Regulating Function Of TNF-α At EPCs Via The Pathway Ofα7nACHR

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells, whichparticipate not only the embryonic vascularization, but also the vascular neogenesis andrepairing process after endothelial injury after born. During inflammation and infection,EPCs are not only the participator, but also as the first damaged target cells, which lead tothe blood capillary damage and microcirculatory dysfunction or the first componentelement of MODS. Many animal and clinical experiments have already certificated thatwhile injuring, the EPCs are activated to proliferate, migrate, differentiate and turn intoendothelial cells (ECs). And then, the new ECs replace the damaged endothelial cells,repair the damaged vessel and cause the microvascular to regenerate. But if the body wasseriously injured, the regulation of EPCs would be disturbed, the damaged vessel andmicrovascular couldn’t be repaired, even further organ damage, eventually could lead toMODS.In recent years, transplantation of endothelial progenitor cell for treatment ofcardiovascular and cerebrovascular diseases, tumor angiogenesis, and traumatizedischemic disease had got great success, and that shows its broad application prospects. Butthere are some limitations in clinical applications: first, limited number of cells can notmeet the needs of the clinical treatment; second, long time to culture may lead to miss thebest time for treatment time.Nicotinic acetylcholine receptors (nAChR) α-7is a ligand gated ion channel receptors,which is an important component of the vagus nerve activated "cholinergicanti-inflammatory pathway". The human body can regulate the levels of tumor necrosisfactor(TNF), high mobility group protein B1(HMGB1) and other cytokines through thecholinergic anti-inflammatory pathway when inflammatory. Recent studies show thatα7nACHR do exist on the surface of the EPCs, and also play a very important role inregulating the function of EPCs.We want to see the difference of EPCs’ function regulated by TNF-α throughintervention of theα7nACHR. Then we can conclude that theα7nACHR is one mediator ofthe regulating of EPCs by TNF-α. So we can provide confidence for the clinical prevention,early treatment of MODS caused by any kinds of inflammation or injury, even we canmake the progress of MODS stop and backward.There are two parts in our study: in the first part, we use the standard method ofisolation, culture, identification and detection the function of proliferation, migration and vascular generating of EPCs from umbilical cord venous blood; in the second part, weobserve that whether the regulating of EPCs by TNF-α would change through addingagonist or inhibitors of α7nACHR. So we can learn the regulatory mechanisms of theTNF-α on EPCs.Part1Isolation, Culture, Identification and Function ofEndothelial Progenitor Cells from Umbilical Cord Venous BloodObjective: To optimize the isolation, culture, amplification and identification of EPCsfrom umbilical cord venous blood for further study.Methods: We isolated mononuclear cells (MC) from umbilical cord venous blood bydensity gradient centrifugation. Then we cultured the MC in accordance with the density of1×10~6/cm~2with specific culture solution for EPCs which contains cytokines and fetalbovine serum (FBS). After about14days, the P4-EPCs would be identified by thecharacteristics of cell ultrastructure, immunohistochemistry testing, flow cytometry testing,taking up Dil-ac-LDL and FITC-UEA-1and vascular generating testing. We used theWestern blot to detectα7nACHR on endothelial progenitor cells.Results: After two days, the cells would become spindle-shaped and attach the bottomwhich were called attaching cells (AT cells). At the fifth day, the At cells gathered andgathered to become colonies, about7-9days later, the AT cells would be covered with thebottom. In P4-EPCs we had seen Weibel-Palade body through electron microscope. Morethan80%EPCs could both take Dil-Ac-LDL and FITC-UEA-1. Immunohistochemicaltesting showed that: CD133(+), CD34(++), CD31(+++), KDR (+++). Flow cytometrytesting showed that: CD13319.14±4.06%, CD34:45.08±6.15%, CD31:79.62±11.24%,KDR:85.32±12.09%.22.92±8.62/HP EPCs could be seen generating vascular. Theadherence rate was53.04±4.80%, and migration rate was14.41±1.65%. Western Blotshowed thatα7nACHR do express on endothelial progenitor cells.Conclusion: We can get sufficient endothelial progenitor cells with morphologicalcharacteristics and typical functions from umbilical cord venous blood, providing technicalsupport for further study.Part2Regulating Function of TNF-α at EPCs via the Pathway ofα7nACHRObjective: To investigate that the nicotinic acetylcholine receptor was the pathway of regulating function of TNF-α at EPCs.Methods: The collected AT cells of P4-EPCs were divided into ten groups with thedensity of1×106/ml, adding TNF-α (100mg/L) at every group. We randomly selected fivegroup, adding nicotine (0,10-8,10-6,10-4,10-2mol/L), marking group A1-A5; the otherfive added methyllycaconitine (0,10-8,10-6,10-4,10-2mol/L), marking group B1-B5. Atthe time of12h,24h and48h later, we would test the EPCs functions of taking upDil-ac-LDL and FITC-UEA-1, proliferation, adhesion and migration.Results: Flow cytometry testing result (positive rate): A1-A5: CD133:18.67±4.06%,CD34:47.08±7.08%, CD31:82.16±8.89%, KDR:89.02±8.28%; B1-B5: CD133:16.19±6.04%, CD34:48.12±6.34%, CD31:80.43±9.01%, KDR:87.24±9.12%. The functionof vascular generating: A1-A5:20.02±4.67/HP; B1-B5:10.38±7.55a/HP. Adhesionfunction (paste the wall rate): A1-A5:48.14±5.78%; B1-B5:29.08±5.12%. Migrationfunction (migration rate): A1-A5:12.82±1.44%; B1-B5:4.87±0.82%. Proliferationfunctions: A1-A5:18.87±4.45×10~4; B1-B5:4.12±1.02×10~4.Conclusion: TNF-α can lower the function of EPCs. And the results can be reinforcedby adding nACHR-α7inhibitors, weakened by addingα7nACHR agonists. Cholinergicanti-inflammatory pathway may be the way of regulatory mechanisms of TNF-α atendothelial progenitor cells.Summary: When the human body is suffered from inflammation or trauma, thedecreasing number of EPCs and their dysfunction may be the essential causing of MODSdevelopment. TNF-α, one of the main inflammatory factors, plays a critical role in thisprocess. The cholinergic anti-inflammatory pathway would be one important pathway ofTNF-α regulating the function of EPCs. Therefore we can infer that at the early time ofinflammation, usingα7nACHR agonists can block the damage of inflammatory cytokineson EPCs. So we can find the way to step down the morbidity and mortality of MODS andprevent the development even occurrence of the MODS.