Primary Culture Human Tenon’s Capsule Fibroblasts And Study P53gene Expression In The Tenon’s Capsule Fibroblasts

Purpose To investigate the cultivation method of human Tenon’s capsule fibroblasts in vitro and simulate the scarring process of filtering channel after glaucoma filtering surgery in vitro and explore the change of the expression of p53 in the scarring process of filtering channel.Materials and Methods Human Tenon’s capsule was received from strabismus surgery patients after we expose the conjunctival sac in operating rooms. The tissue was taken back to laboratory immediately. The primary HTFs were cultured in vitro by tissue culture method and the cells were identified by immunofluorescence assay. The activity and proliferation of the cells were detected by MTT method and show ed on the growth curve. The 3-5 generation cell which in good growth state were selected as the experimental cells. TGF- β 1(10ng/ml, 48h) was used to induce HTFs transform to MFs. Total RNA extraction kit was used to extract total RNA from HTFs and MFs respectively. the total RNA was reversed transcription to c DNA, p53 gene was amplificated in PCR and agarose gel electrophoresis of PCR products were observed in UV spectrometer to observe individual bands and take pictures.Results 1.Primary HTFs were cultured with tissue culture method after 5~10 days,the cells were climbed out from the edge of the visible tissue under the microscope.The cells were round or ova, the nucleus was clear and part of the cells had not been separated from the tissue. After 2-3 weeks of culture, the cells were concentrated around the tissue, the cells were typical long fusiform shape, radially arranged around tissue block. On the other parts of the bottle bottom, the growth state of cells was also good and arrangement spiral. The spindle cells’ surface area was larger, the nucleus was big and clear, the cytoplasm was rich in granular materials. After 3-4 weeks of culture, there were a large number of cells in the culture bottle and monolayer cells cover 80% bottle bottom, the cell growth is more intensive. 2.Cell passage culture was observed under inverted microscope, the morphology of cells was basically consistent with the primary cells and were typical long fusiform shape, the cells were surrounding the tissue and arranging radial or whorled. Cells not only had a strong ability to proliferate and grow rapidly, but also had 80% fusion after 3-4 days, then we could subculture again. Experiments showed that the proliferation ability of 3-5 generation cells was the strongest, after the 6 generation cells, the growth activity was gradually decreased slowly which not abled to use in experiment. 3.The growth state and survival of cells were detected by MTT assay. After measure the OD value and draw the growth curve, the results showed at the time of 1-2 days, OD value was measured showed a small increase, indicating that the cells grow slowly and in cubation period, at the time of 2-6 days, OD value was measured showed a big increase,indicating that the cells grow rapidly and in logarithmic growth phase, the cells proliferation ability was strong. 4. Immunofluore scence staining was used to identify the cells. Under the fluorescence microscope, we could seen the cytoplasm was filled with a lot of colored filaments which constitute a network structure parallel to the vertical axis of cells and the nuclear staining was negative. In the cytoplasm of primary cultured cells, vimentin staining showed strong positive and the cultured cells in vitro were identified to HTFs. 5.Under the inverted microscope we observed TGF- β 1-induce HTFs, after 24 h the cells had no change in morphology, which was in agreement with the normal HTFs shape showing a long spindle shape and radial or spiral arrangement. after 48 h the cells had great change in morphology, which showed more plump and increased the volume of a single cell, the nucleus was not clear and granular materials were incr eased in the cytoplasm, the morphology was irregular shape or long spindle shape, the boundary between cells was not clear and cells arranged in disorder no longer in a radial arrangement. Fibroblasts activation changed to muscle fibroblasts. 6.The expression of p53 protein was observed by semi quantitative PCR.Total RNA of HTFs and MFs were extracted from the same sample respectively. After total RNA reverse transcription into c DNA p53 was amplicated in PCR instrument. After electrophoresis the amplified bands were clearly visible in the gel imaging system,marker was divided into six bands from 100 bp to 600 bp. Positive control group(human GAPDH) was amplified, which product length was 496 bp. P53 gene in HTFs and MFs were amplified, which product length were both 141 bp. The imaging showed that the amplified reaction band of HTFs was more clearly and brighter than the amplified reaction band of MFs and indicated that the expression of p53 in HTFs was higher than the expression of p53 in MFs. The expression of p53 was significantly decreased after TGF-β1 induced HTFs.Conclusions The expression of p53 in TGF-β1-induced-HTFs is significantly decreased compared with the expression of p53 in normal HTFs.