和治疗组相比较,白细胞计数、中性粒细胞百分比、CRP、ESR均治疗组明显低于对照组,P0.01。生长因子在两组治疗前后的变化,PDGF、TGF-β在治疗前对照组和治疗组未见明显差异,P0.05,在治疗14天后,治疗组均显著高于对照组,p0.05。 VEGF在0天时对照组和治疗组在存在非常明显差异,P0.01,在14天,治疗组与对照组未见显著性差异,p0.05。结论：溃疡油干预糖尿病溃疡大鼠能够促进局部肉芽组织生长并促进微血管形成,明显加速创面的愈合,其机制可能与溃疡油能够有效的抑制AGEs和PTPIB,并能够促进PDGF、 VEGF生长相关。溃疡油外敷治疗湿热蕴结型糖尿病溃疡患者取得较好的临床疗效,能够明显提高创面总有效率,有效的控制炎症反应,在创面修复过程中能够明显提高促愈因子PDGF、 TGF-β的浓度,加速创面愈合,对于糖尿病溃疡慢性创面的愈合和组织再生有较好的促进作用。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259

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