摘要
目的探讨砷剂（arsenic trioxide，ATO）和沙利度胺（thalidomide，THAL）单用及联用在人骨髓增生异常综合征（MDS）荷瘤小鼠体内抗瘤作用及其机制。方法（1）建立动物模型：取对数生长期人MDS细胞株MUTZ-1细胞接种于第一代4～6周龄SCID小鼠和裸鼠（BALB／CA-nμde）的肩胛皮下；用组织病理学、免疫组织化学（immunohistochemistry，IHC）、流式细胞术免疫学、染色体分析等方法对荷瘤组织来源及其生物学特性进行鉴定。动物传代，观察第二代61只SCID小鼠和8只裸鼠的成瘤率和成瘤潜伏期。（2）动物实验：随机取56只MDS荷瘤小鼠，分为治疗组40只（ATO、THAL单用及联用）和对照组16只（生理盐水和未治疗）；用病理学、IHC、微血管密度计数（microvessel densitycount，MVD）、DNA凝胶电泳、PI染色、TUNEL染色等多参数方法，观察药物对各组荷瘤小鼠生长、瘤体大小、生存期、存活率及细胞凋亡等影响。结果（1）动物模型的建立：SCID小鼠成瘤率（98．4％，60／61）明显高于裸鼠（62．5％，5／8）（P=0．0027）；SCID小鼠成瘤潜伏期（中位数12d）较短；而裸鼠潜伏期明显延长（中位数26d）（Z=4．605，P〈0．001）。荷瘤组织的来源鉴定结果证实为人源性的、保持了原细胞系（MUTZ．1细胞）生物学特性，表明动物模型建立成功。（2）动物体内实验：与对照组比较，大剂量ATO（7．5μg）单用有较强的抑制荷瘤生长（F=146．94，P=0．000）和诱导凋亡作用（F=30．10，P=0．000），但副作用较大，小鼠难以耐受；小剂量ATO（5．0μg）单用亦有明显的抑瘤作用和诱导细胞凋亡作用、副作用较轻，小鼠平均生存期较长（F=25．11，P〈0．01）、疗效较好。大剂量ATO与THAL联用副作用大、小鼠平均生存期较短（P〈0．01），疗效不佳。THAL单用抑瘤有效，但显效较迟缓，其机制并非诱导细胞凋亡（P〉0．05），而可能与抑制血管生成机制有关。结论（1）成功建立人MDS-SCID荷瘤小鼠模型；（2）小剂量（5．0wg）ATO在MDS荷瘤小鼠体内抑瘤有效、副作用较轻，能延长荷瘤小鼠生存期、提高存活率，其机制与诱导细胞凋亡有关。
Objective To investigate the anti-tumor effect and the possible mechanism of arsenic trioxide（ATO） alone or in combination with thalidomide （THAL） for treatment of SCID mice model transplanted with human myelodysplastic syndrome （ MDS ） cell line MUTZ-1 cells. Methods （ 1 ） The animal model was established in SCID mice;75 SCID mice and 10 BALB/CA-nude mice were studied in this experiment. MUTZ-1 ceils were cultured in vitro and made for mono-ceil suspension （with 1 × 10^8/ml ceil density and in exponential growth behavior） and were subcutancously implanted into 4-6-week-old firstgeneration SCID mice and BALB/CA-nude mice. The biological characteristics of the subcutancous tumor cells were evaluated by the methods of cell morphology, histopathology, immunology by flow cytometer, chromosome analysis and immunohistochemistry （IHC）. Subsequently, the tumor cells from first-generation mice model were respectively subcutaneously implanted into 61 second-generation SCID mice and 8 BALB/ CA-nude mice and the rate of the tumor formation and the latent period of the tumor formation were observed. （2） In vivo, 56 MDS-SCID mice were randomly grouped; 40 of them were used as ATO treated groups [5.0 μg or 7.5 μg/（g ·d） intraperitoneal injection （i p） 5 d a week, ×3 weeks] alone or in combination with THAL 8 μg/（g · d）, ×3 week or THAL alone and 16 mice as the control groups [with 0. 9% NaCl 10μl/ （g·d） i p or untreated]. The mean tumor diameters （MTD） of subcutaneous tumors were measured with slide gauge and the therapeutic effects and the survival period and the rates of survival were evaluated by the methods of histopathology, IHC, microvessel density count （MVD）, DNA ladder, TUNEL and PI with flow cytometry. Results （ 1 ） The rate of the subcutancous tumor formation was higher （98.4%, 60/61 ） in SCID mice than in BALB/CA-nude mice （62. 5%, 5/8 ） （ P = 0. 0027 ）. The latent period of the tumor formation was significantly longer （23-28 d, median 26 d） in BALB/CA-nude mice than that in SCID mice （ 10-17 d, median 12 d） （Z =4. 605 ,P 〈0. 001 ）. The biological characteristics of the tumor cells in the MDS-SCID mice model were evaluated and considered as of anthropo-source and were consistent with that of MUTZ-1 cells, which showed that the MDS-SCID mice model was successfully established. （2） In vivo, the marked inhibitory effect on the subcutaneous tumors growth （ F = 146. 94, P = 0. 000） and the higher rates of ceils apoptosis were seen in the groups treated with ATO 5.0 μg or 7. 5 μg alone or in combination with THAL than in control groups （ F = 30. 10, P = 0. 000）. The longer-survival periods （ F = 25.11, P 〈 0. 01 ） with lower toxicity were only observed in lower-dose group of ATO 5.0 μg than in other treated groups and control groups. THAL alone group had a mild inhibitory effect on the tumors growth （ 〉 2 weeks） with a longersurvival period and higher-rate of survival than controls, but had no events of cell apoptosis. The expression of vascular endothelial growth factor （VEGF） protein and CD34 protein and MVD were markedly down-regulated by THAL compared with control groups （ P 〈 0. 01 ）, suggesting that the possible mechanisms of the inhibitory effect on tumor growth by THAL related to inhibition of vascular endothelial growth. The legs paralysis in 2 MDS-SCID micewas observed after treatment with THAL alone （ 11 d and 15 d, respectively）, which were considered as the side-effect of THAL associated with deep venous thrombotic （DVT） events and the mechanism for these events is unclear. The therapeutic effects were unsatisfied in the group treated with ATO 7. 5 Ixg in combination with THAL due to more intense toxicity and the shorter-survival periods than other treated groups （ P 〈 0. 001 ）. Conclusions （ 1 ） The Hum-MDS-SCID mice model was successfully established, which served as an animal model for studying pathogenesis of MDS and therapeutie agents selection. （2）ATO had marked inhibitory effect on the subcutaneous tumors growth in MDS-SCID mice model in vivo. The longer-survival periods and higher-rates of survival with lower toxicity were observed in lower- dose ATO （5.0 μg） alone than other groups. The mechanisms of the anti-tumor effect of ATO were considered to be related to inducing cells apoptosis, but in the case of THAL, related to inhibition of vascular endothelial growth. （ 3 ） The results do not support the preconceived hypothesis of a synergistic effect of ATO （7.5 Ixg） in combination with THAL for treatment of MDS mice model due to the more intense toxicity and the shorter-survival periods in the treated group. Whether or not this will translate into clinically relevant effect of the ATO or THAL in MDS patients deserves further investigation.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2006年第3期228-233,共6页
Chinese Journal of Pediatrics
基金
浙江省科学技术厅重点资助项目(2003C23013)
