Under Hypoxic Conditions Vecs Nscs Proliferation, Differentiation And Apoptosis

Objective : In vitro studies of hypoxic VECs NSCs proliferation, differentiation and apoptosis. To VECs and NSCs were cultured transplantation in the treatment of hypoxic-ischemic diseases provide a scientific basis.Methods: 1, the first three days of newborn rats brain hippocampus neural stem cells, using immunohistochemical methods to identify the neural stem cells. 2, Retrieve Health ~ Wistar rat brain cultured brain microvascular endothelial cells using immunocytochemical methods of their identification. 3, promotion of purification of brain microvascular endothelial cells with 0.25% trypsin digestion solution into single cells after vaccination in a number of poly-l-ysine coated glass on the cover. Inoculated with three generations of trypsin digestion of neural stem cells to coverage ratio of 10 to 1 completely nerve cells co-cultured medium. 4, paraffin oil with sterile sugar coverage in cell culture liquid surface preparation hypoxia sugar solution. Control groups : normal neural stem cells at 37℃, the volume fraction of 0.05% CO2, saturated humidity culture; A total of hypoxic training group : a total of cells cultured for 3 days, the culture medium was sucked out by the sterile Hank 's buffer cleansing 2nd, Hypoxia sugar solution to a common role after 4h and 28h for fluid, with PBS buffer washed three times, other nerve cells completely medium, cells placed in an incubator reoxygenation culture; Hypoxia alone neural stem cells : from the traditional three-generation neural stem cells with sterile Hanks buffer 2nd after cleaning method same. 5, fluorescence immunohistochemical identification under hypoxic conditions neural stem cell proliferation and apoptosis. Immunohistochemical staining identified under hypoxic conditions neural stem cell differentiation.Results : 1, NSCs and VECs culture and identification : the primary separation NSC4d single nerve cell hyperplasia of the ball, After passage, nestin immunofluorescence staining, cells were positive signal; primary cultured brain microvascular endothelial cells 6-8d bearded bottle, or polygonal cells were flat spindle, nuclear oval. Factor VIII related antigen. 2, the group of neural stem cells form of light microscope features : the control group NSCs soma satiated and strong dioptre surrounded by a glow discharge. Hypoxia alone neural stem cells NSCs soma swelling, refraction poor, a large number of cell debris, and the emergence of suspended cells die. Hypoxic group were cultured NSCs form has improved cell refraction is still good, the majority have yet to see protruding cell retraction, only a few NSCs swelling and necrosis. 3, and newborn hypoxic-time rat cerebral cortex neural stem cell activity : hypoxia , respectively. 2 hours , 4 hours, 8 hours and 28 hours after reoxygenation for 48 h, NSCs with the extension of hypoxia, markedly increased cell death, NSCs survival decreased significantly; hypoxic group were cultured NSCs form has improved cell refraction is still good, Most of NSCs neurite retraction yet, only a few cell swelling, NSCs were cultured group was significantly higher than that of hypoxia NSCs group (P<0.05). 4, under hypoxic conditions brain microvascular endothelial cells to neural stem cell differentiation of : brain microvascular endothelial cells to hypoxia neural stem cells into neurons and glial cells simply divide the number of small nerve hypoxia Compared to cell differentiation, increased significantly, but after the formation of differentiated neurons and glial cells in a very small percentage of impact, through the count, not statistically (P>0.05). 5, under hypoxic conditions brain microvascular endothelial cells to neural stem cell proliferation and apoptosis of : hypoxic cells were cultured cell proliferation significantly higher than the number of hypoxic simple neural stem cells (P <0.05) Apoptosis was lower than the number of cells hypoxia simple neural stem cells (P <0.05).Conclusion : neonatal rat hippocampus detachable train neural stem cells, and have proliferation self-renewal capacity and multiple differentiation characteristics; whose brain microvascular endothelial cells can promote hypoxia neural stem cell proliferation, differentiation, apoptosis decreased.