Effects Of Lipopolysaccharide Preconditioning On The Transplantation Efficiency Of Bone Marrow Mesenchymal Stem Cells And Its Mechanism

Objective: To isolate, culture mesenchymal stem cells (MSCs) from bone marrow and identify MSCs. Then, to study its characteristics and potentiality to differentiate into myocardial cells.Methods: MSCs were isolated from femurs of C57BL/10J mice and then were cultured and passed on. The phenotype of MSCs were assayed by FACScan flow cytometer. Then , MSCs were induced to differentiate into adipogenic and osteogenic cells. MSCs of passage 3 were incubated with10μmol/L 5-Azacytidine(5-Aza ) for 24 hours and then cultured for another 4 weeks. The specific myocardial proteins (cardiac troponin I) expressed in 5-Aza-induced MSCs were assessed using immunocytochemistry with specific antibodies .Results: The MSCs of passage 3 derived from mice bone marrow were negative for CD14,CD34,CD45, and positive for CD29, CD44, CD105 when assayed by a FACScan flow cytometer. MSCs could be induced to differentiated into adipogenic and osteogenic cells.The cultured MSCs of third passage incubated with 5-azacytidine (10μmol/L) for 24 hours and then cultured without 5-azacytidine for another four weeks were able to express cardiac troponin I when detected by immunocytochemistry.Conclusion: MSCs could be isolated from the cells of bone marrow by passing culture. The adherent, fibroblast-like cells that derived from mice bone marrow expressed phenotype of MSCs and could differentiate into several mesodermal lineages. After incubated with 5-Aza, the MSCs were able to differentiate into myocardia-like cells, suggesting the promising perspective of MSCs transplantation after acute myocardial infarction.Objective: To study the effect of lipopolysaccharides on proliferation and VEGF secretion of mesenchymal stem cells in vitro , and investigate the function of TLR4/NF-κB signaling pathway.Methods:①The wMSCs of passage 3 derived from wild-type mice and tMSCs of passage 3 derived from TLR4 deleted mice were co-cultured with different dose of LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)for 48 hours. The proliferation of MSCs was assayed by MTT and then measured by O.D. at 450nm.②The wMSCs of passage 3 were co-cultured with different dose of LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)for 48 hours. The concentration of VEGF in supernatant of medium were measured by ELISA kit.③The cultured cells were divided into 4 groups: 1. wMSCs group;2. wMSCs+ 1.0μg/ml LPS group;3. wMSCs+ 1.0μg/ml LPS+PDTC group;4. tMSCs+ 1.0μg/ml LPS group. The concentration of VEGF in supernatant of medium were measured by ELISA kit and mRNA of VEGF were assessed by RT-PCR.Results:①Different dose of LPS could enhance proliferation of wMSCs, and 1.0μg/ml LPS attained best function. However, LPS could not enhance proliferation of tMSCs.②Different dose of LPS could enhance VEGF secretion of wMSCs, and 1.0μg/ml LPS attained best function.③When TLR4 gene was deleted and MSCs were exposed to inhibitor of NF-κB, MSCs expessed less mRNA and protein of VEGF.Conclusion: LPS of 1.0μg/ml could enhance proliferation of MSCs and stimulate secretion of VEGF at most degree. When TLR4 gene was deleted and exposed to inhibitor of NF-κB, MSCs expessed less mRNA and protein of VEGF. The effect of LPS on VEGF secretion of MSCs was via TLR4/NF-κB signaling pathway.Objective: To investigat whether LPS-preconditioned wild MSCs transplantation can ameliorate cardiac function and explore the underlying mechanisms.Methods: Acute myocardial infarction model was developed by left anterior descending coronary artery ligation. 80 rats were divided into 4 groups randomly and given an intramyocardial injection of one of the following treatments: 30μl PBS (control group), 3×106 wild MSCs/30μl (wMSCs group), 3×106 LPS-preconditioned wild MSCs/30μl (LPS-wMSCs group),or 3×106 LPS- preconditioned TLR4 gene deleted MSCs/30μl (LPS-tMSCs group). After 3 weeks, we used echocardiography to assess cardiac function and TTC methods to test the infarcted area. We used Masson’s trichrome to assess fibrosis, real-time PCR to evaluate the survival of engrafted MSCs, immunohistochemical assay to test the rate of apoptotic cardiomyocytes and density of blood vessel, and Western blot to analyze secretion of VEGF and phosphor-Akt.Result: After 3 weeks, in the 4 groups, LPS-preconditioned wild MSCs transplantation (LPS-wMSCs group) reduced LVDd、LVDs(P<0.01)and elevated LVEF and FS(P<0.01). Conpared with other 3 groups, LPS-wMSCs group reduced fibrosis of infarcted myocardium(6.6±0.5%,P<0.05) and reduced apoptosis of myocardium(14.8±1.5%,P<0.01). Vascular density was markedly increased in LPS-wMSCs group compared with other three groups(45.3±4.3,P<0.01). Survival rate of engrafted MSCs was elevated in infarcted heart in LPS-wMSCs group(as 1.89±0.10 times as wMSCs group,P<0.01) . Expression of VEGF and phospho-Akt was increased in the infarcted myocardium after transplantation of LPS- preconditioned MSCs.Conclusions: LPS preconditioning enhanced survival of engrafted MSCs, stimulated expression of VEGF and activated PI3K/Akt pathway. LPS preconditioning before MSCs transplantation resulted in superior therapeutic neovascularization and recovery of cardiac function. LPS preconditioning provided a novel strategy in maximizing biologic and functional properties of MSCs.