Effect of Hypoxia on the Gene Profile of Rat Neural Stem Cells and Human Bone Marrow-Derived Mesenchymal Stem Cells

Abstract: Hypoxia is the basic environment of life. The devepoment of embryo exits in hypoxia, and the concentration of oxygen both in the organim of brain and bone marrow of adult mammal are about 1~4%, which is also hyoxic environment. Hypoxia can induce the expression of hypoxia inducible fator 1 (HIF-1) in most kinds of cells and tissues, HIF-1 is one of the key transcription fator in response to hypoxia, its targeted genes are involved in cellular homeostasis, cell proliferation and differentiation et al.Neural stem cells (NSCs), is a kind of neural precursor cell, which has the capability of self-renewal and diverse potency of differentiation. It has a potential value on cureing degenerative disease and ischemia disease. Human bone marrow-derived mesenchymal stem cells (hMSC), is a kind of non-haematogenesis stem cell existing in the bone marrow. Because of its characteristic of none or little immunological reaction in the transplantation, it has been used extensively in the cell transplantation of Parkinson's disease and gene therapy.Based on our previous research—–continual hypxia (3%O_2) could promote theproliferation of NSCs and hMSCs in vitro, in the present study, we detected theeffect of genes expression of NSCs and MSCs under the hypoxia environment bygene chips. We also analysed the possible role of the differentially expressed genesby bioinformatics, and the molecular mechanism involved in the proliferation ofstem cells under hypoxia.1. Effect of hypoxia on the gene expression profile of NSCs.1.1. Effect of hypoxia on the number and functional classification ofdifferentially expressed genes in NSCs. We used the gene chip to analyse the effect of hypoxia on the differentially expressed genes during the proliferation.We firstly counted the number of the genes on different hypoxia time points and the number of genes of different function. There were total 5705 genes on the gene chip, and there are 131 differentially expressed genes at 24h and 72h of hypoxia. The number of differentially expressed genes was 71 at 24h, and 62 at 72h. The metabolisic gene class was the majority among all the differentially expressed genes, and the next was transportational、differentiation and adhesional gene class by turns.1.2. RT-PCR verification of the known HIF-1 targeted genes and proliferation related genesIn the 131 differentially expressed genes, we found 7 known HIF-1 targed genes (ldha、aldoa、eno1、gapdh、tpi1、pgk1、bnip3) and 5 genes (dnaja2、ccnb1、tgfβ2、ccng1、rgs7) which were related to proliferation and cell cycle. The concordance rate of the RT-PCR results with the gene chip Ratio of the 12 genes was 58.3%, and the 7 HIF-1 targted genes were mostly up regulated at the both two points.1.3. The bioinformatic analysis of the genes that could be HIF-1 targeted genes.Through the bioinformatically prediction, among the other predicted 124 genes, we found that there were 13 genes having HIF-1 targted sequence at 24h, and 12 genes at 72h, which may be related with signal pathway. We also found that the Ppp1cb has the HIF-1 targted sequence both on the 24h and 72h.From above data, we concluded that verification of 7 known HIF-1 targeted genes that were all up regulated at both 24h and 72h. The results supported conclusion of our lab's previous research -—hypoxia promoted proliferation of NSC through the HIF-1 pathway, and in the earlier period ,hypoxia stimulated NSCs to proliferate ;in the later period,hypoxia may modulated the apoptosis of NSCs.2. Effect of hypoxia on the gene expression profile of hMSCs2.1. Calculation of differentially expressed genes and functional classificationThe number of the differentially expressed genes was counted on different hypoxia time points, and the number of genes for different function was analysized. There were 21329 genes on the total gene chip. Among them, there were 282 differentially expressed genes, and we found that the number of differentially expressed genes was the most at 24h.2.2.RT-PCR verification of the known HIF-1 targeted genes and the continually changed genesWe found 4 known HIF-1 genes (pgk1、igfbp3、bnip3、tgfβ3) and 12 continually changed genes (hac、bnip3、eests、ndrg1、nefl、tfrc、his1、ddr1、trib3、ahnak、tnfrsf11b、txnip) in the 282 differentially expressed genes, they changed mostly at 36h and 72h. The total concordance rate was 84.6%.2.3. The bioinformatic analysis of the genes that could be HIF-1 targted genes.By the way of bioinformatical analysis, we predicted 12 continually changed genes, we found there were 3 genes having HIF-1 targted sequence which were his-1、tfrc、ndrg1.From above data, and we found 4 known HIF-1 targted genes had the same point of the 12 continously changed genes, they changed the most at 36h and 72h; By the prediction of sequence, we found 3 genes having the HIF-1 targted sequence. In summary, HIF-1 might play a kind of role in the proliferation of hMSC under hypoxia…
Key words: hypoxia; neural stem cells; human bone derived Mesenchymal stem cells; gene chip; HIF-1

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