| Experimental allergic encephalomyelitis (EAE) is considered as a primary demyelinated disease,characterized by wide demyelination and relatively reserved axons for long time. Not until the end of last century is it proved that there are axon injures in the EAE focus. And people do extensive research to explore measures to help myeline and axons regenerate,but make little progress. At present, there is no effective therapy.Generally, the therapy of EAE is as the following:(1) to activate the intrinsic repairment mechanism.(2) to provide extrinsic sheath cells.(3)to prevent the impairment of inhibitators in the environment on the nerve cells. Because the clinical trials have not succeeded to promote intrinsic myeline regeneration until now and the effect of intravenous immune globulin to resist harmful factors is not satisfying,people wish that cell transplanation solve the medical problem. Cell transplanation and neurotrophin can improve the micro-environment and promote injured axons and myelines to regenerate,which is the basis of gene therapy. It brings new hope for EAE.Olfactory ensheathing cells (OECs) have the characters during development period and plasticity ,such as promoting mylines and axons to regenerate,assisting regenerated axon to traverse impassable colloidal scar and micro-envioronment in central nerve system(CNS),inducing the nerve to grow in the right place. Moreover, originated from CNS, the course of OECs integrated with CNS and migrating is very natural. Besides, OECs, characterized by availability,easily survival, acceptability and expressing extro-gene for long term is the ideal place for recombination gene multiplication. Therefore,OECs is the ideal supplier of transplanation and the ideal receptor of gene transfection.Neurotrophin-3(NT-3) as the most effective member in the nerve nutrition family has extensive physical function by connected with its receptors in corticospinal tract and big sensory axons,which is the aim in the EAE therapy. Besides, some researches find that NT-3 accelerates axons and myelines to differentiation and prolongation while BDNF accelerates neron body to develop and survive.The study is to apply ectogenetic Human olfactory ensheathing cells(hOECs) to gene project, inducting NT-3 gene to reverse transcription virus,transfecting hOECs to construct active OECs-NT-3 gene project cells and then treating EAE by transplanation. MethodshOECs are attained from olfactory ball of 3-6months abortion embryo by primary cultivation.Then cell positive rate is identified by P75,GFAP and SABC immunochemical dyeing.1. Cell purification is detected by immuochemical methods from different acquired ways, purified ways, cultivation utensils to obtain the most ideal procedure. The purified cells are applied to transplantation.2. PA317-NT-3 packed cell is constructed by constructing reverse transcription carrier PN2A-NT-3, transfecting NT-3 into PA317 with positive ion liposome and screening out positive clone by G418.hOECs are infected with high virus titer upper-liquor which is mensurated by NIH3T3 cell. then hOECs-NT-3 gene engineering cell is built up. 3. NT-3 protein secreted by hOECs-NT-3 along with the time is determined by ELISA. RNA in the upper-liquor is detected by RT-PCR to identify with expected NT-3 line. The cultivation of PC12-TrKC cells is to investigate the biological activity of expressive NT-3. the multiplication of hOECs-NT-3 is to investigate the effect of transfection on the life.4. hOECs-NT-3 cell transplantation is applied to treat EAE in mice,and its distribution and migration is observed in the host.5. The treatment of hOECs-NT-3 is observed from myeline repairment, axon regeneration,change of nerve conduction velocity,regressive protection on neuron and movement function resumption of limbs.6. Explore the repair mechanism of hOECs-NT-3 cell transplantation on EAE and propose the current problems and the settlements.Results1. While the purity of hOECs from human embryo olfactory ball could reach 90% during 2~8 day,.the activity of purified cells vary with the time which reach the highest 9~10 days after purification..After the identification of P75,GFAP immunofluorescence dyeing, the seed cells for transplantation could be confirmed.2. NT-3 of upper-liquor in transfection group is significantly higher than un-transfection group and vacant carrier transfection group, and it last until 28 days after transfection steadily.(conetent value at 3d,8d,13d,18d,23d,28d is 29.274+0.131,30.367+0.136,30.362+0.146,30.460+0.168,30.363+0.144,30.361+0.140ng/106 cell/24h ,P>0.05) two special strips,l774bp and 258bp are detected in the upper-liquor with RT-PCR,which is coherent to the expected. There is no significant difference in content value between transfection group and un-transfection group, indicating the transfection has no effect on the multiplication. The infected hOECs can promote the differentiation of PC12-TrkC,which shows that expressive NT-3 has biological activity. 3. It is found with immunofluorescence and ultrastructure morphology that hOECs-NT-3 surives,diffuses,migrates with axons and spreads in the focus diffusely after transplantation.The effect of tranfered gene group is better than hOECs therapy group,more nerve fibers,better myeline repair,more intact mitochondria and microfilament and more clear myeline structure.inflammatory focus in myeline group are obviously lower than hOECs therapy group(P<0.05).4. The number of the neuron signed by HRP in hOECs-NT-3 transplantation group is significantly higher than hOECs transplantation group at 28th day after transplantation.(1858±112.5,3561.5±296.5,p<0.01).5. The number of the nerve fibers in hOECs-NT-3 group (14d,28.8±2.2; 28d, 32.5±2.8)is significantly higher than hOECs group(14d,34.4±3.2;28d, 38.8±3.4)at 28th day after transplantation(P<0.05). the movement score is also obviously higher. There is positive correlation between them.6. The latent time and amplitude(20.6+0.3ms,74+9) are not significantly different from the normal in transplantation group(P>0.05),while the latent tim(e24.2+0.3ms) is obviously prolonged and the amplitude(21+2) is lower in contrast group(P<0.05).Conclusions1. hOECs-NT-3 gene engineering cells constructed with recombined reverse transcription virus,could express NT-3 protein steadily and exert theirs biological activity in the offspring of human olfactory ensheathing cells.2. hOECs-NT-3 could surive,diffuse,migrate with axons,spread in the focus diffusely and help myeline to renovate after transplantation,and demyelination makes chemotaxis of hOECs.3. hOECs-NT-3 transplanation may alleviate regressive neuron injury in cortex and promote the regeneration of axons in spinal cord in EAE.4. The number of nerve fibers and movement score are elevated and there is positive correlation between them .hOECs-NT-3 transplanation.could promote the resumption of movement function of limbs.5. The results of morphologic and electro-physiological researches show that hOECs-NT-3 transplanation could accelerate myeline to regenerate and improve the nerve conduction velocity.6. Gene mocificated hOECs secrete persistent and adequent NT-3,which play a coordinated role in EAE therapy.7. There are some problems in gene therapy of EAE applied to the clinic. What we do is a try or preparation for the next self-transplantation.
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