Effects Of Melatonin On Proliferation And Differentiation Of Rat Spinal Neural Stem Cells In Vitro

Spinal cord injury is commonly encountered in clinical..Traumatic spinal cord injury can result in severe damage, leading to tetraplegia, or worse. Recently ,the research of SCI have made a great progress. The discovery of NSCs and the introduce of cell transplantion will bring splendid prospective and enhance our confidence in overcoming spinal cord injured-disease. This study is about in vitro cultivating, proliferation and differentiation of spinal cord-derived NSCs, it provide convenience and aliable seed cells. this reaearch lay an experimental foundation to the next research of transplant therapy to SCI .Part 1: To investigate the character and regularity of cultivation and in vitro inducement differentiation of spinal cord-derived NSCs from the embryonic rats. we isolated clone cells from E14 fetus rat's spinal cord through the methods of microscopic dissection,serum-free cell culture,these cells were subcultured in vitro. We also investigated the character of NSCs and its growth condition .The result shows that the spinal cord-derived NSCs are capable of proliferation and float in the culture fluid.repeated subculture.Without serum,NSCs grow rapidly and become neurosphere after48 or 72 hours.Immunohistochemistry shows that the isloate cells are nestin postive.and confirm to be NSCs.Part 2: In order to investigate the effects of melatonin (MT) on the proliferation and differentiation of cultured spinal cord-derived NSCs,Choice the 4th NSCs and.add different concentration of MT into culture fluid. The neural cell phenotypic antigens were identified by means of immune fluorescent assays,and the ratios between phenocytes were calculated. The result we found it that: the high and middle concentration of MT (100μmol/L and 1μmol/L ) enhance the proliferation of NSC,but 10 nmol/L MT have oppositic effect .All of the three concentration of MT can enduce NSCs to differentiate into neuron .The ration of neuron in A ,B and C group is,(45.3±1)%,(42.5±2)%and(50.7±2)%. while the contol group is (23.6±2)%.Part 3:On the base of part one , boasting the 4 th NSCs neurosphere into single cell and seeding it into 25 cm2 glass culture flask with cell density of 105.. Combine all-trans retinoic acid and melatonin and add into it .Afer observation and Immunohistochemistry, and statistics analysis,we make up some conclusion.ATRA only can't enhace the process of differentiation and the ratio of MAP2 positve cell didn't any difference between group A and Control.The combination of ATRA(0.1μmol/L) and MT (100μmol/L) made the most postive influence with the ratio of (53.9±2)%;while the another combine group also increase the neuron ratio .But the latter didn't show any statistical difference to the two group of single MT.