紫杉醇奥曲肽偶合物对A549裸鼠移植瘤的靶向治疗研究

Tumor growth inhibition of paclitaxel-octreotide conjugates on human non small cell lung cancer： experiment with mice

目的研究生长抑素类似物奥曲肽（OCT）与紫杉醇（PTX）的偶合物对人肺腺癌细胞株．4549裸鼠移植瘤的靶向治疗作用。方法构建1分子紫杉醇-1分子奥曲肽偶合物（PTX-OCT）和2分子紫杉醇-1分子奥曲肽偶合物（2PTX-OCT），建立裸鼠腋部皮下移植瘤模型，将荷瘤裸鼠随机分为8组。在1、7、21d尾静脉分别注射150nmol/kg PTX—OCT；2PTX-OCT；OCT、PTX混合物（OP）；OCT；PTX；300nmol/kg PTX（2PTX）和2PTX—OCT[2（2PTX-OCT）]。测各组小鼠体重及肿瘤体积；计算肿瘤倍增时间；眼内眦静脉取血测每组小鼠白细胞数量；组织切片HE染色及核仁区银染（AgNOR）观察肿瘤细胞生长情况；DNA梯度法检测细胞凋亡；RT-PCR法测肿瘤细胞生长抑素受体（SSTR）1至5亚型（SSTR1-SSTR5）mRNA表达情况；免疫组化SP法检测肿瘤微血管密度（MVD）以及生长抑素受体2和5亚型表达情况。结果2PTX-OCT与2（2PTX-OCT）处理组在3次给药后与对照组相比：肿瘤生长显著抑制（均P〈0．01）；肿瘤倍增时间延长（均P〈0．01）。PTX与2PTX-OCT处理组相比，在每次给药后5d裸鼠体重减轻差异有统计学意义（P〈0．05）；PTX与2PTX组的白细胞总数在26d降到最低，与对照组、2PTX-OCT组及2（2PTX-OCT）组相比差异均有统计学意义（均P〈0．05）。2PTX-OCT组及2（2PTX—OCT）组MVD均明显少于对照组（均P〈0．01）。DNA梯度法显示2PTX-OCT处理组有明显的DNA片段化的梯状条带。生长抑素受体1、2、4、5亚型的mRNA在荷瘤小鼠体内表达；免疫组化显示SSTR2主要表达于肿瘤细胞的细胞膜上，SSTR5表达于肿瘤细胞的细胞膜和胞质中。结论2PTX-OCT可显著抑制肿瘤增殖，减少肿瘤微血管形成，显示出良好的治疗作用，有望成为晚期非小细胞肺癌治疗的有效手段之一。

Objective To evaluate the antitumor effects of the conjugates synthesized by coupling eytotoxie paclitaxel （PTX） to somatostatin analog oetreotide （OCT） on human non small cell lung cancer （NSCLC）. Methods Two cytotoxic somatostatin analog, PTX-OCT and 2PTX-OCT, were developed in which FIX was linked to octreotide. Forty-five BALB/c nu/nu nude mice were injected with human NSCLC cells of the line A549 into the right armpit. The nude mice that were xenografted were randomly divided into 8 groups. （1）control group （n =6）,（2） PTX-OCT group （n =5）, injected intravenously with PTC-OCT 150 nmol/kg on days 1,7, and 21,（3） 2PTX-OCT group （ n = 6 ）, injected intravenously with PTC-OCT 150 nmol/kg, （4） OP group （n =6）, injected with mixture of PTX and OCT 150 nmol/kg, （5） OCT group （n = 5） injected with OCT 150 nmol/kg （6） PTX group （n = 6）, injected with PTX 150 nmol/kg, （7） 2PTX group, injected with PTX 300 nmol/kg, and （8）2（PTX-OCT） injected with PTX-OCT 300 nmol/kg, The tumor volume and body weight （BW） were observed regularly. The tumor volume doubling time was calculated. White blood cells were counted by collecting peripheral blood sample. By the end of experiment the mice were killed with the tumors taken out. The expression of mRNA of subtypesl -5 of human somatostatin receptor （SSTR1 -SSTR5 ） were investigated using RT-PCR. Histological apoptosis was detected by DNA ladder. Immunohistochemistry was used to examine the SSTR2 and SSTR5 expression andtumor microvessel density （MVD）. Results The tumor volumes of 2PTX-OCT and 2 （2PTX-OCT） groups were significantly smaller than those of other groups （ all P 〈 0. 01 ）. The tumor doubling times of the 2PTX- OCT and 2（2PTX-OCT） groups were significantly longer than those of the other groups too （ all P 〈0. 01 ）. The MVD levels of the 2PTX-OCT and 2（2PTX-OCT） groups were significant lower than those of the other groups （all P 〈0. 01 ）. The toxicity of the PTX group was more obvious. The WBC count levels of the PTX and 2PTX groups were significantly lower than those of the other groups （ all P 〈 0.05 ）. mRNA expression could be found for SSTR1, 2, 4, and 5 in the A549 xenografts. Immunohistochemistry showed that SSTR2 was mainly found in the tumor cell membrane, and SSTR5 was found in the tumor cell membrane and cytoplasm. More histological apoptosis bands were shown by DNA ladder method in the 2PTX-OCT and 2 （PTX-OCT） groups. Conclusion The targeting conjugate 2PTX-OCT inhibits the proliferation of tumor cells, reduces microvessel, and decreases the toxicity in comparison with the cytotoxic radical PTX.