發(fā)布時間：2018-08-29 08:35

【摘要】：研究背景及目的視網(wǎng)膜母細(xì)胞瘤（RB）是學(xué)齡前兒童最常見的眼部惡性腫瘤，惡性度高，就診時患兒較多已進(jìn)入嚴(yán)重危害視力及生命的時期。目前我國對其治療仍以患眼眼球摘除為主，輔以放射治療、化學(xué)治療、冷凝治療等其他手段。而在對RB治療的研究方面，基因治療已成為其主要方向，其中又以對基因載體的研究，，是其整個領(lǐng)域的關(guān)鍵和瓶頸問題。近年來超聲微泡造影劑成為基因載體研究的熱點(diǎn)之一。Rb基因是公認(rèn)的與RB形成關(guān)系最為密切的抑癌基因，而p53基因被認(rèn)為目前的與人類惡性腫瘤相關(guān)性最高的一種抑癌基因。本研究以RB為研究對象，運(yùn)用超聲微泡造影劑這一新型載體，將wtp53基因及Rb94基因轉(zhuǎn)染至裸鼠RB模型內(nèi)，分別觀察單個基因、聯(lián)合基因?qū)β闶笠暰W(wǎng)膜母細(xì)胞瘤的治療作用。 研究方法：1. HXO-Rb44細(xì)胞培養(yǎng)于含10%FBS的RPMI1640培養(yǎng)基，待至對數(shù)期，收集并調(diào)整細(xì)胞密度為(5.5～6.5)×106/mL備用。40只裸鼠行雙眼視網(wǎng)膜下注射Rb細(xì)胞懸液1ul，術(shù)后每3天觀察腫瘤生長情況。2．將造模成功的裸鼠隨機(jī)分為五組：①空白對照組；②空質(zhì)粒組；③p53質(zhì)粒組；④Rb94質(zhì)粒組；⑤p53+Rb94質(zhì)粒。除組①外，其余四組每天以0.5W/cm2超聲波輻照眼球60s（輻照4s，間隔24s，連續(xù)兩次，共用60s），7天后取出腫瘤組織行RT-PCR及Western-blot證實(shí)轉(zhuǎn)染是否成功。3.取轉(zhuǎn)染后裸鼠腫瘤組織，用免疫組化染色及Western-blot法檢測各組腫瘤組織VEGF表達(dá)，檢測各組腫瘤組織的腫瘤微血管密度及組織細(xì)胞的凋亡情況。 研究結(jié)果：1.40只裸鼠雙眼視網(wǎng)膜下種植瘤細(xì)胞，有32只裸鼠（48只眼）造模成功。2.RT-PCR及WB可見單基因組及聯(lián)合基因組均有外源基因?qū)肽[瘤組織，并在蛋白水平得到表達(dá)。3.對各組瘤組織的檢測可見，單基因組及聯(lián)合基因組對其VEGF、MVD及凋亡均有影響，但聯(lián)合組的效應(yīng)更為明顯，同單基因組的差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 研究結(jié)論：超聲微泡作為一種新型的轉(zhuǎn)染介質(zhì)，可達(dá)到轉(zhuǎn)染單個基因及聯(lián)合基因的目的,通過觀察wtp53聯(lián)合Rb94基因轉(zhuǎn)染腫瘤組織后，對其的抑制作用優(yōu)于單獨(dú)轉(zhuǎn)染其中任一基因，為RB的治療提供更多、更有效的選擇，也對RB發(fā)生發(fā)展的機(jī)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.72

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唐松;視網(wǎng)膜母細(xì)胞瘤自殺基因療法的研究進(jìn)展[J];國外醫(yī)學(xué).眼科學(xué)分冊;2001年04期

2 王成偉,龐琦,張慶林;血管生成與腫瘤的生長及轉(zhuǎn)移[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊);2001年02期

3 刁林瓊;周希瑗;;超聲微泡介導(dǎo)野生型P53基因轉(zhuǎn)染鼠視網(wǎng)膜母細(xì)胞瘤的實(shí)驗(yàn)研究[J];激光雜志;2008年03期

4 管曉翔,范樂明,陳琪,酈明芳,李肖蓉,邵力正;超聲介導(dǎo)白蛋白微泡破裂法促進(jìn)增強(qiáng)型綠色熒光蛋白C3基因在中國倉鼠卵巢細(xì)胞的表達(dá)[J];中國動脈硬化雜志;2003年01期

5 張愛鳳;陳平圣;魯勤;劉東風(fēng);;TUNEL法原位檢測腫瘤組織細(xì)胞凋亡的改進(jìn)[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2008年05期

6 鄭嵩山;吳中耀;楊華勝;顏建華;毛羽翔;;兔眼前房視網(wǎng)膜母細(xì)胞瘤動物模型的建立[J];眼科研究;2008年01期

7 王麗萍;周希瑗;彭周貴;鄭敏明;邱明忠;雷博;;裸鼠視網(wǎng)膜下注射HXO-Rb_(44)細(xì)胞建立視網(wǎng)膜母細(xì)胞瘤動物模型[J];眼科研究;2010年07期

8 畢穎文;陳榮家;;視網(wǎng)膜母細(xì)胞瘤動物模型的研究進(jìn)展[J];中國眼耳鼻喉科雜志;2006年03期

9 羅霽菡;周希瑗;;超聲微泡介導(dǎo)野生型p53基因轉(zhuǎn)染Y79細(xì)胞的實(shí)驗(yàn)研究[J];中國超聲醫(yī)學(xué)雜志;2008年12期

10 吳荒;孫瑩;吳雅臻;張靈;趙雪儉;;視網(wǎng)膜母細(xì)胞瘤中survivin的表達(dá)[J];中國實(shí)驗(yàn)診斷學(xué);2007年11期

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