热休克蛋白70mRNA反义寡核苷酸对荷瘤小鼠膀胱癌的作用

The effect of Heat shock protein 70 antisense oligonucleotides to the bladder cancer in mouse loaded with tumor

目的探讨热休克蛋白70（HSP70）mRNA反义寡核苷酸（ASO）对体内膀胱癌发生发展的影响及其可能的作用机制。方法用培养的人类膀胱癌细胞株BIU-87建立40只BALB／c裸鼠膀胱癌皮下荷瘤模型，待皮下肿瘤体积约为100mm^3时，随机分为4组，每组10只。HSP70 mRNA ASO加丝裂霉素C（MMC）处理组；HSP70 mRNA反义寡核苷酸组；丝裂霉素C组；空白对照组。HSP70 mRNA按10mg／kg，肿瘤局部注射，每周2次；MMC 0．1mg／kg，腹腔注射，每周2次，空白组用生理盐水代替上述处理。治疗的第30天托颈处死裸鼠，剥离出皮下肿瘤，照像并称重量。免疫组织化学法检测肿瘤组织中的微血管密度（MVD），RT—PCR和Western blotting检测HSP70表达；原位末端标记法（TUNEL）检测细胞凋亡。结果HSP70 mRNA ASO加MMC组肿瘤抑制率超过50％，高于HSP70 mRNA ASO组或MMC组，差异有统计学意义（P〈0．05）。HSP70 mRNA ASO和MMC组相比，差异无统计学意义（P〉0．05）。ASO＋MMC组的凋亡指数（AI）分别高于其他三组，差异有统计学意义（P〈0．05）；MMC和ASO组AI分别高于空白组（P〈0．05）；MMC和ASO组相比，差异无统计学意义（P〉0．05）。肿瘤组织中的MVD与上述结果一致。结论HSP70 mRNA ASO局部注射可以抑制肿瘤的生长，其作用机制可能和其抑制微血管的形成及促进细胞凋亡有关。

Objective To investigate The effect of Heat shock protein 70 （HSP70） antisense oligonucleotides （ASO） to bladder carcinoma in mouse loaded with tumor. Methods The 40 mice loaded with tumor subcutaneously were established by cultured BIU-87 cells, and divided into 4 groups randomly when the subcutaneous neoplasms grew to about 100 mm^3, namely, HSP70 mRNA ASO plus mitomycin C （MMC） group; HSP70 mRNA ASO group; MMC and blank control. HSP70 mRNA ASO were injected into neoplasms, 10mmg/kg weight, twice every week, and MMC 0.1mg/kg weight, twice every week, and the above schemes were replaced with normal saline to blank. The neoplasms were peeled off, photograghed and weighed in 30 days. HSP70 expressions were examined with reverse transcription polymerase chain reaction （RT-PCR）, microvascular density （MVD） was evaluated by immunohis to chemical staining and the tumor cells apoptosis was detected by terminal deoxynucleotidyl transferase （TdT）-mediated dUTP-biotin nick end labeling technique （TUNEL）. Results The tumor inhibition rate in ASO ＋ MMC surpassed 50 %, more than ASO or MMC respectively, and the differences were significantly （P 〈0.05）. The ASO and MMC exceeded blank group respectively （P 〈0.05）. The ASO was the same as the MMC （P 〉0.05）. The apoptotic index （AI） in ASO ＋ MMC surpassed the other three groups （P 〈0.05）. The difference between ASO and MMC was not significant （P 〉0.05）, while the AI of ASO or MMC was more than blank respectively （P 〈0.05）. The results of MVD were in accordance with the above results. Conclusion The injection of HSP70 mRNA ASO in tumor locally can inhibit neoplasm growth, and this effect might correlate with the inhibition of apoptosis and microvascular forming resulting from the ASO.