| Objective: In this study,we are going to choose the cell mass of the crypt inside intestinal epithelium as the research object.By culturing cell mass of the crypt in vitro and isolating and culturing single intestinal stem cell,we observe and analyze the reasons for the difference of crypt proliferation in vitro.Besides,we would also to study and discusse the internal relationship among crypt,intestinal stem cell and epithelial cell.Methods:1.General tissue separation,enzyme digestion method,chelation of EDTA were respectively used to separate the intestinal epithelium of fetal rats.The effects of the exposure degree and integrity of the intestinal crypt on the culture effect of the primary intestinal epithelium were observed.2.The crypt cells of adult mice of C57 were isolated by the method of chelation of EDTA,and the three-dimensional culture system of intestinal organoids was established.The growth and differentiation of crypt cells were followed.The Lgr5 cells inside the crypt were stained and identified both in the intestinal epithelium and the cells curltured in vitro.3.At the same time,the cells isolated by the method of chelation of EDTA were recovered.Destroying or digesting the crypt again in order to make stem cells lose contact with the crypt or completely release.The growth of Lgr5 was observed by single Lgr5 cells or cell clusters by immunomagnetic beads.Results:1.Intestinal epithelial cells isolated by different methods showed significant differences in activity,which was possible closely related to the degree of exposure and integrity of the isolated intestinal crypt.A large number of epithelial cells can be grown by the method of enzyme digestion method;the results of general tissue separation would obtained by fibroblasts because the degree of crypt exposure is not enough;Due to the damage cansed by EDTA,the method of chelation of EDTA resulting in the cells would not stick to the wall in the ordinary medium and a large number of epithelial cells won't survived.2.The complete crypt cell mass was successfully isolated,and a long-term stable curlturing system of organoids was formulated in the three-dimensional culture system.At the early stage of isolation,the volume of crypt cells expanded with time,goes and the number of differentiated cells increased gradually in about 3-5 days.The total number of cells increased rapidly and the organoids emerged later.3.Lgr5 cells with magnetic beads were successfully isolated.It can be observed that single Lgr5 cells got to be round and bright at the initial stage,and then undergo asymmetric division.After a certain number of days,the cells would not differentiate or division.but in any case,single Lgr5 cells or Lgr5 cells separated from the crypt cell cluster would not be observed to form crypt or a organoid.Conclusion:1.The culture of intestinal epithelium in common medium depends on cell mass of crypt.Although there is no real cell mass of crypt structure in fetal mice,the broken intestinal epithelial cell mass or single suspension cell usually cannot form intestinal epithelial cell cluster.2.The Lgr5 cells,academic regarded as the intestinal stem cells,have the ability to differentiate into different terminal intestinal epithelial cells under the support of crypt cell clusters.Once lost the connection between the crypt cells group,the differentiation ability of Lgr5 cells will become very limited,and single Lgr5 can not differentiate into a crypt or organoids.3.The number of Lgr5 cells in three-dimensional culture system was significantly higher than that in intestinal epithelium.According to the relevant research reports,the organoids have the characteristic of reliable and long-term stability in genetic material,and the influence of in vitro culturing and passaging of genetic material is very little.Therefore,it can be used as an good carrier for the study of intestinal stem cell function,as well as an excellent carrier of intestine for the study of inflammation,tumor,Intestinal flora and other related research. |
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