Study On The Effect Of Melatonin On Human Mesenchymal Stem Cells And Primary Investigation On The Mechanisms

Objective:To investigate the effects of melatonin(Mel)on proliferation and differentiation of mesenchymal stem cells(MSCs),to optimize the experimental parameters of proliferation and differentiation of MSCs;to research on the expression of Mel receptors subtypes of MSCs induced by Mel,to provide evidence for the experiments in vitro of MSCs.Methods:1.MSCs were purified by density gradient centrifugation method and passage method,and cultivated in L-DMEM containing 15%fetal bovine serum,and identitied with CD45,CD44 and CD 105 using FATS.2.The the sixth passage of MSCs were cultivated with adding different concentration Mel.The proliferation of MSCs was detected using MTT at the 12th hour,3rd day,5th day after adding;the expression of NSE,GFAP was detected by using immunocytochemical method and RT-PCR method,at the 24th hour,3rd day,5th day after adding.3.The expression of mRNA of MT₁ and MT₂,which are subtypes of melatonin receptors,was detected by using RT-PCR:extracting total NRA first,then reverse transcripting, then amplificating,then gel electrophoresising,and then gene sequencing.Results:1..MSCs can adhere the walls of culture flasks after postinoculation.The photonasty of MSCs is strong.Shuttle-like or polygon cells appeared after 3 days.MScs grew like settlements.Most MSCs of The 5th or 6th passage,look like homogeneous smechanocyte.The results of FACS are:CD44⁺,CD105⁺,CD45,which could improve the cells cultivated were pure MSes.2.The difference of the proliferation among the two experimental groups and the control group did not have statistical significance,contrast phase microscope.Observating under the phase microscope,we found the status of MSCs changed, while pole and multipolar cells emerged 24 hours after adding Mel.The results of immunocytochemical method showed the NSE express rate was 82.45±2.05%,and the control group was 5.3±0.15%,the difference of which had statistical significance.All groups did not express GFAP.3.RT-PCR:The total RNA was extracted to carry out RT-PCR.The results of agarose electrophoresis showed a strap,about 370 bp,which is approach to the primer of MT₁.The results of sequence showed the gene order acquired through amplification,is coincidence with the gene order in the human gene bank.Results:1.MSCs cultivate by density gradient centrifugation method and passage method,had high survival rate and could adhere the culture flasks' walls,and could generate more than 15 passage.2.Mel could induce MSCs to differentiate to neuro-like cells,but it could not influence the proliferation of MSCs.3.First time,this experiment proved that MSCs express MT₁mRNA,whose gene order is coincidence with the gene order in the human gene bank.The MT₂mRNA had not been detected.