The Effect Of Basic Fibroblast Growth Factor On Apoptosis-correlated Gene Expression In Chondrocytes Of Tissue Engineering

ObjectiveTo detect the expression of p53, collagen - II , telomerase, Bel - 2 and Bax mRNA in articular cartilage of rabbits with bFGF and to determine the function of bFGF on the apoptosis or aging of chondrocytes.Materials and methods1. Culture and collection of articular cartilageThe rabbit articular chondrocytes were isolated enzymaticcally from cartilage chips scraped from the tibial plateaus and femoral condyles of 3 - week -old Japanese White rabbits. The chondrocytes were released by continuous digestion of 4 hours at 37℃ homoiothermy agitation with 2mg/ml collagenase - II and cultured in Dulbecco' s Modified Eagle' s Medium ( DMEM) with 20% FBS (test - group including 10ng/ml rhbFGF). 3 days later the culture medium was exchanged for the first time, and then exchanged every two days. When the chondrocytes were grown to 90% confluence;, we transfered the chondrocytes from one flask to two flasks and another was collected into eppendorf tubes. After repeating the above procedures until the seventh passage, we collected the chondrocytes of all the flasks into eppendorf tubes.2. RT - PCRTotal RNAs were extracted from chondrocytes by TRIZOL reagents, and were reverse - transcripted to the first strand of cDNA using reverse transcriptase system. The cDNA was amplified by PCR. The expression of p53, collagen -II , telomerase, Bel - 2 and Bax genes were detected with β - actin as an internal control in tissue engineering chongdroc;ftes of test - group and control -group. Primers were designed using Primer 3.0 software.3. Observing indexMicroscope and invertoscopeResult1. Microscope and invertoscopeAfter culting 4 th passage, in the control - group, the variety of morphology could be observed on some chondrocytes. From 5th to 6th passage, most cartilages showed the characters of aging. Moreover, it was found that there were some representative features of apoptosis on a lot of chondrocytes in 7th passage. On the other hand, in the test - group, we observed morphologic change from 3rd passage, directing flbroblast. However, it was not showed until 7l passage that tissue - engeering chondrocytes had the character of apoptosis. The cells in test - group proliferated faster than control - group.2. RT - PCRThe two groups differed in the index of objective gene of p53, collagen -II , telomerase, Bcl - 2 and Bax, which indicated the expression of mRNA of each one had altered with adding bFGF. In the test - group, we detected that the indexes of collagen - II , telomerase, Bcl - 2 ascended, while the ones of p53 and Bax declined. The differences between two groups were more prominent before 4th or 5th passage about collagen - II , telomerase, Bcl - 2 and p53.DiscussionThree basic factors are needed in the study of tissue engineering: first, e-nough seeded cells with normal functions; next, suitable extracellular matrices; the third, cell factors to modulate cell generation and keeping their phenotypes. In this study we pay attention to cell factors.Basic fibroblast growth factor (bFGF) is a polypeptide binding with hepa-...