Experimental Study On Overexpression Of Lentivirus Transfected Hair Follicle Neural Crest Stem Cells

Objective:Glioma is a common malignant tumor in the brain.Its invasive growth characteristics pose great challenges for surgical treatment.Due to the existence of the blood-brain barrier,many chemotherapy drugs and targeted therapeutic drugs cannot fully play.Efficacy.Sema4D is a member of the Semaphorins signaling family.It relies on binding to the transmembrane protein Plexin-B1 to affect cell frame stability,promote cell migration,induce T cell activation,affect B cell survival,and promote vascular endothelial cell production.Sema4D and Plexin-Bl Interactions have been shown to affect the migration and proliferation of a variety of tumor cells.VX15/2503 is a human-derived IGg4 monoclonal antibody,which has been widely used in anti-tumor therapy in recent years.It has achieved gratifying results in Phase I clinical trials in Europe.VX15/2503 acts on the binding site between Sema4D and Plexin-B1,competitively inhibits the binding of Sema4D to Plexin-B1,thereby inhibiting the dispersion and distant metastasis of tumor cells.Hair follicle neural crest stem cells(hfNCSCs)are located in the bulge region of the outer root sheath of the hair follicle in the skin and are stem cells derived from neural crest.Most mammalian hair follicles are widely distributed,so the source of the material is not a problem for hfNCSCs.The hair follicle bulge is easier to use than other sources of stem cells,making it easy to carry out large-scale cell culture.Moreover,hfNCSCs are derived from the neural crest structure and are more advanced than other adult stem cells,so their canceration tendency is also smaller.Because of its material in the body hair follicles,immune reactivity and ethical issues are relatively small.For this reason,hfNCSCs can be an ideal seed cell for gene therapy,tissue engineering,and new drug development.In this study,hfNCSCs were transfected with the over-expressed lentiviral vector CD-100 containing the VX15/2503 gene sequence,and the morphology of the cells after transfection was observed to identify the expression products of the transfected cells.Gene therapy for stromal tumors provides a new way of thinking.Methods:(1)Primary culture of the extracted hair follicle neural crest stem cells was carried out by using the hair bud Bugle area tissue block adherent culture method,and the cell type was identified by immunofluorescence after cell passage.(2)The transfected hair follicle neural crest stem cells were transfected with CD-100 overexpression lentiviral vector containing EGFP green fluorescent protein gene constructed by Jikai Gene Chemical Technology Co.,Ltd.,and the transfection was observed under inverted fluorescence microscope.The cell culture supernatant after transfection was collected.(3)The cell culture supernatant after transfection was subjected to enzyme-linked immunosorbent assay to identify its expression product.Results:(1)The hair follicle neural crest stem cells cultured from the tissue block extracted from the Bugle region of the outer root sheath of rat hair follicles have good activity,fast cell migration speed,strong proliferative ability,and diverse cell morphology,which are compared with slender spindle and triangle.More common,immunofluorescence identification showed Nestin,p75 positive.(2)Lentiviral transfection was carried out on the passage cells.Under the inverted fluorescence microscope,the green fluorescent protein of the transfected cells was clear and the transfection efficiency was high.The cell activity was less affected after transfection.(3)The cell culture supernatant after transfection was identified by enzyme-linked immunosorbent assay(ELISA).The results showed that the protein expression of the experimental group was significantly up-regulated compared with the control group,and the transfection was successfully confirmed and the product expression was successful.Conclusion:(1)The tissue blocks extracted from the Bugle region of the outer root sheath of the rat hair follicle can culture hair follicle neural crest stem cells.(2)The CO-100 overexpressing lentiviral vector constructed based on the monoclonal antibody VX15/2503 protein sequence can successfully transfect hair follicle neural crest stem cells.(3)The hair follicle neural crest stem cells can be successfully transfected to express the product having the monoclonal antibody VX15/2503 protein sequence.