舒尼替尼及联合顺铂对小儿睾丸卵黄囊瘤荷瘤鼠模型的抗肿瘤作用

Antitumor effects of Sunitinib or Sunitinib combind with cis-diamminedichloroplatinum on an athymicmouse human testicular yolk sac tumor xenograft model

目的探讨舒尼替尼（Sunitinib）、顺铂（CDDP）及两者联合用药对小儿睾丸卵黄囊瘤（TYST）异种移植荷瘤鼠模型的抗肿瘤作用及相关作用机制。方法肿瘤标本来自本实验室的小儿,睾丸卵黄囊瘤裸鼠第17代模型，并接种在雄性裸鼠单侧腹股沟皮下区，成瘤后随机分成4组（n=5）：对照组、CDDP组、Sunitinib组和Sunitinib＋CDDP组。绘制肿瘤体积和裸鼠体重变化曲线图，计算肿瘤消退率；HE染色观察肿瘤组织形态学变化；免疫组织化学法检测AFP、Ki-67、Glypican-3、CD105在各组肿瘤中的表达：CD105测定微血管密度（MVD），Ki-6表示细胞增殖率（PI）；TUNEL法检测肿瘤细胞凋亡率（AI）；实时荧光定量PCR（RT-qPCR）验证靶向因子的mRNA表达变化。结果各治疗组均能显著抑制肿瘤生长，并能消退肿瘤体积。在治疗后肿瘤体积上，除顺铂组与舒尼替尼组间无统计学差异外（41．61±7．61比67．15±5．39，P〉0．05），其余各组间都有统计学差异：对照组与顺铂（651．72±121．16比41．61±7．61，P＜0．05），对照组与舒尼替尼组（651．72±121．16比67．15±5．39，P〈0．05），对照组联合舒尼替尼±顺铂组（651．72±121．16比23．03±2．37，P〈0．05），舒尼替尼组与联合舒尼替尼＋顺铂组（67．15±5．39比23．03±2．37，P〈O．05），顺铂组与联合舒尼替尼＋顺铂组（41．61±7．61比23．03±2．37，P％0．05）；在裸鼠体重上，相比对照组，除舒尼替尼组无统计学差异外（25．90±0．75比26．66±0．65，P〉0．05），其余各组间差异均有统计学意义：对照组与顺铂组（25．90±0．75比18．90±0．63，P〈O．05），对照组与联合舒尼替尼＋顺铂组（25．90±0．75比18．26±1．20，P〈0．05）；AFP、Glypican-3在各治疗组阳性表达面积（Pixels）均少于对照组（AFP：对照组与顺铂组，1．26×10＾6±1．48×10＾5比5．54×10＾5±8．14×10＾4，P〈0．05；对照组与舒尼替尼组，1．26×10＾6±1．48×10＾5比7．09×10＾5±6．64×10＾4，P〈0．05；对照组与联合舒尼替尼＋顺铂组，1．26×10＾6±1．48×10＾5比3．62×10＾5±4．83×10。，P〈0．05。Glypican-3：对照组与顺铂组，9．68×10＾5±7．63×10＾比4．04×10＾5±5．04×10＾，P〈0．05；对照组与舒尼替尼组，9．68×10＾5±7．63×10＾4比4．59×10＾5±2．32×10＾4，P〈O．05；对照组与联合舒尼替尼＋顺铂组，9．68×10＾5±7．63×10＾4比1．89×10＾5±2．55×10＾4，P〈0．05）。两者在顺铂组与舒尼替尼组的比较中差异无统计学意义（P〉0．05）；PI和AI在各治疗组中相比对照组，差异都具有统计学意义（PI和AI：对照组与顺铂组，58．97±1．381；L42．36±1．28和1．69±0．201比54．62±2．49，P〈O．01；对照组与舒尼替尼组，58．97±1．38比43．48±1．00和1．69±0．20比47．32±2．00，P〈0．01；对照组与联合舒尼替尼＋顺铂组，58．97土1．38比33．34±1．19和1．69±0．20比63．41±2．23，P〈O．01）。顺铂组相比联合舒尼替尼＋顺铂组，PI：P=0．001，AI：P=0．002；舒尼替尼组相比联合舒尼替尼＋顺铂组，PI和AI：P〈0．001；顺铂组相比舒尼替尼组，PI：P=0．597，AI：P=0．059；RT-qPCR证实M-CSFR、PDGFR-β、RET、VEGFR．2的mRNA表达受到抑制。结论Sunitinib能显著抑制小儿睾丸卵黄囊瘤的生长，并消退肿瘤体积：主要通过直接抑制肿瘤细胞的生长和肿瘤血管的新生，从而诱导肿瘤细胞凋亡，同时伴有直接细胞毒作用引起坏死；Sunitinib相比CD-DP具有更轻的毒副作用，且联合CDDP具有增强抗肿瘤作用。

Objective To study the antitumor effects of Sunitinib or Sunitinib combind with cis - diamminedichloroplatinum （CDDP） on an athymic mouse human testicular yolk sac tumor xenograft model. Methods The athymic mouse human testicular yolk sac tumor xenograft model was established by subcutaneous injection of 17th passage pediatric testicular yolk sac tumor cells in the unilateral inguinal region of the male nude mice. The mice of control group didn＇t receive any treatment. The tumor bearing mice were treated with either CDDP, or Sunitinib group, or Sunitinib combined with CDDP. The tumor-bearing nude mice were divided into 4 groups （5 in each） according the treatment they underwent. The tumor volumes and mice weight were measured to calculate the regression rate of tumor. The tumor was collected for H＆E staining and immunohistochemical staining of AFP, Ki-67, Glypican-3 and CD105. Microvessel density （MVD） was measured by analyzing the CD105 expression. The tumors＇ proliferation index （PI） was studied by analyzing Ki-67 expression. The apoptosis of the tumor was quantitated using TUNEL staining. The mRNA expressions of cytokines were determined by quantitative real-time PCR （RT-qPCR）. Results The tumor volumes were significantly decreased after chemotherapy. No difference of tumor volume was found between CDDP group and Sunitinib group （41.61 ± 7. 61 vs. 67. 15 ±5.39, P〉0. 05）. Significant differences of tumor volumes were found between CDDP group and CDDP＋ Sunitinib group （41.61± 7. 61 vs. 23. 03 ± 2. 37, P〈0. 05）, and between Sunitinib group and CDDP＋ Sunitinib group （67. 15 ± 5.39 vs. 23.03 ±2. 37, P 〈0. 05）. Of the body weight of tumor-bearing mice, no difference was found between controls and Sunitinib group （25.90 ±0. 75 vs. 26. 66 ± 0. 65, P〉0. 05）. And significant differences of the body weight were noted between controls and CDDP group （25.90 ± 0. 75 vs. 18. 90±0. 63,P〈0. 05）, controls and CDDP＋ Sunitinib group （25.90 ±0. 75 vs. 18. 26± 1.20,P〈0. 05）. The positive areas （pixels） of AFP in the mice with chemotherapy were less than those of control mice （Control vs. CDDP.. 1.26× 10^6 ±1. 48× l0^5 vs. 5. 54×10^5 ± 8. 14 × 104 , P〈0. 05. Control vs. Sunitinib： 1.26 ×10^6 ± 1.48 × 10^5 vs. 7. 09× 10^5 ± 6. 64× 10^4, P〈0, 05. Control vs. CDDP ＋ Sunitinib： 1.26 ×10^6±1.48 X 10s VS. 3. 62× 10^5 ±4. 83 × 104, P 〈0. 05）. The positive areas （pixels） of Glypican-3 in the mice with chemotherapy were less than those of control mice （Control vs. CDDP： 9. 68 × 10^5± 7. 63 ×10^4 vs. 4. 04 × 10s ±5.04 ×10^44 , P〈0. 05. Control vs. Sunitinib： 9. 68 × 10^5 ± 7. 63 × 10^4 vs. 4. 59 × 10^5 ± 2. 32 × 10^4 , P〈0. 05. Control vs. CDDP ＋ Sunitinib： 9. 68 ×10^5±7. 63 ×10^4vs. 1. 89× 10^5 ± 2. 55 × 104 , P〈0. 05）. However, there were no statistical differences of the positive areas （pixels） of AFP and Glypiean-3 between CDDP and Sunitinib groups （P〉0. 05）. The PI was significantly decreased after chemotherapy （Control vs. CDDP.. 58. 97 ±1.38 vs. 42. 36 ± 1.28, P%0. 01. Control vs. Sunitinib： 58. 97 ±1.38 vs. 43.48 ± 1.00, P〈0. 01. Control vs. CDDP＋ Sunitinib： 58. 97 ±1.38 vs. 33. 34 ± 1.19, P〈0. 01）. The apoptosis index （AI） was also significantly increased after chemotherapy （Control vs. CDDP： 1.69 ±0. 20 vs. 54. 62 ± 2. 49,P〈0. 01. Control vs. Sunitinib： 1.69 ± 0. 20 vs. 47.32±2.00, P〈0.01. Control vs. CDDP＋Sunitinib： 1.69±0.20 vs. 63.41±2.23, P〈 0. 01）. Significantly differences of PI and AI were found between CDDP and CDDP ＋ Sunitinib groups （P〈0. 01）, and Sunitinib and CDDP ＋ Sunitinib （P〈0. 01）. RT-qPCR study confirmed that the mRNA expressions of M-CSFR, PDGFR-β, RET and VEGFR-2 were decreased in the mice underwent chemotherapy. Conclusions Sunitinib is effective to suppress the pediatric testicular yolk sac tumor growth, and reduce the tumor volume. Sunitinib can inhibit the angiogenesis in tumor, induce apoptosis of tumor cells, and kill the tumor cells directly. Sunitinib is less toxic than CDDP, and synergistic with the antitumor effect of CDDP.