视网膜母细胞瘤基因联合放射处理对裸鼠种植瘤Rb蛋白和Ki67蛋白表达的影响

The effects of retinoblastoma gene therapy combined with radiotherapy on the retinoblastoma protein and Ki67 protein in carcinoma cells of tumor- bearing nude mice

目的探讨视网膜母细胞瘤Rb94基因与放射联合治疗后裸鼠食管癌细胞中Rb蛋白和Ki67蛋白的表达情况。方法将食管癌K150细胞接种裸鼠建立肿瘤模型,实验分为5组,对照组未进行处理,Ad-Lac Z组为含Lac Z基因但不含Rb94基因的对照腺病毒,分别于处理后即刻、处理后的第3、7天以Ad-Lac Z感染瘤体,Ad-Rb94组分别于处理后即刻、处理后的第3、7天以Ad-Rb94感染瘤体,照射组分别于第1、4、8天进行4 Gyγ射线局部照射瘤体,联合组在Ad-Rb94感染后进行4 Gy局部照射。采用免疫组化方法观察肿瘤组织Rb蛋白和Ki67蛋白的变化,采用TUNEL法检测细胞凋亡。结果 Ad-Rb94组[(28.96±3.35)%]和联合组[(33.13±1.59)%]Rb蛋白阳性表达增加,明显高于对照组,差异有统计学意义(q=9.96、13.35,P<0.01);Ad-Rb94组和联合组Rb蛋白表达均高于照射组,差异有统计学意义(q=12.72、15.36,P<0.01)。Ad-Rb94组[(29.15±1.65)%]、照射组[(38.16±2.53)%]和联合组[(25.18±3.06)%]Ki67蛋白阳性表达减少,与对照组比较差异有统计学意义(q=6.36、5.59、7.52,P<0.01);联合组Ki67蛋白表达显著低于照射组(q=6.49,P<0.01)。AdRb94组[(14.1±3.2)%]、照射组[(17.2±3.0)%]和联合组[(24.3±3.2)%]细胞凋亡率显著增加,与对照组比较差异有统计学意义(q=6.62、7.29、10.54,P<0.01);联合组细胞凋亡率明显高于照射组和Ad-Rb94组,差异有统计学意义(q=6.36、7.89,P<0.01)。结论 Rb94基因与放射联合应用协同抑瘤的机制之一是通过增强Rb蛋白表达和抑制抗凋亡蛋白Ki67表达。

Objective To reveal the effects of retinoblastoma 94 gene therapy combined with radiotherapy on the expression of Rb protein and Ki67 protein in esophageal carcinoma cells of tumor- bearing nude mice. Methods The model of tumor- bearing nude mice was established by K150 cells inoculation. The cohorts were divided into five groups,blank control（ no treatment）,Ad- Lac Z group（ control adenovirus including Lac Z gene but not Rb94 gene,Ad- Lac Z was transfered into tumor xenograft on 0d,3d,7d therapy separately）,Ad- Rb94 group（ tumor xenograft was transfected with Ad- Rb94 on 0d,3d,7d therapy separately）,radiation group（ tumor xenograft was irradiated with 4Gy γ- radiation on 1d,4d,8d therapy separately） and Ad- Rb94 combined with radiation group（ combining group for short,tumor xenograft was irradiated with 4Gy after transfected with Ad- Rb94）. The expression of Rb protein and Ki67 protein in esophageal carcinoma was detected by immunohistochemical analysis. Cell apoptosis was examined by TUNEL assay.Results The expression of Rb protein in Ad- Rb94 group（28.96±3.35%） and combining group（33.13 ±1.59%）was significantly increased compared with that in control group（ q=9. 96 and 13. 5,P〈0.05）. The expression of Rb protein in Ad- Rb94 group and combining group was significantly higher than that in radiation group（q=12.72 and15.36,P〈0.01）. The expression of Ki67 protein in Ad- Rb94 group（29.15±1.65%）,radiation group（ 38.16±2.53%） and combining group（25.18±3.06%） was significantly reduced compared with that in control group（q=6.36,5.59 and7.52,respectively,P〈0.01）. The expression of Ki67 protein in combining group was significantly less than that in radiation group（q=6.9,P〈0.01）. The ratios of cell apoptosis in Ad- Rb94 group（14.1 ± 3.2%）,radiation group（17.2±3 0%） and combining group（24.3±3.2%） were significantly increased compared with that in control group（q=6.62,7.29 and 10. 54,respectively,P〈0.01）. The ratio of cell apoptosis in combining group was significantly higher than that in radiation group or Ad- Rb94 group（q=6.36 or 7.89,P〈0.01）. Conclusion One mechanism of the synergism effects of retinoblastoma 94 gene therapy combined with radiotherapy on the inhibition of tumor growth is through increasing Rb protein expression and decreasing Ki67 protein expression.