| Objectives：Cervical cancer is one of the most common malignant tumorswhich threats both mental and physical health in female. Its morbidity andmortality is on the fifth in human malignant tumors and second in femalecancer. TPX2is a nuclear proliferation related protein and strictly regulated bythe cell cycle. It major function is participated in the assembly of mitoticspindle and microtubule in cell mitotic. Evidences showed that theamplification or over-expression of TPX2induced the amplification ofcentrosome, abnormal segregation of chromosome and leaded to DNAheteroploid and polyploidy. Now researches confirmed that the phenomenonof the amplification or over-expression of TPX2exists in many tumor tissuesand cell lines. In our former in vitro experiment, our research group hadverified that TPX2is significantly over-expressed in cervical cancer andTPX2-siRNA can decrease the expression level of TPX2mRNA and itsprotein, reduce the ability of proliferation and invasion in Hela cell line, whichbenefit for inhibition even elimination of tumor. By using the TPX2-siRNAand its stably transfected Hela cell line, our research will get further in vivostudy of its inhibition of the expression of TPX2protein and growth of tumoras well as its therapeutic value.Method：Choosing four-week-old female nude mice (n=16). Then separatedthem into four groups at random: TPX2-siRNA transfected group,TPX2-siRNA therapy group, blank control group and negative siRNA controlgroup. Each group injected Hela cell in nude mice's right hind armpit in orderto build cervical cancer models. TPX2-siRNA transfected group was injectedby Hela cell line which transfected by TPX2-siRNA. Other three groups wereinjected by untreated Hela cell line. For the sixth day after injection, localinjecting TPX2-siRNA-Lipofectamine2000complex0.1ml (contain TPX2-siRNA10ug) to TPX2-siRNA therapy group's transplanted tumors,while negative siRNA control group local injected with control siRNA-Lipofectamine2000complex0.1ml (contain control siRNA10ug) and blankcontrol group with culture medium0.1ml. TPX2-siRNA transfected group hadno further treatment but only for observation. Treatments are given as aboveonce every four days (six times as total). Then all groups of the nude micewere breaking the neck to death on the30th day. During the observation in the30days, we recorded the emerge time of transplanted tumors, measured thelong and minor axis of the transplanted tumors using slide caliper, drew thegrowth curve of them and made statistic analysis. After the death of nude mice,we measured the weight, long and minor axis of each groups' transplantedtumor samples. Using statistics to analysis the transplanted tumor samples'volumes and weights. The inhibition effect of the TPX2-siRNA intransplanted tumor samples of each group was evaluated byimmunohistochemistry.Results：The time of tumorigenesis and the tumors' growth condition innude mice: The emerge of tumors in TPX2-siRNA transfected group,TPX2-siRNA therapy group, blank control group and negative siRNA controlgroup are at9.37±0.75days,4.37±0.48days,4.50±0.07days and4.12±0.25days. The rate of tumorigenesis in each group was100%. Compared withTPX2-siRNA therapy group, blank control group and negative siRNA controlgroup, the time of tumorigenesis in TPX2-siRNA transfected group is delayed,and has significant difference in statistic (P＜0.05). The growth tendencybetween TPX2-siRNA transfected group and TPX2-siRNA therapy group hasno significant difference in statistic (P＞0.05), as well as it between blankcontrol group and negative siRNA control group. Compared with blankcontrol group and negative siRNA control group, the growth rate oftransplanted tumors in TPX2-siRNA transfected group and TPX2-siRNAtherapy group is significantly decreased and has significant difference instatistic (P＜0.05)(Table2). The growth curve of nude mice's transplantedtumors: We can see the volumes and growth rate of the transplanted tumors in TPX2-siRNA transfected group and TPX2-siRNA therapy group areobviously decreased than them in blank control group and negative siRNAcontrol group. The growth rate of transplanted tumors in TPX2-siRNA therapygroup is low and stable throughout the whole experiment. In TPX2-siRNAtransfected group, the grow rate is lower than treat group at the beginning,however, in the later period of the experiment its grow rate has an accelerationtrend, but still lower than blank control group and negative siRNA controlgroup.The expression of TPX2affects the volumes and weights of nude mice:The final volume of transplanted tumors in TPX2-siRNA transfected group is(61.875±25.702) mm3, with its weight is (52.925±18.892) g. The finalvolume of transplanted tumors in TPX2-siRNA therapy group is(58.6175±27.231) mm3, with its weight is (41.950±12.972) g. The finalvolume of transplanted tumors in blank control group is (608.250±359.378)mm3, with its weight is (407.800±174.339) g. The final volume oftransplanted tumors in negative siRNA control group is (569.625±196.221)mm3, with its weight is (452.700±111.722) g. The final weight and volumebetween TPX2-siRNA transfected group and TPX2-siRNA therapy group,blank control group and negative siRNA group had no significant difference instatistic (P＞0.05). Compared with blank control group and negative siRNAcontrol group, the final weight and volume in TPX2-siRNA transfected groupand TPX2-siRNA therapy group had significantly decreased and hadsignificant difference in statistic (P＜0.05). Analysis the expression of TPX2protein in each groups' transplanted tumor samples by usingimmunohistochemistry: The staining positive rate of TPX2in TPX2-siRNAtransfected group's tissue slices is＜10%. The staining positive rates ofTPX2in TPX2-siRNA therapy group, blank control group and negativesiRNA control group are0%,40.3%and42.2%. In TPX2-siRNA therapygroup's tissue slice, there's no brown granules substance in its nuclei. Tissueslices both in blank control group and negative siRNA control group haveTPX2positive staining mainly located in cell nuclei which present browngranules substance in nuclei. Conclusion: Nude mice transplanted tumor in vivo experiment shows thatTPX2-siRNA can effectively reduce the time of tumorigenesis, inhibit thegrowth of transplanted tumor in nude mice and decrease the expression levelof TPX2protein. TPX2has the potentiality to be a novel target for the therapyof cervical cancer. |
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