水凝胶递送碱性成纤维细胞生长因子对氧糖剥夺后NIH3T3细胞功能的影响

Effect of hydrogel-based delivery of bFGF on function of NIH3T3 cells after oxygen-glucose deprivation

目的:探讨水凝胶递送碱性成纤维细胞生长因子(bFGF)对氧糖剥夺(OGD)后小鼠成纤维NIH3T3细胞功能的影响,并阐明负载bFGF的水凝胶对OGD条件下成纤维细胞氧化应激反应的改善作用。方法:制备bFGF明胶水凝胶,将对数生长期NIH3T3细胞分为空白组、20%明胶组和20%明胶+戊二醛组,采用CCK-8法检测6、12和24 h水凝胶浸出液共培养的NIH3T3细胞活性。将NIH3T3细胞分为对照组、OGD组和OGD+不同质量bFGF负载组(OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组)。采用Western blotting法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平。检测各组细胞中丙二醛(MDA)水平和乳酸脱氢酶(LDH)及超氧化物歧化酶(SOD)活性。采用酶联免疫吸附试验(ELISA)法检测bFGF体外释放率。结果:CCK-8法检测,与空白组比较,不同浸出时间20%明胶组和20%明胶+戊二醛组NIH3T3细胞活性差异无统计学意义(P>0.05)。Western blotting法检测,与对照组比较,OGD组NIH3T3细胞中α-SMA和Ⅰ型胶原蛋白表达水平明显升高(P<0.05或P<0.01);与OGD组比较,OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组NIH3T3细胞中α-SMA蛋白表达水平均明显降低(P<0.01),Ⅰ型胶原蛋白表达水平均明显降低(P<0.05或P<0.01),并呈剂量依赖性。与对照组比较,OGD组NIH3T3细胞中MDA水平和LDH活性升高(P<0.05),SOD活性降低(P<0.05)。与OGD组比较,OGD+1 ng bFGF组NIH3T3细胞中MDA水平和LDH活性降低(P<0.05),SOD活性升高(P<0.05)。ELISA法检测,在4 h内约10%bFGF由水凝胶中释放,在12 h内约15%bFGF由水凝胶中释放,在24 h内约21%bFGF由水凝胶中释放。结论:负载bFGF的水凝胶可以调控OGD条件下成纤维细胞的转化,抑制成纤维细胞Ⅰ型胶原蛋白表达,并改善细胞氧化应激反应,且该系统具有一定的持续释药能力。

Objective:To discuss the effect of hydrogel-based delivery of basic fibroblast growth factor(bFGF)on the function of the NIH3T3 fibroblast cells after oxygen-glucose deprivation(OGD),and to clarify the improvement effect of bFGF-loaded hydrogel on the oxidative stress responses in the fibroblasts under the OGD condition.Methods:The bFGF-containing agarose hydrogel was prepared,and the NIH3T3 cells at logarithmic growth phase were divided into blank group,20%agarose group,and 20%agarose+glutaraldehyde group.The viabilities of the NIH3T3 cells after co-cultured with the hydrogeleluted fluid for 6,12,and 24 h were detected by CCK-8 assay.The NIH3T3 cells were divided into control group,OGD group,and OGD+different masses of bFGF-loaded groups(OGD+1 ng bFGF group,OGD+10 ng bFGF group,and OGD+100 ng bFGF group).The expression levels ofα-smooth muscle actin(α-SMA),typeⅠcollagen,and typeⅢcollagen proteins in the NIH3T3 cells in various groups were detected by Western blotting method;the content of malondialdehyde(MDA)and activities of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)in the NIH3T3 cells were detected by assay kits;the in vitro release rate of bFGF in the NIH3T3 cells in various groups was detected by enzyme-linked immunosorbent assay(ELISA)method.Results:The CCK-8 assay results showed that compared with blank group,the viability of the NIH3T3 cells between blank group,20%agarose group,and 20%agarose+glutaraldehyde group at different elution times had no significant difference(P>0.05).The Western blotting results showed that the expression levels ofα-SMA and typeⅠcollagen proteins in the NIH3T3 cells in OGD group were significantly higher than that in control group(P<0.05 or P<0.01).Compared with OGD group,the expression levels ofα-SMA and typeⅠcollagen proteins in the NIH3T3 cells in OGD+1 ng bFGF,OGD+10 ng bFGF,and OGD+100 ng bFGF groups were significantly decreased(P<0.05 or P<0.01)in a dose-dependent manner.Compared with control group,the level of MDA and activity of LDH in the NIH3T3 cells in OGD group were increased(P<0.05),while the activity of SOD was decreased(P<0.05).Compared with OGD group,the level of MDA and the activity of LDH in the NIH3T3 cells in OGD+1 ng bFGF group were decreased(P<0.05),while the activity of SOD was increased(P<0.05).The ELISA results showed that about 10%bFGF was released from the hydrogel within 4 h,about 15%bFGF was relleased from the bydrogel with in 12 h,and about 21%bFGF was released from the hydrogel within 24 h.Conclusion:The bFGF-loaded hydrogel can modulate the transformation of the fibroblasts under the OGD condition,inhibit the expression of typeⅠcollagen protein,and improve the oxidative stress responses in the fibroblasts.Moreover,this system exhibits a sustained drug-release ability.