| Objective: To analyze the curative effect of joint treatment of preconditioned hypoxic neural stem cells of bone marrow and BDNF on the model rats with infarction, and to study the characteristics of BMSCs-NSCs in vitro and in vivo. The study would provide animal experimental bases for cell-based transplantation therapy of cerebral infarction in plateau areas.Methods:Seventy-two SD rats were randomly allocated into 2 groups, hypoxia group and normoxia pretreatment group. The Rats in hypoxia-treatment group were indwelled in hypobaric cabin for 3 hours each day for 3 days, which simulates an acute hypoxia circumstance at an altitude of 5000 ms in height. The model rats with well-established cerebral artery MCAO/R were divided into 3 groups, each consisting of 12 rats. Group A: transplanted with MSCs-NSCs combined with BDNF; Group B: transplanted with MSCs-NSCs; Group C: injected only with DMEM/F12 culture medium as contrast. Under the guide of stereotaxy neuronavigation, they were injected into the ipsilateral caudate nucleus of occultation lesions respectively. Neurological function scoring was performed on 3d, 7d, 14 d, 21 d, 28 d and 35 d, including balance beam test, walking test and grasping test. The rats were then sacrificed to collect the brain for morphological and histological study of the differentiation of bone marrow derived neural balls, to evaluate the therapy efficacy.Results:1. MSCs-NSCs were identified by CD133 and Nestin staining. The cells derived from bone marrow mesenchymal stems in vitro were cultured and induced with basic fibroblast growth factor, epidermal growth factor and B27 induced from neural balls.2. MSCs-NSCss expressed positively, after being stained by immunofluorescent staining of Nestin, MAP-2, GFAP and Galc antigens, which indicated MSCs-NSCss of SD rats could be differentiated as neurons, astrocyte and oligodendrocyte in vitro.3. The contents of FBS in culture medium correlated with the speed of cell division. The number of neurons in culture medium containing 10% FBS and 10% FBS+50ng/m L BDNF were 22±2.4 and 1±1.7(P < 0.01), indicating BDNF being able to improve the ratio of neuron differentiation.4. On the 14 th day after transplantation, neural function in the rats was significantly improved, and on the 21 st day after transplantation, the number of the immigrated Badu(+) cells reached peak in the occultation lesions. The cells were mainly composed of GFAP(+) astrocytes.5. There was no significant difference between the hypoxia group and the corresponding oxygen group(P>0.05)Whether hypoxia group, and normal oxygen group, BMSCs-NSCs+ BDNF group between the detection indexes were significantly higher than those of BMSCs-NSCs group(P<0.05); BMSCs-NSCs+BDNF group, BMSCs-NSCs graft group, the indexes were higher than those of the control group(P<0.05).Conclusions:1. BMSCs can be induced to differentiate into MSCs-NSCs in vitro. They have multilineage properties in vitro and in vivo, thus can be induced differentiation into neuro, oligodendrocyte and astrocytes.2. There existed a correlation between MSCs-NSCs’ division rate and FBS concentration in culture medium in vitro. BDNF can significantly improve differentiation proportion of neurons both in vitro and in vivo, and can increase the MSCs-NSCs synapses in vitro, the number and length of synaptic connections, and can promote functional synaptic connections in vivo.3. On the 14 th day after transplantation, nerve function of the rats acquires obvious improvement, while on the 21 st day differentiated MSCs-NSCs gather most in the infarct area. Addition of BDNF can significantly improve the efficacy of transplantation.4. Hypoxia preconditioning does not promote the differentiation of exogenous MSCs-NSCs, but can significantly improve the neurological function of rats. While hypoxic preconditioning treatment may promote the proliferation and differentiation of endogenous MSCs. |
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