Effects Of Pressures And Brain-Derived Neurotrophic Factor (BDNF) On Cultured Bovine Trabecular Meshwork (BTM) Cells In Vitro

PUPROSE:1. To improve the cell culture method of BTM cells in vitro.2. To study the effects of pressures on cultured BTM cells in vitro.3. To study the effects of BDNF on cultured BTM cells in vitro.4. To study the effects of BDNF on cultured BTM cells under different pressures in vitro.METHODS:Part 1: Establishment of the cultured BTM cells in vitroBovine eyes were enucleated and carried to the lab under 4 ℃ . We separated TM tissues from bovine eyes under sterile conditions, inoculated to 25 cm2 culture bottles, or digested the TM tissues with 2 ml mixture which was composed of 0.25% Trypsin and 0.3 % Collagenase equally. Collected the cells and seeded into 50 ml culture bottle with 104 cell/cm2. The culture medium was DMEM containing 20% fetus serum, lOOU/ml penicillin, 100 U/ml streptomycin and 2.5 ng/ml amphotericin B. The cells were passaged whenthey came to be confluent.Part 2: The effects of pressures on cultured BTM cells in vitroThe third passaged BTM cells were seeded into 10 ml culture bottles with 104 cell/cm2, and about 1 week later they became to be confluent. We changed the culture medium, blocked the culture bottles and injected sterilized airs into the bottles through a T-shaped tube whose another entrance connected with a baresthesiometer. Four experimental groups were blocked and raised air pressures to 1.33, 2.67, 5.33 and 10.67Kpa (1 Kpa=7.5 mmHg) respectively. In control group, cells were only blocked in bottles. 6 bottles were repeated in every group.Part 3: The effects of BDNF on cultured BTM cells in vitro The cells of experimental groups were cultured in the culture medium with 50 ng/ml BDNF, on the contrary the cells of the control group were cultured without BDNF. The cells' growth curve was drawn. Next, all groups were observed under the pressures of 5.33 KPa and 10.67 KPa for 24 h.RESULTS:1. BTM cells grew well and passaged normally by our culture method.2. Compared with the control group, the cells under the pressure of 1.33 KPa or 2.67 KPa lasted 48 h had noremarkable difference. There were slight changes in the group under 5.33 KPa pressure lasted 24 h. The cellular damages appeared more severe or even the cells appeared to death if the pressure was 5.33 KPa lasted 48 h or elevated to 10.67 KPa lasted 24 h.3. The culture medium containing 50 ng/ml BDNF could obviously improve the third passaged BTM cells to be confluent.4. Compared with the control group, BDNF could protect the cells' quantities and activities under different pressures. There was significant difference between the control group and the experimental groups (P<0.05).CONCLUSIONS:1. Not only digestion culture method but also tissue culture method could obtain well BTM cells. Compared with tissue culture method, digestion culture method was easier and more convenient to harvest enough cells for studies.2. BTM cells kept alive only under a certain pressure level, and the cells would be damaged if the pressure was too high or the exposure time was too long.3. BDNF could greatly improve BTM cells growth.4. BDNF could protect the quantities and activities of BTM cells under a certain high pressure, thus could protect their normal physical functions and protect the oculartissues from being damaged by too high IOP.