| Research on neural stem cells (NSCs) is one of the current hot-spots in neuroscience. NSCs are thought to be the most appropriate candidate for neural transplantation and there is a great hope to rebuild the lost function of central nerverous system by cell replacement using them. Treating neurodegeneratiove diseases by NSCs' transplantation has made great progresse while the corresponding applications in cerebral vascular diseases (CVD), especially in ischemic CVD (iCVD), just begin. How does the neuons in ischemic areas affect the extrinsic NSCs' proliferation and differentiation? Researches on it are few. The experiment present here was arranged to co-culture NSCs with injuried neocortical nerons in ischemic-like conditions, to find out the effects neocortical neurons imposed to NSC. This experiment is a feasibility study.This experiment can be divided into two parts. The first part contained Section 1 and Section 2 and focused on the technique to culture NSCs in vivo. The second part, Section 3 and Section 4, was mainly about the co-culture of neo-cortical neurons and NSCs.In Section 1, the mouse fetuses (El2-16) were removed from pregnantKun-Ming mice, and their brains were dissected into portions of the striatum, cortex and brain stem before primary culturing in a chemically defined medium consisting of DMEM/F12, N2 supplement, EOF and bFGF. 3-5 days after inoculation, neurospheres were seen in each culture of the three portions. Some neuropheres were selected and dissociated into single cells and inoculated in 96-well plates in a very low density (5-10 cells/mL). Another 3-5 days later, new monoclonal neurospheres formed from single cells.In Section 2, some of the new-born monoclonal neurospheres aquired in Section 1 were moved to 24-well or 48-well plates pretreated by poly-L-lysine (PLL), cultured 2 hours and fixed for immunofluorescence staining of nestin, a molecular marker for NSCs. Some the new-born neuropheres were moved into PLL pretreated 24-well plates, culture medium were replaced by DMEM containing 10% fetul bovine serum (FBS).Four days later, lots of cells plately growthed on the bottoms of wells with their processuses crossing like a web and immunocytochemistry stainings showed that NSE+, GFAP+, GalC+ and p-tubulinlll4 cells existed in them.In Section 3, neo-cortical neural cells from mouse fetues (Kun Ming race, El8) were primary cultured in Millicell (see Discussion of Part 2), 3 days later aracytidine was add to inhibit the growth of glia and 7 days later neurons were dominant in culture. These neurons were treated by ischemia-like conditions (no-glutamine-and-hypoxia) for 15 minutes and co-cultured with NSCs Section 1 provided (Ischemic+Millicell group). NSCs co-cultured with neurons as contral group (Millicell group) and cultured alone as another contral group (Normal group). Proliferation of NSCs in three groups was evaluated by MTT assay and their growth curves were drawnmeanwhile the differentiation was observed by cell-immunostaining and positive-cell-countting (see Section 4). From Section 3, we found that: NSCs' proliferation accelerated when co-cultured with ischemia-injuried neocortical neurons and the relative growth rate was 0.0575 and 7.59 days later arrived the most growth rate while Millicell group was 0.0344 and 8.47, Normal group was 0.0387 and 8.51. The differences between the results from Ischemic+Millicell group and the two control groups were significant (P<0.05) while between the two-control groups was not (P>0.05).From Section 4, we found: Differentiated cells adhered to every slip and well distributed. In this condition, neurospheres were found also. Co-cultured for 4d, anchored cells yet few and there was no significant difference between experimental group and control. Eight days for co-culture, the number of anchored cells in experimental group slips and the ratio of NSE positive cells were higher than control group (p<0.05).From the results, I conclude:1. Stable NSCs can be isolated from stratum of El 3 mouse.2.
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