An Dose-dependence Study Of FK506 On Schwann Cells

With the development of tissue engineering of peripheral nervous system, the lack of autologous nerve possibly could be solved by artificial nerve. Artificial nerve is composed of seed cell and biomaterials which can be degrade. Schwann cells are the most important seed cells which can synthesize and secrete extracellular matrix and other elements constitute the substrate, and enveloping the axons to form myelinated nerve fibers myelin and non-myelinated nerve fibers in endometrium. Schwann cells can secrete many nerve nutrition factor such as NGF(nerve growth factor). These nerve nutrition factor which are secreted by SCs play a very important role during the regeration of peripheral nerve. Nerve regeneration distance will increase if there is Schwann cells, otherwise, will be shortened.FK506 is separated from streptomycete at the region of Tsukuba, Japan in 1984. FK506 is the laboratory names. its immunosuppression function probably is 10 times of the CsA's. In recent years a series of studies have shown that FK506 has very strongly role of nerve growth-promoting besides of the immunosuppression function. Its receptor protein-FKBP (FK506 Binding Pro-tein) not only exists in the immune system, but also in the nervous system extremely, probably the content of the later is 10～50 times of the former's.If FK506 stability and long-lasting exist in the artificial neural, the foreign Schwann cells will be protected.Objective:1.To found a good approach to obtain more population and more purification of SCs from new-born SD rats. Experimental studied on culture of SCs in vitro, while we observed SCs growth cases and the percentage of SCs purified. 2.To study the effects of different concentrations of FK506 on the growth of Schwann cell in vitro in order to lay the practical foundation for the construction of tissue-engineered bridging substances.Methods:1.To dissect the sciatic and brachial nerve of new-born SD rats. The nerve fascicles were then extracted under microscope. The nerve was cut to be 1mm3 tissue pieces. Two enzymes were used to digest the sciatic nerve speciments. Low density trypsin quickly digested to passage SCs. Then we adopted morphology observation, S-100 protein assessment.2. Primary Schwann cells were counted and were observed about their proliferation. Schwann cells of the second passage were cultured under different conditions according to group assignment. In the experiment groups, Schwann cells were cultured in DMEM media supplemented with 10-6mol/L,10-7 mol /L,10-8mol/L FK506 respectively, while in the control group the cells were cultured in the same culture media without FK 506. The OD of different intervals was determined by MTT assay to obtain the growth curves.ELESA(enzyme linked immunosorbent assay)was performed to determine how FK506 influenced nerve growth factor(NGF)expression level of Schwann cells. To observate the growth, activity and secretion of functional status of Schwann cells dynamicly.Results:1.Through the above-mentioned method we isolated and cultured prim- ary Schwann cells which proliferated rapidly. Schwann cells were identifi- ed by S-100 protein. And we got high purified Schwann cells of the second passage, The purity of SCs was beyond 90%.2.Schwann cells of each group proliferate significantly after 3 to 5 days cultured. There is no significant difference of cell activity between FK506 groups and the control group from 1d to 3 d. The viability of the Schwann cells of FK506groups was significantly better than those of control group from 3d to 9d (P <0.05).3.ELISA assay results showed that FK506 could significantly promote Schwann cells secrete NGF, gradually increased, and maintain a high secretion level from 4d to 8d. The highest secretion is at 6d. low dosage (10-8mol/L) administration of FK506 was observed to promote proliferation and NGF expression of Schwann cells more significantly.Conclusions:1.The sciatic nerves and brachial plexuses of 3～5d old rats are less fibrous tissue components and relatively easy to cut. Schwann cells obtain- ed from the method adhere to culture dish well and proliferate fast.2.FK506 can enhance the proliferation of Schwann cells cultured in vitro.This effect of FK506 was evident in low dose level. low dosage (10-8 mol/L) administration of FK506 could significantly protect Schwann' s cell.3. Excluding the impact of the immune,neurotrophic role of FK506 may be the key to promote injury nerve regeneration and to accelerate nerve growth.