[The study of apoptosis factors released from laser injured cartilage inducing apoptosis of chondrocyte].
OBJECTIVE
To explore the role of pro-apoptotic signals following tissue injury and how these may promote a progression of further cell death.


METHODS
Laser treated porcine articular cartilage disks were maintained in culture media. The collected media at various time periods (3, 6, 9, 12, 24 and 48 h), was called treated conditioned media (TCM). Non-laser treated cartilage disks were used to create control conditioned media (CCM). Each disk was subsequently maintained for 28 days and used in confocal microscopic assessment to document the progression of the damaged area. Isolated porcine chondrocytes were cultured in monolayer, and were exposed to TCM, CCM or normal culture medium (NM). As a positive inducer of apoptosis, the monolayer cells were exposed to UV radiation for 10 min and cultured in NM. Following 24 h exposure, the cells were harvested and stained with the appropriate combination of fluorescent dyes and processed via flow cytometry.


RESULTS
All cultured cells exposed to TCM displayed a caspase-3 positive subpopulation, a loss of CMXRos, and with a reduced or lost NO signal. CCM exposure signals were comparable to the NM treatments with all having retained CMXRos, NO and without evidence of caspase-3 activity. UV treatment also induced a reduction in NO, but both CMXRos and caspase-3 positive, representing an earlier stage of apoptosis and suggesting that the mode of cell death via UV and TCM exposure are via different processes. The investigation of a dose (100%, 50%, 25% and 12.5%) and time (0.5, 1, 3, 9, 12 h) response to TCM exhibited that all treatments observed an increase in caspase-3 positive cells and a reduction in NO and CMXRos.


CONCLUSIONS
The usefulness of FCM can be used in the study of cell viability and apoptosis. Such a system may be useful in the study of mechanisms of disease such as osteoarthritis, thus may be of practical use for the pharmaceutical industry for screening associated drugs.
Zheng
Min
M
Department of Gynecology and Interventional Radiology, Cancer Center, Sun Yat-sen University, Guangzhou 510060, China.
Grogan
Sp
S
Schaffner
T
T
Mainil-Varlet
P
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D002356:Cartilage / Q000378:metabolism / Q000528:radiation effects*; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D066298:In Vitro Techniques; D007834:Lasers; D009569:Nitric Oxide / Q000378:metabolism; D009930:Organic Chemicals / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014466:Ultraviolet Rays