Investigation On Nicotine Regulating The Function Of HuMSCs Via The Pathway Of α7nACHR

Multiple Organ Dysfunction Syndrome (MODS) is generally recognized as the mostcommon cause of death in the surgical intensive care unit (SICU)，and the severity ofMODS correlates with mortality and length of hospitalization signiifcantly. The damage oftissues and systematic inlfammatory response syndrome (SIRS) are the most importantpathogenesis of MODS which remains to further research. Therefore，we aim to investigateeffective treatment on MODS in this study.Mesenchymal stem cells (MSCs) are mesoderm-derived stem cells and in a suitablemicro-environment, MSCs have the ability of self-renewal and multi-potency, which coulddifferentiate into cells of the mesodermal lineages and other embryonic lineages, includingadipocytes, osteocytes, chondrocytes, hepatocytes, neurons, muscle cells and epithelialcells.Furthermore, MSCs have the property of immunomodulatory. Therefore MSCs couldinhibit the production of pro-inlfammatory cytokines and promote the release ofanti-inflammatory cytokines.Because MSCs have several advantages, such as easyavailability as well as few ethical concerns and low immunogenicity. In addition, MSCsalso have extensively proliferative properties in vitro while maintaining theirundifferentiated multipotent status.These properties make MSCs an ideal candidate celltype for tissue engineering, regenerative medicine and autoimmune disease treatment.Nicotinic acetylcholine receptors a-7(a7nACHR) is a ligand gated ion channelreceptors, which is an important component of the vagus nerve activated “cholinergicanti-inflammator"y pathway. When it is activated, a7nACHR could signiifcantly inhibitthe production of pro-inflammatory cytokines, including TNF-a, IL-1，IL-6，High mobilitygroup protein B1(HMGB1) and promote the release of anti-inflammatory cytokines,including TGF-P，IL-10. Nicotine could activate the both signal pathway which could playan important role in anti-inlfammatory during the phase of MODS.Nicotine is speciifc agonist of a7nACHR. Nicotine is more eiffcient thanacetylcholine in aspects of inhibition of the production of pro-inlfammatory cytokines andin aspects of promotion of the release of anti-inflammatory cytokines.So far, there is no related research reports have proved that a7nACHR exists on thesurface of the MSCs.Moreover, there is no related research reports have proved thata7nACHR also play a important role in regulating the function of MSCs. Our study is theifrst to test the existence of a7nACHR on the surface of the MSCs. In addition, our studyhave tested that a7nACHR also play a important role in regulating the function ofMSCs.When MSCs are pretreated by small does of nicotine, its immunomodulatoryactivity and anti-inflammatory activity strengthen. Our study provides a new theoreticalbasis for MSCs used in the treatment of MODS.There are two parts in our study: in the ifrst part, we use the standard method ofisolation, culture, identiifcation to test the function of proliferation and differentiationability of MSCs from human umbilical cord; In the second part, we have proved theexistence of a7nACHR on the surface of the MSCs by Western blot. We have also provedthat when MSCs are pretreated by small does of nicotine, its immunomodulatory activityand anti-inlfammatory activity strengthen. Nicotine could regulate the function of MSCs via the pathway of a7nACHR.Part1Isolation, Culture, Identification and Function ofMesenchymal stem cells from Human umbilical cordObjective: To optimize the isolation, culture, ampliifcation and identiifcation ofMSCs from human umbilical cord for further study.Methods:We isolated Mesenchymal stem cells from human umbilical cord bytissue isolation. The morphological characteristic of the cells was investigated by lightmicroscope and their expression of mesenchymal surface markers were analyzed by lfowcytometry.Their multi-differentiation potential was examined in vitro too.Results:MSCs could be successful obtained by tissue isolation.Mesenchymal stemcells derived from human umbilical cord were spinal-shaped adherent cells.They havestrong proliferation ability.They were strongly positive express for CD29, CD44, CD90andCD105，but negative expression for CD34，CD45and HLA-DR. After induction, oilstaining were positive and also could be induced to differentiate into adipocytes,osteoblasts.Conclusion:Mesenchymal stem cells can be isolated and cultured. Mesenchymalstem cells can be isolated from human umbilical cord. The cells can be cultured well invitro, and have the typical characteristics of the mesenchymal stem cell morphology, inaddition, the cells could be induced to differentiate into adipocytes, osteoblasts.Part2Investigation on Nicotine Regulating the Function ofUCMSCs via the Pathway of a7nACHRObjective: To prove that the a7nicotinic acetylcholine receptor exists on thesurface of MSCs. After pretreated by nicotine，how the functions of MSCs would be changincluding immunomodulatory activity, proliferation ability, migratory ability and adhesionfiinction.To lay further foundation of the application of mesenchymal stem cells in clinicaldisease.Methods:we tested the existence of a7nACHR on the surface of the MSCs by Westernblot, The collected AT cells of P3-MSCS were divided into3groups and we randomly addednicotine (0，5umol/L, lOumol/L,15umol/L,20umol/L,50umol/L) to the culture medium. Atfer pretreated2hours by nicotine,we collected MSCs respectively.Then we inoculated cells in24-wellplate. Atfer the donor signed informed consent, we collected peirpheral venous blood10ml fromthe healthy donor under sterile conditions. We isolated mononuclear cells rfom peirpheral blood.Atfer mesenchymal stem cells attached to the culture plate,we added to each well with humanperipheral blood mononuclear cells (2x106) and phytohemagglutinin (PHA)2ul. After a total of72hours of incubation, we centrifuged medium and collected supernatant. The IL-1and TNF-a levelsof the co-culture supernatant were defected using the ELISA. The collected AT cells of P3-MSCswere divided into6groups and we randomly added nicotine (0，5umol/L, lOumol/L,15umol/L,20umol/L,50umol/L) to the culture medium. Atfer pretreated2hours by nicotine, we collectedMSCs respectively. Then we inoculated cells in24-well plate, and detected the proliferation abilityof MSCs. The collected AT cells of P3-MSCs were divided into6groups and we randomly addednicotine (0，5umol/L, lOumol/L,15umol/L,20umol/L,50umol/L) to the culture medium. Atferpretreated2hours by nicotine, we collected MSCs respectively. Thus, we tested the migratoryability and adhesion function of MSCs.Results I We proved the existence of a7nACHR on the surface of the MSCs by Westernblot. We proved that atfer pretreated by small does of nicotine, MSCs can inhibit peripheral bloodmononuclear cells stimulated by PHA to secret TNF-a and MSCs can promote peripheral bloodmononuclear cells stimulated by PHA to secret IL-lO.The pretreatment condition could enhanceproliferation ability,the migratory ability and adhesion function of MSCs. As the dose ofnicotine increases, the function of MSCs is suppressed. This phenomenon indicates thatlarge does of nicotine may produce toxic effects on MSCs.Conclusion：We proved the existence of a7nACHR on the surface of MSCs.Thepretreatment condition of small does of nicotine could enhance proliferation ability, the migratoryability and adhesion function of MSCs, in a dose-dependent manner. As the dose of nicotineincreases, the function of MSCs is suppressed. This phenomenon indicates that large doesof nicotine may produce toxic effects on MSCs.Slimmary： The damage of tissues and systematic inflammatory responsesyndrome (SIRS) are the most important pathogenesis of MODS which remains to furtherresearch. Under the induction of local microenvironment, MSCs have the property ofimmunomodulatory.Therefore，MSCs could inhibit the production of pro-inlfammatory cytokines and promote the release of anti-inlfammatory cytokines.There are goodprospects for the application of mesenchymal stem cells in the prevention and treatment ofMODS, a7nACHR exists on the surface of the MSCs. Nicotine is speciifc agonist of a7nACHR. When MSCs are pretreated by small does of nicotine, its immunomodulatoryactivity and anti-inflammatory activity strengthen. And then, the effectiveness of thetreatment of MODS could be improved.