| 1. To observe the growth, proliferation and differentiation of stem cells derived from the cat marrow.2.To study the possibility of neural stem cells derived from cat marrow as resource cells.Materials and Methods Materials20 adult cats with health by qualified quarantine, each half of male and female, age limit from one to two years, body weight limit from 2.5 to 4.0 kg. MethodsAfter anesthetizing by muscle injection of ketamine, 3~5ml marrow was drawed out from the inferior extremity of hindlimb femur by bone puncture. The adult cat BMSCs harvested by grads centrifugation plus 3~4ml cyte separating medium were cultured in petri dish with "Cytokines ?NSCM", giving various culture conditions in order to induce the special differentiation.Based on the different additions such as serum and factors, the cells were divided into the experimental groups including: 1. the simplicity NSCM, 2.the NSCM +FCS(fmal concentration 20%), 3. the NSCM +LIF(20ng/ml)+ bFGF(20ng/ml), 4. the NSCM + PCS (final concentration 20%) + LIF(20ng/ml)+bFGF(20ng/ml).Based on the different time getting rid of the no-attached cells after cell culture, the cells were divided into the experimental groups including the 4 h, 24h, 48h, controlgroup. The observation and photography of the growth and the immunocytochemical stain of the cells at the different culture situations was performed by Type CK2 Inverted Microscope.ResultThe majority of cells were rapidly in apoptosis without serum in medium, even if LIF and bFGF were added in the medium during the culture process, and, there were no obvious difference of cell growth in the medium with serum whether LIF or bFGF was in the culture medium. After getting rid of the no-attached cells in 4 hours, there were one or two ellipse attached cells, with a tiny process. It was found out that the cells sharply disappeared without proliferation. After 24 hours, the number of attached cells obviously increased. Cell processes increased and longer, and there were obvious cell proliferations in the highdensity petri dish. After 48 hours, the attached cells divided and proliferated; after one or two weeks, the cells could fully pave on the petri dish bottom and go down to the future generation or change into spherical cell fallowing shaped into cell clone.After 24 hours, the big spherical cell gradually came into cell processes and grew into attached cells which would shaped into cell clone cluster like island. If given proper differentiation condition, they could differentiate into sorts of cells with long processes.At the 6~7th day after culture, these cells which have proliferation ability could presented Nestin antigen, a specific antigen on the fetal neural epithelium, NSE antigen, and NeuN antigen by immunocytochemistry, and could have been induced to neuron-like cells.ConclusionThe cat BMSCs, as stem cells, have the characteristics of proliferation, differentiation and going down, and could normally develop with considerable cell density in Cytokines ?NSCs medium with calf serum, and could induced to the cells which presented NSE antigen and NeuN antigen in proper conditions. So, it is possible for the adult cat BMSGs to be induced into NSCs under certain experimental conditions.Part 2. Experimental study on the therapy of injured visualcortex with NSCs derived from cat BMSCsObjectives1. To build the cat model of injured visual cortex.2. To probe into the possibility of the treatment of injured visual cortex with NSCs derived from cat marrow.Materials and Methods Materials10 adult health cats (no neurobiological abnormality after examination). Methods1.After general anesthetizing, the right occipitoparietotemporal cortex, such as 5, 7, 17, 18, 19, 20a, 20b, 21a , 21b, DLS, VLS, PS, PMLS, PLLS, AMLS, ALLS and SVA were lesion with electric coagulation following the operation of disclosed cranium. After operation, the fields of vision were observed by neurobiology examination.2.After drew out bone marrow for the established hemiano... |
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