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目的探讨砷剂(arsenictrioxide,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(microvesseldensitycount,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(5.0μg)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 mechanism of arsenictrioxide (ATO) and thalidomide (THAL) alone and in combination on human myelodysplastic syndrome (MDS) tumor-bearing mice. Methods (1) Establishment of animal model: MUTZ-1 cells from logarithmic growth phase were inoculated into the scapular skin of first-generation SCID mice and nude mice (BALB / CA-nμde) Pathology, immunohistochemistry (IHC), flow cytometry immunology, chromosome analysis and other methods to identify the source of tumor-bearing tissues and their biological characteristics. The animals were passaged, the second generation of 61 SCID mice and 8 nude mice were observed for tumor formation rate and tumorigenic potential. (2) Animal experiments: 56 MDS tumor-bearing mice were randomly divided into treatment group 40 (ATO, THAL single and combined) and control group 16 (saline and no treatment); with pathology, IHC , Microvessel density (MVD), DNA gel electrophoresis, PI staining and TUNEL staining were used to observe the growth, tumor size, survival, survival rate and apoptosis of the tumor-bearing mice in each group influences. Results (1) Establishment of animal model: The rate of tumor formation in SCID mice was significantly higher than that in nude mice (98.4%, 60/61) (62.5%, 5/8) (P = 0.0027) The median latency was 12d). However, the latent period of nude mice was significantly longer (median 26d) (Z = 4.605, P <0.001). The source identification of tumor-bearing tissues proved to be human-derived, maintaining the biological characteristics of the original cell line (MUTZ-1 cells), indicating that the animal model was successfully established. (2) In vivo experiments in animals: Compared with the control group, the high dose of ATO (7.5μg) alone had stronger inhibition of tumor growth (F = 146.94, P = 0.000) and induction of apoptosis (F = 30.10, P = 0.000) .However, the side effects were more difficult to tolerate in mice. The low dose of ATO (5.0μg) alone also had obvious antitumor and apoptosis-inducing effects with less side effects and longer mean survival (F = 25.11, P <0.01), the effect is better. High-dose ATO combined with THAL side effects, the average survival time of mice was shorter (P <0.01), poor efficacy. THAL alone was effective in inhibiting tumor, but its effect was slow. Its mechanism was not to induce apoptosis (P> 0.05), but may be related to the inhibition of angiogenesis. Conclusion (1) MDS-SCID tumor-bearing mouse model was established successfully. (2) Small dose (5.0μg) of ATO could effectively suppress tumor in MDS tumor-bearing mice with less side effects, prolong the survival of tumor- Improve survival rate, the mechanism of apoptosis and induction.