Construction And Identification Of Antibacterial Mesenchymal Stem Cells

Sepsis is a systemic inflammatory response syndrome(SIRS) attributed to an infection. Sepsis is an extremely fast-paced and high mortality disease. It is the leading cause of death in sever burns, trauma and an advanced stage cancer patients, there is no effective therapy for sepsis yet. The current treatment plan, according to the Surviving Sepsis Campaign, is consist of anti-infection and supportive care that almost related to all organs.The good news is that the mesenchymal stem cells(MSCs) may be a promising therapeutic measure for sepsis. Many evidences confirmed that MSCs do not only have immunomodulatory and anti-inflammatory function, but also have definitive regeneration and tissue repair function. Applying MSCs transplantation to treating spinal cord injury, myocardial ischemic injury, acute lung injury, acute brain injury and femoral head necrosis tissue injury disease showed a good effect. In addition, it has been found that MSCs transplantation was also effective in treating sepsis. MSCs can secrete cytokines such as TGF-β, IL-10, IL-13 and TSG-6 which can regulate inflammation; On the other hand, MSCs also can interact with dendritic cells, NK cells, T and B lymphocytes, then control behavior of these cells in inflammatory circumstance. In animal experiment, MSCs transplantation showed a significant treatment effect for sepsis. The mortality is decreased and inflammatory reaction is limited.Bactericidal/ permeability-increasing protein(BPI) is an anti-bacterial peptide existing in the the primary granules of neutrophils with a molecular weight about 55 kDa. It can kill Gram negative bacterial and neutralize LPS. rBPI21 is a 21 kDa recombinant protein composed of the N? end part of BPI. Comparing to the wild type BPI, r BPI21 is more stable and powerful the anti-bacterial effect. According to the studies based on animal models, rBPI21 helps animals to against pneumonia, sepsis and other infectious diseases. Furthermore, the anti-infection effect has been ascertainin clinical trials.LL-37 is the only member of Cathelicidin protein family in human beings and can be secret by a variety of tissue and cells. It has been found can kill pathogenic micro organisms including both Gram positive and negative bacteria, fungi and viruses. Moreover, immune regulation and wound healing functions have been found in recent studies.Based on the characteristic and current treatmentsituation of sepsis, we propose a treatment strategy that “anti-bacterial, anti-endotoxin anti-inflammation and organ protection in one-shot ” in this study. And we are going to seek a promising drug that correspond with this strategy.Research Purpose:1. Construct a fusion protein composed of r BPI21 and LL-37. We suppose this fusion protein possess anti-bacterial, anti-endotoxin and immunomodulation capability.2. Using the fusion protein to modify MSCs, We suppose the genetically modified MSCs possess anti-bacterial, LPS neutralizing, anti-inflammatory and tissue repair capacity.Method:Amplified the BPI and LL-37 gene from cDNA, then modified them into a correct fragment through the PCR method. Using restriction enzyme and DNA ligase construct the fusion expression vector. In this fusion protein, BPI protein and LL-37 protein are linked by a flexible fragment(GGSGG). Then transfected the expression vector into the A549 cells. Detected the fusion protein expression through Western blot. Tested the anti-bacterial ability of fusion protein through the antibacterial assay.Once the fusion protein was constructed successfully, Cloning the fusion protein gene from expression vector to lentiviral vectors. Then packaged the lentivirus. Using the virus infected the human umbilical cord mesenchymal stem cells(h U-MSCs) to construct the genetically modified MSCs. Identifying the surface marker of modified MSCs with flow cytometry. Testing the expression of fusion protein with Western blot. Usinganti-bacterial assayto identify the antibacterial ability of modified MSCs.Results:1. Determined by restriction enzyme, DNA electrophoresis and DNA sequencing, successfully construction of the fusion protein expression vector(p LVX-BPI-LL-37) was confirmed; Western blot showed that r BPI21-LL37/LL37-r BPI21 fusion protein can be expressed at secretory mannerin A549 cells, fusion protein molecular weight ranged from 25 to 30 k Da; Anti-bacterial assayprove the fusion protein can inhibit the growth of bacteria.2. h U-MSCs was successful separation and culture. And its surface marker match the relevant standards. And these cells has multi-differentiation potential.3. The fusion protein expression lentivirus was packaged successfully. And h U-MSCs can be infected by this lentivirus. The genetically modified MSCs have the standard MSCs phenotype measured by flow cytometry. Western blot showed that the modified MSCs expressed fusion protein at secrectory manner, and the supernatant possess antibacterial ability.Conclusion:1. Construction of r BPI21-LL37/LL37-r BPI21 fusion protein expression vector is successfully. The fusion protein can be expressed at secrectory manner after the vector transfected into the A549 cells. And a in vitro experiment confirm that this fusion protein possess anti-bacterial effect.2. Genetically modified MSCs have a normal stem cell phenotype, can express and secrete antibacterial fusion protein. The MSCs culture supernatants containing secreted substances possess antibacterial ability.