【作者】 张萍；

【导师】 程玉峰；

【作者基本信息】 山东大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的：视网膜母细胞瘤(Retinoblastoma, Rb)基因Rb94为全长型视网膜母细胞瘤基因N末端缺失112个氨基酸残基的剩余部分,编码分子量为94KD的抑癌蛋白,其与Rb110基因相比,具有更强的肿瘤抑制效应。前期研究已表明,Rb94基因转染人肺腺癌细胞系A549后可抑制鞘氨醇激酶1(sphingosine kinase1, SphKl)表达、增强鞘氨醇激酶2(sphingosine kinase2, SphK2)表达、抑制细胞增殖、促进细胞凋亡、调控细胞周期,对A549细胞具有放射增敏作用。在前期体外实验的基础上,本研究构建Rb94基因转染裸鼠肺腺癌皮下移植瘤的动物模型,观察Rb94的抑瘤作用及对鞘氨醇激酶(SphK)表达的影响,进一步探讨SphK在抑制细胞增殖、促进细胞凋亡等方面可能发挥的重要作用；进一步研究Rb94基因的放射增敏作用,探讨其作用机制,为以Rb94为靶点进行肿瘤放射增敏提供理论依据。方法：将由齐鲁医院中心实验室保存的空载体pIRES、质粒pIRES-Rb94分别转化感受态大肠杆菌DH5α,挑取单克隆,提取质粒,XhoI酶切鉴定后交由南京金思特有限公司进行大量提取,以供后续的动物实验应用。每只裸鼠皮下接种5×106数目的A549细胞,待肿瘤生长至180mm3大小时随机分为空白对照组、脂质体组、空载体组、转染Rb94基因组,各组又分为未照射组和照射组。各组裸鼠瘤体内分别注射0.01M磷酸盐缓冲液(Phosphate Buffered Saline, PBS)、脂质体(lipofectamine)、pIRES、pIRES-Rb94;照射组加照一定剂量X线。观察肿瘤生长情况并计算抑瘤率；采用实时RT-PCR和Western bolt法检测瘤组织中Rb94基因表达；实时RT-PCR和免疫荧光染色法观察Rb94基因对裸鼠肺腺癌皮下移植瘤中SphK表达的影响；Hoechst染色观察细胞凋亡情况；实时RT-PCR检测hTERT、Bcl-2mRNA的表达；免疫组化SP法检测细胞周期阻滞。结果：转染Rb94基因组移植瘤被明显抑制,平均抑瘤率达51.99%,与空白对照组、空载体组、脂质体组(抑瘤率分别为0、7.86%、11.78%)相比差异有统计学意义(P<0.01),而对照组间差异无统计学意义；联合治疗组抑瘤率为95.84%,与单独转染组或单独放射组相比,差异具有统计学意义(P<0.01)；转染Rb94基因组移植瘤中Rb94基因在mRNA和蛋白水平表达增高；SphK1在mRNA和蛋白水平表达下调而SphK2表达上调；Hoechst染色法示转染组细胞凋亡增加；hTERT、Bcl-2 mRNA表达下降(三个对照组间相比,P>0.05；转染组与对照组相比,P<0.05)；免疫组化SP法示转染组G2／M期细胞阻滞(三个对照组间相比,P>0.05；转染组与对照组相比,P<0.05)。结论：成功构建了裸鼠皮下移植瘤模型；Rb94基因抑制SphKl的表达,增强SphK2的表达；Rb94基因对裸鼠皮下移植瘤具有明显的抑制作用,联合射线抑瘤效果更好,其放射增敏作用机制可能与Rb94基因抑制SphK1的表达,增强SphK2的表达以及引起细胞凋亡、G2／M期周期阻滞、下调hTERT、Bcl-2 mRNA的表达有关,这就进一步从体内方面证实了Rb94基因对A549细胞的放射增敏作用,并为下一步研究提供理论依据。更多还原

【Abstract】 OBJECTIVE:A NH2-terminal truncated RB protein of 94 kDa (Rb94), lacking 112-amino acid residues, has been found to have greater efficacy than Rb110 in tumor suppression. Former studies have showed that Rb94 could enhance radiosensitivity by inhibiting the expression of SphKl, enhancing the expression of SphK2, suppressoring cell proliferation, promoting apoptosis and regulating cell cycle in A549 cell line after transfection. To construct a mouse xenograft model of Rb94 gene transfering A549 cells, and to investigate the Rb94’s inhibition in mice tumors. To observe the effect of Rb94 on the expression of SphK and the functions of SphK in the regulation of cell proliferation and apoptosis in cancer cells. To research the possible mechanisms of radiosensitising enhancement, thus to offer theorical proof for Rb94 enhancement radiosensitivity in A549 cells.METHODS:PIRES and pIRES-Rb94, which preserved by the laboratory of QiLu Hospital, were transformed into competent E.coli. respectively. To pick the positive colony, and then to extract the plasmid after amplification. After identification with XhoI, plasmid pIRES and pIRES-Rb94 were amplified by Genscript Company for ulterior study. 5×106 cells suspension were subcutaneously injected into flanks of every nude mouse. Then the inoculated mice were devided into 4 groups after tumors arriving 180mm3: treated with PBS, lipofectamine, pIRES empty vector and pIRES-Rb94 respectively. Then to give halves of the mice with X-ray. The tumor inhibiting rate was observed and the expression of Rb94 gene was detected by Real time RT-PCR and Western blot. The expression of SphK was determined by real-time RT-PCR and immunofluorescence(IF). The number of apoptosis cells was measured by Hoechst staining. Expression of hTERT and Bcl-2mRNA were detected by real-time RT-PCR. Cell cycle distribution was measured by Immunohistology(IH).RESULTS:Treatment of pIRES-RB94, which inhibited ratio was 51.99%, had statistically differences compared with no treatment, liposome treatment and pIRES treatment(P<0.01). There were no statistical differences between no treatment, liposome treatment and pIRES treatment. The inhibited ratio of tumor in combination group was 95.84%, and statistically significant differences on the inhibition ratios were found between the combination group and the other groups(P<0.01). Real-time RT-PCR and Western blot analysis showed that Rb94 gene could be effectively expressed in the treatment of pIRES-RB94. The expression of SphKl was down-regulated, while the expression of SphK2 was up-regulated. Hoechst staining analysis demonstrated that the number of apoptosis cells was increased. The expression of hTERT and Bcl-2 mRNA were down-regulated by real-time RT-PCR. Immunohistology analysis showed that the number of cells of G2/M phases was increased in treatment of pIRES-RB94.CONCLUSION:The mouse xenograft model has been successfully constructed and analysis suggest that Rb94 has significant effects for inoculated tumor in nude mice. The tumor inhibition rate of combination group seems to be higher than that of the other groups. The possible mechanisms of radiosensitising enhancement of Rb94 gene are about down-regulation of SphKl,up-regulation of SphK2,promoting apoptosis, G2/M phase blocking and down-regulation of hTERT and Bcl-2 mRNA expression.更多还原