| Objective: Pleomorphic adenoma is also named salivary mixed tumor. It is the most common tumor in salivary gland. The statistical results according to the department of oral pathology in six schools suggested that pleomorphic adenoma occupied over 50% of salivary epithelial tumor, and over 87% of all oral benign tumor. Though the tumor is benign, grows slowly, has no obvious symptom, it tends to plant and to relapse. Relapse repeatedly, it could malignant change and be difficult to be treated. Right now in our country the cell line of pleomorphic adenoma has not been established. So, establishment of the cell line of pleomorphic adenoma is necessary for reserch further. Materials and Methods: 1.Cell culture in vitro: (1) Sample collection: The sample was obtained from a male patient, 37 years old, with right parotid bump. Clinical diagnosis is parotid mixed tumor. He had undergone partial superfical parotidectomy for his neoplasm. Histopathologically is pleomorphic adenoma. Prior operation, the patient had not received any chemotherapy and radiotherapy. (2) Cell culture process: under aseptic condition,tumor tissue was obtained from operation sample . After operation, for primary culture, pieces of the resected tumor was placed in RPMI1640 supplemented with 20% fetal calf serum (FCS), streptomycin(500ug/ml) and penicillin (1000U/ml) for 30 minutes, removed normal parotid tissue and necrotic tissue, plasma clot, mucosa. The materials were minced into fine pieces with scissors and washed several times with Hank's solution containing antibiotics. Then, fragmental tissue were planted in 25ml tissue culture flasks, stood for 10 min, turned over and incubated in 5% CO2 /95% air at 37℃ for 2 h. Then they were turned over again, and added a little culture medium. (RPMI1640 /20% FCS, 100ug/ml streptomycin, 100U/ml penicillin). After 24 h, a little culture medium supplied until fragmental tissue were just submerged. After 3 days, the first observation had been done, and then observated once a day. The medium was added or changed according to the variation of PH. The 4th day, very few fibroblast-like cells were observed. On the 7th day, cells grew from partial fragmental tissue, a few cells were free from the clusters. Cells around fragmental tissue were polygonal -shaped and free cells were spindle-shaped. The medium was changed every 3 days. On the 17th day cells reached 80% confluency. They were harrested by trypsinization with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. The method of trypsinization was adopted to get rid of fibroblast through 2~3 times, the pure tumor cells wereobtained. The time of primary culture was the longest to over 60 days. The attached cells were not digested easily. Results:1. Growth state: In primary culture, tumor cells formed "cell clusters" surrounding the explant and spreaded gradually. Overlapped growth phenomenon was investigated. In 2nd passage of culture, within 48 h of planting most of cells had attached to the surface of flakes, for a short time, cells began to spread and proliferate. Cells were clusters or free from the cluster. Last, confluent monolayers were obtained when one glass of cells were subcultured into the two, within 5~7 days, cells could reach 80% confluency and confluent monolayers were obtained. Concerned index as follow: (1) Growth curve: The 5th passage cells were diluted into cell suspension, and inoculated to tissue culture flasks, culture medium was changed every one day. Two flasks were took every day, and counted, adding up to 8 days, linked curve. Because at the 1st, 2nd day, cells had not attached to the surface of the flasks, the curve was fallen. On the 3rd day, the curve began to raise. On the 5th, 6th day, the curve was reached a peak. (2) Growth state after frozen and resuscitated: The 7th passage of cells were recovered by centrifngation, the supernatant was removed, the resulting cells were transferred into RPMI1640 containing 10%DMSO, preserved into liquid nitrogen. Two months later, t...
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