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Objective To investigate the feasibility of human neuroblastoma xenograft model in C57BL/6j mice. To study inhibitory effects of proliferative effect of Gallic acid （GA） on human neuroblastoma xenograft model, as well as the synergistic effect of cyclophosphamide （CP） chemotherapy, and to discuss that GA protected testicular cytoskeleton protein vimentin induced by CP.Methods 3day,7day,3week,4week,5week,6week and 7week age C57BL/6j and 6week age BALB/C nude mice were subcutaneously inoculated 1×107cell/ml huaman neuroblastoma SK-N-SH cells which were logarithmic phase, respectively. The tumor generation rate, relative tumor volume （RTV）, relative growth rate （K）, relative doubling time （Td）, survival time and pathology were tested. The 3-week-old immunocompetent C57BL/6 mice were transplanted with SK-N-SH cells, distribution of randomized block design, 20 of each, the control groups:①PBS group, 0.5ml per bearing tumor mouse, intraperitoneally （i.p）, as a negative control;②normal control group （NC）, as the reproductivity of mice;③CP group, 75mg/kg per bearing tumor mouse, i.p, twice a week, total two weeks, as a positive control. Experimental groups:①GA group, administered gallic acid 250mg/kg i.p per bearing tumor mouse, every other day, continuous two weeks;②CP+GA group, GA and CP independent administration, dose and time with the above. All drugs are administrated at 9:00. Monitoring the relative tumor volume growth curves （RTV）, body weight weekly, and measureing relative tumor volume growth rate. Tumor cell proliferation was detected by bromouracil deoxyriboside labeling （BrdU）. To compare the absolute weight and the relative organ index of the testis, epididymis and seminal vescle. Immunohistochemistry, reverse transcriptase polymerase chain reaction （RT-PCR） and western blotting （WB） were used to detect testicular cytoskeleton protein vimentin mRNA expression and protein distribution.Results①The continuing tumor generation rate of C57BL/6j mice and BALB / C nude mice was 100% （10/10） in 3day, 100% （10/10） in 7day, 90% （9/10） in 3week and 70% （7/10） in 4week, 60%（6/10） in 5week, 0% （0/10） in 6week, 0% （0/10） in 7week and 100% （10/10） in 6week, respectively. There were no difference in RTV, K, Td, and survival time （P>0.05）, compared to 3week C57BL/6j mice and BALB/C nude mice.②After administration of the 2nd-5th week, compareing each group RTV, GA+CP group<CP group<GA Group<PBS group, there were significant differences （P<0.01）. After the administration of the 2nd week, GA group, CP group, GA + CP group of T/C （%） were: 74.83%, 33.11%, 10.98%, a significant difference （P<0.01）. After the administration 5w, GA group, CP group, GA + CP group of T/C （%） were: 35.46%, 1.16%, 0, a significant difference （P<0.01）. It was reallized that GA inhibited human neuroblastoma xenografts, and the synergistic effect of CP chemotherpy.③After administration of the 2nd week, the tumor cell proliferation index （LI） in the tumor tissues of mice treated with PBS-treated, GA-treated, CP-treated, and CP plus GA-treated groups were 0.23±0.08, 0.12±0.02, 0.11±0.02, 0.04±0.01, respectively. Compared with the PBS group was significant difference （P<0.01）. The LI of the mice treated with CP combined with GA was significantly smaller than that the CP or GA group （P<0.01）.④After administration of the 2nd week, PBS group body mass, testis, epididymis and seminal vesicle absolute weight lower than NC group （P<0.01）, organ index was no significant difference （P>0.05）. CP Group testis, epididymis, seminal vesicle organ index decreased compared with NC group （P<0.01）, GA + CP group of organ index is higher than CP group （P<0.05）, it realized that CP induced the damage of testis, epididymis, seminal vesicle in mice, but GA aginsted its toxicity. After the administration the 7th week, organ index of each group no significant difference （P>0.05）, this showed that there may have a role in mice compensatory growth after CP chemotherapy.⑤After administration of the 2nd and 7th week, Immunohistochemistry showed that the distribution and expression of testicular cytoskeletal protein vimentin were similarly in GA group, GA+CP group and PBS group. CP group caused testicular cytoskeletal protein vimentin to collapse, which was disrupted and decreased the expression in the basement membrrane. After the administration of the 2nd week, GA group and the GA + CP group of testicular cytoskeletal protein vimentin mRNA and protein expression were increased, there was significant difference （P<0.01） compared with CP group. After the administration the 7th week, GA group and the GA + CP group of testicular cytoskeletal protein vimentin mRNA and protein expression increased, there were significant differences （P<0.05） compared with CP group, it showed that GA may against the effect of testicular cytoskeletal protein vimentin induced by CP.Conclusions①Human neuroblastoma xenograft model were successfully established the in immunecopetent C57BL/6j mice, as well as the most appropriate vaccination in 3 week age.②Gallic acid can inhibit tumor proliferation and synergism CP anti-tumor growth in human neuroblastoma xenograft.③GA may against the testis, epididymis and seminal vesicle toxicity induced by CP, and maintain normal distribution and expression of testicular cytoskeletal protein vimentin in vivo.
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