先天性巨结肠血浆外泌体分子生物学及生物信息分析的初步研究

Molecular biology and bioinformatics of plasma exosomes in Hirschsprung's disease

目的探究先天性巨结肠(Hirschsprung’’s disease, HD)血浆外泌体参与HD发生、发展的可能机制。方法收集2020年4月至2021年7月嘉兴市妇幼保健院收治的14例男性患儿血液样本, 将其中7例HD患儿作为实验组, 年龄为(18.14±18.17)个月, 范围为2~48个月;将年龄范围与实验组患儿相匹配的7例男性非HD患儿作为对照组, 年龄为(27.14±13.81)个月, 范围为6~48个月。收集14例患儿外周血各8 ml, 采用超速离心法分离血浆后提取外泌体。经过透射电镜观察、样品粒径分析、蛋白指标检测和RNA浓度检测鉴定外泌体后, 进行外泌体miRNA测序, 最后对差异表达的miRNA及其靶基因采用GO富集分析和KEGG富集分析。结果 14例样本中均成功分离出外泌体, 透射电镜下可观察到形态完整、球形、大小均一的典型外泌体结构。外泌体粒径分析结果显示, 实验组与对照组血浆外泌体粒径分别为(144.22±33.98 )nm和(122.99±21.24)nm, 两组间的差异无统计学意义(P=0.186)。血浆外泌体浓度分析结果显示, 对照组与实验组血浆外泌体浓度分别为(3.39± 3.90)×109和(3.47±4.04)×1010 Particles/ml, 两组间的差异具有统计学意义(P=0.002)。外泌体蛋白指标检测结果显示:①对照组与实验组的外泌体蛋白浓度分别为(0.42±0.17)μg/μl和(0.13± 0.12)μg/μl, 对照组浓度显著高于实验组, 差异具有统计学意义(P=0.004);②对照组和实验组的外泌体特异性阳性标志物CD9和CD81均有不同程度的表达, 阴性标志物IgG基本没有表达。外泌体miRNA测序结果显示:①实验组RNA浓度高于对照组, (0.66±0.54 )ng/μl比(0.24±0.10 )ng/μl, 两组间的差异具有统计学意义(P=0.011);②共筛选出3个差异性表达的miRNA, 包括miR-382上调, miR-29B2和miR-29A下调。运用KEGG分析差异表达的miRNA结果显示, miR-382主要富集于PI3K-Akt信号通路, miR-29A富集于癌症中的蛋白聚糖和Hippo信号通路, 而miR-29B2则富集于细胞周期通路。分析miR-382、miR-29B2和miR-29a的靶基因, 取完交集后分别得到73、782和770个靶基因。结论 HD患儿血浆外泌体中差异性表达的miRNA及其靶基因参与肠神经节细胞的生物学功能、细胞周期等调节, 可能与HD的发生、发展机制相关。

Objective To explore the possible role of plasma exosomes in the development and progression of Hirschsprung's disease(HD).Methods Blood samples were collected from 14 boys hospitalized from April 2020 to July 2021.They were divided into experimental and control groups(n=7 each).The age range was(18.14±18.17)(2-48)and(27.14±13.81)(6-48)months respectively.Eight milliliters of peripheral blood was harvested and exosomes were extracted after plasma separation by ultracentrifugation.After transmission electron microscopy,sample particle size analysis,protein index detection,RNA concentration detection and exosome miRNA sequencing,differentially expressed miRNA and target genes were examined by GO/KEGG enrichment analysis.Results Typical exosomes with intact morphology,spherical shape and uniform size could be observed under transmission electron microscope.Particle sizes in experimental group and control group were[(144.22±33.98)vs(122.99±21.24)nm]and inter-group difference was not statistically significant(P=0.186).And plasma exosome concentrations in control and experimental groups were(3.39±3.90)×109 and(3.47±4.04)×1010 Particles/ml and inter-group difference was statistically significant(P=0.002).Protein concentrations was significantly higher in control group than that in experimental group[(0.42±0.17)vs(0.13±0.12)μg/μl]and difference had statistical significance(P=0.004).Exosome-specific positive markers CD9 and CD81 were expressed to different extents in both groups.Negative marker IgG was basically not expressed.RNA concentration was higher in experimental group than that in control group[(0.66±0.54)vs.(0.24±0.10)ng/µl]and inter-group difference was statistically significant(P=0.011).A total of three differentially expressed miRNA were selected,including miR-382 up-regulation and miR-29B2 and miR-29A down-regulation.KEGG analysis of differentially expressed miRNA indicated that miR-382 was predominantly enriched in PI3K-Akt signaling pathway,miR-29A became enriched in proteoglycan and Hippo signaling pathways in cancer while miR-29B2 was enriched in cell cycle pathway.The target genes of miR-382,miR-29B2,and miR-29A yielded 73,782 and 770 target genes after taking intersections.Conclusions Differentially expressed miRNA and their target genes in plasma exosomes of HD children are involved in regulating biological functions and cell cycles of intestinal ganglions.It is probably correlated mechanically with the occurrence and development of HD.