| Neuroblastoma ( NB) is a paediatric tumor originating from sym-pathoadrenal progenitors derived from the neural crest. After the treatment of chemotherapy and radiotherapy, the survival rate of NB is still less than 30%. 90% tumors from long - term survival patients have good differentiation, belonging to stage I or stage II ,only 10% tumors have poor differentiation, belonging to stage El or stage IV- As NBs have been observed to regress spontaneously in vivo to benign gangli-onneuromas, how to induce differentiation of NB cells becomes a problem noticed by scholars all over the world.Recent studies have revealed that growth, differentiation and survival of NB cells are strongly regulated by neurotrophins and their receptors. The receptors of neurotrophins include TrkA, TrkB and TrkC. TrkA is a high - affinity receptor for NGF, and also binds neu-rotrophin - 3 ( NT - 3 ) , while TrkB is a high - affinity receptor for brain - derived neurotrophic factor( BDNF) and also binds NT -3 and NT -4/5. TrkC is a high - affinity receptor for NT -3. Trk was origi-nally found as an oncogene fused with the tropomyosin gene in the extracellular domain in a colon cancer. In NB, TrkA was expressed in tumors with favorable prognosis, and N - myc amplification strongly down - regulated its expression. The low or absent expression of TrkA in tumor tissue is strongly associated with an unfavorable prognosis. In contrast to TrkA, TrkB is preferentially expressed in aggressive NB, especially in those with N - myc amplification.The NGF signaling pathway appears in NB, and especially in favorable tumors expressing high level of TrkA receptor. Primary culture studies have shown that exogenous NGF induces differentiation of NB cells depending their survival on NGF. In contrast, the cells obtained from unfavorable NB with N - myc amplification do not respond to NGF, perhaps due to the absence of TrkA expression.In this study, IFN - r, NGF and the combination treatment of them were used as induction drugs respectively to induce the differentiation of SMS - KCNR cells. The TrkA mRNA expression was measured for the differences.MaterialHuman NB cell line SMS - KCNR was donated by professor Carol J. Thiele (NIH, USA).Methods1 ,SMS - KCNR Cell Culture; The NB cell line SMS - KCNR was cultured in RPMI - 1640 supplemented with 15% fetal calf serum, 100IU/ml penicillin and 100ug/ml streptomycin at 31C in 5%CO2.2 Experiment Group: IFN -ry and NGF was dissolved in aqua liquid at concentrations of 5000IU/ml and 4. 5 x 105ng/ml and reserved at -80C for future use. Cells were divided into four groups, control group used routine medium, A group used medium containing 25IU/ml IFN -7,6 group used medium containing 100ng/ml NGF, C group used medium containing both 25IU/ml IFN - r and 100ng/ml NGF.3 Cell Counts; KCNR cells were plated in 24 -well dishes at a concentration of 1 x 105/ml. After 24 hours, cells were treated with medium, medium containing 25IU/ml IFN - r, 100ng/ml NGF, 25IU/ml IFN - r and 100ng/ml NGF. Living cells were counted after the treatment of 0,3,6,10 days. Cell counts were performed using a hemocytometer and viability was assessed by trypan blue exclusion.2 Morphological Studies: Human SMS - KCNR cells were plated at a density of 1 x 10 /cm . Cells were respectively treated with medium, medium containing 25IU/ml IFN - r, 100ng/ml NGF, 25IU/ml IFN - r and 100ng/ml NGF. Morphological differentiation was observed under the phase - contrast microscope after the treatment of 10 days. For determination of neurite outgrowth, the number of cells extending a neurite longer than the cell body were counted and expressed as a percentage of the total number of 200 cells counted.5 TrkA mRNA Expression: Expression of TrkA oncogene on day 10 after treatment of 25IU/ml IFN -r, 100ng/ml NGF, 25IU/ml IFN - r and 100Ong/ml NGF was analyzed using RT - PCR. The products obtained were analyzed and visualized by electrophoresis, using a 1. 5% agarose gel staining with ethidium bromide.Result... |
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