微囊化内皮抑素球周注射的实验研究

Experimental research of Endostatin microcapsulized by periocular injection

目的检测乳化-内部凝胶化制得的海藻酸钙-壳聚糖（CAC）内皮抑素微囊的体外释放情况，大鼠眼球周注射后眼内组织分布情况及生物相容性。方法实验研究。采用乳化-内部凝胶化方式制备微囊，紫外分光光度法、考马斯亮蓝法检测内皮抑素囊外释放情况；32只SD大鼠随机分成4组，每组8只。A组：微囊化ES组，球周注射微囊化内皮抑素20μl（2．5g/L）；B组：单纯内皮抑素组，球周注射内皮抑素蛋白20μl（2．5g／L）；C组：空微囊组，球周注射空微囊20μl；D组：生理盐水组，球周注射生理盐水20μl。免疫组织化学法、ELISA法、Western—blot法检测内皮抑素分布、浓度和蛋白表达；观察SD大鼠的日常活动，进食情况和体重，以断颈法将大鼠处死取心、肝、脾、肺、。肾及球周组织（球周注射部位周围组织）行病理学检查。采用独立样本设计定量资料t检验对数据进行分析。结果十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测可见内皮抑素蛋白条带，3、7、14、21d各时点上清液中内皮抑素蛋白质浓度逐渐增加。A组：球周注射后7、14d，内外核层和RPE层见内皮抑素蛋白阳性表达；B组：7d时，以上组织可见内皮抑素蛋白阳性表达，14d后无蛋白阳性表达；C组和D组7、14d以上组织均未见内皮抑素蛋白阳性表达，表明内皮抑素蛋白经CAC微囊化后通过球周注射可以穿过巩膜壁进入球内。A组7、14d，眼球组织匀浆内皮抑素蛋白浓度分别为每只眼（63．16±7．64）μg/L和（33．2±5．77）μg/L，而B组分别为（33．2±2．89）μg/L，（15．73±2．08）μg/L，A组明显大于B组且差异具有统计学意义（t=6．364、4．920，P=0．003、0．008）；大鼠日常活动和进食情况均正常，14d后病理学检查心肝、脾、肺、肾及球周组织未见异常。结论微囊化内皮抑素具有缓控释功能，球周注射微囊化内皮抑素，能穿过巩膜壁并分布在视网膜组织内，微囊对机体无明显毒副作用。

Objective To detect the release of endostatin in microcapsules of calcium alginate gel by emulsification-internal gelatification technology, to observe the distribution of Endostatin microcapsulized in the eye by periocular injection and biocompatibility. Methods The calcium alginate gel microcapsules encapsulating endostatin were prepared by emulsification-internal gelatification technology, the release of endostatin in microcapsules in vitro was examined by uv-Spectrophotometry. 32 SD mice were divided into randomly four groups： group A, periocular injection of endostatin microcapsulized 20μl（2.4 g/L） ; group B, periocular injection of endostatin protein 20 μl （2. 5 g/L）; group C, periocular injection of null- Microcapsules 20 μl ;group D, periocular injection of saline 20μl. Western blot, immunohistochemical and enzyme-linked immunosorbent assay were used. Protein expression and pathology of the heart, liver, spleen, lung, kidney and periocular tissue were examed. Daily activities and eating condition were observed, t-test was wsed to analyze the data. Results SDS polyacrylamidedel electrophoresis showed strap of ES protein. Concentration of ES protein in superior schedule fluid gradually increased at 3,7,14,21 days. A group： ES protein expression were observed in inner nuclear layer,outer nuclear layer and RPE lamina after periocular injection 7 or 14 days. B group： ES protein expression were observed in above tissues at 7 days, but not at 14 days. C and D group： ES protein expression were not observed in above tissues at 7 or 14 days. The results showed that Endostatin microcapsulized was able to pass through the sclera and spread out in various retinal tissues. Concertration of endostatin in the homogenates of eyeball was （63.16 ± 7.64）μg · L-^-1 · eye^-1 and （ 33.2 ± 5.77 ） μg · L^- 1 ·eye^ -1 respectively after one and two weeks in A group, but （33.2 ± 2. 89 ） μg · L^- 1 ·eye^ -1 and （ 15.73 ±2.08 ） μg · L^- 1 ·eye^ -1, The concertration of endostatin was significant difference in two group, A group was obviously larger than B group（t =6. 364 and 4. 920,P =0. 003 and 0. 008 respectively ）. Eriocular administration. Daily activities and eating condition were normal. The abnormality in important organs were not detected by pathologic examination. Conclusions Endostatin microcapsulized is able to release persistently, to pass through the sclera and spread out in various retinal tissues. There were good biocompatibility and not ill effect remarkably.