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目的制备一种新型-βNGF缓释系统,通过Sandwich ELISA法建立-βNGF标准释放曲线及无细胞存在下体外释放曲线,检测缓释系统能否延缓释放-βNGF。方法利用纤维蛋白作为释放系统基质,加入肝素、肝素结合肽、-βNGF及含Ca2+的凝血酶制成-βNGF缓释系统,加入24孔组织培养板内,37℃,95%相对湿度,5%CO2培养箱中培育5~10 min,然后每天加500μL/孔TBS液轻轻冲洗,小心吸取冲洗液收集于EP管内,共6 d,最后加100μL/孔含纤溶酶的TBS液将基质完全溶解,收集基质溶解液于EP管中,用Sandwich ELISA法测定每天冲洗液含有的-βNGF的量和最后基质溶解液中剩余-βNGF的量,建立-βNGF浓度-时间释放曲线。实验分为2组,实验组:fibrin+heparin+peptide+100 ng/mL-βNGF;对照组:fibrin+100 ng/mL-βNGF。结果第1天,对照组有98.23%的NGF释放出来,而NGF缓释系统组只释放21.37%,明显少于对照组(P<0.01);对照组最后的基质溶解液中无NGF剩余,而实验组仍有48.92%的NGF剩余(P<0.01)。结论在体外无细胞存在情况下,实验组能明显延缓NGF的释放,延缓时间在6 d以上。
OBJECTIVE To prepare a new -βNGF sustained-release system, the standard release curve of -βNGF and the in vitro release curve in the absence of cells were established by Sandwich ELISA to detect whether delayed release system could delay the release of -βNGF. Methods Fibrin was used as substrate for release system. Heparin, heparin binding peptide, -βNGF and Ca2 + -containing thrombin were added to make the -βNGF sustained release system. The cells were added into 24-well tissue culture plates and incubated at 37 ℃, 95% RH and 5% CO2 incubator incubated for 5 ~ 10 min, and then every day plus 500μL / well TBS light wash gently, carefully absorb the rinse collected in the EP tube, a total of 6 d, and finally add 100μL / well plasminogen-containing TBS solution to the matrix completely Solubilize and collect the matrix lysate in an EP tube. Measure the amount of -βNGF contained in the daily washings and the amount of remaining -βNGF in the final matrix solution by Sandwich ELISA to establish the -βNGF concentration-time release curve. The experiment was divided into two groups, experimental group: fibrin + heparin + peptide + 100 ng / mL-βNGF; control group: fibrin + 100 ng / mL-βNGF. Results On the first day, 98.23% of the NGF was released in the control group, while only 21.37% in the NGF sustained-release system group was significantly less than that of the control group (P <0.01); in the control group, no NGF remained in the final matrix solution 48.92% of NGF remained in experimental group (P <0.01). Conclusion In the absence of cells in vitro, the experimental group can significantly delay the release of NGF, the delay time of 6 d or more.