The Effect Of Lps On The Expression Of G-CSFR In Sk-N-Sh Cell And The Apoptosis Of Human Nuroblastoma

Objectives:The purpose of the study was to study the expression of granulocytecolony-stimulating factor (G-CSF) and its receptor in children solid tumors,and to determine the effect of lipopolysaccharide (LPS) in culture mediumon the expression of G-CSFR in SK-N-SH cells and the apoptosis of humanneuroblastoma tumor tissue in vitro.Methods:①We gathered81specimens of children solid tumors from Children’sHospital of Chongqing Medical University, including27specimens Wilm’stumor,25specimens neuroblastoma,11specimens rhabdomyosarcoma，6specimens teratoma,5specimens lymphoma,4specimens clear cellsarcoma and3specimens ganglioneuroma from September2008to June2011;②Immunohistochemistry was used to detect the expression ofgranulocyte colony-stimulating factor and its receptor in children solidtumors;③Cultured human neuroblastoma SK-N-SH cells were stimulated with different concentrations of LPS (0ug/ml,1ug/ml and10ug/ml) for6h,12h and24h;Cultured fresh tumor tissue from neuroblastoma patient werestimulated with different concentrations of LPS (0ug/ml,50ug/ml,100ug/mland200ug/ml) for24h,48h and72h;④To detect the expression of G-CSFR in tumor cells,immunohistochemistry and reverse transcription PCR were performed;⑤TUNEL was be used to test apoptosis after intervention with LPS.Results:①81tumor samples of G-CSF and G-CSFR immune staining results:G-CSF-positive rate was64.19%, G-CSFR-positive rate was67.90%;②Granulocyte colony-stimulating factor and its receptor expressionrate in Wilm’s tumor specimens were62.96%,66.67%, respectively.Co-expression G-CSF and G-CSFR in the same samples were16cases(59.26%);③Granulocyte colony-stimulating factor and its receptor expressionrate in neuroblastoma specimens were68.00%,72.00%, respectively.Co-expression of G-CSF and G-CSFR in the same samples were12cases(48.00%);④Granulocyte colony-stimulating factor and its receptor expressionrate in rhabdomyosarcoma specimens were72.70%, co-expression ofG-CSF and G-CSFR in the same samples were5cases (45.45%),interatoma were50.00%,83.30%, respectively, co-expression were2cases (33.33%),in lymphoma were40.00%, co-expression was1case (20.00%),in clear cell sarcoma were75.00%, co-expression were3cases (75.00%),in ganglioneuroma were66.67%,33.33%, respectively, co-expression was1cases (33.33%);⑤G-CSF and G-CSFR expression were no significant correlation withgender, age, clinical stage and pathology types in neuroblastoma andWilm’s tumor;⑥Immunohistochemistry showed that the G-CSFR expression was nosignificant difference in all groups when the concentration of LPS was0ug/ml, the expression of G-CSFR was decreased in a time-dependedmanner when the concentration of LPS were1ug/ml and10ug/ml;⑦RT-PCR indicated that the G-CSFR expression was no significantdifference in all groups when the concentration of LPS was0ug/ml, theG-CSFR expression was decreased in a time-depended manner when theconcentration of LPS were1ug/ml and10ug/ml. In6h group, compared0ug/ml with1ug/ml, there was no significant difference P=0.056,compared0ug/ml with10ug/ml, there was significant difference P=0.000,compared1ug/ml with10ug/ml, there was significant difference P=0.027.In12h group,0ug/ml,1ug/ml and10ug/ml each concentration groupcompared with each were statistically significant, P <0.01. In24h group,compared0ug/ml with1ug/ml, there was significant difference P=0.01, P<0.05, compared0ug/ml with10ug/ml, there was no significant difference P=0.001, P <0.05, compared1ug/ml with10ug/ml, there was nosignificant difference P=0.164, P>0.05;⑧Cultured neuroblastoma tumor tissue in vitro, there was nosignificant statistical difference in all time groups when the concentrationof LPS were0ug/ml and50ug/ml, P=0.417, P=0.059, respectively，butwhen the concentration of LPS were100ug/ml and200ug/ml, it was asignificant statistical difference in all time groups, P=0.002, P=0.009,respectively. After intervention with LPS24h, there was no significantstatistical difference in different concentration groups, P=0.247;but both in48h and72h groups, there was a significant statistical difference indifferent concentration groups, P=0.000, P=0.0013, respectively.Conclusions:①G-CSF and G-CSFR are highly expression in children solid tumors.Regardless of gender, age, clinical stage and pathology types of tumorshould be detected G-CSF and G-CSFR expression. For theG-CSFR-positive patients, it is recommended to use G-CSF should becarefully considered its impact on the tumor;②Lipopolysaccharide down-modulates granulocyte colony-stimulat-ing factor receptor on SK-N-SH cells;③LPS can promote the human neuroblastoma cell apoptosis, but thedose and intervention time could effect the expression of G-CSFR and cellapoptosis.