重组蛛丝蛋白支架材料生物相容性的研究
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摘要
重组蛛丝蛋白作为一种新型的生物材料在组织工程领域得到广泛关注。利用本室先期已构建含有细胞黏附位点RGD的基因工程菌pNSR-32/BL21(DE3)pLysS进行高密度发酵,建立适合pNSR-32基因工程菌表达蛋白的纯化方法。确定的最终纯化方案为:按1:5(m/v)的比例混悬湿菌体和磷酸盐缓冲液,70%功率冰浴超声10min,脉冲9s,脉停2s。80℃加热超声液10min,调节pH至5.5。离心,上清用15%饱和度硫酸铵溶液沉淀1.5h,调上清液pH至蛋白等电点6.5。离心后收集沉淀,透析冻干保存。按上述方法进行纯化,目的蛋白纯度达到85%以上,得率约在1.5～2.5mg/g湿菌体。
将纯化的重组蛛丝蛋白作为原料,按本室所建立的方法制备支架材料,评价其细胞相容性。采用MTT的方法讨论重组蛛丝蛋白支架材料的细胞毒性,并通过NIH-3T3细胞与支架材料复合培养研究支架材料对细胞黏附、生长和分化的影响。MTT实验结果表明,重组蛛丝蛋白支架材料浸提液对NIH-3T3细胞的生长和增殖影响较小,根据细胞毒性实验毒性评定标准,判定重组蛛丝蛋白支架材料毒性等级在1级以下,符合合格生物材料的要求。电子扫描电镜(SEM)和常规HE染色观察发现,含有RGD三肽的重组蛛丝蛋白支架材料更利于NIH-3T3细胞黏附,细胞在材料表面形成多层结构且深入材料内部生长。碱性成纤维生长因子(bFGF)免疫组化实验结果说明复合培养的NIH-3T3细胞可以表达bFGF因子。
通过建立大鼠背部深度烫伤模型,研究重组蛛丝蛋白支架材料—细胞复合物对深度烫伤创面愈合的影响。用pNSR-16和pNSR-32两种材料与NIH-3T3细胞复合物修复烫伤创面,两者的羟脯氨酸含量均高于其余各组。HE染色结果显示,在创面愈合初期,pNSR-16和pNSR-32两种材料与细胞复合物的创面修复情况比其余各组活跃。免疫组化结果发现,bFGF因子在细胞增殖活跃的创面修复部位均有较高表达,pNSR-16和pNSR-32两种材料与细胞复合物组比其余组表达bFGF明显。以上结果初步表明,重组蛛丝蛋白支架材料—细胞复合物组优于单纯材料组和烫伤对照组,对促进深度烫伤创面愈合有一定作用。
The recombinant spider silk protein as a new biomaterial had caused wide attention in tissue engineering. In our previous work, a new plasmid pNSR-32 containing RGD cell adhesion sites was constructed to express the higher molecular weight recombinant spider silk protein by E. coli BL21(DE3)pLysS. We optimized the purification conditions of the recombinant spider silk protein in this study. The conditions are ultrasound broken cells for 10min, heating for 10min at 80℃, adjusted to pH 5.5, sulfates saturation at 15%. The final purity of protein was above 85% and yield 1.5～2.5mg/g wet cells.
The recombinant spider silk protein were made the scaffolds and evaluated the cytocompatibility. MTT assay was used to investigate the scaffolds cytotoxicity. The effects of cell growing, adhesion and differentiation on the scaffolds were discussed by culturing NIH-3T3 cells on scaffolds. The MTT experimental results were shown that the toxic rank of scaffolds below 1 level, were the qualified biomaterials. The SEM and HE staining showed the scaffolds with RGD had the better cell adhesion Cells can grow in the scaffolds and form the multi-layered cells on the surface of scaffolds. Immunohistochemistry results showed the cells on the scaffolds can proliferate and secret bFGF.
By the back depth scalded of rat model, we studied the depth scald wound healing using scaffolds with NIH-3T3 cells. When the scald wound was repaired with pNSR-16 or pNSR-32 protein scaffold-NIH-3T3 cell complexes,the hydroxyproline contents of these two groups were more than others. HE staining showed that the scald wound repair of these two groups were more active than others in the initial wound healing. Immunohistochemistry showed that bFGF had the high expression on cell reproduction active areas during the scald wound healing. The bFGF expression of these two groups were higher than others. The preliminary results indicated that the recombinant spider silk protein scaffold-cell complexes group were better than simple material group and the control group. It had effect on promoting the deep scald wound healing.
将纯化的重组蛛丝蛋白作为原料,按本室所建立的方法制备支架材料,评价其细胞相容性。采用MTT的方法讨论重组蛛丝蛋白支架材料的细胞毒性,并通过NIH-3T3细胞与支架材料复合培养研究支架材料对细胞黏附、生长和分化的影响。MTT实验结果表明,重组蛛丝蛋白支架材料浸提液对NIH-3T3细胞的生长和增殖影响较小,根据细胞毒性实验毒性评定标准,判定重组蛛丝蛋白支架材料毒性等级在1级以下,符合合格生物材料的要求。电子扫描电镜(SEM)和常规HE染色观察发现,含有RGD三肽的重组蛛丝蛋白支架材料更利于NIH-3T3细胞黏附,细胞在材料表面形成多层结构且深入材料内部生长。碱性成纤维生长因子(bFGF)免疫组化实验结果说明复合培养的NIH-3T3细胞可以表达bFGF因子。
通过建立大鼠背部深度烫伤模型,研究重组蛛丝蛋白支架材料—细胞复合物对深度烫伤创面愈合的影响。用pNSR-16和pNSR-32两种材料与NIH-3T3细胞复合物修复烫伤创面,两者的羟脯氨酸含量均高于其余各组。HE染色结果显示,在创面愈合初期,pNSR-16和pNSR-32两种材料与细胞复合物的创面修复情况比其余各组活跃。免疫组化结果发现,bFGF因子在细胞增殖活跃的创面修复部位均有较高表达,pNSR-16和pNSR-32两种材料与细胞复合物组比其余组表达bFGF明显。以上结果初步表明,重组蛛丝蛋白支架材料—细胞复合物组优于单纯材料组和烫伤对照组,对促进深度烫伤创面愈合有一定作用。
The recombinant spider silk protein as a new biomaterial had caused wide attention in tissue engineering. In our previous work, a new plasmid pNSR-32 containing RGD cell adhesion sites was constructed to express the higher molecular weight recombinant spider silk protein by E. coli BL21(DE3)pLysS. We optimized the purification conditions of the recombinant spider silk protein in this study. The conditions are ultrasound broken cells for 10min, heating for 10min at 80℃, adjusted to pH 5.5, sulfates saturation at 15%. The final purity of protein was above 85% and yield 1.5～2.5mg/g wet cells.
The recombinant spider silk protein were made the scaffolds and evaluated the cytocompatibility. MTT assay was used to investigate the scaffolds cytotoxicity. The effects of cell growing, adhesion and differentiation on the scaffolds were discussed by culturing NIH-3T3 cells on scaffolds. The MTT experimental results were shown that the toxic rank of scaffolds below 1 level, were the qualified biomaterials. The SEM and HE staining showed the scaffolds with RGD had the better cell adhesion Cells can grow in the scaffolds and form the multi-layered cells on the surface of scaffolds. Immunohistochemistry results showed the cells on the scaffolds can proliferate and secret bFGF.
By the back depth scalded of rat model, we studied the depth scald wound healing using scaffolds with NIH-3T3 cells. When the scald wound was repaired with pNSR-16 or pNSR-32 protein scaffold-NIH-3T3 cell complexes,the hydroxyproline contents of these two groups were more than others. HE staining showed that the scald wound repair of these two groups were more active than others in the initial wound healing. Immunohistochemistry showed that bFGF had the high expression on cell reproduction active areas during the scald wound healing. The bFGF expression of these two groups were higher than others. The preliminary results indicated that the recombinant spider silk protein scaffold-cell complexes group were better than simple material group and the control group. It had effect on promoting the deep scald wound healing.
引文
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