新生小鼠肠道类器官的发育成熟过程

Development and maturation of intestinal organoids in neonatal mice

目的探究新生小鼠肠道类器官的发育成熟过程,为围产期胎儿/新生儿肠道上皮发育及相关疾病的研究提供新模型。方法取3日龄C57BL/6小鼠肠道组织,利用标准条件培养肠道类器官,传代培养至第5代。利用倒置相差显微镜观察并记录各代肠道类器官形态变化。利用实时荧光定量聚合酶链反应和免疫荧光技术检测各代肠道类器官中肠道干细胞及肠道上皮各类型分化细胞标志物的表达及组织细胞定位(选取的标志基因:肠道干细胞:Lgr5;胎儿期肠道祖细胞:Tpm2和Gja1;肠道上皮细胞:Villin;帕内特细胞:Lyz1;杯状细胞:Muc2;内分泌细胞:Chga;选取的各细胞标志蛋白:肠上皮细胞:绒毛蛋白;杯状细胞:黏蛋白2;内分泌细胞:嗜铬粒蛋白A;帕内特细胞:溶菌酶)。采用单因素方差分析和Bonferroni检验进行统计学分析。结果新生小鼠肠道类器官中存在不成熟的球状体和具有隐窝-绒毛结构的成熟型类器官这2种类型。原代培养物中以球状体为主要形态,占(96.61±1.36)%;从原代至传代第2代间,类器官形态变化表现为球状体比例明显下降[第2代占比下降至(8.93±1.50)%],且面积缩小(F=12.88,P<0.001);第2代至第5代类器官的形态以成熟类器官为主,占比从(91.07±1.50)%升至(95.56±2.14)%。肠道干细胞标志基因Lgr5的表达在从原代传代到第2代之间降低(F=76.75,P<0.001),第2代Lgr5表达为原代的0.40±0.06,在第2代之后上升。胎儿期肠道祖细胞标志基因Tpm2表达在传代中明显下降(原代1.00±0.11,第5代0.003±0.001,F=148.00,P<0.001);Gja1的表达在原代(1.00±0.14)至第2代(0.06±0.04)间下降(F=197.10,P<0.001),在第2代后保持稳定低表达(F=2.20,P=0.13)。肠道上皮内各类型分化细胞(肠上皮细胞、杯状细胞、内分泌细胞和帕内特细胞)的标志基因表达在传代第2代之后呈现升高趋势(Villin:第2代0.46±0.11,第5代1.02±0.05;Muc2:第2代0.68±0.29,第5代8.79±0.61;Chga:第2代2.53±0.16,第5代4.32±0.45;Lyz1:第2代0.98±0.21,第5代3.81±0.36;P值均<0.05)。免疫荧光染色结果显示,在原代及第5代培养物中,肠上皮细胞标志蛋白绒毛蛋白均沿肠道类器官绒毛侧分布。杯状细胞标志蛋白黏蛋白2及内分泌细胞标志蛋白嗜铬粒蛋白A在原代中表达很低,而到第5代中染色明显。在原代培养物中帕内特细胞标志蛋白溶菌酶弥散性均匀分布在类器官细胞内,而在第5代中可见高荧光强度点状表达。结论新生小鼠肠道类器官的连续培养可模拟未成熟肠道上皮的发育成熟过程。原代至传代第2代类器官也许可作为胎儿至新生儿期肠道上皮发育的研究模型,传代第2~5代类器官也许可作为新生儿期肠道疾病研究的新模型。

Objective To investigate the development and maturation process of intestinal organoids in neonatal mice so as to provide a new model for research on perinatal/neonatal intestinal epithelial development and related diseases.Methods Intestinal tissue of 3-day-old C57BL/6 mice were collected and cultured for mouse intestinal organoids(MIOs)under standard conditions down to the fifth generation.The morphological changes of MIOs were observed and recorded using inverted phase contrast microscope.Real-time fluorescent quantitative polymerase chain reaction and immunofluorescence technique were used to detect the expression and location of markers of intestinal stem cells and differentiated cells of intestinal epithelium among different generations of MIOs(Selected marker genes:Lgr5 for intestinal stem cells,Tpm2 and Gja1 for fetal intestinal progenitor cells,Villin for intestinal epithelial cells,Lyz1 for Paneth cells,Muc2 for goblet cells,Chga for endocrine cells;Selected marker proteins:villin for intestinal epithelial cells,mucin 2 for goblet cells,chromaffin A for endocrine cells,lysozyme for Paneth cells).One-way analysis of variance and Bonferroni test were adopted for statistical analysis.Results Two types of MIOs were observed,immature spheroid and mature organoids with crypt-villus structure.Spheroid was the main form in the primary culture.From primary to the second generation,the proportion of spheroids decreased from(96.61±1.36)%to(8.93±1.50)%,and so did the size(F=12.88,P<0.001).During the second to the fifth generation,mature organoid,as the main form,increased from(91.07±1.50)%to(95.56±2.14)%.The expression of intestinal stem cell marker Lgr5 in the second generation decreased to 0.40±0.06 times of the primary one(F=76.75,P<0.001)and then increased after this period.The expression of fetal intestinal progenitor markers Tpm2 decreased significantly during the passage(primary generation:1.00±0.11,the fifth generation:0.003±0.001,F=148.00,P<0.001);And the expression of Gja1 decreased from primary generation(1.00±0.14)to the second generation(0.06±0.04)(F=197.10,P<0.001),but kept stable from the second to fifth genetation(F=2.20,P=0.13).The expressions of gene markers of differentiated cells in intestinal epithelium,including enterocytes,goblet cells,endocrine cells,and Paneth cells,increased after the second generation(the second generation:Villin:0.46±0.11;Muc2:0.68±0.29;Chga:2.53±0.16;Lyz1:0.98±0.21;the fifth generation:Villin:1.02±0.05;Muc2:8.79±0.61;Chga:4.32±0.45;Lyz1:3.81±0.36;all P<0.05).Immunofluorescence showed that villin,the intestinal epithelial cell marker protein,was distributed along the villus-side of MIOs in primary and the fifth generation culture.Mucin 2 from goblet cell and chromaffin A from endocrine cell expressed at a very low level in the primary generation,while higher in the fifth generation.In the primary culture,lysozyme from Paneth cell was evenly distributed in organoid cells,and high fluorescent dot-shaped expression was observed in the fifth generation.Conclusions The development and maturation of immature intestinal epithelium can be simulated by continuous culture of neonatal MIOs.MIOs between the primary and second generation could be used as a research model for development of perinatal intestinal epithelium,and the second to the fifth generation as a model for neonatal intestinal diseases studies.