發(fā)布時間：2018-06-30 16:50

  本文選題：絲素蛋白 + γ射線��； 參考：《蘇州大學》2013年碩士論文

【摘要】：【目的】 為研究γ射線對絲素蛋白的生物相容性及降解性的影響，本研究通過對γ射線照射后絲素蛋白力學性能的測試，篩選出能進行生物學評價的照射劑量，并利用細胞及動物實驗的方法對照射后絲素蛋白的生物相容性及降解性進行研究。 【方法】 1.γ射線照射后絲素蛋白膜的力學結(jié)構(gòu)變化情況：將制作好的絲素蛋白膜，加入分別為0、25、50、100、200、300、500、1000kGy的γ射線照射組。（1）絲素蛋白膜斷裂強度和斷裂伸長率變化：將絲素蛋白膜用特制的模具，制成啞鈴狀條帶，對各組絲素蛋白膜的斷裂強度和斷裂伸長率進行測試。（2）絲素蛋白膜結(jié)構(gòu)變化的測試：將照射后的絲素蛋白膜剪碎制成粉末，用KBr壓片，經(jīng)Nicolet5700FTIR型傅立葉變換紅外光譜儀檢測。2. γ射線照射后的絲素蛋白生物相容性研究：將SD大鼠乳鼠頸椎脫臼處死后，取其背部皮膚，分離原代真皮細胞培養(yǎng)傳至3、4代。（1）真皮細胞在絲素蛋白膜上的生長曲線測定：將原代培養(yǎng)的真皮細胞以每孔1×105細胞，接種于鋪在24孔板中的輻照絲素蛋白膜上，培養(yǎng)7d，每天各取6孔，使用CCK-8法檢測細胞增殖活力，酶標儀于450nm波長處，，測得OD值，并計算細胞量，求得生長曲線。（2）絲素蛋白浸提液細胞毒性試驗：用制備好的絲素蛋白浸提液培養(yǎng)原代真皮細胞，以每孔5000個，接種于3塊96孔板中，待細胞貼壁后移去普通培養(yǎng)基，加入絲素蛋白膜浸提液，100μL/孔，培養(yǎng)1、3和6d，每個時間點各取一板，加入CCK-8溶液反應(yīng)，使用酶標儀測得各孔OD值。（3）溶血試驗：取志愿者全血制成稀釋血。設(shè)生理鹽水、雙蒸水、絲素蛋白浸提液及含絲素蛋白膜的生理鹽水4個組。每個試管加入0.2mL稀釋血，37℃水浴60min。反應(yīng)后的溶液以1000r/min離心5min，取上清液移入96孔板中，使用酶標儀于545nm波長處測定吸光度。3.生物相容性及降解作用的體內(nèi)研究：將不同劑量（25kGy、50kGy、100kGy、200kGy）γ射線照射后的絲素蛋白膜植入SD大鼠皮下，植入后7、14、28、56、84天，剖殺取材。對植入部位的皮下組織進行病理檢查，通過ELISA法檢測絲素蛋白植入后的大鼠血清中IL-6和TNF-α含量；取出植入后的絲素蛋白稱重，計算質(zhì)量減少率并繪制質(zhì)量減少曲線。 【結(jié)果】 1.γ射線照射后絲素蛋白膜的力學結(jié)構(gòu)變化情況：經(jīng)過0、25、50、100、200、300、500、1000kGy的γ射線照射后，絲素蛋白的斷裂強度及斷裂伸長率隨照射劑量的增加，均呈現(xiàn)出下降趨勢。紅外光譜儀檢測發(fā)現(xiàn)，γ射線照射后的絲素蛋白Silk II的吸收峰1628cm-1、1526cm-1并未發(fā)生明顯移動，說明γ射線對絲素蛋白的二級結(jié)構(gòu)影響不明顯。2. γ射線照射后的絲素蛋白生物相容性研究：原代真皮細胞在絲素蛋白膜上的生長情況良好，增殖表現(xiàn)出相同的趨勢，均在生長96h達到峰值，而后下降，對每個時間點各組細胞增殖活力進行單因素方差分析，各組吸光度值均具有方差齊性，且各組細胞增殖活力之間不存在顯著性差異(p0.05)。（2）細胞用絲素蛋白浸提液培養(yǎng)24、72和144h，分別測定增殖情況，并按照國家標準，其毒性在分級標準的1級（RGR:75%－99%）以內(nèi)，經(jīng)SPSS17.0統(tǒng)計分析，各時間點各組細胞RGR差異無統(tǒng)計學意義（p0.05）。細胞增殖趨勢也具有一致性。（3）絲素蛋白浸提液及含絲素蛋白的生理鹽水，溶血率均小于國家標準所規(guī)定的5%，SPSS17.0軟件統(tǒng)計結(jié)果顯示，各組溶血率之間差異均無統(tǒng)計學意義（p0.05）。3.生物相容性及降解性的體內(nèi)研究：將各組絲素蛋白膜植入SD大鼠背部皮下，并以空白植入作為對照，排除手術(shù)引起炎癥反應(yīng)的影響，發(fā)現(xiàn)植入的絲素蛋白均未引起明顯的炎癥反應(yīng)，并且隨時間變化，較高劑量照射組的絲素蛋白裂解較為明顯。各照射劑量絲素蛋白膜植入組的SD大鼠，IL-6和TNF-α分泌沒有顯著性差異（p0.05），與空白植入組（Blank）比較，發(fā)現(xiàn)絲素蛋白并未引起明顯炎癥反應(yīng)（p0.05）。絲素蛋白在體內(nèi)降解性表現(xiàn)為，較高劑量照射組的絲素蛋白在體內(nèi)降解隨時間的延長碎片形成越明顯，質(zhì)量下降也越快。 【結(jié)論】 1.在0-1000kGy的照射劑量范圍內(nèi)，γ射線能改變絲素蛋白機械性能，但是對其二級機構(gòu)改變不明顯。 2.經(jīng)0-200kGy的γ射線照射后，絲素蛋白膜具有良好的細胞相容性和血液相容性。 3.經(jīng)0-200kGy的γ射線照射的絲素蛋白膜，在體內(nèi)具有良好的生物相容性，其降解性也隨受照劑量的增大有所提高。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R144.1

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