The Experimental Research Of VEGF Transfected Neural Stem Cells For The Treatment Of Cerebral Palsy In The Neonatal Rats

Cerebral palsy(CP) is a major disabling disease in motor function of the children.Currently CP has no specific treatment and management is limited only to the symptomatic support,so the CP is still a very important issue in pediatric neuro-developmental research.Neural stem cell (NSCs) has strong abilities of self renewal,proliferation and differentiation.Recently NSCs is considered to be an ideal carrier for gene therapy due to low-immunogenicity,integration into the host cell and expression of exogenous gene with prolonged stability.Vascular endothelial growth factor (VEGF) can act on vascular endothelial cells specifically and accelerate their proliferation, promote the formation of new blood vessels and increase permeability of blood vessels, in addition it is also act directly on nerve cells and displayed to neurotrophy and neurogenesis.Previous research has shown that the therapy of VEGF transfected NSCs transplantation has been used to treat limited cerebral ischemia of adult rat model.It was found that NSCs can express the VEGF gene products, and the therapy has protective effect on local blood vessels and nerves.So far,there is no report about research of VEGF transfected neural stem cells for the treatment of cerebral palsy in the neonatal rats. Objective:to study the Neuro-protective effect on the way of VEGF transfected neural stem cells for the treatment of cerebral palsy in the neonatal rats.Methods:Our laboratory has been successfully established recombinant lentiviral vector called pGC-FU-VEGF165 carrying VEGF165 and infected the NSCs which isolated and cultured in vitro.In this experiment,we constructed CP neonatal rat models as Rice way at first,and then identify the models.The 7-day SD rats were randomly divided into 5 groups:Controlled group(CON group),cerebral palsy animal model group(CP group),cerebral palsy animal model group with Buffer-transplanting(PBS group),cerebral palsy animal model group with the treatment of VEGF-NSCs transplanting(VEGF+NSCs group),cerebral palsy animal model group with the treatment of NSCs transplanting(NSCs group).PBS group,VEGF+NSCs group and NSCs group were separately injected into the 2μl buffer,recombinant lentiviral vector carrying VEGF transfected with NSCs suspension (multiplicity of viral infection being 50TU/cell and NSC cell number being 5*10^4/ul) and NSCs suspension(cell number being 5*10^4/ul)with stereotaxic instrument in the left sensorimotor cortex in rats after 3 days of the CP model constructed.Compare the daily weight gain and detect the expression of VEGF protein in model cerebral tissues thought VEGF immunohistochemistry method in 14-day old animals.In 3-4 weeks old of the animals,observe their nerve behavioral testing by Searching-water test in Radiational-maze,holding test and Foot faults test.In 35 days old animals,we observed the brain pathology by HE stain.Results:PART 1:The way of establishing CP animal model by ligating of unilateral common carotid artery combined with hypoxia in neonatal rats was succeed;the experimental results have shown varying degrees of brain damage in CP groups which leading to changes in the rats'behavior and pathology in brain tissue.PART 2:1.The weight growth rate determination:VEGF+NSCs group and NSCs body weight growth rate was significantly higher than CP group and PBS group(P<0.05);2.VEGF Immunohistochemistry result:The positive cells number of VEGF Immunohistochemistry in VEGF+NSCs group and NSCs group are significantly higher than the CP group and PBS group(P<0.05),it is the same in VEGF-NSCs group than NSCs group;3.Searching-water test in Radiational-maze:①Searching-water time comparing:The time seeking-water in VEGF+NSCs group and NSCs group are significantly shorter than the CP group and PBS group(P<0.05),VEGF-NSCs group shorter than NSCs group;②Error-times comparing:VEGF+NSCs group and NSCs group are significantly less than CP group and PBS group (P<0.05), VEGF+ NSCs group less than NSCs group(P<0.05);③Repetition-times comparing:VEGF+NSCs group and NSCs group are significantly less than CP group and PBS group(P<0.05);VEGF+NSCs group has no statistically significant difference from CON group;4.Holding test:The holding-time of VEGF+NSCs group and NSCs group are significantly longer than CP group and PBS group(P<0.05); 5.Foot faults test:The fault-times in VEGF+NSCs group and NSCs group are much lower than CP group and PBS group(P<0.05);6.HE staining:brain cells in CP group and the PBS group aligned disorderly,nerve cells have decreased, degeneration,necrosis and gliosis; nerve cells in VEGF+NSCs group and NSCs groups degenerated and necrosis reduced,karyopyknosis can be seen occasionally.Conclusion:The approach to NSCs transplant with VEGF-transfecting treated CP neonatal rats can increase the expression level of VEGF protein in neonatal rats,alleviate brain damage after ischemia and hypoxic, ameliorate long-termed functions of learning and memory,and had a neuroprotective function.