γ射线照射对丝素蛋白生物相容性及降解性的影响

发布时间：2018-06-30 16:50

本文选题：丝素蛋白 + γ射线　；参考：《苏州大学》2013年硕士论文

【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(p0.05)。（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（p0.05）。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（p0.05）。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（p0.05），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（p0.05）。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。
[Abstract]:[Objective]
In order to study the effect of gamma ray on the biocompatibility and degradability of silk fibroin, this study screened the biological evaluation of the biological evaluation of the biological compatibility and degradation of silk fibroin through the test of the mechanical properties of fibroin protein after gamma ray irradiation.
[method]
The change of the mechanical structure of silk fibroin membrane after 1. gamma ray irradiation: a good silk fibroin membrane was made and added to the gamma ray irradiation group of 0,25,501002003005001000kGy, respectively. (1) the breaking strength and elongation at break of the silk fibroin membrane were changed: the silk fibroin membrane was made into dumbbell shaped strip with a special mould, and the fibroin protein was made to each group of silk fibroin The fracture strength and elongation at break of the membrane were tested. (2) test of the changes of silk fibroin membrane structure: the silk fibroin membrane was cut into powder after irradiation, and the Nicolet5700FTIR Fu Liye transform infrared spectrometer was used to detect the biocompatibility of silk fibroin after.2. gamma ray irradiation, and the cervical vertebra dislocations of SD rats were dislocated. After death, take the skin of its back and isolate the original dermal cell culture to the 3,4 generation. (1) the growth curve of dermis cells on the silk fibroin membrane: the original cultured dermal cells were inoculated on the irradiated silk fibroin membrane of 24 Kong Banzhong by 1 x 105 cells per pore, and cultured for 6 holes each day, and the cell proliferation was detected by CCK-8 method. Strength, enzyme labeling at 450nm wave length, measured the OD value, and calculated the cell volume and obtained the growth curve. (2) the cytotoxicity test of the silk fibroin extract: using the prepared fibroin extract to cultivate the original dermal cells, 5000 of the pores per pore, inoculated to 3 96 Kong Banzhong, and then removed to the ordinary medium after the cell adhered to the wall and added the silk fibroin membrane extract. 100 mu L/ hole, culture 1,3 and 6D, each time point to take one plate, add the CCK-8 solution reaction, use the enzyme labeled instrument to measure the orifice o value. (3) hemolysis test: take the volunteers whole blood to make the diluted blood. 4 groups of physiological saline, double water, silk fibroin extract and fibroin membrane. Each test tube is added to 0.2mL dilute blood and 37 centigrade water bath After 60min. reaction, the solution was centrifuged by 1000r/min for 5min, and the supernatant was removed to 96 Kong Banzhong. The biocompatibility and degradation of absorbable.3. was measured at 545nm wavelength at 545nm wavelength. The silk fibroin membrane of different doses (25kGy, 50kGy, 100kGy, 200kGy) was implanted subcutaneously in SD rats. After the implantation, the 7,14,28,56,84 days were implanted. The subcutaneous tissue of the implanted parts was examined by pathological examination. The content of IL-6 and TNF- alpha in the serum of the rat after fibroin implantation was detected by ELISA, and the weight of silk fibroin after implantation was removed, and the quality reduction rate was calculated and the quality reduction curve was plotted.
[results]
The change of the mechanical structure of silk fibroin membrane after 1. gamma ray irradiation: after 0,25,501002003005001000kGy gamma ray irradiation, the breaking strength and elongation at break of silk fibroin were decreased with the increase of irradiation dose. The absorption peak of Silk II after gamma ray irradiation was found to be 162. 8cm-11526cm-1 did not move obviously, indicating that gamma ray irradiation on the two grade structure of silk fibroin did not affect the biocompatibility of silk fibroin after.2. gamma ray irradiation. The growth of original dermal cells on the silk fibroin membrane was good, and the proliferation showed the same trend, and the growth of 96h reached the peak, then decreased. The cell proliferation activity of each group was analyzed by single factor variance. The absorbance was homogeneous in each group, and there was no significant difference between the proliferation activity of each group (P0.05). (2) the cells were cultured 24,72 and 144H with silk fibroin extract, and the proliferation was measured respectively, and the toxicity of the cells was 1 grade according to the national standard. (RGR:75% 99%), by SPSS17.0 statistical analysis, there was no significant difference in the RGR difference between each time point (P0.05). The proliferation trend of cell proliferation was also consistent. (3) the physiological saline of silk fibroin extract and fibroin protein were less than 5% of the national standard, SPSS17.0 software statistics showed that the hemolysis rate of each group was between each group. In vivo studies on the biocompatibility and degradability of the difference (P0.05).3.: the fibroin membrane was implanted subcutaneously into the back of the SD rat and the blank implantation was used as the control. The effect of the operation on the inflammatory reaction was excluded. There was no significant difference in the secretion of silk fibroin in the irradiated group. There was no significant difference in the secretion of IL-6 and TNF- alpha in the SD rats of the fibroin membrane implantation group (P0.05). Compared with the blank group (Blank), the fibroin protein did not cause obvious inflammatory reaction (P0.05). The degradation of silk fibroin in the body was manifested in the higher dose irradiation group. The degradation of protein in vivo is more obvious with the extension of time, and the quality decreases faster.
[Conclusion]
1. in the radiation dose range of 0-1000kGy, gamma ray can change the mechanical properties of fibroin, but the change of the second level mechanism is not obvious.
2. after irradiation with 0-200kGy, the silk fibroin membrane has good cytocompatibility and blood compatibility.
3. the silk fibroin film irradiated by 0-200kGy by gamma rays has good biocompatibility in vivo, and its degradability also increases with the increase of dose.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R144.1

【参考文献】