碱性成纤维细胞生长因子团聚体对糖尿病大鼠外周神经病变的治疗作用
发布时间：2018-08-18 11:05
【摘要】：目的探究新型生物材料碱性成纤维细胞生长因子(bFGF)团聚体对糖尿病大鼠坐骨神经病变的治疗作用。方法将聚乙烯精氨酸天冬氨酸甘油二酯(PEAD)、肝素和bFGF按50∶10∶1(m/m/m)的比例混合,制备成bFGF团聚体。Western蛋白印迹法检测该团聚体中bFGF含量,ELISA法检测团聚体中bFGF释放速率。雄性SD大鼠ip给予链脲佐菌素制备糖尿病大鼠模型,继续饲养8周使之发生外周神经病变,随机分为空载体组、bFGF治疗组和bFGF团聚体治疗组。bFGF治疗组每天im给予bFGF 200μg·kg~(-1),连续3 d;bFGF团聚体治疗组一次性im给予含等量bFGF的团聚体12.2 mg·kg~(-1);空载体组给予等体积的空载体(PEAD+肝素)。每周测量后肢足步印迹进行坐骨神经功能指数(SFI)的行为学评分。给药后第30天处死大鼠,收集两侧坐骨神经,HE染色进行病理学观察,DAPI荧光染色检测神经组织内细胞凋亡,Western蛋白印迹法检测细胞增殖标志物Ki67和增殖细胞核抗原(PCNA)蛋白水平。结果 PEAD、肝素和bFGF按50∶10∶1配比制备的bFGF团聚体结合bFGF良好;ELISA释放曲线结果表明,该团聚体可缓释bFGF。大鼠行为学评价和病理指标检测结果显示,与正常对照组比较,空载体组大鼠SFI显著减小(P0.01),神经纤维排列紊乱且内部脱髓鞘情况严重,而且组织内部细胞凋亡明显,Ki67和PCNA蛋白表达水平显著下降(P0.01)。与空载体组比较,bFGF治疗组和bFGF团聚体治疗组SFI升高(P0.05,P0.01),神经纤维排列紊乱和内部脱髓鞘现象改善,组织内部细胞凋亡减少,且Ki67和PCNA蛋白表达显著增加(P0.01)。与bFGF治疗组相比,bFGF团聚体治疗组SFI在治疗后第28天升高更加明显(P0.05),神经纤维排列规整,脱髓鞘现象基本消失,组织内部细胞核的荧光强度与形态基本接近正常,Ki67和PCNA蛋白表达水平显著升高(P0.05)。结论新型生物材料PEAD能有效地结合bFGF并缓控其释放。该团聚体治疗糖尿病大鼠坐骨神经病变效果可能优于单纯bFGF给药。
[Abstract]:Objective to investigate the therapeutic effect of basic fibroblast growth factor (bFGF) aggregates on sciatic neuropathy in diabetic rats. Methods Polyethylene arginine aspartate diester (PEAD), heparin and bFGF were mixed at 50:10:1 (m/m) ratio to prepare bFGF aggregates. Western blot was used to detect bFGF content in the agglomerates and Elisa was used to detect bFGF release rate in the aggregates. Male SD rats were treated with streptozotocin for 8 weeks to induce peripheral neuropathy. They were randomly divided into two groups: the empty carrier group and the bFGF agglomerate treatment group. The bFGF 200 渭 g kg ~ (-1) was given daily in the bFGF treatment group, and the same volume of PEAD heparin was given to the PEAD heparin in the empty carrier group after 3 consecutive days of single im administration of the aggregates containing the same amount of bFGF (12.2 mg kg ~ (-1). The behavioral score of sciatic nerve function index (SFI) was measured weekly. The rats were killed on the 30th day after administration. The histopathological observation was performed by HE staining of sciatic nerve on both sides. Apoptosis in nerve tissue was detected by DAPI fluorescence staining and the levels of proliferative marker Ki67 and proliferating cell nuclear antigen (PCNA) protein were detected by Western blot. Results the bFGF aggregates prepared by 50:10:1 ratio of PEAD, heparin and bFGF combined with good bFGF release curve showed that the agglomerates could release bFGF sustainably. The results of behavioral evaluation and pathological examination showed that compared with the normal control group, the SFI of the empty carrier group decreased significantly (P0.01), and the nerve fibers were disordered and demyelinated seriously. Moreover, the expression of Ki67 and PCNA protein decreased significantly (P0.01). Compared with the empty vector group, the SFI of the treated group and the bFGF agglomeration group were increased (P0.05, P0.01), the arrangement of nerve fibers and internal demyelination were improved, the apoptosis of cells in the tissue decreased, and the expression of Ki67 and PCNA protein increased significantly (P0.01). Compared with the bFGF treatment group, the SFI increased more obviously on the 28th day after treatment in the bFGF group (P0.05), the nerve fibers were arranged regularly, and the demyelinating phenomenon disappeared basically. The fluorescence intensity and morphology of the nuclei in the tissues were similar to the normal expression levels of Ki67 and PCNA (P0.05). Conclusion the new biomaterial PEAD can effectively combine bFGF and slow control its release. The effect of this agglomeration on diabetic rats with sciatic neuropathy may be better than that of bFGF alone.
【作者单位】： 温州医科大学药学院浙江省生物技术制药工程重点实验室;
【基金】：国家自然科学基金(81372112)~~
【分类号】：R587.2;R745
，
本文编号：2189275
[Abstract]:Objective to investigate the therapeutic effect of basic fibroblast growth factor (bFGF) aggregates on sciatic neuropathy in diabetic rats. Methods Polyethylene arginine aspartate diester (PEAD), heparin and bFGF were mixed at 50:10:1 (m/m) ratio to prepare bFGF aggregates. Western blot was used to detect bFGF content in the agglomerates and Elisa was used to detect bFGF release rate in the aggregates. Male SD rats were treated with streptozotocin for 8 weeks to induce peripheral neuropathy. They were randomly divided into two groups: the empty carrier group and the bFGF agglomerate treatment group. The bFGF 200 渭 g kg ~ (-1) was given daily in the bFGF treatment group, and the same volume of PEAD heparin was given to the PEAD heparin in the empty carrier group after 3 consecutive days of single im administration of the aggregates containing the same amount of bFGF (12.2 mg kg ~ (-1). The behavioral score of sciatic nerve function index (SFI) was measured weekly. The rats were killed on the 30th day after administration. The histopathological observation was performed by HE staining of sciatic nerve on both sides. Apoptosis in nerve tissue was detected by DAPI fluorescence staining and the levels of proliferative marker Ki67 and proliferating cell nuclear antigen (PCNA) protein were detected by Western blot. Results the bFGF aggregates prepared by 50:10:1 ratio of PEAD, heparin and bFGF combined with good bFGF release curve showed that the agglomerates could release bFGF sustainably. The results of behavioral evaluation and pathological examination showed that compared with the normal control group, the SFI of the empty carrier group decreased significantly (P0.01), and the nerve fibers were disordered and demyelinated seriously. Moreover, the expression of Ki67 and PCNA protein decreased significantly (P0.01). Compared with the empty vector group, the SFI of the treated group and the bFGF agglomeration group were increased (P0.05, P0.01), the arrangement of nerve fibers and internal demyelination were improved, the apoptosis of cells in the tissue decreased, and the expression of Ki67 and PCNA protein increased significantly (P0.01). Compared with the bFGF treatment group, the SFI increased more obviously on the 28th day after treatment in the bFGF group (P0.05), the nerve fibers were arranged regularly, and the demyelinating phenomenon disappeared basically. The fluorescence intensity and morphology of the nuclei in the tissues were similar to the normal expression levels of Ki67 and PCNA (P0.05). Conclusion the new biomaterial PEAD can effectively combine bFGF and slow control its release. The effect of this agglomeration on diabetic rats with sciatic neuropathy may be better than that of bFGF alone.
【作者单位】： 温州医科大学药学院浙江省生物技术制药工程重点实验室;
【基金】：国家自然科学基金(81372112)~~
【分类号】：R587.2;R745
，
本文编号：2189275
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