Established And Identified Cell Lines From Human Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors(GIST) are the most common mesenchymal tumors of gastrointestinal tract. The major pathogenesis of GIST is gain-of function mutations of c-kit gene. Mutations of the c-kit gene are known to be present in over 85% of GIST, most commonly in exon 11, exon9, exon13 and exon17. But the mechanism of c-kit mutations make c-KIT activated has not been known. A subset of GIST dose not have c-kit mutations, and about 7% of GIST have PDGFRA gene mutations. In addition to this, some proteins and genes also participate the pathogenesis and development of GIST, for example HMGB1, COX-2, Hsp90, CA-RP, but their mechanisms are not distinct. GIST is not sensitive to radiotherapy and common chemotherapy, and the major therapy device is operation, but 50% of GIST would relapse and metastasize after operation. Glivec(Imatinib mesylate, STI571) is a specific tyrosine kinase inhibitor that acts with BCR-ABL, PDGFR and c-KIT, and has been used successfully in unresectable or metastatic GIST patiernts. But secondary resistanse to STI571 in GIST patients has been reported, but the cellular mechanisms of resistance to STI571 have not been identified.Therefore, to establish human gastrointestinal stromal tumor cell lines in vitro culture make for research pathogenesis of GIST and mechanisms of resistance to STI571. There are three gastrointestinal stromal tumor cell lines that have been cultured in the world : GIST882, GIST-T1, GIST780, and GIST882 is a classic GIST cell line among them. But it is very difficult to culture GIST882 cells in vitro. The GIST cell lines from Chinese GIST patients have not been cultured. Therefore, we established human gastrointestinal stromal tumor cell lines in vitro culture, and identified their biological characters.Obejective: To establish agastrointestinal stromal tumor (GIST) cell lines fromGIST specimen, and identified their biological characters. Methods:1. To optimize a series of conditions that is suitable to culture primary human gastrointestinal stromal tumor cells in vitro.To optimize a separation method: to use dispase digestion n pamcreatin digestion and dissection to small tissue piece respectively to separate cells from fresh GIST specimen;To optimize a cultivation condition: to use DMENLRPMI1640 respectively as medium, to use GIBCO FCS and domestic-made FCS respectively.2. To immortalize human gastrointestinal stromal tumor cellsTo immortalize the GIST cell line GIST-1 by ectopic expression of telomerasereverse transcriptase QiTERT) and simian virus 40 Large T(SV40LT) antigen.3. To identify the biological characters of the cell lines(1) To detect the expression of c-kit and c-abl through Western blot.(2) To detect mutation of c-kit gene through RT-PCR and DNA sequencing.4. To detect the drug susceptibility of the GIST-1 cell lineWe detected respectively the inhibition effect of paclitaxeh cisplatim gemcitabine and STI571 to the cell line GIST-l through MTT dye. Results:1. Optimization a series of conditions that is suitable to culture primary human gastrointestinal stromal tumor cells in vitroWe found that the optimal culture condition was dispase digestion and RPMI1640 which contain 20% GIBCO FCS. Four lines of GIST cells had been established through this method, and were named GIST-1 > GIST-2x GIST-3^ GIST-4.2. The phenotype and growth condition of GIST cell linesThe cells showed fibroblast-like phenotype, fusiform shape, and formed monolayer in vitro culture. After cultured 7-8 PDs, the growth velocity of GIST cells steped down, the doubing time increased, the cells crimpled, seniled and apoptosised.The immortalized GIST-1 didn't show fibroblast-like phenotype, and showed short fusiform shape > anomalistic phenotype, which were different from GIST^ GIST-3> GIST-4. The GIST-1 grew persistently and had been cultured to 30th passage.3. The biological characters of the GIST cell lines(1) Results of Western blot: c-kiU c-abK hTERT> SV40LT antigen expressed by the transfected cells. The cells owned characters of GIST.(2) Results of RT-PCR and DNA sequencing: The sequencing results revealed that GIST-3 cell line had a point mutation which located at exon 11(GTT>GAT), GIST-2 cell line had a deletion of 12 nucleotides(AAAGGTAACAAC)which located at exon9.4. The drug susceptibility of the GIST-1 cell line in vitroSTI571 could significantly inhibited the proliferation of the cells. The rate of cell growth inhibition is above 90% treated with 0.1/miol/L STI571 for 48 hours. Above lOmg/ml paclitaxeh cisplatin^ gemcitabine enhanced the growth suppression of GIST-1 cells. Cisplatin had little inhibition of GIST-1 cells at the range of 0.1-10 mg/ml in a dose-dependent pattern, and the minimal effective concentration was 10 mg/ml. Paclitaxel and gemcitabine could significantly inhibited the proliferation of the cells above 0.1 mg/ml. Conclusion:1. Four lines of human gastrointestinal stromal tumor cells were successfully established, and a optimal culture condition was optimized. The optimal culture condition was dispase digestion and RPMI1640 which contain 20% GIBCO FCS.2. GIST cells could be successfully immortalized through ectopic expression of telomerasereverse transcriptase (hTERT) and simian virus 40 Large T(SV40LT) antigen.3. The immortalized GIST cells expressed c-kit> c-abl antigen, and were extremely sensitive to STI571.4. The GIST cell lines had different mutation of c-kit gene.