Experiment Studies On Biological Behaviours Of Olfactory Ensheathing Cells Intensified By Ginsenoside Rg1

Objective:1?To establish economical and practical methods of culturing and purifying olfactory ensheathing cells(OECs) to shorten culturing period and to increase the yield. 2?To explore whether ginsenoside Rg1 would influence the biological behaviours of OECs such as proliferation, mRNA expressing and neurotrophin secretion of relevant neurotrophic factors(NTFs),which would be served as clinically acceptable up-regulating factor of OECs. All of the above-mentioned, to deal with the source and biological activity of OECs,the promising transplantation cells for spinal cord injury(SCI).Methods: 1?Simplified methods to isolate, culture and purify olfactory ensheathing cells: The olfactory bulb were dissected, then pia mater and capillaries were peeled and central white matter were removed under a dissecting microscope. The gray matter of the bulb was retained in DMEM/F-12 containing 10% fetal bovine serum medium at 4°C, rapidly cut for 5 minutes then blown gently for 15–20 times. Instead of trypsinization, the homogeneous suspension was directly seeded and incubated at 37°C/5% CO2. The culture medium was semi-substituted every three days. Until the cells 80% converged,the primary cultures were purified by three sequential steps as follows: step 1, to trypsinize with a low concentration of trypsin; step 2, to remove short-time preferential adherent cells;step 3,to inhibit mitosis with the anti-mitotic agent Ara-C. The result of each purifying step was assessed by confocal immunofluorescence identification and purity detection. 2?To determine the effect of Rg1 on cell proliferation of OECs MTT assays: Purified OECs were counted and incubated in 96-well plate, treated with Rg1 in concentration gradient of 0 (control), 5, 10, 20, 40, and 80?g/ml, and incubated respectively for 24, 48, 72, 96 h. The MTT absorbance was read at OD 570 nm. The chart of growth tendency was drawn,and the optimal concentration and action time was determined, which was performed in the succedent experiments. BrdU assay: Cell proliferation was measured by detecting the incorporation of BrdU. Purified cells were distributed to 3 groups: 1, blank contral; 2, Rg1 treated; 3, 2?M Forskolin treated as reference. The 24 h cell proliferation index was evaluated. 3 To explore whether Rg1 would influence mRNA expressing and secretion of relevent NTFs RT-PCR:Purified cells were distributed to 2 groups, one for control, another for Rg1 treated with optimal concentration and action time. RT-PCR of GDNF, BDNF and NGF were performed, and the PCR products were analyzed after electrophoresis on a 2% agarose gel. The relative intensities of PCR products of the 3 NTFs were analyzed. ELISA assay: Culture medium of the two groups mentioned above was collected to detect the protein content of GDNF, BDNF, and NGF using ELISA kits according to the manufacturer's instructions. All data are expressed as mean±SD. Statistical significance was evaluated by the Student's t-test and defined as P < 0.05.Result:1?With our methods, the procedure of isolating and culturing OECs is simplified dramatically from 60-90 min, multipul steps to 8-10 min, single step. The OECs grew dominantly in cultures. The purity of OECs increased gradually during the three steps purification with minimum cellular loss, and the ultimate purity reached upto 95% then maintained for a long time. 2?Rg1 promoted cell proliferation of OECs, the effect was optimal at 40?g/ml,72 h. The 24 h proliferation indexs of control, Rg1, Forskolin group were 9%, 17%, and 20%, respectively,difference between control and Rg1 group was statistically significant(p<0.05), and difference between Rg1 and Forskolin group was statistically nonsignificant(p>0.05). 3?RT-PCR analysis demonstrated that Rg1 up-regulated mRNA expression of GDNF, BDNF and NGF((p<0.05). Moreover, Rg1 increased the protein secretion of the three NTFs(p<0.05), which was confirmed by ELISA analysis. OECs, with shortened the culturing period, are inexpensive, easy to repeat,heavy producing and low cellular loss, which provides sufficient OECs of controllable purity and facilitates their use in further research. 2?Rg1 is a clinically available intensifying factor of OECs, which promotes cell proliferation as well as up-regulates mRNA expression and neurotrophin secretion of GDNF, BDNF and NGF. 3?The research studied on source and biological behaviour of OECs, which would provide more preferable transplantation cells for repairing spinal cord injury.