【摘要】：目的探究新型生物材料堿性成纖維細胞生長因子(bFGF)團聚體對糖尿病大鼠坐骨神經(jīng)病變的治療作用。方法將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,制備成bFGF團聚體。Western蛋白印跡法檢測該團聚體中bFGF含量,ELISA法檢測團聚體中bFGF釋放速率。雄性SD大鼠ip給予鏈脲佐菌素制備糖尿病大鼠模型,繼續(xù)飼養(yǎng)8周使之發(fā)生外周神經(jīng)病變,隨機分為空載體組、bFGF治療組和bFGF團聚體治療組。bFGF治療組每天im給予bFGF 200μg·kg~(-1),連續(xù)3 d;bFGF團聚體治療組一次性im給予含等量bFGF的團聚體12.2 mg·kg~(-1);空載體組給予等體積的空載體(PEAD+肝素)。每周測量后肢足步印跡進行坐骨神經(jīng)功能指數(shù)(SFI)的行為學評分。給藥后第30天處死大鼠,收集兩側(cè)坐骨神經(jīng),HE染色進行病理學觀察,DAPI熒光染色檢測神經(jīng)組織內(nèi)細胞凋亡,Western蛋白印跡法檢測細胞增殖標志物Ki67和增殖細胞核抗原(PCNA)蛋白水平。結(jié)果 PEAD、肝素和bFGF按50∶10∶1配比制備的bFGF團聚體結(jié)合bFGF良好;ELISA釋放曲線結(jié)果表明,該團聚體可緩釋bFGF。大鼠行為學評價和病理指標檢測結(jié)果顯示,與正常對照組比較,空載體組大鼠SFI顯著減小(P0.01),神經(jīng)纖維排列紊亂且內(nèi)部脫髓鞘情況嚴重,而且組織內(nèi)部細胞凋亡明顯,Ki67和PCNA蛋白表達水平顯著下降(P0.01)。與空載體組比較,bFGF治療組和bFGF團聚體治療組SFI升高(P0.05,P0.01),神經(jīng)纖維排列紊亂和內(nèi)部脫髓鞘現(xiàn)象改善,組織內(nèi)部細胞凋亡減少,且Ki67和PCNA蛋白表達顯著增加(P0.01)。與bFGF治療組相比,bFGF團聚體治療組SFI在治療后第28天升高更加明顯(P0.05),神經(jīng)纖維排列規(guī)整,脫髓鞘現(xiàn)象基本消失,組織內(nèi)部細胞核的熒光強度與形態(tài)基本接近正常,Ki67和PCNA蛋白表達水平顯著升高(P0.05)。結(jié)論新型生物材料PEAD能有效地結(jié)合bFGF并緩控其釋放。該團聚體治療糖尿病大鼠坐骨神經(jīng)病變效果可能優(yōu)于單純bFGF給藥。
[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.
【作者單位】： 溫州醫(yī)科大學藥學院浙江省生物技術(shù)制藥工程重點實驗室;
【基金】：國家自然科學基金(81372112)~~
【分類號】：R587.2;R745
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本文編號：2189275