The Effect Of OPN On The Immune System Of The Pups And The Establishment Of A Stable Cell Line Of PTPRU Knockout Malignant Glioma

Previous studies have shown that Osteopontin(OPN)can enhance infant resistance to infection.However,the underlying mechanisms are remained to be explored.Here we study the effects of OPN on the development and functions of immune cells in infants fed with OPN-enriched formula milk compared with those with regular formula milk.The postnatal(P)7 rats were assigned randomly into 3 groups,whom are fed with OPN-enriched formula milk(OF),regular formula milk(RF)and breast milk(BF)respectively.After 21 days feeding,flow cytometry was used to analyze the different T-cell and B-cell subsets in the central and periphery lymphoid organs to check the development of the infant rat immune system.After immunization with thymus independent antigen LPS or thymus dependent antigen OVA,Indirect Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the plasma concentration of Lipopolysaccharide(LPS)or Ovalbumin(OVA)specific immunoglobulin to observe the immune response of the three infant rat groups.The results have shown that the proportion of B220+ B cells and CD3+ T cells of peripheral immune organs in OF group is closer to that in BF group compared with RF group.OF group exhibits a more similar T cell maturation process to that of the BF group compared with RF group.The proportion of two major T cells subsets namely CD4+ or CD8+ T cells in the periphery immune organs keeps intact in the OF group.As to the immune response to TI antigen,there are no significant differences in the concentrations of LPS-IgA,IgG and IgM between OF,RF and BF groups.After TD antigen OVA immunization,the concentration of OVA specific IgA and IgG in RF group is significant lower than that in BF group.Adding OPN in RF milk can alleviate this tendency.So OPN can affect the development of immune cells and improve the T cell dependent humoral immune responses in infant rats.Infant formula with high OPN content can function closer to breast milk than regular formula.Malignant glioma is the most common and most aggressive tumor in the central nervous system,but is still lack of effective treatments.Receptor type protein tyrosine phosphatase U(PTPRU)is a R2B transmembrane receptor protein belonging to the PTPs superfamily.Our previous studies have showed that PTPRU is required for glioma growth and motility,but the underlying mechanisms remain to be further studied.In present study,we used the CRISPR Cas9 to knockout PTPRU gene in malignant gliomal cell line U87 in order to study the specific effects of this gene on the occurrence and progression of malignant glioma.We designed the target sequence of PTPRU using Zhang Feng's sgRNA design website,and then transfected three plasmids linked to sgRNA together with a plasmid containing Cas9 into U87 cells using transfection reagent lipo3000.After the transfection,puromycin was used to screen out the monoclonal cells which successfully knocked out PTPRU gene.From the gene sequencing peak map,we find that the front and rear peak maps of the target sequence have undergone significant changes.By aligning the DNA sequence of the monoclonal U87 cells with the sequence of the PTPRU gene,it was found that the target sequence was knocked out.These results indicate that the CRISPR Cas9 system successfully knocked out the PTPRU gene in U87 cells.Taken together our work will provide a stable U87 cell line with PTPRU gene knock out to explore the function and mechanism of PTPRU on the occurrence and progression of malignant gliomas.