【作者】 金洹宇；

【导师】 安艳；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2013， 硕士

【摘要】 【目的】为研究γ射线对丝素蛋白的生物相容性及降解性的影响，本研究通过对γ射线照射后丝素蛋白力学性能的测试，筛选出能进行生物学评价的照射剂量，并利用细胞及动物实验的方法对照射后丝素蛋白的生物相容性及降解性进行研究。【方法】1.γ射线照射后丝素蛋白膜的力学结构变化情况：将制作好的丝素蛋白膜，加入分别为0、25、50、100、200、300、500、1000kGy的γ射线照射组。（1）丝素蛋白膜断裂强度和断裂伸长率变化：将丝素蛋白膜用特制的模具，制成哑铃状条带，对各组丝素蛋白膜的断裂强度和断裂伸长率进行测试。（2）丝素蛋白膜结构变化的测试：将照射后的丝素蛋白膜剪碎制成粉末，用KBr压片，经Nicolet5700FTIR型傅立叶变换红外光谱仪检测。2. γ射线照射后的丝素蛋白生物相容性研究：将SD大鼠乳鼠颈椎脱臼处死后，取其背部皮肤，分离原代真皮细胞培养传至3、4代。（1）真皮细胞在丝素蛋白膜上的生长曲线测定：将原代培养的真皮细胞以每孔1×105细胞，接种于铺在24孔板中的辐照丝素蛋白膜上，培养7d，每天各取6孔，使用CCK-8法检测细胞增殖活力，酶标仪于450nm波长处，测得OD值，并计算细胞量，求得生长曲线。（2）丝素蛋白浸提液细胞毒性试验：用制备好的丝素蛋白浸提液培养原代真皮细胞，以每孔5000个，接种于3块96孔板中，待细胞贴壁后移去普通培养基，加入丝素蛋白膜浸提液，100μL/孔，培养1、3和6d，每个时间点各取一板，加入CCK-8溶液反应，使用酶标仪测得各孔OD值。（3）溶血试验：取志愿者全血制成稀释血。设生理盐水、双蒸水、丝素蛋白浸提液及含丝素蛋白膜的生理盐水4个组。每个试管加入0.2mL稀释血，37℃水浴60min。反应后的溶液以1000r/min离心5min，取上清液移入96孔板中，使用酶标仪于545nm波长处测定吸光度。3.生物相容性及降解作用的体内研究：将不同剂量（25kGy、50kGy、100kGy、200kGy）γ射线照射后的丝素蛋白膜植入SD大鼠皮下，植入后7、14、28、56、84天，剖杀取材。对植入部位的皮下组织进行病理检查，通过ELISA法检测丝素蛋白植入后的大鼠血清中IL-6和TNF-α含量；取出植入后的丝素蛋白称重，计算质量减少率并绘制质量减少曲线。【结果】1.γ射线照射后丝素蛋白膜的力学结构变化情况：经过0、25、50、100、200、300、500、1000kGy的γ射线照射后，丝素蛋白的断裂强度及断裂伸长率随照射剂量的增加，均呈现出下降趋势。红外光谱仪检测发现，γ射线照射后的丝素蛋白Silk II的吸收峰1628cm-1、1526cm-1并未发生明显移动，说明γ射线对丝素蛋白的二级结构影响不明显。2. γ射线照射后的丝素蛋白生物相容性研究：原代真皮细胞在丝素蛋白膜上的生长情况良好，增殖表现出相同的趋势，均在生长96h达到峰值，而后下降，对每个时间点各组细胞增殖活力进行单因素方差分析，各组吸光度值均具有方差齐性，且各组细胞增殖活力之间不存在显著性差异(p>0.05)。（2）细胞用丝素蛋白浸提液培养24、72和144h，分别测定增殖情况，并按照国家标准，其毒性在分级标准的1级（RGR:75%－99%）以内，经SPSS17.0统计分析，各时间点各组细胞RGR差异无统计学意义（p>0.05）。细胞增殖趋势也具有一致性。（3）丝素蛋白浸提液及含丝素蛋白的生理盐水，溶血率均小于国家标准所规定的5%，SPSS17.0软件统计结果显示，各组溶血率之间差异均无统计学意义（p>0.05）。3.生物相容性及降解性的体内研究：将各组丝素蛋白膜植入SD大鼠背部皮下，并以空白植入作为对照，排除手术引起炎症反应的影响，发现植入的丝素蛋白均未引起明显的炎症反应，并且随时间变化，较高剂量照射组的丝素蛋白裂解较为明显。各照射剂量丝素蛋白膜植入组的SD大鼠，IL-6和TNF-α分泌没有显著性差异（p>0.05），与空白植入组（Blank）比较，发现丝素蛋白并未引起明显炎症反应（p>0.05）。丝素蛋白在体内降解性表现为，较高剂量照射组的丝素蛋白在体内降解随时间的延长碎片形成越明显，质量下降也越快。【结论】1.在0-1000kGy的照射剂量范围内，γ射线能改变丝素蛋白机械性能，但是对其二级机构改变不明显。2.经0-200kGy的γ射线照射后，丝素蛋白膜具有良好的细胞相容性和血液相容性。3.经0-200kGy的γ射线照射的丝素蛋白膜，在体内具有良好的生物相容性，其降解性也随受照剂量的增大有所提高。更多还原

【Abstract】 Objective: To evaluate the biocompatibility and biodegradation of silk fibroin withγ-irradiation， we test the mechanical properties and molecular structure and selectedthe dose range for the next experiments. The cells experiments and rats surgery wereused to evaluate the biocompatibility and biodegradation.Methods:1.Mechanical and molecular structure changes of gamma ray irradiatedsilk fibroin: The silk fibroin films were divided into different groups which were0,25,50,100,200,300,500,1000kGy, respectively.(1) Changes of break strength andelongation of silk fibroin: Silk fibroin films were made into special shape with mould ofinternational standard and tested the break strength and elongation in every group.(2)Molecular structure change: The silk fibroin were made into powder, tableting withKBr, and tested the structure change with FTIR.2. Biocompatibility of gamma rayirradiation silk fibroin films in vitro: We got the skin of back of newborn SD rat,detached the hypodermal cell from the skin, cultured and subcultured the cells to the3rdgeneration.(1) Growth curve of dermal cells on silk fibroin films: The dermal cellswere cultured into24-well plates,1×105cells per well. Cultured the cells for7days,and then, we tested the grown situation of cells everyday with CCK-8kit, testing theOD value of6wells of every group on the microplate reader at the wave of450nm.(2)The extract toxicity of silk fibroin: Dermal cells were cultured with the extracts of silkfibroin in the96-well plates,5000cells per well. After cells adherence, we exchangedthe normal culture media with extracts,100μL per well and tested the OD value of cellat the1st,3rd,6thday on the microplate reader at the wave of450nm.(3) hemolysis: Weget the blood of volunteer and diluted it. The experiments had4groups which werenegative group, positive group, extract group and the normal saline with silk fibroinfilms group. Every test tube was injected10ml normal saline, distill water, extracts ornormal saline with silk fibroin film and0.2ml diluted blood,37℃water bath for60min.We had the tubes centrifuged with1000r/min for5min, moved the supernate into 96-well plate and test the OD value at the wave of545nm on the micoplate reader.3.Biocompatibility and biodegradation of silk fibroin films in vivo: We implanted the silkfibroin films with different doses(25,50,100,200kGy) of gamma ray irradiation in theback of SD rat subcutaneously. At the date of7th,14th,28th,56th,84th, we got the skintissue where the silk fibroin films implanted, blood of experiment animals. We madeparaffin sections with the tissue and stained them with HE staining kit. We test theconcentration of IL-6and TNF-αwith the ELISA kit. The silk fibroin films gotten fromthe tissue were weight and compared with the mass weighted before, getting the massdecreasing curve.Results:1. Mechanical and molecular structure changes of gamma ray irradiatedsilk fibroin: The break strength and elongation of silk fibroin was decreased afterirradiated by gamma ray. And the extent of decrease was more with the increasing of thedose. The FTIR test results showed that the special peaks of Silk II, which is at1628cm-1,1526cm-1, had not moved significantly. It indicated that the gamma ray wouldnot change the secondary structure of silk fibroin.2. Biocompatibility of gamma rayirradiation silk fibroin films in vitro:(1)The dermal cells showed similar trend in growthcurve. We analyzed the OD value of cells in different groups with variance analysis andfound that there was no significant on their growth(p>0.05).(2)We tested the growthactivity of dermal cells cultured with extracts of silk fibroin with different dosesirradiated by gamma ray at the date of1st,3rdand6thday after cells adherence. Thetoxicity of extracts were within the range of the1stlevel of toxicity grades, which meansthat the RGRs of extracts were between75%-99%. And there was no significantdifferent among the groups after analyzed by SPSS17.0(p>0.05). The hemolysis testshowed there was no significant different among the groups(p>0.05).And the hemolysisrate in all these groups were less than5%, which is specified by national standard.3.Biocompatibility and biodegradation of silk fibroin films in vivo: The subcutaneousexperiment did not occurred obviously inflammatory responses in all these groups.Along the periods after implantation, silk fibroin films in the higher dose groupsfracture more obviously. The concentrations of IL-6and TNF-α in different groupshad no significant differences compared with the Blank(p>0.05). We could not find outthat the post-irradiated silk fibroin occurred obviously inflammatory responses. Thebiodegradation was showed by the fragments and mass decrease. They all showed obviously in the higher dose groups.Conclusions:1. The break strength and elongation of silk fibroin showed a significant downtrendand the molecular structures were not obviously changed in the0-1000kGy dose range.2． Silk fibroin films with0-200kGy dose of γ-irradiation had goodcytocompatibility and Blood compatibility.3. In the0-200kGy dose range, silk fibroin films in all these groups showed thegood biocompatibility and the biodegradation rate increased more significantly with theincrement of the absorbed doses and implantation time.更多还原