摘要
乳腺癌是女性最常见恶性肿瘤,其死亡率仅次于肺癌居第二位,发病率呈逐年上升趋势,内科保守治疗除了传统的放化疗和内分泌治疗外,包括抗肿瘤血管生成治疗在内的生物靶向治疗成为乳腺癌新的治疗热点。1971年,Folkman提出的肿瘤血管生成理论认为,如果没有血管的生成,肿瘤直径不会超过1-2mm。乳腺癌是实体肿瘤,肿瘤的生长离不开新生血管,而血管内皮基因稳定,针对血管内皮的治疗不易产生耐药性,较之传统的化学治疗方案理应有更多优势。内皮抑素是已知的抗血管生成药物之一,由184个氨基酸组成,是相对分子量为22kD的多肽。国产重组人内皮抑素(恩度,endostar)业已上市,我所参加的Ⅱ、Ⅲ期多中心临床试验表明,该药联合化疗在非小细胞肺癌取得良好疗效。但内皮抑素联合化疗方案对乳腺癌的治疗,目前尚缺少实验研究。本课题旨在研究国产重组人内皮抑素联合化疗方案对人乳腺癌(MCF-7)祼鼠模型血管生成及肿瘤生长的影响,为临床上采用内皮抑素与化疗药物联用治疗乳腺癌提供实验依据。
实验方法:
1、建立人乳腺癌裸鼠原位模型,每只裸鼠右侧乳垫下接种1×10~7/0.3ml人乳腺癌MCF-7细胞。
2、实验分组,48只荷瘤鼠随机分为A、B组,两组各随机分为4个实验组,分别为联合用药组(联合组),单一化疗组(化疗组),单一内皮抑素组和阴性对照组(生理盐水组),每组6只,分别进行一个周期的药物干预,联合组给予重组人内皮抑素+化疗(TP方案,紫杉醇+顺铂):紫杉醇20mg·kg~(-1),腹腔注射,d1、d7、d14,顺铂5 mg·kg~(-1),腹腔注射,d1、d7。重组人内皮抑素(恩度)10 mg·kg~(-1),尾静脉缓慢静脉推注,1/日,自第1天起连续用药14天;单一化疗组只按上述TP方案给予化疗药物;内皮抑素组给予单药重组人内皮抑素,用药方法同前;阴性对照组给予等剂量无菌生理盐水。A组观察各项免疫及生化指标,B组观察生存期。
3、观察肿瘤体积、瘤体质量,计算抑瘤率。
4、病理组织技术,观察肿瘤组织结构,免疫组化技术观察移植瘤血管内皮CD34的表达,TUNEL法观察肿瘤组织细胞凋亡。
5、ELISA法检测裸鼠血清血管内皮生长因子(VEGF)。
6、观察一周期用药结束,各组荷瘤鼠生存时间,绘制生存曲线。实验结果:
1、成功建立人乳腺癌MCF-7裸鼠模型,成瘤率100%。
2、肿瘤体积(mm3):联合组343.6057±214.55573,化疗组809.2204±196.46286,内皮抑素组1085.8786±157.46491,生理盐水组1227.9323±350.45055,联合组与其它各组比较有统计学差异(P<0.05),瘤体质量(g):联合组0.5233±0.11656,化疗组0.8633±0.11448,内皮抑素组1.055±0.17986,生理盐水组1.3633±0.3793。抑瘤率:联合组61.62%,化疗组36.676%,内皮抑素组22.61%。
3、各组微血管密度:联合组22.1667±5.98052,化疗组37.3333±6.80196,内皮抑素组32.0000±3.34664,生理盐水组48.0000±3.40588。各实验组间两两比较,联合组肿瘤微血管密度显著低于其它各组(P<0.05),化疗组与内皮抑素组比较无明显差异(P>0.05)。
4、肿瘤细胞凋亡指数(%):联合组60.0000±5.32917,化疗组38.0000±7.21110,内皮抑素组33.8333±7.83369,生理盐水组11.5000±3.78153。联合组与其它3组比较,明显增加(P<0.05)。
5、裸鼠血清VEGF(pg·mL~(-1)):联合用药组33.5±2.167 95 ,化疗组45.666 7±3.559 03,内皮抑素组41.3333±4.92612,对照组73.666 7±3.50238,联合用药组裸鼠血清VEGF水平较其它各组显著降低(P<0.05)。
6、对各组荷瘤鼠进行一个周期连续14天的药物治疗,联合组与化疗组比较,生存期无统计学差异(P>0.05),与阴性对照组比较,生存期延长(P<0.05)。
结论:
重组人内皮抑素(恩度)联合紫杉醇加顺铂化疗方案,治疗人乳腺癌荷瘤裸鼠,可显著增强化疗药物的抗肿瘤生长作用,其机制可能与人内皮抑素降低血清VEGF浓度,减少肿瘤血管生成,以及增加肿瘤细胞凋亡有关。本实验结果为临床开展国产重组人内皮抑素(恩度)联合化疗在乳腺癌治疗中的应用提供了实验依据。
Breast cancer is the second leading cause of cancer deaths in women today ,after lung cancer, and is the most common cancer among women. From year to year, the incidence and the morbility of breast cancer are increasing. The conservative treatments for breast cancer include radiotherapy, chemotherapy, and endocrine therapy. Recent years, bio-targeted therapy becomes becomes a novel strategy In 1971, Folkman reported the theory of angiopoiesis. If there is no blood vessel neogenesis, tumor growth will be inhibited within 2-3mm in diameter. As solid cancer, the grwoth of breast cancer depends on blood vessel neogenesis, which supplys nutrient. The gene of blood vessel endothelium is stable, and drug resistance seldom occurs . It is known that endostatin is one of the antiangiogenesis, which is formed by 184 amino acids, 22KD of molecular weight. China-made rhES has been commercialized under the trade name“Endostar”. Our institute carried out clinical trial (Phase II and III), demonstrating a good effect of Endostar combined with chemotherapy on treating non small cell lung cancer. However, there is a lack of experimental research on ES combined with chemotherapy in treating breast cancer. In this study, MCF-7 breast cancer cell line and nude mice with human breast cancer were treated with recombinant human endostatin. Then we observed the inhibition of tumor proliferation, the apoptosis of cancer cells, and the blood vessel neogenesis of the tumors. The inhibitive effect of rhES combined with Paclitaxel-cisplatin on human breast cancer was evaluated by comparison with Group single chemotherapy, Group single rhES, and Group sodium,respectively.
Methods
1. The cultured MCF-7 human breast cancer cells were subcutaneously injected into the breast of each nude mouse in situ.
2. 48 nude mice bearing human breast carcinoma were devided into Team A and Team B randomly, then every team were subdivided 4 groups. Group combined-drug were given espectively both rhES and TP: paclitaxel, 20mg/kg, ip, d1, d7, d14; DDP, 5mg/kg, d1, d7; rhES, 10mg/kg, d1-14. Group single chemotherapy, were given paclitaxel, 20mg/kg, ip, d1, d7, d14; DDP, 5mg/kg, d1, d7. Group single rhES were given rhES 10mg/kg. And Group sodium were given equal volume sodium.
3. Microvessel density (MVD) in tumors were detected by immunohistochemistry. And apoptosis was tested by TUNEL staining.
4. Serum VEGF was determined by ELISA.
5. Survial time was observed in Team B.
Results
1. 10 days after implantation, the mice were all observed tumor appearancing.
2. Tumor volume: tumors of Group combined-drug grew more slowly than other groups(P<0.05). Group combined-drug, 343.6057±214.55573; Group single chemotherapy, 809.2204±196.46286; Group single rhES, 1085.8786±157.46491; Group sodium 1227.9323±350.45055.
3. The expression of MVD of Group combined-drug is lower than that of other groups (P<0.05). Group combined-drug, 22.1667±5.98052; Group single chemotherapy, 37.3333±6.80196; Group single rhES, 32.0000±3.34664; Group sodium, 48.0000±3.40588.
4. Serum VEGF of Group combined-drug is lower than other groups (P<0.05). Group combined-drug, 33.5±2.16795; Group single chemotherapy, 45.6667±3.55903; Group single rhES, 41.3333±4.92612; Group sodium, 73.6667±3.50238.
5. Apoptotic index was increased in Group combined-drug(P<0.05).
6. There was no significantly difference of survival time between Group combined-drug and Group single chemotherapy (P>0.05). But the survival time of Group combined-drug is siginificantly longer than that of Group sodium (P<0.05). Conclusion
rhES combined TP chemotherapy on nude mice bearing human breast cancer can inhibit tumor growth more siginificantly than single TP chemotherapy. The reason may be that rhES can decrease serum VEGF, reduce microvessel growth inhibition, and induce apoptosis of tumors. Our study provides the evidence on endostar combined chemotherapy in clinical treatment of breat cancer.
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