Research On MTOR Pathway Through The CD44to Promote Neuronal Regeneration In The Inner Mechanism

Its progression of diseases of the nervous system injuries often exhibits thedisease itself has a high degree of complexity. It is a major problem troubling themedical profession to treat the diseases of the nerve damage. Currently, there are twomethods of treating the diseases of the nerve cells in general, including alternativetreatments and improved local inhibitory nerve damage microenvironment. Becauseof the reason of the failed central nervous system regeneration is complex, the effectsof the treatment often cannot fundamentally solve the problem. Therefore, the key totreatment of diseases of nerve injury to clarify the intrinsic regenerative capacity ofneural mechanisms and explore related nerve axon regeneration pathways. In thisexperiment，the SY5Y cell is induced in vitro, make its have the feature of matureneurons, which is used as the study of neural cells in vitro. By using a RNAinterference technique to cut the PTEN gene expression of SH-SY5Y cells, whichmakes mTOR pathway activation, detect whether CD44molecule expression growsand its internal connection with the axon growth, further validates the CD44moleculerelated with the axon growth can pass the pI3K/Akt/mTOR pathway promoting axongrowth and improve the regeneration ability of neurons.Methods: SH-SY5Y cells is cultivated in vitro and induced to differentiationwith10μM ATRA (RTRA) for3days. The SH-SY5Y cell of the induced groupmorphological changes is observes by an inverted microscope and the control groupsis set up. The transcription and expression of the microtubule associatedprotein(MAP2) in the SH-SY5Y cells of the induced group and control group isdetected by RT-PCR and the immunohistochemical staining techniques and furtherchecked the differences in cell differentiation between the experimental group and thecontrol group. Then the PTEN （Chromosome10missing homologue gene proteinphosphatase and stress） RNAi is carried out for differentiation of mature cells, cutting the PTEN expression of the experimental groups and the negative controlgroup and the blank control group isestablished. Using the fluorescence microscope toobserve the cells of the experimental groups, the fluorescent transfection efficiency ofthe experimental groups is preliminary validated. The transcription of the PTEN inexperimental groups interfered after24h is tested by RT-PCR technology. The Ps6K(Phosphorylation ribosomal proteins) and mTOR (Rapamycin target protein)expression is further tested by western blot and at the same time the control groups isestablish for testing. The CD44transcription is tested by RT-PCR and compares withcontrol group. CD44expression level, whether present positive correlationrelationship with the axon growth is observed. CD44molecule, whether throughPI3K/Akt/mTOR pathway signal promote axon growth is validated by the observationresults. Finally, we will make some negative experiments， the induced cells aftersimultaneously CD44and PTEN genes interference, and observing the cell axonlength after cutting the two gene transcription.Result: RT-PCR results confirm that the MAP2transcription of induced cellsafter3days compared with control groups raises obviously and theimmunohistochemical method confirm that the number of positive cells in the inducedcells after3days compared with control groups also increase obviously. And then, theRNAi is used to operate the PTEN gene interfering for the induced SH-SY5Y cells,the transcription of the PTEN gene is tested by RT-PCR after24h. Compared withcontrol groups, the transcription is falling obviously. The expression of the mTOR andPs6K protein in cells after interfering is tested again after36h. Compared withnegative control group and blank control group, the expression of the mTOR andPs6K protein raise obviously, it is significant difference (p<0.05). After interfering thePTEN gene for24h, after repeating, results shows that the transcription of CD44protein raises in the interfering group. It is significant difference (p<0.05) comparedwith negative control group and induced group. And then the cell axon length indifferent groups is measured by using imageproplus6.0and the measurement data isstatistical analyzed. The results show that the cell axon length of the cells interfered after3days is longer than the control groups, it is significant difference(p<0.05).The opposite validation is to use the common interference of PTEN RNAiand CD44RNAi. The results show that the cell axon length of the PTEN interferedafter3days is longer than the common interference and induced groups, it issignificant difference (p<0.05).Conclusion: The activation of mTOR signaling pathway can through the CD44topromote neuronal regeneration, in order to promote neuronal regeneration.