中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
10期
908-913
,共6页
高睿骐%周希瑗%杨映雪%王志刚
高睿騏%週希瑗%楊映雪%王誌剛
고예기%주희원%양영설%왕지강
基因转染/Rb94、野生型p53%超声微泡造影剂%视网膜母细胞瘤%凋亡
基因轉染/Rb94、野生型p53%超聲微泡造影劑%視網膜母細胞瘤%凋亡
기인전염/Rb94、야생형p53%초성미포조영제%시망막모세포류%조망
Gene transfection/Rb94,wild-type p53%Ultrasound microvexicle%Retinoblastoma%Apoptosis
背景 研究表明,野生型p53(wtp53)和Rb94基因对人视网膜母细胞瘤(RB)的生长均有抑制作用,这2个基因是诱导和维持细胞衰老信号通路中重要的参与基因,因此2个基因联合应用是否对RB的生长抑制效果更好是近来关注的问题. 目的 观察超声微泡介导Rb94联合wtp53基因转染裸鼠RB后对其凋亡的影响.方法 将HXO-Rb44细胞悬液种植至40只雌性SPF级BALB/c裸鼠视网膜下腔建立RB动物模型,造模成功的裸鼠按随机数字表法平均分为模型对照组、wtp53质粒组(含wtp53质粒的微泡悬液)、Rb94质粒组(含Rb94质粒)和wtp53 +Rb94质粒组(联合组)(含wtp53质粒及Rb94质粒),其中模型对照组不做任何处理,其他3个组造模后第7天均由鼠尾静脉注入含相应基因的微泡悬液后,每天以0.5 W/cm2超声波辐照眼球4s,间隔24 s,循环2次.转染基因超声辐照7d摘除肿瘤组织,利用半定量逆转录PCR(RT-PCR)法检测肿瘤组织中wtp53 mRNA及Rb94 mRNA的表达；采用Western Not法检测基因转染的肿瘤组织中wtp53及Rb94蛋白的表达；用免疫组织化学法测定肿瘤组织中血管内皮生长因子(VEGF)的表达；采用TUNEL法检测基因转染后肿瘤组织的凋亡情况,计算凋亡指数(AI). 结果 HXO-Rb44细胞悬液视网膜下腔注射后移植瘤构建的成功率为80％(32/40),常规组织病理学检查提示检测样本的细胞异型性明显.基因转染后7d,模型对照组无wtp53 mRNA及Rb94 mRNA的表达条带；wtp53组wtp53 mRNA相对值为0.65±0.07,wtp53+Rb94组为0.32±0.02,差异有统计学意义(t=11.743,P=0.000)；Rb94组Rb94 mRNA相对值为0.42±0.03,wtp53 +Rb94组为0.23 ±0.03,差异有统计学意义(t=5.041,P=0.001).wtp53、Rb94或wtp53+ Rb94转染后,各组可见与转染相应的蛋白表达条带,wtp53+ Rb94组可同时检测到wtp53及Rb94蛋白的表达条带,但模型对照组无任何基因反应条带.免疫组织化学染色表明,wtp53+Rb94组肿瘤细胞中VEGF阳性反应强度明显弱于wtp53组、Rb94组和模型对照组.wtp53 +Rb94组AI为37.35±2.14,明显高于模型对照组的0.46±0.05、wtp53组的5.05±0.80和Rb94组的6.43±1.02,差异均有统计学意义(t=-34.395、-28.206、-26.006,P＜0.01).结论 超声微泡造影剂可介导双基因联合转染RB移植瘤,且Rb94联合wtp53基因对RB细胞的促凋亡作用较单基因转染增强.
揹景 研究錶明,野生型p53(wtp53)和Rb94基因對人視網膜母細胞瘤(RB)的生長均有抑製作用,這2箇基因是誘導和維持細胞衰老信號通路中重要的參與基因,因此2箇基因聯閤應用是否對RB的生長抑製效果更好是近來關註的問題. 目的 觀察超聲微泡介導Rb94聯閤wtp53基因轉染裸鼠RB後對其凋亡的影響.方法 將HXO-Rb44細胞懸液種植至40隻雌性SPF級BALB/c裸鼠視網膜下腔建立RB動物模型,造模成功的裸鼠按隨機數字錶法平均分為模型對照組、wtp53質粒組(含wtp53質粒的微泡懸液)、Rb94質粒組(含Rb94質粒)和wtp53 +Rb94質粒組(聯閤組)(含wtp53質粒及Rb94質粒),其中模型對照組不做任何處理,其他3箇組造模後第7天均由鼠尾靜脈註入含相應基因的微泡懸液後,每天以0.5 W/cm2超聲波輻照眼毬4s,間隔24 s,循環2次.轉染基因超聲輻照7d摘除腫瘤組織,利用半定量逆轉錄PCR(RT-PCR)法檢測腫瘤組織中wtp53 mRNA及Rb94 mRNA的錶達；採用Western Not法檢測基因轉染的腫瘤組織中wtp53及Rb94蛋白的錶達；用免疫組織化學法測定腫瘤組織中血管內皮生長因子(VEGF)的錶達；採用TUNEL法檢測基因轉染後腫瘤組織的凋亡情況,計算凋亡指數(AI). 結果 HXO-Rb44細胞懸液視網膜下腔註射後移植瘤構建的成功率為80％(32/40),常規組織病理學檢查提示檢測樣本的細胞異型性明顯.基因轉染後7d,模型對照組無wtp53 mRNA及Rb94 mRNA的錶達條帶；wtp53組wtp53 mRNA相對值為0.65±0.07,wtp53+Rb94組為0.32±0.02,差異有統計學意義(t=11.743,P=0.000)；Rb94組Rb94 mRNA相對值為0.42±0.03,wtp53 +Rb94組為0.23 ±0.03,差異有統計學意義(t=5.041,P=0.001).wtp53、Rb94或wtp53+ Rb94轉染後,各組可見與轉染相應的蛋白錶達條帶,wtp53+ Rb94組可同時檢測到wtp53及Rb94蛋白的錶達條帶,但模型對照組無任何基因反應條帶.免疫組織化學染色錶明,wtp53+Rb94組腫瘤細胞中VEGF暘性反應彊度明顯弱于wtp53組、Rb94組和模型對照組.wtp53 +Rb94組AI為37.35±2.14,明顯高于模型對照組的0.46±0.05、wtp53組的5.05±0.80和Rb94組的6.43±1.02,差異均有統計學意義(t=-34.395、-28.206、-26.006,P＜0.01).結論 超聲微泡造影劑可介導雙基因聯閤轉染RB移植瘤,且Rb94聯閤wtp53基因對RB細胞的促凋亡作用較單基因轉染增彊.
배경 연구표명,야생형p53(wtp53)화Rb94기인대인시망막모세포류(RB)적생장균유억제작용,저2개기인시유도화유지세포쇠로신호통로중중요적삼여기인,인차2개기인연합응용시부대RB적생장억제효과경호시근래관주적문제. 목적 관찰초성미포개도Rb94연합wtp53기인전염라서RB후대기조망적영향.방법 장HXO-Rb44세포현액충식지40지자성SPF급BALB/c라서시망막하강건립RB동물모형,조모성공적라서안수궤수자표법평균분위모형대조조、wtp53질립조(함wtp53질립적미포현액)、Rb94질립조(함Rb94질립)화wtp53 +Rb94질립조(연합조)(함wtp53질립급Rb94질립),기중모형대조조불주임하처리,기타3개조조모후제7천균유서미정맥주입함상응기인적미포현액후,매천이0.5 W/cm2초성파복조안구4s,간격24 s,순배2차.전염기인초성복조7d적제종류조직,이용반정량역전록PCR(RT-PCR)법검측종류조직중wtp53 mRNA급Rb94 mRNA적표체；채용Western Not법검측기인전염적종류조직중wtp53급Rb94단백적표체；용면역조직화학법측정종류조직중혈관내피생장인자(VEGF)적표체；채용TUNEL법검측기인전염후종류조직적조망정황,계산조망지수(AI). 결과 HXO-Rb44세포현액시망막하강주사후이식류구건적성공솔위80％(32/40),상규조직병이학검사제시검측양본적세포이형성명현.기인전염후7d,모형대조조무wtp53 mRNA급Rb94 mRNA적표체조대；wtp53조wtp53 mRNA상대치위0.65±0.07,wtp53+Rb94조위0.32±0.02,차이유통계학의의(t=11.743,P=0.000)；Rb94조Rb94 mRNA상대치위0.42±0.03,wtp53 +Rb94조위0.23 ±0.03,차이유통계학의의(t=5.041,P=0.001).wtp53、Rb94혹wtp53+ Rb94전염후,각조가견여전염상응적단백표체조대,wtp53+ Rb94조가동시검측도wtp53급Rb94단백적표체조대,단모형대조조무임하기인반응조대.면역조직화학염색표명,wtp53+Rb94조종류세포중VEGF양성반응강도명현약우wtp53조、Rb94조화모형대조조.wtp53 +Rb94조AI위37.35±2.14,명현고우모형대조조적0.46±0.05、wtp53조적5.05±0.80화Rb94조적6.43±1.02,차이균유통계학의의(t=-34.395、-28.206、-26.006,P＜0.01).결론 초성미포조영제가개도쌍기인연합전염RB이식류,차Rb94연합wtp53기인대RB세포적촉조망작용교단기인전염증강.
Background Researches showed that wild-type p53(wtp53)and Rb94 genes inhibit the growth of retinoblastoma(RB),and these genes are involved in signal pathway in the induction and maintenance of cellular senescence.Thus the combination of two genes to inhibit growth of RB is concerned.Objective This study was to observe the inhibitory effect of the co-transfection of Rb94 and wtp53 gcnc into subretina on RB with ultrasound microbubble.Methods HXO-Rb44 suspension was subretinally injected to establish the RB model in 40 SPF female Balb/c nude mice.The RB models were randomized into model control group,wtp53 group,Rb94 group and wtp53+Rb94 group,and 0.1 ml relevant gene microvesicle suspension was injected via caudal vein in the different groups,but no any gene was used in the model control group.Seven days after modeling,followed by 0.5 W/cm2ultrasonic wave irradiated the eyeballs immediately for 4 seconds ×2 times and interrupted for 24 seconds.Eyeballs were extracted 7 days after gene transfection,and the expressions of wtp53 mRNA and Rb94 mRNA in tumor tissuc were detected by RT-PCR,and wtp53 and Rb94 protein in tumor tissue were assayed using Western blot.Immunochemistry was used to exam the VEGF expression,and TUNEL was used to evaluate the apoptosis of the tumor cells.Results The model successful rate after HXO-Rb44 suspension was 80％ (32/40)and obvious malformation cells were seen under the light microscope.In 7 days after gene transfection,no response band for wtp53 mRNA and Rb94 mRNA were found.The relative expression valuc of wtp53 mRNA was 0.65±0.07 in the wtp53 group,and that in wtp53+Rb94 group was 0.32±0.02,showing a significant difference between them (t =11.743,P =0.000).Rb94mRNA relative value was 0.42 ±0.03 in Rb94 group,and that in the wtp53 + Rb94 group was 0.23 ± 0.03,with a significant difference(t=5.041,P=0.001).The response bands of wtp53 and Rb94 proteins were seen in wtp53group,Rb94 group and wtp53+Rb94 group.Immunochemistry showed that the positive reactive intensity for VEGF in tumor tissue was obviously weaker in wtp53+Rb94 group than that in the wtp53 group,Rb94 group and model control group.Apoptotic index(Al) was 37.35±2.14 in the wtp53+Rb94 group,showing a significant increase in comparison with model control group (0.46 ± 0.05),wtp53 group (5.05 ± 0.80) and Rb94 group (6.43 ± 1.02) (t =-34.395,-28.206,-26.006,P＜0.01).Conclusions Ultrasound microvesicle enable double gene transfecting into RB tumor tissue,and Rb94 gene cooperation with wtp53 gene enhance the inhibitory effect on RB by promoting the apoptosis of RB cells.
