Study Of Shh Signaling Pathway On Machnism Of MSCs Differentiating Into Neural Cells At Rat

Marrow stromal cells (MSCs) contain some multi-potent, mesenchymal progenitor cells that can differentiate into neuron and glia both in vitro and in vivo. The ability to differentiate into a neural lineage attracts much interest in the possible applications of MSCs in cell and gene therapy for neurological diseases. MSCs can be obtained from bone marrow easily and expanded rapidly in vitro. However, application of MSCs was confined because we cannot obtain adequate neural cells from MSCs in vitro by the current technique. Key point of resolving this problem is to study and interpret mechanism of MSCs directional differentiation.The Sonic hedgehog(Shh) signaling pathway is consisted of Shh, Ptc, Smo, and downstream zinc finger transcription factor Gli protein. When there is no Shh, Ptc inhibits activity of Smo, and thus inhibits the expression of target genes. Once the Shh protein binds to Ptc, the inhibition of Smo is relieved and allows Gli protein to enter cell nucleus and induce the expression of target genes.During organisma multicellularis development process, Shh signaling pathway participates the regulation of cell proliferation and differentiation. It is still unknown whether the Shh signaling pathway has similar effect on MSCs? As a result, this experiment aims to study mechanism of MSCs differentiation into neural cells by means of Shh signaling pathway.In part I, MSCs were isolated and cultured in vitro by the whole bone marrow adherence method. Biological characteristics of MSCs were then identified. The results were as following:Proliferation activity of the third generation MSCs were already stabilized. 95.18% and 94.36% of the third generation cells showed positive CD54 and CD44 (MSCs surface markers) expression respectively. The percentage of hematopoietic lineage marker CD14 decreased obviously from 23.19% in primal culture of the cells to 2.37% in the third generation.The result showed that the third generation cells can be used as the seed-cells for study of cells induction and differentiation and as the resource of cells transplantation.In part II, effect of bFGF and EGF, inducing MSCs to differentiate into neural cells was studied by morphology, immunofluorescence, and Western Blot. The results were as following:1. Large non-adherent spherical cell masses were noticed on day 5 after induction by bFGF and EGF. We also noticed that single cell, which was separated from primary spherical masses , proliferated and formed secondary spherical masses. Interestingly, some cells migrated to the periphery of spheroid masses after a few hours. More importantly, the migrating cells appeared to comprise a network of cells within five days. On day 5, those cells seemed to exhibit neural characteristics with multi-polar processes and growth cone-like features.2. Nestin was detected by immunofluorescence method, whileβ-tublin III, NSE, MAP-2, GFAP, and GalC were not. However, nestin expression appeared to decrease with prolonged culture. In contrast, differentiated cells derived from neurospheres expressed several specific neuronal markers, includingβ-tubulin III, NSE, and MAP-2, as well as the astrocyte marker GFAP, and the oligodendrocyte markerβ-tubulin III, NSE GalC. A considerable portion of cells (15-55%) expressed and MAP-2 on day 5. However, GalC was detected in relatively small number of cells. We also noticed that the NSE-positive cells usually had smaller cell bodies, with one or two long processes and a growth cone.3. Western blot study showed that level of nestin protein rapidly decreased, but NSE, MAP-2 ,and GFAP protein level increased ,β-tubulinw?ith time after plating neurosphere in differentiated medium. However III protein level increased at day 1, but decreased at day 5.The results indicate that MSCs can be effectively induced to form neurospheres by bFGF and EGF. The neurospheres may amplify and differentiate into neurons, glial cells, and oligodendrocytes.In partⅢ, expression of Shh and correlated signaling molecules was studied by immunofluorescence, Western Blot and real-time PCR methods. The results were as following:There were strong expression of Shh, Ptc1, and Gli-1 mRNA and protein for MSCs.The result shows Shh signaling pathway exists in MSCs.In part IV, effect of MSCs differentiation into neural cells on Shh signaling pathway was studied by immunofluorescence, Western Blot and quantitative real-time PCR methods in vitro. The results were as following:1.The expression of Shh, Ptc, Gli-1, Nestin, and NSE mRNA was enhanced significantly in induced group than control group(P <0.01, P <0.05).2.The expression of Gli-1 protein was located in cytoplasm in control group, and nucleus in induced group by immunofluorescence. The result indicates that Gli-1 protein can generate nucleus translocation.3. Western Blot method showed that there was expression of Shh, Ptc, Gli-1,Nestin, and NSE protein before MSCs induced. It was increased significantly after MSCs induction than before induction(P<0.01). Gli-1 protein in cytoplasm decreased significantly(P<0.01)and increased significantly(P<0.05) in nucleus after MSCs induction than before MSCs induction.These results show that Shh signaling pathway was activated when MSCs were induced to differentiate into neural cells.In part V, effect of MSCs differentiation for Shh and cyclopamine was studied by Western Blot and quantitative real-time PCR .The results were as following:1.The expression of nestin mRNA in induced group and solvent group was increased than normal group after MSCs were induced for 48 hours(P <0.01).It was decreased significantly in Shh and cyclopamine group than induced group(P<0.01),but there was increased in cyclopamine group than Shh group(P<0.05).2. The expression of NSE mRNA in induced group, Shh group, and solvent group was increased than normal group after MSCs were induced for 48 hours(P<0.01).It was also increased in cyclopamine group than normal group(P<0.05). It was increased significantly in Shh than induced group(P <0.01),but there was decreased significantly in cyclopamine group than induced group(P<0.01). There was decreased obviously in cyclopamine group than Shh group(P<0.01).3. The expression of nestin protein in induced group, cyclopamine group, and solvent group was increased than normal group after MSCs were induced for 48 hours(P<0.01). There was no difference in Shh group and normal group. It was decreased significantly in Shh and cyclopamine group than induced group(P<0.01),but there was increased in cyclopamine group than Shh group(P<0.05).4. The expression of NSE protein in induced group and Shh group was increased more obviously than normal group after MSCs were induced for 48 hours(P<0.01). It was also increased in solvent group than normal group(P<0.05). There was no significant difference in cyclopamine group and normal group. It was increased significantly in Shh group than induced group(P<0.01), but there was decreased significantly in cyclopamine group than induced group(P<0.01). There was decreased obviously in cyclopamine group than Shh group(P<0.01).The results show there are expression of nestin, NSE mRNA and protein after MSCs were induced 48 hours. Shh can promote MSCs to differentiate into neural cells, which is inhibited by cyclopamine.In partⅥ, cell vitality was assayed with CCK-8. LDH, MDA, GSH, NO and SOD were assayed to observe the role of Shh signaling pathway in the anti-oxidative effect of directionally induced MSCs. The results were as following:1. Cell vitality in groups after MSCs ischemic-anoxaemia was decreased than normal control group and induced group(P<0.05); cell vitality in Shh group was increased than normal-ischemic group and induced-ischemic group (P<0.05); cell vitality in Cyclopamine group was decreased than normal-ischemic group and induced-ischemic group (P <0.05). The results show Shh may protect cells against ischemic- anoxaemia damage, and cyclopamine may attenuate the protective effect of Shh.2.NO and LDH release rate, level of GSH, MDA and SOD in normal ischemic group, induced group, solvent group and cyclopamine group were increased than that of the normal control group and induced group (P<0.01). There is no significant difference between Shh group, normal control group, and induced group for NO release rate, LDH release rate, and level of MDA(P>0.01),but they were significantly decreased compared with the normal ischemic group(P<0.01).GSH and SOD in Shh group were increased than all other groups(P<0.01).The result showed Shh may inhibit cells to release NO, LDH and MDA, but promote cells to release GSH and SOD. NO, LDH, and MDA in Cyclopamine group increased significantly, but GSH and SOD did not increase significantly. The result showed Cyclopamine may promote cells to release NO, LDH, and MDA, but inhibit cells to release GSH and SOD.These results indicate that the activation of Shh signaling pathway may relieve oxygen radicals induced cells damage and increase cell vitality; the inhibition of Shh signaling pathway may increase oxygen radicals induced cells damage and decrease cell vitality.