Proteomic Effects Of Homocysteine On The Proliferation Of Fetal Neural Stem Cells In Vitro

ObjectiveThis research focus on two directions:(1) observing the effects of homocysteine on proliferation of fetal neural stem cells, the proteome changes and the potential mechanism in vitro;(2) building HuSH shRNA Plasmid-mediated RNAi technique system.MethodsThe NSCs taken from hippocampal tissues of rat fetuses (14-16days) were cultured in serum-free medium in vitro and divided into four groups:vehicle control, folic deficiency group (Folic-D, medium contained0.65μg/mL folic and no Hey), middle-dose Hey group (Hcy-M,4μg/mL folic and100μmol/L Hey), and high-dose Hey group (Hcy-H,4μg/mL folic and300μmol/L Hey). The proliferation of NSCs was determined by MTT test. The number of newly formed neurospheres per well was counted under phase microscopy72h after seeding. To measure neurosphere size, the area of the newly formed neurospheres (at least50neurospheres/well were measured) was estimated. Percentages of dead cells among the neurospheres were determined24and72h after seeding.The NSCs were collected at6th day of culture to extract the protein.2D electrophoresis plus mass spectrum were used to detect proteome change in different groups.Real-time PCR was used to determine the mRNA level of the protein which level have been changed in proteome. The activities of ATP, SOD, MDA, ROS and mitochondrial enzyme complexes respiratory chain NADH-Q oxidoreductase (CⅠ), butanedioic acid-Q oxidoreductase (CⅡ), cell pigment oxidoreductase (CⅢ) and cell enzyme cytochrome oxidase (CⅣ) were determined. Two complementary oligonucleotides were designed with hairpin loop for MTHFR siRNA according to the design principle.Transfection, cloning and screening of HEK293cell. MTHFR, CBS mRNA and protein expression were detected by Real-time PCR and Western Blotting.ResultsThe morphological results showed that NSCs were damaged after administration of Hey. MTT test showed that in different time point, proliferation of NSCs in control group was much higher than in Hcy-M and Hcy-H group (P<0.05). Treatment of cultures with100μmol/L and300μmol/L Hey significantly reduced the size of the neurospheres formed without affecting their number. After disaggregation of the neurospheres, the total number of cells was significantly lower in Hcy-treated cultures (P<0.05). The effect of Hcy on neurosphere size and total number of cells was concentration-dependent (P<0.05). However, the percentage of nonviable cells was not changed by treatment with Hcy (P>0.05). Proteome essay showed the protein that NSCs expressed have a great change after administration of Hcy. From the protein which expression level have been changed, we choosed seven protein to do identification and analysis by HPLC-ChiP-MS/MS. Six protein were identified successfully. Two protein were cytochrome b-c1complex subunit2and aconitate hydratase, which have involved in respiratory chain and tricarboxylic acid cycle in mitochondria; which prompted us that Hcy might affect NSCs proliferation by interferencing mitochondria function. We further examined ATP, SOD, MDA and ROS level; the level of ATP and SOD were decreased by treatment of Hcy (P<0.05), while the product of MDA and ROS were elevated by treatment of Hcy (P<0.05). Meanwhile the activity of mitochondrial respiratory chain enzyme complexes NADH-Q oxidoreductase (CⅠ), butanedioic acid-Q oxidoreductase (CⅡ), cell pigment oxidoreductase (CⅢ) and cell enzyme cytochrome oxidase (CⅣ) were significantly decreased by treatment of Hcy (P<0.05). We built a HuSH shRNA Plasmid-mediated RNAi technique system.ConclutionThis research showed Hcy could inhibit the proliferation of NSCs, interference the form of neurosphere and proteome of NSCs. Hcy could reduce the expression level of cytochrome b-c1complex subunit2and aconitate hydratase both in mRNA and protein level. Hcy could reduce the product of ATP. Hcy could reduce antioxidative capacity of NSCs by decreasing SOD activity but elevating MDA and ROS level. Hcy interferenced mitochondria function by decreased respiratory chain enzyme complexes. Build a HuSH shRNA Plasmid-mediated RNAi technique system.