姜黄素壳聚糖脂质体治疗兔角膜碱烧伤的研究

发布时间：2018-10-30 07:00

【摘要】：目的:制备姜黄素壳聚糖脂质体,检测该药在眼部应用安全基础上,建立兔角膜碱烧伤模型,评价该脂质体对兔角膜碱烧伤的治疗作用,并初步探讨其作用机制,为姜黄素壳聚糖脂质体滴眼液的临床应用提供实验依据。方法:(1)姜黄素壳聚糖脂质体的制备和评价。采用薄膜分散法制备姜黄素壳聚糖脂质体(curcumin-loaded chitosan liposomes,CCLP),测定其粒径和Zeta电位,离心法测定其包封率。(2)姜黄素壳聚糖脂质体眼部应用安全性评价。6只兔随机分为姜黄素壳聚糖脂质体组和生理盐水组,每组3只,右眼为实验眼。姜黄素壳聚糖脂质体组滴姜黄素壳聚糖脂质体滴眼液,生理盐水组滴生理盐水(0.9%Nacl),4次/天,连续给药14天,通过肉眼观察兔子反应、裂隙灯微观观察、采用Draize眼刺激性实验评分、病理切片,评价姜黄素壳聚糖脂质体滴眼液对正常兔眼有无明显毒性。(3)姜黄素壳聚糖脂质体治疗兔角膜碱烧伤的研究。35只新西兰大白兔随机分为:正常对照组3只、生理盐水组8只、空白壳聚糖包衣脂质体组8只、地塞米松组8只和姜黄素壳聚糖脂质体组8只,除正常对照组外,均选用右眼建立角膜碱烧伤模型,各组分别给予相应药物治疗,4次/天,连续给药14天。裂隙灯观察角膜新生血管(corneal neovascularization,CNV)生长情况及角上皮愈合情况,并用裂隙灯进行眼前节照相;分析CNV的长度及面积,角膜上皮愈合率。于碱烧伤后1、4、7、14天,取兔房水检测白介素-8(interleukin-8,IL-8)的含量。碱烧伤后7、14和28天随机抽取各组兔取角膜,行病理切片观察角膜形态;免疫组化法观察血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达情况。结果:(1)姜黄素壳聚糖脂质体的性质。成功制备出姜黄素壳聚糖脂质体滴眼液,测得平均粒径为96.6±14.7 nm,平均Zeta电位为58.8±2.3 m V,平均包封率为51.41±1.05%,符合眼用制剂要求,可作为滴眼液用于动物实验。(2)姜黄素壳聚糖脂质体安全性好。短期局部应用姜黄素壳聚糖脂质体滴眼后兔子无明显挠抓眼部情况;裂隙灯检查,结膜、角膜透明,虹膜无渗出,晶体透明;两组角膜、虹膜及结膜Draize评分比较无差异(p0.05);病理切片示角膜无炎性细胞。(3)姜黄素壳聚糖脂质体、地塞米松(dexamethasone,DX)和生理盐水组相比均能有效抑制CNV的生长(p0.05),空白壳聚糖包衣脂质体组和生理盐水组CNV面积和长度没有显著差异(p0.05)。与地塞米松组相比,在治疗早期,姜黄素壳聚糖脂质体组的CNV长度和面积大(p0.05);在治疗后期,地塞米松组和姜黄素壳聚糖脂质体组的CNV长度和面积无统计学差异(p0.05)。姜黄素壳聚糖脂质体组与生理盐水组相比,能促进角膜上皮恢复(p0.05),减轻炎症,有效降低角膜组织中VEGF的表达(p0.05),降低房水中IL-8的表达(p0.05)。结论:姜黄素壳聚糖脂质体药物包封率较高,安全性好,可明显减轻兔角膜碱烧伤炎症,抑制角膜新生血管生长,促进角膜上皮修复,降低角膜VEGF表达和房水中IL-8表达。姜黄素壳聚糖脂质体有望成为新型的治疗角膜碱烧伤的局部用药制剂,为角膜碱烧伤治疗提供新思路。
[Abstract]:Objective: To prepare curcumin chitosan liposome, and to test the effect of the liposome on rabbit corneal alkali burn, and to discuss its mechanism of action. It provides experimental basis for clinical application of curcumin chitosan liposome eye drops. Methods: (1) Preparation and evaluation of curcumin chitosan liposomes. The entrapment efficiency of curcumin chitosan liposomes (CCLP) was determined by thin-film dispersion method and its particle size and Zeta potential were measured. (2) The safety evaluation of curcumin chitosan liposome eyes was evaluated. Six rabbits were randomly divided into the curcumin chitosan liposome group and the normal saline group, each group had 3 eyes, and the right eye was the experimental eye. The curcumin chitosan liposome group drops curcumin chitosan liposome eye drops, physiological saline group drops normal saline (0.9% Nacl), 4 times per day, continuously administrated for 14 days, the rabbit reaction and slit lamp microscopic observation are observed by naked eyes, the Draize eye irritation experiment score and pathological section are adopted, To evaluate the toxicity of curcumin chitosan liposome eye drops to the eyes of normal rabbits. (3) The effects of curcumin chitosan liposome on corneal alkali burn in rabbits were studied. Thirty-five New Zealand white rabbits were randomly divided into three groups: normal control group (n = 3), saline group (8), blank chitosan coated liposome group (8), dexamethasone group (8) and curcumin chitosan liposome group (8). In addition to the normal control group, the right eye was used to establish the corneal alkali burn model, each group was given the corresponding drug treatment, 4 times/ day, and the continuous administration was 14 days. slit lamp was used to observe the growth of corneal neovascularization and angle epithelial healing, and the anterior segment was photographed with slit lamp; the length and area of the cornea were analyzed, and the corneal epithelial healing rate was analyzed. The levels of interleukin-8 (IL-8) and interleukin-8 (IL-8) were detected on 1, 4, 7 and 14 days after alkali burn. The corneal morphology was observed on 7, 14 and 28 days after alkali burn, and the expression of vascular endothelial growth factor (VEGF) was observed by immunohistochemistry. Results: (1) The properties of curcumin chitosan liposomes were studied. In this paper, curcumin chitosan liposome eye drops were successfully prepared. The average particle size was 96. 6, 14. 7 nm, the average Zeta potential was 58. 8, 2. 3 m V, the average entrapment efficiency was 51. 41% 1. 05%, which was consistent with the requirements of eye preparations and could be used as eye drops for animal experiments. (2) curcumin chitosan liposome has good safety. In short-term topical application of curcumin chitosan liposome, the rabbit did not scratch the eyes obviously; slit lamp examination, conjunctival, cornea transparency, iris did not exude, crystal clear; two groups of cornea, iris and conjunctival draft score were not different (P0.05); pathological section showed no inflammatory cells of cornea. (3) There was no significant difference between curcumin chitosan liposome, dexamethasone (dexamethasone, DX) and saline group (P0.05). Compared with the dexamethasone group, in the early stage of treatment, there was no significant difference in length and area between the dexamethasone group and the curcumin chitosan liposome group (P0.05). Compared with normal saline group, curcumin chitosan liposome group can promote corneal epithelial recovery (A0.05), reduce inflammation, effectively reduce the expression of VEGF in corneal tissue (P0.05), and decrease the expression of IL-8 in the water. Conclusion: The entrapment efficiency of curcumin chitosan liposome is high, the safety is good, the corneal alkali burn inflammation can be obviously reduced, corneal neovascularization can be inhibited, corneal epithelium repair is promoted, corneal VEGF expression and IL-8 expression in house water can be reduced. The curcumin chitosan liposome is expected to be a novel topical medicine preparation for treating corneal alkali burn, and provides a new idea for the treatment of corneal alkali burn.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R772.2

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