The Isolation Of Two Kinds Of Mesenchymal Stem Cells And The Modification Of Cxcr2 Were Used In The Experimental Study Of LPS-induced Sepsis In Mice

Sepsis is a life-threatening organ dysfunction caused by a dysregulated immune response to infection.It is the leading cause of death from infection worldwide.More than30 million people worldwide suffer from sepsis,and as many as one in four or more die.The treatment of sepsis has placed a huge burden on the global health care industry,especially in low-income and middle-income countries.Currently,the treatment of sepsis is mainly the use of antibiotics and supportive therapies which have limited therapeutic effects in systemic inflammation and organ dysfunction caused by sepsis.Therefore,there is an urgent need for clinical and scientific researchers to explore and develop more effective treatments.In the past decade,cell therapy has become one of the new hopes for the treatment of sepsis.Mesenchymal stem cells(MSC)are adult stem cells that are widely present in almost all tissue matrices such as peripheral blood,bone marrow,fat,umbilical cord,cord blood,and placenta.They have the ability to rapidly proliferate,differentiate,and self-renew and replicate,and induce differentiation.MSC can induce differentiation into a variety of human cells,including osteoblasts,chondrocytes,fat cells,muscle cells and so on.The most striking feature of MSC is its strong immunomodulatory effect,and its abundant source,easy separation and in vitro expansion make it an ideal seed cell for cell therapy.In recent years,a number of experimental studies have shown that MSC can regulate the pathophysiological process of sepsis,and MSC-based cell therapy is a new means of treatment for sepsis.The main mechanism is that MSC acts as a potent regulator of immune response,which restores the balance between innate and adaptive immune responses;inhibits bacterial proliferation and cytokine storms in sepsis through antibacterial and anti-inflammatory properties;reduces cell death and tissue damage.Although MSC showed a certain potential in the treatment of sepsis,the apoptosis of MSC would be induced when MSC were transplanted into the body under disease conditions,such as hypoxia,serum deprivation,inflammation,heat shock or oxygen free radicals.In addition,only a small fraction(0.001-1%)of intravenous MSC can reach the site of inflammation.The decrease in homing ability may be due to imperfect culture conditions in vitro or multiple passages of MSC,resulting in decreased expression of chemokine receptors on the surface of MSC.In view of the limited number of homing target tissues after MSC transplantation,the therapeutic and anti-inflammatory molecules produced from MSC are not sufficiently potent.Recently,a number of studies reported that the genetically modified MSC could increase the therapeutic efficacy of MSC.CXC type chemokine receptor 2(CXCR2)belongs to the G protein which is a coupled receptor family and is a ligand for the chemokines CXCL1,CXCL2,CXCL3,CXCL5,CXCL6,CXCL7 and CXCL8.Many chemokines have been upregulated in the inflammation site.In recent years,some studies have shown that MSC can effectively recruit inflammatory sites toexecute the corresponding biological effects after overexpressing chemokine receptors.In this experiment,mouse bone marrow and human umbilical cord-derived MSC were isolated and modified with cxcr2 gene to explore whether they could enhance MSC homing ability,and then observe the therapeutic effect in LPS-induced sepsis animal model.The main experimental methods and results of this study are as follows:1.Evaluation of mouse bone marrow MSC isolation,cxcr2 gene modification and treatment of sepsis induced by LPSBone marrow mesenchymal stem cells(BMSC)of BALB/c mice were isolated by whole bone marrow adherence method.The third generation BMSC were obtained by passage and purification.BMSC were identified by morphological observation,flow cytometry,and osteogenic differentiation.The results showed that the isolated BMSC reach the standards made by International Stem Cell Association.At the same time,the RNA of whole bone marrow cells was extracted and reverse transcribed into cDNA as a template.The cxcr2 fragment was amplified by PCR using the primer designed by mouse cxcr2 in Genbank,and inserted into the lentiviral vector pLenti-GZ constructed in our laboratory.It was identified by enzyme digestion and submitted to the company for sequencing.The recombniant whose sequence was correct was named by pLenti-cxcr2-GZ.The pLenti-cxcr2-GZ was co-transfected into HEK293 T cells with the helper plasmids psPAX2 and pMD2.G.The lentiviral supernatant was collected,and the lentivirus was concentrated and purified with 20% sucrose as a cushion.BMSC were infected by centrifugation and screened to obtain CXCR2 stably transfected with BMSC(CXCR2-BMSC).Flow cytometry and RT-PCR results showed that CXCR2 was highly expressed in CXCR2-BMSC.Transwell experiments show that the cxcr2 gene-modified BMSC has better migration ability than the unmodified BMSC.In 6-8 weeks old BALB/c male mice,an animal model of sepsis was established by intraperitoneal injection of LPS.The expression levels of chemokines cxcl1,cxcl2,cxcl5,cxcl9,cxcl10,and cxcl12 mRNA were significantly up-regulated in multiple organs of the LPS-induced sepsis model.After treatment with MSC in the tail vein,compared to GZ-BMSC,the number of tissues reaching the lung tissue of the CXCR2-MSC transplantation group was significantly higher,indicating better tissue targeting.GZ-BMSC,CXCR2-BMSC transplantationgroup can alleviate inflammatory factor levels,pathological damage and lung dry-to-wet ratio to some extent,but no significant enhancement effect was observed in the CXCR2-BMSC transplantation.2.Evaluation of human umbilical cord MSC isolation,cxcr2 gene modification and treatment of sepsis induced by LPSThe umbilical cord of the full-term newborn were obtained from the pregnant based on informed consent of maternal and family members,and the umbilical cord mesenchymal stem cells(hUCMSC)were isolated and cultured according to the tissue block adherence method.When cultured to the third generation,MSC was identified by morphological observation,flow cytometry,and three-line differentiation.The results showed that the isolated hUCMSC reached the standard of mesenchymal stem cells established by the International Cell Therapy Association.The hUCMSC was infected by the above-prepared pLenti-cxcr2-GZ virus,and the expression of CXCR2 in CXCR2-hUCMSC was detected by flow cytometry and real-time PCR,which the positive cell expressing CXCR2 was about 78%.The proliferation of CXCR2-hUCMSC was improved,the cell cycle was unchanged,and the molecular markers were consistent with the characteristics of mesenchymal stem cells.The results of transwell experiments showed thatthe migration ability of CXCR2-hUCMSC was significantly enhanced,and the number of CXCR2-hUCMSC reaching the target tissues was also increased in vivo,compared to hUCMSC.The 6 to 8-week-old BALB/c male mice were injected intraperitoneally with LPS to prepare the sepsis model.GZ-hUCMSC and CXCR2-hUCMSC both reduced inflammatory factor levels and lung tissue damage.CXCR2-hUCMSC group was slightly obvious.In summary,two kinds of MSC were isolated in this study.Their biological characteristics are consistent with international stem cell standards.MSC modified by cxcr2 gene do not change the biological characteristics of stem cells,both in vitro and in vivo experiments showed that MSC from two different sources significantly increased stem cell homing to target tissues after CXCR2 modification.In LPS-induced sepsis animal experiments,both BMSC and hUCMSC improved sepsis-related symptomscompared to LPS group,while CXCR2-modified BMSC showed no significant enhancement compared to BMSC alone,it may be related to the decrease of immunomodulatory property of BMSC during passage;CXCR2-hUCMSC enhances its therapeutic effect compared to hUCMSC alone.This study provides an experimental basis for the treatment of sepsis with CXCR2 modified MSC,and its therapeutic efficacy needs to be further improved.