Intervention Of BMSCs Transfected With VEGF And Ang1 Gene On Hypoxic-ischemic Induced Neonatal Mice PVL Model

OBJECTIVE: To investigate the protective effect of BMSCs transfected with VEGF and Ang1 gene on hypoxic-ischemic induced neonatal mice PVL model.METHODS: The study include two parts.Part I: Establishment of hypoxic-ischemic induced neonatal mouse model of PVL and the change of VEGF and Ang1 levels in model brainIn this section,50 cases 7-day-old KUNMING neonatal mice were randomly divided into model group(30cases)and sham group(20 cases).We ligatured the right carotid artery of the mice in model group and raise them in a box,which filled with 6 % O2 + 94% N2,for 6 hours to constructing the PVL model.And the mice in sham group was separated the ipsilateral carotid artery only.Then,after 24 hours,48 hours,72 hours and 7 days,two groups of mice were sacrificed to take brain tissue for HE staining,immunohistochemical staining and RT-PCR test,to identification whether the modeling was successful and the ralationaship between the brain pathological and VEGF & Ang1 level.Part II: Intervention of BMSCs transfected with VEGF and Ang1 gene on hypoxicischemic induced neonatal mice PVL modelIn this section,100 cases of the PVL model mice were randomly divided into BMSCs-VEGF,BMSCs-Ang1,BMSCs-A+V,BMSCs and model-saline group(n = 20),in addition,selecting 20 cases same-day-old mice were isolated carotid artery as air-saline group.Each group of mice was injected whith relevant factor into right lateral ventricle under stereotaxic instrument within 24 hours.After 24 hours,48 hours,72 hours and 7 days of injection,6 groups of mice were sacrificed to take brain tissue for HE staining,immunohistochemical staining and RT-PCR test,to investagate the change of pathological,myelination levels,and VEGF & Ang1 mRNA levels respectively.RESULTS: The first part: After unilateral carotid artery ligation combined with hypoxia induced making mice PVL model,mouse from model group have slow weight gain,hair is coarse and lacklustre,brain specimens could seesoftening oven,and then become into the lumen of the liquefaction necrosis gradually.HE staining showed the model group of nerve cells vary in size,nuclear condensation,fragmentation,confirmed the presence of PVL brain changes in pathology.RT-PCR show after surgery recovery,the model brain VEGF and Ang1 expression gradually increased at 48 hours.Both of VEGF and Ang1 mRNA levels were higher than the sham group.Immunohistochemical showed both of VEGF and Ang1 expression of two groups of mice were increased with time,but the model group,the average optical density(AVD)of VEGF and Ang1 was significantly higher than the sham group(P <0.05).The second part: 1.Mouse in BMSCs-V+A and BMSCs-Ang1 group have ideal weight growth after anesthesia recovery,pathology examination confirmed brain damage was alleviate obviously,BMSCs and BMSCs-VEGF group weight growth is slow,no statistical differences compared with model group(P > 0.05),and the improvement of pathology in brain is no statistical differences compared with model-saline group.2.I Immunohistochemical: VEGF value increases in BMSCs-V+A and BMSCs-VEGF group,but no difference between BMSCs –VEGF and model-saline group of AVD of MBP(P > 0.05).Both of the AVD of Ang1 and MBP were increased in BMSCs-V+A,BMSCs-Ang1 and BMSCs group with significant difference compared with model control group(P < 0.05),the AVD of MBP of BMSCs-V+A group was close to airsaline group,and no statistical difference(P > 0.05).3.RT-PCR: BMSCs-VEGF and BMSCs-V+A group VEGF mRNA rise rapidly,have significant difference with the model-saline group(P < 0.05),Ang1 mRNA increased significantly in BMSCs-V+A and BMSCs-Ang1 group,have significant difference with the model-saline group(P < 0.05).CONCLUSION: 1.We can construction the ideal model of PVL in neonatal mice induced by hypoxic-ischemic,both of VEGF and Ang1 secretion were increased in early stage of brain injury.2.The combination of VEGF and Ang1 gene can effectively repair damaged NVU,protect nerve cells,promote myelination of nerve fibers,reduce PVL brain injury,so that provide a new way for effective intervention of PVL.