超声微泡介导野生型p53基因联合Rb94基因转染裸鼠视网膜母细胞瘤的实验研究

发布时间：2018-08-29 08:35

【摘要】：研究背景及目的视网膜母细胞瘤（RB）是学龄前儿童最常见的眼部恶性肿瘤，恶性度高，就诊时患儿较多已进入严重危害视力及生命的时期。目前我国对其治疗仍以患眼眼球摘除为主，辅以放射治疗、化学治疗、冷凝治疗等其他手段。而在对RB治疗的研究方面，基因治疗已成为其主要方向，其中又以对基因载体的研究，，是其整个领域的关键和瓶颈问题。近年来超声微泡造影剂成为基因载体研究的热点之一。Rb基因是公认的与RB形成关系最为密切的抑癌基因，而p53基因被认为目前的与人类恶性肿瘤相关性最高的一种抑癌基因。本研究以RB为研究对象，运用超声微泡造影剂这一新型载体，将wtp53基因及Rb94基因转染至裸鼠RB模型内，分别观察单个基因、联合基因对裸鼠视网膜母细胞瘤的治疗作用。研究方法：1. HXO-Rb44细胞培养于含10%FBS的RPMI1640培养基，待至对数期，收集并调整细胞密度为(5.5～6.5)×106/mL备用。40只裸鼠行双眼视网膜下注射Rb细胞悬液1ul，术后每3天观察肿瘤生长情况。2．将造模成功的裸鼠随机分为五组：①空白对照组；②空质粒组；③p53质粒组；④Rb94质粒组；⑤p53+Rb94质粒。除组①外，其余四组每天以0.5W/cm2超声波辐照眼球60s（辐照4s，间隔24s，连续两次，共用60s），7天后取出肿瘤组织行RT-PCR及Western-blot证实转染是否成功。3.取转染后裸鼠肿瘤组织，用免疫组化染色及Western-blot法检测各组肿瘤组织VEGF表达，检测各组肿瘤组织的肿瘤微血管密度及组织细胞的凋亡情况。研究结果：1.40只裸鼠双眼视网膜下种植瘤细胞，有32只裸鼠（48只眼）造模成功。2.RT-PCR及WB可见单基因组及联合基因组均有外源基因导入肿瘤组织，并在蛋白水平得到表达。3.对各组瘤组织的检测可见，单基因组及联合基因组对其VEGF、MVD及凋亡均有影响，但联合组的效应更为明显，同单基因组的差异有统计学意义（P0.05）。研究结论：超声微泡作为一种新型的转染介质，可达到转染单个基因及联合基因的目的,通过观察wtp53联合Rb94基因转染肿瘤组织后，对其的抑制作用优于单独转染其中任一基因，为RB的治疗提供更多、更有效的选择，也对RB发生发展的机制研究拓展了新思路。
[Abstract]:Background and objective retinoblastoma (RB) is the most common ocular malignant tumor in preschool children. At present, the main treatment in China is ophthalmectomy, plus radiotherapy, chemotherapy, condensation therapy and other means. In the research of RB therapy, gene therapy has become the main direction, and the research of gene vector is the key and bottleneck problem in the whole field. In recent years, ultrasound microbubble contrast agent has become one of the hotspots in gene vector research. RB gene is recognized as the most closely related tumor suppressor gene to RB formation, while p53 gene is considered to be one of the most relevant tumor suppressor genes in human malignant tumors. In this study, RB was used as a novel vector to transfect wtp53 gene and Rb94 gene into RB model of nude mice. The therapeutic effects of single gene and combined gene on retinoblastoma in nude mice were observed. Research method: 1. HXO-Rb44 cells were cultured on RPMI1640 medium containing 10s, and were cultured in logarithmic phase. The cell density was (5.5 ~ 6.5) 脳 106/mL. 40 nude mice were injected with Rb cell suspension 1 ul. the tumor growth was observed every 3 days after operation. The successful nude mice were randomly divided into five groups: control group (n = 5) and control group (n = 40). 3p53 plasmid group: 4 Rb94 plasmid group: 5 p53 Rb94 plasmid. In addition to group 1, the other four groups were irradiated with 0.5W/cm2 ultrasound for 60 s (4 s, 24 s, 2 times, 60 s) 7 days later, the tumor tissues were taken out for RT-PCR and Western-blot to confirm whether the transfection was successful or not. The expression of VEGF was detected by immunohistochemical staining and Western-blot method. The tumor microvessel density and the apoptosis of tumor cells in each group were detected. Results in 1.40 nude mice implanted tumor cells under the retina, 32 nude mice (48 eyes) were successfully modeled. 2. RT-PCR and WB showed that exogenous genes were introduced into tumor tissues and expressed at protein level. The detection of tumor tissues in each group showed that both single genome and combined genome had an effect on VEGF,MVD and apoptosis, but the effect of combination group was more obvious, and the difference was statistically significant compared with single genome (P0.05). Conclusion: as a new transfection medium, ultrasound microbubbles can transfect a single gene or a combined gene. By observing the inhibitory effect of wtp53 combined with Rb94 gene transfection on tumor tissue, the inhibitory effect of ultrasound microbubble on one of these genes is superior to that of single transfection. It provides more and more effective options for the treatment of RB and extends new ideas for the study of the mechanism of the occurrence and development of RB.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.72

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