课题背景：课题为国家自然科学基金资助项目（30640088）。葡萄球菌生物被膜引起的人工关节感染治疗棘手，最新的研究表明RNA Ⅲ抑制肽可以是葡萄球菌一种有效的全面抑制剂，其作用机制与传统抗生素明显不同。本课题组在国内首次合成RNA Ⅲ抑制肽，并对其作用机制和防治葡萄球菌感染的能力进行了初步的研究。

应用要点：实验在前期研究的基础之上，制备PLGA/RNA Ⅲ抑制肽缓释微球，选择急性全身毒性实验、MTT细胞毒性实验、肌肉内植入实验、过敏实验和热源实验, 对PLGA/RNA Ⅲ抑制肽的组织容性和安全性进行初步评价，为其应用于临床奠定基础。

同行评价：实验将可防治葡萄球菌引起的感染的RNA Ⅲ抑制肽包载于PLGA微球中，并根据国际标准化组织(ISO)和国家标准GB/T16886-1规定的实验考察了PLGA/RIP微球的组织相容性，研究表明材料具有良好的组织相容性。该研究可为材料的进一步动物实验，甚至临床实验提供依据。

摘要
目的：在前期实验证实可防治葡萄球菌感染的RNA Ⅲ抑制肽（RNAⅢ inhibiting peptide, RIP）具有良好的组织相容性和安全性的基础上，进一步评价聚乳酸乙醇酸/ RIP(PLGA/ RIP)缓释微球的组织相容性。
方法: 实验于2005-10/2007-10在解放军总医院骨科研究所、临床药理研究所及医学动物实验中心完成。①PLGA/RIP微球制备：采用Fmoc法由C端至N端先合成粗品肽；采用反相液相色谱法对RIP粗品进行纯化分析,按紫外吸收峰收集组分,冷冻干燥,得到RIP纯品。再采用液相复乳法制备直径50~70 μm的PLGA/RIP微球。②组织相容性实验：以急性全身毒性实验观察 PLGA/RIP的全身毒性反应；MTT细胞毒性实验观察PLGA/RIP对细胞增殖的影响；肌肉内植入实验观察材料有无肌肉刺激反应；过敏实验观察PLGA/RIP的致敏情况；热原实验观察材料对体温的影响。
结果：①急性全身毒性实验结果：腹腔注射PLGA/RIP洗提原液和50%的PLGA/RIP洗提液后动物无中毒反应，无死亡，体质量无明显变化。②MTT细胞毒性实验结果：两种洗提液的平均细胞增殖率均大于85%，细胞毒等级1级，不具有细胞毒性。③肌肉内植入实验结果：RIP和PLGA/RIP植入4 周后，组织未见明显充血、变性或坏死。材料周围未见明显炎症细胞浸润, 材料已被纤维囊包裹。④过敏实验结果：3组动物的平均原发刺激指数分别为0.38，0.33和0.31，3组间无显著差别。⑤热原实验结果：各组动物体温升高均在0.5 ℃以下, 证实材料无热源性，符合热原实验的评价标准。
结论: PLGA/RIP不引起全身毒性反应，无细胞毒性，未见免疫排斥，不引起过敏反应，无热源性，具有良好的组织相容性和安全性。
关键词：PLGA/RIP微球；组织相容性；动物实验；生物材料

张小斌，郝立波，王继芳，姚琦，梁茂华. 聚乳酸乙醇酸/RNA Ⅲ抑制肽缓释微球组织相容性评价[J].中国组织工程研究与临床康复，2008，12(1):39-42 [www.zglckf.com/zglckf/ejournal/upfiles/08-1/1k-39(ps).pdf]

Histocompatibility of polyaiticglycolic acid/RNAⅢ inhibiting peptide sustained release microspheres

Abstract

AIM：RNAⅢ inhibiting peptide (RIP) has been previously proved to possess good histocompatibility and safety for preventing and curing staphylococcal infection, and this study evaluated histocompatibility of polyaiticglycolic acid/RIP (PLGA/RIP) sustained release microsphere.
METHODS: The experiment was performed in the Orthopedic Institute, Pharmacologic Research Institute and Animal Experimental Center of General Hospital of Chinese PLA from October 2005 to October 2007.①Preparation of PLGA/RIP: The solid-phase synthesis (Fmoc) method was used to synthesize RIP from C end to N end, then the synthesized peptide was purified by the reverse phase high performance liquid chromatography, and composition was collected by means of ultraviolet absorption peak. The purified RIP was obtained after freezing and drying. Liquid-phase multiple emulsion method was used to synthesize PLGA/RIP microsphere of 50-70 μm diameter.②Acute general toxicity test was studied in PLGA/RIP. Effect of PLGA/RIP on the cell proliferation was detected with cytotoxicity test by MTT method. Intramuscular implanting test was used to observe the irritation reaction of muscles by implantation materials. Sensitivity test was used to observe the sensitization of PLGA/RIP. Changes of animal's body temperature were determined with pyrogen test.
RESULTS: ①Acute general toxicity test: Neither toxicosis reaction nor animal death was found after animals were injected with 100% and 50% eluents of PLGA/RIP peritoneally. Animal's body weight was not changed significantly.②Cytotoxiciity test by MTT method: The average proliferation rate of cell in two kinds of eluents exceeded 85% and cytotoxicity was graded in 1 rank, indicating no cytotoxicity.③Intramuscular implanting test: At 4 weeks after RIP and PLGA/RIP were implanted into the animals, there was not obvious synathresis, denaturation or necrosis in tissues. No inflammatory cell infiltration occurred around the materials. There had been the fibrous capsules around the materials.④Sensitivity test: Average primary irritation index of three groups were 0.38, 0.33 and 0.31 respectively. There was no significant difference among three groups.⑤Pyrogen test: Fervescence of each animal in the experiment was under 0.5 ℃, confirming that the materials had no pyrogenic characteristics. This was in coincidence with evaluation criterion of pyrogen test.
CONCLUSION: PLGA/RIP has good histocompatibility and safety, without general toxic reaction, cytotoxicity, immunological rejection, hypersensitive response or pyrogenic characteristics.

Zhang XB, Hao LB, Wang JF, Yao Q, Liang MH.Blood compatibility of polyaiticglycolic acid/RNA Ⅲ inhibiting peptide microspheres.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(1):39-42(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-1/1k-39(ps).pdf]