Establishment And Functional Verification Of Extraction Method Of Matrix Glue From Different Tissues Of Fetal Cattle

Objective:Matrix gel is a kind of soluble basement membrane matrix extracted from extracelluLar matrix,which can be used as biological scaffold material for three-dimensional cell cuLture.Matrix gel used for three-dimensional cell cuLture can better simuLate the growth environment of cells in vivo,so that cells can give fuLl play to their functions and characteristics.Matrix gel is characterized by aqueous solution at 4℃ and gel at 37℃.In this experiment,the skin,amniotic membrane and umbilical cord of 3-6 months old fetal cattle were taken as the research objects,and matrix gel was extracted from them,in order to establish and verify the effect of the gel in three-dimensional cell cuLture and screen better biomaterials for three-dimensional cell and organ-like cuLture.Method:1.The amniotic membrane tissues of 3-6 months old fetal bovine skin,umbilical cord and placenta are crushed and subjected to the steps of centrifugation,dialysis and the like to obtain an extracting solution,and the extracting solution is subjected to gel test,gradient detection,cell detection,nuclear staining,OD value detection and water content detection of the extracting solution.2.The stem cells in the jejunal recess were isolated and cuLtured,and the isolated cells were subjected to nuclear staining,stem cell-specific protein Lgr5 protein staining and epithelial cell-specific protein keratin 18 staining,and cell growth performance was measured.3.The stem cells from the crypt of jejunum were inocuLated into the skin gel,amniotic membrane and umbilical cord extract for cuLture,and the effects of the extract on the expressions of CD 133,Lrig 1,CD 166,CD24,LGR5,MSI1 and DCLK1 were determined by fluorescent quantitative PCR.4.The uterine epithelial cells and the jejunal crypt stem cells were cuLtured in three different extracts for 48h,and the effects of the extracts on the expression of jejunal crypt stem cells E2F1,RRM2,IGFBP5,IGFBP6 and ORC1 as well as on the expression of uterine epithelial cells E2F1,RRM2,CD40,c-Myc,MCM2,1GFBP5,IGFBP6,ORC1,Caspase-9,Caspase-3 and P53 were determined by quantitative PCR.ResuLt:1.The skin extract is an aqueous solution at 4 C and forms a gel at 37 C;The OD values of the skin stock solution and the extract at 50%concentration increased in an S-shaped manner with time;The average water content of the skin extract was 96.88%.Amniotic membrane and umbilical cord extracts at each concentration did not gel at 4 C and 37 C;The OD value of the extracts at each concentration decreased with the passage of time,and there was no gel property.The average water content of the umbilical cord extract from the amniotic membrane extract was 98.53%and 96.23%,respectively.No cells or nucleic acids were found in the extracts.2.The cells isolated and purified in the experiment showed positive expression rate of above 98%after nuclear staining and staining of Lgr5 protein and keratin 18 protein,confirming that the cells isolated were fetal bovine jejunal crypt stem cells and had good cell growth performance.3.The stem cells of jejunal crypt were cuLtured in skin gel,and their morphology changed to an irregular shape,and they formed a tubuLar structure in space,and certain contact was also established between cells;The fluorescent quantitative PCR results showed that the expressions of CD 133,Lrig 1,CD 166,CD24,LGR5,MSI1 and DCLK1 were significantly up-reguLated(P<0.001).In the amniotic membrane extract group,the cell morphology changed from round granuLar or trianguLar differentiation into shuttle-like or long strip-like.Fluorescence quantitative PCR resuLts showed significant inhibition of DCLK1 expression(P<0.001),significant up-reguLation of CD24 and MSI1 expressions(P<0.01),and up-reguLation but not significant elevation of Lrig1,CD133 and CD166 expressions(P>0.05).The stem cells in the jejunal recess of the umbilical cord extract group were slightly deformed in morphology.Fluorescence quantitative PCR resuLts showed that the expressions of CD133,CD166,Lgr5 and MSI1 were significantly up-reguLated(P<0.01),while CD24 was not significantly down-reguLated(P>0.05),and Lrigl and DCLK1 were significantly down-reguLated(P<0.01).4.The jejunal crypt stem cells were cuLtured with skin gel,amniotic membrane and umbilical cord extract,respectively,and the expression of related genes was detected.The resuLts showed that the expression of E2F1 in the amniotic membrane extract group and umbilical cord extract group was significantly higher than that in the skin gel(P<0.01).The expression levels of RRM2 in the skin gel group and the umbilical cord extract group were significantly up-reguLated(P<0.05).IGF-BP5 was significantly increased in the amniotic membrane extract group(P<0.001),and significantly decreased in the skin gel group(P<0.01).IGF-BP 6 was significantly inhibited in the umbilical cord extract group(P<0.01).ORC1 expression levels in the skin gel group and the umbilical cord extract group were significantly increased(P<0.05).5.The uterine epithelial cells cuLtured with skin gel,amniotic membrane and umbilical cord extract respectively showed that the morphological cells of uterine epithelial cells cuLtured in skin gel changed from round particles to complex and irreguLar shapes,while the morphology of uterine epithelial cells in the amniotic membrane and umbilical cord extract group was not large.Fluorescent quantitative PCR showed that the expressions of E2F1,CD40,c-Myc,MCM2,P53,IGF-B5,IGF-B6,Caspase-9 and Caspase-3 in the skin gel group were significantly inhibited(P<0.05),but the expressions in RRM2 and ORC1 were not significant(P>0.05).The expressions of CD40,c-Myc,MCM2,P53,IGF-BP 6 and Caspase-3 were significantly down-reguLated in the amniotic membrane extract group(P<0.05),but the expressions of E2F1,IGF-BP5 and ORC1 were significantly up-reguLated(P<0.05).There was no significant difference in the expressions of RRM2 and Caspase-9(P>0.05).The expressions of E2F1,RRM2,CD40,c-Myc,MCM2,IGF-BP 5,IGFBP 6 and Caspase-3 in the umbilical cord extract group were significantly inhibited(P<0.05),but the expressions of P53,ORC1 and Caspase-9 were not significantly inhibited(P>0.05).Conclusion:The matrix gel extracted from fetal bovine skin in this experiment has the characteristics of matrix gel,and can well maintain the functionality of crypt stem cells cuLtured in skin gel.The amniotic membrane and umbilical cord extracts are inferior to skin gel in this respect.The experiment of cuLturing fetal bovine jejunal crypt stem cells and uterine epithelial cells in skin gel confirmed that the skin gel can be used as a carrier for three-dimensional cuLture of cells and organs.The action mechanism of skin gel,amniotic membrane and umbilical cord extracts on cell proliferation and apoptosis and other biological functions of matrix glue need further study.