Isolation,Identification And Subculture Of Fetal Bovine Jejunal Crypt Stem Cells

The crypt plays a very important role in the regeneration of intestinal epithelial cells,but it is difficult to establish a method for the cultivation of intestinal crypt stem cells.At present,methods for isolation and culture of mouse intestinal stem cells have been reported,and the passage cell lines thereof have not been successfully obtained.The isolation and culture system of large animal intestinal crypt stem cells has not been reported yet.In addition,due to the complexity of intestinal microbes in adult animals,the isolation of intestinal stem cells is seriously affected.Therefore,this study established a primary culture model of Fetal bovine jejunal crypt stem cells and obtained a biologically stable fetal jejunal crypt stem cell line.In this experiment,3-6 months old fetal cattle were selected as the research object,and a new primary intestinal stem cell culture model was successfully established.The biological function stable fetal jejunal crypt stem cell line was obtained and successfully passed to the first The 40th generation laid the foundation for the establishment of an isolated culture model and its stem cell line in vitro for other animals.The in vitro 3D culture method was preliminarily established to lay a foundation for further study on the effects of EGF,LPS and VD on the division,proliferation and differentiation of j ej unal crypt stem cell model through Wnt/β-catenin signaling pathway.The main test contents and results are as follows:1.Morphological analysis.The morphological structure of duodenum,jejunum,ileum,colon and cecum was observed by transmission electron microscopy,scanning electron microscopy and HE staining.The crypt cells were located,which laid a foundation for the subsequent isolation of jejunal crypt stem cells;2.Fetal bovine jejunal crypt stem cells were isolated and subcultured.The proximal jejunum(approximately 10 cm long)of 3-6 month old fetal calf was selected as the test sample.Under sterile conditions,the jejunal mucosa was scraped with a surgical blade or a sterile coverslip to remove the upper mucosa and villi.After scraping again,the cells were filtered with a 70 μm cell sieve to obtain unfiltered cells.DMEM cultured with different concentrations of FBS was added.The effect of base on fetal jejunal crypt stem cells.Four FBS concentrations were used:5%,10%,15%,20%DMEM medium.When 5%FBS was added to DMEM medium,the cultured fetal jejunal crypt stem cells had weak proliferation ability and colony.The addition of 10%FBS showed strong proliferation,colony,high purity and biological traits.Stable cells;cells with strong proliferative ability,obvious colony,high purity and stable biological characteristics when added with 15%FBS;cells with strong proliferative ability,obvious colony,high purity and stable biological characteristics when added with 20%FBS,But mixed with many miscellaneous cells.The results showed that when the concentration of FBS added in DMEM medium was 15%,it was the best concentration of fetal jejunal crypt stem cell culture;the isolated fetal jejunal crypt stem cells and their passage cells had good developmental characteristics,and Successfully passed the passage cells to the 40th generation so far;3.Identification of fetal jejunal crypt stem cells.By immunohistochemistry and transcriptome sequencing analysis,it was shown that Lgr5,Bmi1,LRIG1,CD 166,MSI1,DCLK1,CD133,CD24 have different degrees of expression in fetal jejunal crypt cells,so the cultured cells are jejunum.Crypt stem cells;4.Isolation and culture of myofibroblasts and their subculture.The proximal jejunum(approximately 10 cm long)of 3-6 month old fetal calf was selected as the test sample.The jejunal mucosa was scraped with a surgical blade or a sterile coverslip under sterile conditions to remove the upper mucosa and villi,and the tissue was shredded to 1 mm3 after scraping again.The results showed that the isolated fetal jejunal crypt cells and their passage cells had good morphological and biochemical characteristics and successfully passed the passaged cells to the 10th generation;5.Effects of different additives on fetal jejunal crypt stem cells.The addition of 0,10,50 ng/ml EGF,0,10,100,1000 ng/ml LPS and 0,10,100,1000 nM VD DMEM complete culture was compared by cell scratch test and Western Blot technique.The effect of base on fetal jejunal crypt stem cells.The results showed that when 10 ng/ml of EGF was added,the recovery rate of damaged jejunal crypt cells was accelerated,which promoted the repair of damaged cells.Adding 10 ng/ml of LPS inhibited the growth of fetal jejunal crypt cells.The addition of 10,100,1000 nM VD could inhibit the growth of fetal jejunal crypt cells,and the addition of 1000 nM VD inhibited the growth rate of fetal jejunal crypt cells;6.In vitro 3D culture.Matrigel is extracted from the amniotic membrane and mixed with the crypt suspension to form a matrix gel droplet.The results showed that the jejunal organs formed and grew well after 7 days of culture.This study has initially established an in vitro 3D culture method.By locating and detecting crypt cells,in vitro isolation and culture,subculture,immunohistochemistry and transcriptome sequencing analysis,the cultured cells were identified as fetal jejunal crypt stem cells,and fetal jejunal crypt stem cells and their passages were obtained.Cell line;the effects of different additives on fetal jejunal crypt stem cells were compared by cell scratch test and Western Blot technique;the in vitro 3D culture method was established by extracting Matrigel from amniotic membrane.To lay a foundation for the establishment of isolated culture models and stem cell lines in vitro for other animals.