小鼠腔前卵泡在淋巴结脱细胞支架中发育的研究

The Study of Preantral Follicles Development in the Decellularized Lymph Node

目的探讨小鼠腔前卵泡在淋巴结脱细胞支架中的发育情况。方法10周龄C57BL/6J小鼠的腋下淋巴结,使用0.5%的十二烷基硫酸钠(SDS)进行脱细胞处理,HE染色和扫描电子显微镜(scanning electron microscope,SEM)观察支架形态,DAPI染色和琼脂糖凝胶电泳和DNA含量检测观察细胞和DNA残留情况。中国仓鼠卵巢细胞(CHO)与支架共培养后,CCK-8验证脱细胞支架对细胞活力的影响。将小鼠腔前卵泡注射到支架内进行体外培养,HE染色观察卵泡的生长情况。免疫组化染色观察CYP-17/A1和FSHR的表达情况,ELISA检测细胞支架培养液中雌激素分泌水平。结果与新鲜淋巴结相比,脱细胞后淋巴结外观变得透明,形态保持完整。HE和SEM发现支架内密集分布大量的三维纤维网架结构。DAPI染色未发现细胞的残留,琼脂糖凝胶电泳未发现明显DNA残留。DNA定量分析表明DNA的去除率为88%[(1390.0±248.6)ng·mg^(-1) vs.(342.6±116)ng·mg^(-1)](P<0.05)。CCK-8检测结果表明,在24、48、96 h时,加支架组与对照组的细胞活力差异均无统计学意义(P均>0.05)。腔前卵泡植入后,在支架中生长,卵泡直径增大,颗粒细胞(FSHR阳性细胞)和膜细胞(CYP-17/A1阳性细胞)增殖,并分泌雌激素。结论淋巴结脱细胞后,可形成细胞外基质支架,可支持小鼠腔前卵泡的生长并具有内分泌功能。

Objective To investigate the preantral follicles development in decellularized lymph node.Methods Axillary lymph node was collected from 10 weeks old C57BL/6J mice and decellularized by 0.5%SDS.HE and scanning electron microscope(SEM)were used to evaluate morphology of decellularized lymph nodes.DAPI,agarose gel electrophoresis(AGE)and DNA content analysis were used to detect residual cells and DNA in decellularized lymph nodes.CCK-8 test was used to test cytocompatibility after decellularized lymph cocultured with CHO.Then,the preantral follicles were injected in decellularized scaffolds and cultured in vitro.HE staining was used to evaluate morphology of growth follicles.IHC staining were performed to observe the expression of FSHR and CYP-17/A1.ELISA was used to detect the secretion of estradiol in scaffold cultural medium.Results Compared to fresh lymph nodes,the appearance of decellularized lymph node became transparent,and the shape remained integral.The results of HE and SEM showed fibrillar network were distributed in scaffold.DAPI staining showed no cell residue be found,AGE showed no significant DNA residue be found,and DNA content confirmed that the DNA removal rate was 88%[(1390.0±248.6)ng·mg^(-1)vs.(342.6±116)ng·mg^(-1)](P<0.05).At 24,48,96 hours CCK-8 test results showed that there was no significant difference in cell viability between the cellular scaffold group and the control group(P all>0.05).After the preantral follicles were injected in decellularized scaffolds,FSHR cell amount gradually increased,CYP-17/A1 cell increased accompanied and estrogen was secreted with follicles development.Conclusion After decellularization,lymph node can form extracellular matrix scaffold that supports the growth of preantral follicles and has function of endocrine.