| Objective:To build a new cell line of NB and to establish tumor-bearing nude mice mode of human neuroblastoma and to study mechanisms of neuroblastoma incidence and metastasis.Furthermore, to provide a good experimental animal models. To screen the differentially expressed microRNAs(miRNAs) between metastatic neuroblastoma(NB) and primary neuroblastoma using miRNA microarray and to explore the relationship between pathogenesis and miRNAs aberrantly expressed in metastatic neuroblastoma to primary neuroblastoma.Methods:We do the experiment from primary culture ourselves using the NB tumor tissue of our inpatient and establish a pure NB cell line through separate other cells that we don't need. Cells were grown in a humidified incubator at 37℃in a 5% carbon dioxide atmosphere and maintained in DMEM medium supplemented with 10% heat-inactivated fetal calf serum,100IU/mL,100μg/Ml streptomycin. In vitro culture human neuroblastoma cell line and take 1×107/0.1ml exponential phase of growth cell suspension take into nude mice subcutaneous Shoulder right forelimb and watch biological characteristics of tumors. Meanwhile, histopathological examination, DNA microarray analysis, detection of subcutaneous tumor and tumor cell lines express NSE were taken. The tissue of metastatic neuroblastoma and primary neuroblastoma were obtained from Nude model. Total RNA, including the small RNAs, was extracted and isolated respectively. Hybridization was carried out on miRNA nficroarray chip. Real-time quantitative RT-PCR was performed to confirm results obtained by microarray analysis.Result:The way of digestion culture is better than that of tissue culture. We can see the cells digested by enzyme the same time if we culture cells by the way of digestion culture, all of them have the morphology of round shape, they became little shuttle shape the second day. The NB cells have the same shapes approximately, small shuttle shapes; tear shapes; and triangle shapes. All these are infantile cells. Some NB cells that have a long prominence are mature cells. The cell line we founded was identified as NB cell line by a series test. A total of 48 cases in 36 cases of tumor-bearing was successed, with a total rate of 75% of tumor-bearing; 10 cases of metastasis, the metastasis rate was 21%. Five cases in which tumor growth, a large tumor volume, even to the rat's own body weight 1/2, but the metastasis did not happen; four cases of injection site without tumor growth, but have distant metastasis; 6 cases of both local tumor growth, but also distant metastasis. In metastatic neuroblastoma,54 miRNAs have been identified,35 of them were upregulated and the remaining 19 were down—regulated in primary neuroblastoma. The differential expression of miR-483-3p,miR-99b,miR-145,miR-602,miR-26,miR-125a-3p,let-7f was significantly confirmed by microarray analysis. Conclusions:We have the ability to establish NB cell line in our laboratory. The cell line we founded was identified as NB cell line by a series test. We have successfully establish tumor-bearing and stable passage human neuroblastoma nude mice model. The transplanted tumor growth rate and metastatic rate was high. This model was ideal for the simulation of natural cell growth,local infiltration and distant metastasis process of human neuroblastoma in vivo. At the same time, this experiment confirm that neuroblastoma is heterogeneous, and this heterogeneity may be important reasons of neuroblastoma metastasis, he aberrant expression of miRNA genes in this study may be involved in the pathogenesis of human neuroblastoma metastasis.
|
PDF Full Text Request