Study On The Co-culture Of Schwann Cells And Vascular Endothelial Cells In Vitro

Objective: After the peripheral nerve injuryed, the defect repair which is an alternative to the clinical attention has not yet been fully resolved. For a long time, autologous nerve graft has been recognized as the "gold standard" of the repair of peripheral nerve defects. However, the search for an ideal alternative to autologous nerve defects in peripheral nerve repair materials become the focus of peripheral nerve research in recent years due to the autologous nerve for transplantation is limited, and the area for transplantation is often with some degree of side-injury, and for areas are likely to cause sensory or motor dysfunction, traumatic neuroma formation, the difficulty of surgery and the dislocation of axon growth and other issues. The artificial neural transplantation can not be compared to the autologous nerve graft yet. The effect of repair is poor if wore than a certain length of nerve defect repair of the nerve or the required diameter, or a tendon, bone blood supply such as poor wound exposed. The reasons may be for tissue engineering of artificial neural graft revascularization delay can not be established in the early and effective blood circulation, part of the seed cell death as ischemia and hypoxia, affecting the quality of nerve regeneration. Therefore, the solutions to the artificial neural transplantation vascularization are critical to the artificial nerve graft transplantation.Schwann cells (SCs) as the peripheral nervous system glial cells, is the peripheral nervous system myelin-forming cells, with support, protection, segregation, nutrition, chemotaxis and promote regeneration of nerve fibers and mature, at the same time can produce some neurotrophic factor, through various channels in damaged peripheral nerve function, thus contributing to the recovery of neurological function in the repair of peripheral nerve regeneration process plays a key role. Thus increasing the local damage of Schwann cell proliferation and volume are better able to promote functional recovery of damaged peripheral nerves. Vascular endothelial cells (VECs) to cover the inner surface of the monolayer in the vascular flat cells, not only in the blood and vessel wall permeability barrier between an optional and is an important part of the capillary. Normal neural tissue is rich in blood vessels, nerves within the distribution of endometrial capillary network, is the nerve tissue of the bridge and nutrients, and to ensure the generation of nerve fibers and the normal function of the play.Purpose of this study is to observe Schwann cells and endothelial cells co-cultured for tissue engineering of artificial neural formation of blood vessels close to the natural conditions, and further evidence of joint training of two kinds of cells can promote angiogenesis, based on the growth of Schwann cells, Also found two cell co-culture of the optimal ratio, and then try to find an ideal promotion of nerve and blood vessel tissue engineering approach for the eventual realization of artificial neural transplantation of tissue engineering to provide an initial basis for the clinical application of theoretical basis.Methods:1 Fetal Schwann cells in primary culture and subcultured for 28 days get pregnant New Zealand white rabbits were sacrificed ear vein into the air, conventional disinfection, sterile shop towels, rabbit fetus rapid Caesarean section, in sterile conditions, whichever is sciatic nerve, and removal of epineurium and perineurium, Hank's solution wash 5 times, cut into pieces, obtained by enzymatic digestion using the two-SCs, double 30min differential adhesion process of law and culture to join the Arab-C (terminal concentration to 10-5mmol/l) removal and inhibited fibroblasts, after replaced with NGF (40ng/ml) for SCs growth medium, observed under inverted microscope every day, the second generation of SCs cultured with S-100 antibody staining to identify and calculate the concentration of cells covered dish to be removed after the culture medium, after digestion with trypsin the cells were collected by cell scraping spoon stand in the culture medium.2 Fetal rabbit vascular endothelial cells in primary culture and passage to remove the fetal rabbit the same way, under sterile conditions, remove the fetal rabbit thoracic aorta, PBS repeatedly washed inside and outside the lumen, the traditional enzyme digestion VECs, in was added with vascular endothelial growth factor (Vascular endothelia growth factor, VEGF) to promote the proliferation of VECs, was observed under inverted microscope every day, the second generation of VECs cultured with Fâ…§RA antibody staining to identify and calculate the concentration of cells to be After the culture dish covered with a removable medium with trypsinized cells were collected by cell scraping spoon stand in the culture medium.3 Schwann cells and endothelial cells were cultured directly take the 3rd passage of the fetal rabbit SCs and VECs, the SCs and the VECs joint direct proportion to 1:1,2:1,4:1 were cultured in RPMI 10 % FBS DMEM and M199 in the mixed medium, is set to A, B, C three groups, while SCs, VECs cultured alone as control group D, E groups. Cultured for 1 day, 3 days, 5 days, 7 days, through the SCs cell count, growth curve test, testing the impact of VECs on the SCs.Results:1 Schwann cells culture and results: The 6cm culture dish was filled with a confluent monolayer cells in about 7 days. Observed under an inverted microscope: SCs'form is spindle, owned 2-3 dendrites, mostly bipolar, occasionally tripolar; nucleus is obviously oval; cells grow end to end, side by side, whirlpool-like or palisading, and confirmed by immunocytochemistry for the SCs; occasionally, a small amount of fibroblasts were "fried egg"-like, cell bodies of Schwann cells than the large, flat, irregular in shape, protruding more short, prominent nucleoli, about 2 to 3 a, Schwann cells in color and light.2 Vascular endothelial cells results observed under inverted microscope every day VECs growth: VECs early as round or oval-shaped, three-dimensional and refractive index is strong, ranging from the number of aggregation, mostly small groups Colony, 6 8 days fully converge to form a continuous monolayer, confluent after the appearance of characteristic "cobblestone"-like shape, arrangement of dense, abundant cytoplasm; passage of VECs smaller individuals, for a short spindle, square or polygonal cells, refractive index of small, cell-dimensional shape enhanced the formation of microscopic "cobblestone"-like change, and confirmed by immunocyto- chemistry for the VECs.3 Schwann cells and vascular endothelial cells were cultured directly the result of the number of SCs were observed with the prolonging of culture time increased, which in co-culture on day 1, the experimental group and control group, no difference in cell number (P>0.05) , were cultured from day 3 until day 7, the experimental group A, C cell count with the control group D were significantly different (P<0.05), experimental group B (SCs and VECs ratio of 2: l) and control group D no significant difference (P>0.05), while the other two experiments A, C group were significantly different (P<0.05).Conclusion:1 SCs in vitro process, the use of double differential attachment method can remove most of the fibroblasts, the initial use in cell culture Arab-C can inhibit fibroblast growth, the latter was added with NGF can promote the growth of SCs to get a lot of purity of more than 90% SCs.2 VECs in vitro process, the traditional enzyme digestion method for VECs, VEGF added to the culture medium can promote the proliferation of VECs, VECs in hypoxia inhibition of apoptosis, but also effectively inhibit smooth muscle cell growth, while control the appropriate time to collagenase digestion, resulting in a large number of more than 90% purity of VECs.3 Fetal rabbit aorta in vitro source of VECs to promote differentiation and proliferation of fetal rabbit SCs; SCs and VECs ratio of 2:1, VECs promote the differentiation and proliferation of SCs strongest. Fetal rabbits SCs were cultured with VECs as nerves and blood vessels for tissue engineering is one of the methods for the eventual realization of tissue engineering of artificial neural transplantation a viable clinical application of basic research.