常规化疗联合节拍化疗对乳腺癌裸鼠移植瘤的实验研究
摘要
目的:观察环磷酰胺常规化疗联合节拍化疗对乳腺癌裸鼠移植瘤的抑瘤效应,探讨其对血管新生和细胞增殖、凋亡的影响。
方法:1.体外培养MCF-7乳腺癌细胞,调整细胞浓度为6×10~7/ml,以0.2ml/只接种于裸鼠右侧胸壁乳垫下,建立原位移植瘤模型。2.20只裸鼠随机分为4组,每组5只:环磷酰胺小剂量节拍化疗组(LDM组-CTX 20mg·kg~(-1)·d~(-1),qd×21d)、常规最大耐受剂量联合小剂量节拍化疗组(联合组- CTX 150mg·kg~(-1)·d~(-1),qod×3次+ CTX 20mg·kg~(-1)·d~(-1),qd×14d)、常规最大耐受剂量化疗组(MTD组- CTX 150mg·kg~(-1)·d~(-1),qod×3次)及生理盐水对照组(等体积,qd×21d)。3.治疗期间观察裸鼠一般状况、隔日称重并测量皮下移植瘤体积,每周尾静脉采血计数外周血白细胞数(WBC),21天治疗结束后观察5天颈椎脱臼处死小鼠,无菌条件下取瘤称重、计算抑瘤率。4.免疫组化检测各组移植瘤组织中微血管密度(MVD)、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、凝血酶敏感蛋白1(thrombospondin-1,TSP-1)、细胞内增殖细胞核抗原(proliferating cell nuclear anti-gen,PCNA)表达,TUNEL法检测肿瘤细胞凋亡。
结果:1.20只裸鼠全部成瘤并且顺利完成实验,用药前后各组小鼠一般状况良好,未发现进食水、排便、活动异常,未见明显消瘦、淤血等。2.CTX三种化疗方案均可不同程度的抑制移植瘤生长,其中LDM组和MTD组生长曲线类似,联合组移植瘤生长明显缓于其他各组,实验结束处死裸鼠,对照组同CTX各治疗组移植瘤体积、质量差异均有统计学意义(P<0.05),联合组同LDM组、MTD组移植瘤体积、质量差异也有统计学意义(P<0.05),而LDM组和MTD组无明显差异(P>0.05),各化疗组抑瘤率分别为:LDM组32.95%、联合组69.15%、MTD组41.57%。3.对移植瘤血管生成影响:LDM组和联合组MVD、VEGF相对低表达、TSP-1相对高表达,和其余两组比较差异有统计学意义(P<0.05),而两组间无明显差异(P>0.05),MTD组和对照组MVD、VEGF和TSP-1表达无明显差异(P>0.05)。4.对移植瘤细胞增殖、凋亡影响:联合组和MTD组PCNA相对低表达,和其余两组比较差异有统计学意义(P<0.05),而两组间无明显差异(P>0.05),LDM组和对照组PCNA表达无差异(P>0.05);联合组凋亡指数(apoptosis index,AI)明显高于其余各组,差异有统计学意义(P<0.05),LDM组和MTD组诱导细胞凋亡无明显差异(P>0.05)。
结论::环磷酰胺常规化疗联合节拍化疗兼有抗血管生成、抑制细胞增殖、促进肿瘤细胞凋亡作用,抑瘤作用较单一传统化疗和节拍化疗更为明显,毒副作用不明显。
Objective: To compare the antitumor effects in nude mice with human breast cancer with cyclophosphamide different chemotherapy regimens and to observe two balances: between pro-angiogenesis and anti-angiogenesis and between tumor cell proliferation and apoptosis.
Methods:1.To establish breast orthotopic transplantation tumor model.2.20 nude mice bearing carcinoma were divided into 4 groups randomly: metronomic chemotherapy group(LDM)、conventional chemotherapy group(MTD)、combined group(LDM+MTD) and control group.3.Observe the general condition of mice and weight them every other day, at the same time measure the volume of subcutaneous tumor. WBC was counted every week. In the end, tumors were taken off and weighted to calculate inhibitory rates of each group.4.Immunohistochemistry was used to detect the expressions of MVD、VEGF、TSP-1 and PCNA.TUNEL was used to detect the apoptosis of the tumor cells.
Results:1.All nude mice were in good condition throughout the trial period without significant side effects.2.Three chemotherapy regimens can inhibit the growth of xenografts in different degrees, whose inhibitory rates were 32.95%、41.75% and 69.15%,respectively.LDM and MTD had similar tumor growth curve. While the xenografts in combination group appeared to grow significantly slower than the other groups. 3. In the combination group and LDM group, xenografts expressed lower level VEGF and MVD and higher level TSP-1 than those in the other groups(p<0.05), but there is no significant difference between the two groups (p>0.05).4. There was lower expression of PCNA in the combination group and MTD group than its in the LDM and control group (p<0.05), but there is no statistically significant difference between the two groups (p>0.05). The apoptosis index (AI) in three chemotherapy groups had statically significant difference with control group (p<0.05). Furthermore, the AI in the combination group was highest.
Conclusion: Conventional chemotherapy combined with metronomic chemotherapy has more powerful anti-cancer effects compared to the single conventional or metronomic chemotherapy without obvious side effect. The probable mechanism may be related with suppressing angiogenesis、inhibiting tumor cell proliferation and inducing apoptosis.
方法:1.体外培养MCF-7乳腺癌细胞,调整细胞浓度为6×10~7/ml,以0.2ml/只接种于裸鼠右侧胸壁乳垫下,建立原位移植瘤模型。2.20只裸鼠随机分为4组,每组5只:环磷酰胺小剂量节拍化疗组(LDM组-CTX 20mg·kg~(-1)·d~(-1),qd×21d)、常规最大耐受剂量联合小剂量节拍化疗组(联合组- CTX 150mg·kg~(-1)·d~(-1),qod×3次+ CTX 20mg·kg~(-1)·d~(-1),qd×14d)、常规最大耐受剂量化疗组(MTD组- CTX 150mg·kg~(-1)·d~(-1),qod×3次)及生理盐水对照组(等体积,qd×21d)。3.治疗期间观察裸鼠一般状况、隔日称重并测量皮下移植瘤体积,每周尾静脉采血计数外周血白细胞数(WBC),21天治疗结束后观察5天颈椎脱臼处死小鼠,无菌条件下取瘤称重、计算抑瘤率。4.免疫组化检测各组移植瘤组织中微血管密度(MVD)、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、凝血酶敏感蛋白1(thrombospondin-1,TSP-1)、细胞内增殖细胞核抗原(proliferating cell nuclear anti-gen,PCNA)表达,TUNEL法检测肿瘤细胞凋亡。
结果:1.20只裸鼠全部成瘤并且顺利完成实验,用药前后各组小鼠一般状况良好,未发现进食水、排便、活动异常,未见明显消瘦、淤血等。2.CTX三种化疗方案均可不同程度的抑制移植瘤生长,其中LDM组和MTD组生长曲线类似,联合组移植瘤生长明显缓于其他各组,实验结束处死裸鼠,对照组同CTX各治疗组移植瘤体积、质量差异均有统计学意义(P<0.05),联合组同LDM组、MTD组移植瘤体积、质量差异也有统计学意义(P<0.05),而LDM组和MTD组无明显差异(P>0.05),各化疗组抑瘤率分别为:LDM组32.95%、联合组69.15%、MTD组41.57%。3.对移植瘤血管生成影响:LDM组和联合组MVD、VEGF相对低表达、TSP-1相对高表达,和其余两组比较差异有统计学意义(P<0.05),而两组间无明显差异(P>0.05),MTD组和对照组MVD、VEGF和TSP-1表达无明显差异(P>0.05)。4.对移植瘤细胞增殖、凋亡影响:联合组和MTD组PCNA相对低表达,和其余两组比较差异有统计学意义(P<0.05),而两组间无明显差异(P>0.05),LDM组和对照组PCNA表达无差异(P>0.05);联合组凋亡指数(apoptosis index,AI)明显高于其余各组,差异有统计学意义(P<0.05),LDM组和MTD组诱导细胞凋亡无明显差异(P>0.05)。
结论::环磷酰胺常规化疗联合节拍化疗兼有抗血管生成、抑制细胞增殖、促进肿瘤细胞凋亡作用,抑瘤作用较单一传统化疗和节拍化疗更为明显,毒副作用不明显。
Objective: To compare the antitumor effects in nude mice with human breast cancer with cyclophosphamide different chemotherapy regimens and to observe two balances: between pro-angiogenesis and anti-angiogenesis and between tumor cell proliferation and apoptosis.
Methods:1.To establish breast orthotopic transplantation tumor model.2.20 nude mice bearing carcinoma were divided into 4 groups randomly: metronomic chemotherapy group(LDM)、conventional chemotherapy group(MTD)、combined group(LDM+MTD) and control group.3.Observe the general condition of mice and weight them every other day, at the same time measure the volume of subcutaneous tumor. WBC was counted every week. In the end, tumors were taken off and weighted to calculate inhibitory rates of each group.4.Immunohistochemistry was used to detect the expressions of MVD、VEGF、TSP-1 and PCNA.TUNEL was used to detect the apoptosis of the tumor cells.
Results:1.All nude mice were in good condition throughout the trial period without significant side effects.2.Three chemotherapy regimens can inhibit the growth of xenografts in different degrees, whose inhibitory rates were 32.95%、41.75% and 69.15%,respectively.LDM and MTD had similar tumor growth curve. While the xenografts in combination group appeared to grow significantly slower than the other groups. 3. In the combination group and LDM group, xenografts expressed lower level VEGF and MVD and higher level TSP-1 than those in the other groups(p<0.05), but there is no significant difference between the two groups (p>0.05).4. There was lower expression of PCNA in the combination group and MTD group than its in the LDM and control group (p<0.05), but there is no statistically significant difference between the two groups (p>0.05). The apoptosis index (AI) in three chemotherapy groups had statically significant difference with control group (p<0.05). Furthermore, the AI in the combination group was highest.
Conclusion: Conventional chemotherapy combined with metronomic chemotherapy has more powerful anti-cancer effects compared to the single conventional or metronomic chemotherapy without obvious side effect. The probable mechanism may be related with suppressing angiogenesis、inhibiting tumor cell proliferation and inducing apoptosis.
引文
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