| 1 The studies on isolation and cultivation of rat marrow mesenchymal stromal cells (MSC) and their biological characteristics in vitro.Objective: To explore a new method for isolation, purification, cultivation of MSC in vitro and analyze their biological characteristics further.Methods: 1 MSC were collected from degermed femurs, tibias and Sternum of 4 to 6-week-old SD rat by flushing the shaft with buffer (D-Hank's, PH 7.2) using a syringe with a No. 26 G needle. Cells were disaggregated by gentle pipetting several times and plated in culture flask and re-fed every 2-3 days (L-DMEM with 10% FBS and 1% penicillin-streptomycin). When 95% fusion, cells were digested with 0.125% trypsogen and 0.02% EDTA 2 min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. 2 The surface ultrastructural feature of MSC was observed with scanning electron microscope (SEM). 3 The morphologic characteristics of MSC were studied by HE staining. 4 The growth curve of P2, P4, P6 were drawn with MTT. Results: The components of primarily cultured MSC were very complex. The marrow cells were round in the beginning and a few cells adhered to flask at 1-2 days, which were in irregular shape such as fusiform, polygon and so on. After changing half of the medium, the cell clone began to proliferate immediately. About 7-9 days later, cells might overgrow the bottom of culture flask and reached over 95% fusion. The cells arranged regularly as a whirlpool. Generated cells stuck on the wall more quickly than primary cells. From the 2nd hour, they began to adhered and completely adhered within 24h. The cell morphology was more uniform and all the cells arranged more regularly. Cells could spread the full flask bottom for 6 or 7 days. After subcultured 10 passages, the cells proliferated rapidly and kept the morphology unchanged. The HE staining result showed that the cell body was fusiform, and some of them had processes. The big and deep staining basophilia nucleus were in the middle of the cell body, and there were 1-5 acidophilia nucleoluses in nucleus. The growth curve of P2, P4, P6 were quite similar and the cells biological character kept stable. The result of growth curve showed that cell growth phase was composed of latency phase, logarithmic phase. Under SEM we could find three kinds of cell morphologies: the most were the large flattened cells and there were a few spindle-shaped and globe cells. The large flattened cells extended process like arborescence and overlapped each other. Their nucleus were big, round or ellipse and contained 1-5 nucleolus. It was interesting that on the cells surface we could see a large amount of short and thick microvillus.Conclusion: 1 In this part a simple new method which was used for the isolation, purification and cultivation in vitro of MSC from SD rat bone marrow by total marrow culture associating with adhering to flash has been established. The MSC could proliferate immediately and keep their biological character stable in vitro. 2 The cells were noted to have a large expansive potential and a fusiform morphology. The growth curves of P2, P4, P6 were quite similar and the cell biological character kept stable. 3 Three kinds of morphology of cells were found: the large flattened cells,the spindle-shaped and globe cells. There were a large amount of short and thick microvillus on the cells surface which might be beneficial to substance exchange between cell and cell matrix.2 The research of the bone marrow mesenchymal stromal cells differentiate into neuron in vitroObjective: To induce bone marrow mesenchymal stromal cells to differentiate into neuron in vitro.Methods: 1 According to the method of the part one that the MSC were isolated, purified, cultured and passaged. 2 MSC were treated by dimethylsulfoxide (DMSO) and basic fibroblast growth factor (bFGF) , then the change of morphology and the number of cells were observed during differentiation. Immunocytochemistry and western blot were used to detect the expression of neurone specific enolase (NSE), glial fibrillary acidic protein (GFAP) and microtubule-associated protein-2 (MAP-2).Results: The morphology of cells in control group was not changed in pre-inducation and induction. Immunocytochemistry showed that the expression of NSE was negative. With phase contrast microscope we found that in 24h's pre-induction the cells did not obviously change in experimental group. 1h after formal inducing, the cells bodies began to retract like a round. 3 hours later the round cells began to extend 1-3 processes. After 5 hours the processes grew more longer and began to branch off and overlapped each other. When they were induced at 7h, the cells bodies were round and extended long 2-3 grade processes and overlapped each other like a network. Cell death was present in induced group containing blood serum 5h after inducation, but not in induced group without serum. Immunocytochemistry showed the inductivity of groups without pre-inducation which contained serum or not were 71.857±7.08249 % and 72.597±6.98511% respectively. The expressions rate of NSE in group which was pre-induced with L-DMEM containing 1% DMSO and 10 ng/ml bFGF for 24h, then induced with L-DMEM containing 10% DMSO and 10ng/ml bFGF for 7h, was 86.67±5.5148%. GFAP and MAP-2 were negative at any groups. Western blot showed that the expression of NSE at 0h, 3h, 5h, 7h (the ratio of NSE toβ-actin) were 0.232±0.00364, 0.3141±0.00414, 0.321±0.00186 and 0.4683±0.00447 respectively.Conclusion: 1 MSC was induced into neuron-like cells 7h after addition of inducing medium which contained 10 ng/ml bFGF, 10% DMSO and 86.67% was the maximum induction rate. 2 The inductivity of group which pre-induced with medium containing 10 ng/ml bFGF and 1% DMSO was improved significantly. 3 Live time of cells was prolonged with inducing medium without serum.3 The research of labeling the MSC with Bromodeoxyuridine (BrdU) in vitro.Objective: To explore the best period, best time and best dose for labeling the MSC with BrdU in vitro, so as to attain the best tracing aim of applying MSC to future research.Methods: 1 According to the method of the part one that the MSC were isolated, purified, cultured and passaged. 2 Chose different passage of the MSC, traced them with BrdU in different period, time and dose, and then immunocytochemistry was used to detect the expression of BrdU, so as to know whether our labeling was successful and analyze the labeling rate. Results: 1 The labeling rate of P2, P4, P6 MSC labeled by BrdU were 94.868±3.739%, 95.696±3.952% and 94.774±3.565% respectively. 2 The labelling rate of MSC on the 2nd, 4th and 6th day were 60.286±6.197 % , 96.175±3.689 % and 65.017±5.982% respectively. 3 The labeling rate of MSC at 24h, 48h and 72h after labelling were 58.895±10.025%, 95.375±4.136% and 94.835±2.970% respectively. 4 The labelling rate of MSC labelled with 5μmol/L, 10μmol/L and 20μmol/L BrdU were 47.192±5.459%, 95.382±4.363% and 95.993±3.344%respectively.Conclusion: 1 The effect of MSC labelled with BrdU was steady among passage cells. 2 10μmol/L BrdU, which incubated MSC in logarithms to living for 48h, could attain the maximum labeling rate. |
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