LMP-1 Recombinant Lentiviral Vector Infection On Induction And Differentiation Of Human Placental-derived Mesenchymal Stem Cells(HPMSCs)

Purpose:Human placental-derived mesenchymal stem Cells is a new type of adult mesenchymal stem cells(MSCs)derived from human term placental tissue.HPMSCs have attracted many attention from scholars in recent years due to their convenient materials acquisition and abundant sources,their multi-directional differentiation potential such as osteogenesis and adipogenesis,and immunosuppression.As an intracellular non-secretory protein,LMP-1 has different levels of expression in the bone,lung,spleen,leukocyte and human periodontal ligament cells,dental pulp cells,and odontoblasts in humans and animals.It plays an important role in the mineralization of bone matrix and osteoblast differentiation.However,the specific mechanism of action of LMP-1 on osteogenic differentiation of HPMSCs remains unclear.This study intends to achieve long-term stable expression of LMP-1 in HPMSCs,and probe into the signal pathways of cytokines released during LMP-1promote bone formation.Methods:1.Tissue culture method to isolate and culture HPMSCs,to obtain relatively pure HPMSCs,and to test their osteogenic and adipogenic induction to determine their multi-differentiation ability.2.HPMSCs were infected with adenovirus and lentiviral vector systems respectively.The expression of GFP protein was observed under fluorescence microscope after 3 days and 7 days.The recombinant LMP-1 lentiviral expression system was constructed,HPMSCs were infected after packaging and purification,andstable transformation strains were screened.The expression of LMP-1 mRNA and protein in HPMSCs was detected by real-time quantitative PCR and Western Blotting.3.CCK-8 assay was used to detect the effect of LMP-1 on the proliferation of HPMSCs.One week after osteogenic induction,the expression of TGF-β1,Smad2,and Runx2 were detected by real-time quantitative PCR and Western Blotting.The differences in osteogenic differentiation ability of the three groups of cells in the blank control group,the negative control group,and the LMP-1 high expression group were compared.Results:1.Microscopic examination of HPMSCs showed a fibroblast-like morphology after passage to the third passage;14 days after osteogenic induction,alkaline phosphatase staining was observed in the blue-stained area;21 days later,alizarin red staining showed mineralized nodules,and lipid droplet formation was seen by oil red staining after adipogenic induction.2.The adenovirus infection rate was slightly higher than that of the lentivirus at 3days of infection,and the lentivirus still had a high expression rate at the 7th day,but the adenoviral vector was almost not expressed.The recombinant LMP-1 lentiviral expression vector was successfully constructed.After being packaged and purified,a stable transgenic strain was selected.The mRNA and protein levels of LMP-1 were highly expressed.3.The results of CCK-8 showed that the cells with high expression of LMP-1 had a decreased ability to proliferate,and the high expression of LMP-1 group had mineralization ability,ALP activity,osteogenic cytokines TGF-β1,Smad2,and Runx2 expression levels increased.Conclusions:1.The HPMSCs were successfully isolated and cultured,and they were identified as having multilineage differentiation capabilities,consistent with stem cellcharacteristics.2.The lentiviral vector has a higher infection rate expression than the adenovirus vector-infected HPMSCs.The recombinant LMP-1 lentiviral vector was successfully constructed,and the HPMSCs-LMP1 stable transfectant cell strain was selected.3.LMP-1 may promote osteogenic differentiation of HPMSCs by increasing the expression of TGF-β1,Smad2,and Runx2 in TGF-β1/Smad2 signaling pathway.