Construction Of The Combinative Implant Of Neural Stem Cells Of Rat In Vitro

Objective:To construct the combinative implant of neural stem cells which consists of neural stem cells (NSCs) and vascular endothelial cells(VECs) and extracellular matrix by choosing appropriate extracellular matrix.Methods:The experiment is divided into two passages .1.The advantage of serum free and clone culturing technology is performed to isolate,culture and passege neural stem cells from neonatal rat (1 day to 3 days), Immunofluocytochemistry and Immunofluorescence label are used for identification of nestin pigmentation. The advantage of M199 culture medium was performed to culture VECs from neonatal rat (1 day to 7days). Immunofluocytochemistry is used for identification.The different extracellular matrix included poly-L-Lysine, Laminin(LN) and matrigel and NSCs and VECs are co-cultured separately. The states of livability, proliferation and differentiation of NSCs in three different extracellular matrixs under microscope; Immunofluocytochemistry is used to inspect proliferation and apoptosis and differentiation of NSCs to nurons and astrcytes after 3 days, 7days and 14 days. 2.To construct the combinative implant of neural stem cells and to observe: Passeged VECs are assimilated 20 minutes by 0.25% trypsin and inoculated in culturing bolt at the density of 3×10⁷L^-1.VECs'culturing media is exhaled after 12 hours . The extracellular matrix is poured on VECs and then NSCs which are assimilated to single cell by 0.125% trypsin at the density of 6×10⁸L^-1. NSCs'culturing media are put in. NSCs'culturing media are exhaled after NSCs all cohere to wall observing by miscrope about 3 hours to 6 hours. The extracellular matrix is poured and VECs are inoculated at same density .They are co-cultured 12 hours to 24 hours after NSCs'culturing media are put in. The best extracellular matrix and NSCs and VECs are co-cultured to form samdwich and to construct the combinative implant of NSCs by blowing it. Immunofluorescence label is to observe the struture of the combinative implant that VECs are labeled VIII factor and NSCs are labeled nestin. the combinative implants which contain different extracellular matrixs separately are inoculated on coverslids in six poles plates. NSCs'culturing media are put in. Growth of NSCs is observed under microscope after 3 days, 7days and 14 days.Results:1 To identity and observe states of NSCs and VECs:Primary isolated single NSC proliferate into neurosphere after 4 days. After passaging, neurosphere is positive when Immunofluorescence label is used for nestin. Isolated VECs grow to monolayer after 6 days to 8 days of external cultivation. The prese polygonal and arrange like pavement stones under light microscope. Correlation antigens of VIII factor are positive by fluorescent stain.2 The effects of three extracellular matrixs to differentiation of NSCs: At 3^rd day MAP₂-positive cells of LN group is more than that of matrigel group and poly-l-lysine group(P<0.05). At 7^th day the difference is obviously significant (P<0.01). There is obvious difference between LN group and matrigel group, but they are more tnan poly-l-lysine group at 14^th day. GFAP-positive cells of LN group is more than that of matrigel group and poly-l-lysine group at 3^rd day (P<0.05). There is not obvious difference among 3 groups. There is not obvious difference between poly-l-lysine group and matrigel group, but but they are more tnan LN group at 14^th day(P<0.05). MAP₂ +GFAP-positive cells of LN group is more than that of matrigel group and poly-l-lysine group at different time point (P<0.01).3 The adhersive ablity of LN is good . There are many NSCs and VECs to construct the combinative implant of NSCs. There are 6 to 9 cells in the combinative implant of NSCs. Its diameter is about 28μm.4 To observe proliferation and apoptosis of NSCs: NSCs increase following various time point in 3 groups, but Brdu-positive cells of LN group and matrigel group increase strikingly at 3rd day(P<0.05), reach a peak value at 7^th day and 14^th day (P<0.01). Caspase+Brdu- positive cells of LN group and matrigel group is less than that of poly-l-lysine group at different time point (P<0.01).5 To observe the cells of the combinative implant under miscrope and Immunofluorescence label of the combinative implant: The instructure of NSCs semi- encircled or adhered by VECs can be established by storied co-culturing and blowing. The combinative implant is irregular under miscrope.NSCs are encircled by VECs or cohered each other. The nuclei of VECs is big and the bodys are irregular and are bigger than that of NSCs. NSCs have characteristic of reflective rays.There are averge 8 to 15 cells in the combinative implant through one hundren combinative implants at random, which contain averge 2.8 VECs.Red immunofluorescence label VIII factor positive VECs encircle green nestin positive NSCs or cohere each other.Conclusions:1 The adhersive ablity of LN is good. It can cohere to NSCs and VECs to form the right combinative implant of neural stem cells.2 LN can improve differentiation of NSCs and enhance NSCs to nurons and decrease NSCs to astrcyte. It is superior to matrige and poly-L-Lysine. This is advantage of transplanting the combinative implant to replace injured neurons.3 LN can improve survive of NSCs of the combinative implant and enhance proliferation of NSCs and VECs of the combinative implant, and then it enhance proliferation of NSCs again through enhance proliferation of VECs.4 The ability of LN to cause to grow axon is superior to matrige and poly-L-Lysine, and it can make NSCs to imgrate.5 LN is a ideal extracellular matrix to the combinative implant of neural stem cells. The combinative implant of neural stem cells whose supportive cells are VECs and extracellular matrix is LN can be constructed successfully though three-dimensional co-culture(VECs-LN-NSCs-LN-VECs).