Antitumor Effect And Mechanism Of Sheep Umbilical Cord Mesenchymal Stem Cells On Melanoma Cells

Mesenchymal stem cells are a type of pluripotent stem cells that can be isolated from bone marrow,umbilical cord,fat and other tissues.It has the advantages of multidirectional differentiation,low immunogenicity,tissue repair,homing to damaged tissues and tumor sites,etc.,and has been applied to the treatment of various diseases.At present,it has been proved that MSC can secrete cytokines in vivo and in vitro,and regulate the relevant signal pathways of tumor cells and the physiological characteristics of tumor microenvironment to affect tumor occurrence and development.Malignant melanoma is a malignant tumor in which melanocytes become cancerous and produce local infiltration.The incidence is low,but the incidence is rapid,the rate of distant metastasis is high,and the prognostic survival rate is low.After traditional surgical resection,tumor tissue is easy to remain,and the recurrence rate is high.Melanoma will quickly develop resistance to commonly used chemotherapeutics and gene-targeted drugs.It can only increase the amount of drug and use it in combination,which is expensive and has strong side effects.Put a great burden on the quality of life of patients.Many experiments have confirmed that homologous MSC can inhibit tumor cells through paracrine effects.However,the research and use of human-derived MSC has a lot of ethical controversy,and the source and clinical application have many limitations.In this paper,sheep umbilical cord MSCs were isolated and cultured,and sheep umbilical cords were used.Mesenchymal stem cells and exosomes extracted from the culture supernatant of the cells,to study the effects of sheep umbilical cord MSCs on the cell biological phenotypes of human melanoma cells A375 and murine B16 through the paracrine pathway,The transcriptome was sequenced to analyze the potential mechanism of action,and exosomes were extracted by the ultra-isolation method to analyze whether they played a major role in the impact on tumors.The specific conclusions are as follows:1.Sheep umbilical cord MSC inhibits the proliferation and invasion of heterologous melanoma in vivo and vitroPrepare sheep umbilical cord MSC conditioned medium to interfere with malignant melanoma,and explore the effects of sheep umbilical cord MSC conditioned medium on the activity,proliferation,apoptosis,migration and invasion of murine and human melanoma cells.Edu and cell colony results show that the conditioned medium of sheep umbilical cord MSC can inhibit the proliferation of melanoma;Annexin V/PI and Tunel experiments show that the conditioned medium of sheep umbilical cord MSC can promote the apoptosis of melanoma and significantly increase the proportion of late apoptosis;The results of cell scratches and Transwell experiments show that the conditioned medium of sheep umbilical cord mesenchymal stem cells can inhibit the migration and invasion of melanoma.B16 cells were injected into the right axillary skin of nude mice to observe the effect of sheep umbilical cord MSC paracrine on tumor formation in mice.After 7 days,the tumor size was measured every 3 days until the end of the survival period.The tumors were collected for frozen section and HE staining.Results The nude mouse animal model found that sheep umbilical cord MSC can also inhibit tumor formation in vivo and change the internal structure and composition of tumor tissue.The above results indicate that sheep umbilical cord MSC can inhibit the activity,proliferation,migration and invasion of murine and human melanoma cells through paracrine,and promote the apoptosis of melanoma cells.2.Screening of target genes for sheep umbilical cord MSCs to inhibit the development of melanoma B16.RNA from B16 cells treated with sheep umbilical cord MSC and control group was collected to explore the potential mechanism and target of action of sheep umbilical cord MSC in inhibiting the occurrence and development of melanoma through transcriptome sequencing combined with bioassay,so as to provide a new mechanism and idea for the inhibition of tumor by heterologous MSC.Using RNA-seq sequencing technology analysis found that sheep umbilical cord MSCS promote cell autophagy and inhibit the MAPK signaling pathway to promote apoptosis of cultured B16,raw letter analysis of RTK,Rab7,CTSB selection,DAPK,TP53INP2,CTSD,GF may participate in regulating the process of invasion of sheep umbilical cord MSCS inhibit B16 value-added.3.Extraction of MSC-derived exosomes and its effect on melanoma B16.The EXO in the supernatant of sheep umbilical cord MSC cells was extracted by the ultra-isolation method,and the effect of EXO at different concentrations on the proliferation and invasion of melanoma B16 was observed.To study whether it plays a major role in sheep umbilical cord MSC inhibiting the occurrence and development of melanoma through the paracrine pathway,provides a theoretical basis and application strategy for the treatment of melanoma by MSC.The results found that low concentration of exocrine extract promoted the proliferation and migration of B16,and with the increase of concentration,the proliferation and migration of B16 increased;until the inoculation concentration of exosomes reached 50uL/mL,the inhibitory effect on melanoma began to appear,indicating that the sheep umbilical cord MSC inhibits the occurrence and development of melanoma not through the secretion of exosomes,but through the synergistic effect of multiple cytokines.In summary,this paper confirms that sheep umbilical cord MSCs inhibit human and murine melanoma cell invasion and promote apoptosis through paracrine pathways in vivo and vitro.The anti-tumor signal pathway and related target genes of sheep umbilical cord MSC were discussed.At the same time,it was verified that the anti-tumor effect is not the main effect of exosomes,but the final result of the cross-action of multiple signaling pathways and cytokines.This study is expected to provide a new method for the clinical treatment of melanoma and improve the survival rate of patients.