Study On The Effect And Mechanism Of Bone Mesenchymal Stem Cell Transplantation On 2,5-Hexanedione Induced Axonal Injury In Rats

Background:N-hexane is a kind of petroleum product,as well as an important kind of industrial organic solvent,which is widely used in the areas of mechanical cleaning preparation,industrial adhesive dispensing,printing,furniture manufacturing and electrical appliance manufacturing.N-hexane is volatile at room temperature,and has no obvious irritation or fat-solubility,so being exposed to a low concentration of n-hexane will lead to chronic poison with sensory-motory peripheral polyneuropathy as the main clinical characteristic.The issues of occupational health problems caused by n-hexane have been continuously reported in places like America,Canada,Brazil,South Africa and so on.However,the group-chronic-n-hexane-poisoning issues happen in our country from time to time.In 2010,workers of touch screen manufacturing department in the the Chinese factory,which belonged to Apple Inc.in the USA,were poisoned for the long-term access to the n-hexane,and this issue was thus widely reported in the society.Chronic n-hexane intoxication has become one of the most serious occupational diseases,which caused the high attention of the society and the media.The chronic n-hexane intoxication mainly caused harm to PNS,and the harm was resulted from HD,the final metabolite and ultimate toxicant of n-hexane.The symptoms are usually limbs anesthesia,pain,too weak to walk and quadriplegia might occur in serious cases.Many people thus have to stay on bed and lost the self-care ability of daily living,which causes the agony not only to the patients but also to their families,and severe economic burden to the family at the same time.But the prophylaxis and treatment of chronic n-hexane are confronted with great challenges now.Since there is no early-warning methods or effective preventive intervening means of chronic n-hexane at present,how to repair its niduses of nerves and how to restore its neruologic functions have been the difficulties in professional medical research.Stem cell is a kind of multipotential cell that has the ability of self-replicating.And under certain circumstances,the stem cells can differentiate into many different kinds of functional cells.BMSCs have the features of both stem cells and matrix cells,which means that they can be induced to differentiate into all kinds of histiocyte under suitable condition of culture and can secrete different cytokines.And BMSCs are easy to acquire and cause little harm to normal tissues,so they have become the major source of the cells for the study of MSCs.In 2001,a scholar used suspensions of bone marrow cells to restore the injured myelin sheath of axon,and this proved that there is possibility for restoring the damage of nervous system with MSCs.In 2009,another scholar successfully restored the brain damage of the rats caused by CO by using MSCs.The result was that the rat’s learning ability was greatly improved,showing that MSCs might help to restore the function of impaired nervous system.So,can MSCs help to restore the impaired nervous system of the patients caused by n-hexane intoxication? If so,what is their restoring mechanism? And all of those mentioned above are the concerns of the contents of this research.The first part: The restoring function of BMSCs transplantation to the damage of axons of the rats exposed to HD.Objective: To discuss the restoring function of BMSCs transplantation to the damage of axons of the rats t exposed to the HD.Method S: In this experiment,there are altogether five groups: Control group,BMSCs group,HD group,HD+NS group,HD+BMSCs group.The experiment evaluated the restoring function of BMSCs transplantation to the damage of axons of the rats that are exposed to the HD through the detection of behavioral change and electrophysiology of the coccygeal nerve of the rats in each group;as well as the observation of the morphological change of their spinal cord and scatic nerve with the help of HE staining,immunofluorescent staining,and TEM.Results:Gait scores,incubation period of conduction and conduction velocity of their coccygeal nerve,the fluorescence intensity of SMI312 as well as the diameter of axons of their spinal cords and sciatic nerves of the rats in HD+BMSCs group increased significantly compared with HD group(p<0.05).Conclusions: 1.BMSCs have the ability to restore the axonal injury of the rats that are exposed to HD.2.BMSCs have the ability to restore the abnormal neurological functions of the rats exposed to HD.3.The role of BMSCs may be related to the ability of restoring the axonal injury of the rats that are exposed to HD The Second Part: BMSCs has the ability to restore the axonal injury of the rats that are exposed to HD through a NGF-dependent manner.Objective: To discuss that BMSCs can promote NGF secretion which has the ability to restore the axonal injury of the rats that are exposed to HD.Methods: The gene and protein expressions of NGF and GAP-43 of spinal cord and scatic nerve in each group were detected by PCR and western blot.The concentration of NGF were detected by ELISA.The protein expressions of Trk A/p-Trk A were detected by western blot.The influence of anti-NGF and K252 a to the protein expression of GAP-43 of DRG were also detected by western blot.The influence of anti-NGF and K252 a to the axon length of DRG were detected by immunofluorescent staining.Results: The gene and protein expressions of NGF and GAP-43 in spinal cord and scatic nerve,the concentration of NGF as well as the protein expression of p-Trk A of the rats in HD+BMSCs group were higher compared with HD group(p<0.05).The protein expression of GAP-43 as well as the axon length of DRG of HD+CM+anti-NGF group and HD+CM+ k252 a group were lower compared with HD+CM group(p<0.05).Conclusions: 1.BMSCs can greatly improve the level of NGF of the rats that are exposed to HD.2.BMSCs can activate the NGF specific receptor(Trk A)of the rats that are exposed to HD 3.BMSCs can repair the axonal injury of the rats that are exposed to HD,which is mediated by NGF.The third part: The study on the mechanism of the repair effect of BMSCs in a NGF-dependent manner on the axonal injury of the rats exposed to HD.Objective: To discuss the mechanism of the repair effect of BMSCs in a NGF-dependent manner on the axonal injury of the rats exposed to HD.Methods: The protein expressions of AKT/p-AKT,m TOR/p-m TOR and CREB/ p-CREB of spinal cord and scatic nerve were detected by western blot.In vitro study,the influence on the protein expressions of AKT/p-AKT,m TOR/ p-m TOR,CREB/ p-CREB,GAP-43 and the axon length of DRG by k252 a,MK-2206 and rapamycin were detected by western blot and immunofluorescence staining.Results: The protein expressions of p-AKT,p-m TOR,p-CREB of spinal cord and scatic nerve of HD+BMSCs group were higher compared with HD group(p<0.05).The protein expression of p-AKT and GAP-43 as well as the axon length of DRG of HD+CM+ k252 a group was lower compared with HD+CM group(p<0.05).The protein expressions of p-m TOR and GAP-43 as well as the axon length of DRG of HD+CM+MK-2206 group were lower compared with HD+CM group(p<0.05).The protein expressions of p-CREB and GAP-43 as well as the axon length of DRG of HD+CM+rapamycin group were lower compared with HD+CM group(p<0.05).Conclusions: 1.BMSCs can greatly improve the level of p-AKT,p-m TOR and p-CREB of the rats exposed to HD.2.BMSCs can activate AKT/m TOR/CREB signaling pathway.3.BMSCs can repair the axon damage of the rats exposed to HD through NGF dependent AKT/m TOR/CREB signaling pathway.