【摘要】：目的:利用體外基因重組技術(shù),構(gòu)建組成型及組織特異型啟動(dòng)子調(diào)控的Luc光學(xué)基因分子探針,對(duì)比研究無靶向及靶向腫瘤光學(xué)分子成像的特點(diǎn)、區(qū)別及各自技術(shù)局限;根據(jù)CRISPR/Cas9基因編輯系統(tǒng)原理,借助該系統(tǒng)的精準(zhǔn)靶向性及在體基因編輯特點(diǎn),設(shè)計(jì)并實(shí)現(xiàn)利用該系統(tǒng)對(duì)失活Luc基因進(jìn)行在體基因編輯介導(dǎo)的光學(xué)基因分子成像,為將本研究設(shè)計(jì)的在體基因靶向成像策略用于活體內(nèi)多靶點(diǎn)條件下的靶向光學(xué)基因成像奠定基礎(chǔ)。方法:1、以不同轉(zhuǎn)染試劑及腺病毒為遞送載體,將體外重組基因p CMV-Luc轉(zhuǎn)染前列腺癌腫瘤細(xì)胞后進(jìn)行光學(xué)成像,探討非靶向光學(xué)基因細(xì)胞成像效果;將上述轉(zhuǎn)染后的細(xì)胞繼續(xù)培養(yǎng)后注入小鼠皮下,完成光學(xué)成像以評(píng)價(jià)活體細(xì)胞示蹤效果,同時(shí)構(gòu)建前列腺癌小鼠模型,以瘤內(nèi)注射的方式向其腫瘤內(nèi)直接注射腺病毒載體Ad.p CMV-luc后進(jìn)行活體光學(xué)成像;2、利用含前列腺癌組織特異性啟動(dòng)子的體外重組基因p DD3-Tf R-Luc的腺病毒載體感染正常細(xì)胞、前列腺癌細(xì)胞及其他腫瘤細(xì)胞,完成細(xì)胞光學(xué)成像,并用Western-blotting方法檢測(cè)基因探針處理后Tf R的表達(dá)水平,評(píng)估該類基因介導(dǎo)的細(xì)胞靶向光學(xué)成像特點(diǎn);3、構(gòu)建前列腺癌小鼠模型,向其腫瘤內(nèi)注射組織特異性表達(dá)探針Ad.p DD3-Tf R-Luc后進(jìn)行光學(xué)成像,分析圖像熒光強(qiáng)度及范圍的變化,進(jìn)而評(píng)估基于靶向基因探針的活體內(nèi)腫瘤靶向成像效果,對(duì)比第一、二部分成像結(jié)果討論體外基因重組用于基因成像的優(yōu)勢(shì)與不足;4、設(shè)計(jì)并合成survivin啟動(dòng)子序列、stop sequence(SS)序列及靶向SS序列的g RNA(SS)序列;以質(zhì)粒PX459和p SG-target為模板構(gòu)建CRISPR/Cas9系統(tǒng)的載體質(zhì)粒p U6-g RNA(SS)-pβactin-Cas9、p U6-g RNA(N)-pβactin-Cas9和失活Luc基因質(zhì)粒p CMV-Luc L-SS-Luc R、p Sur-Luc L-SS-Luc R;5、將質(zhì)粒p U6-g RNA(SS)-pβactin-Cas9、p U6-g RNA(N)-pβactin-Cas9分別與p Sur-Luc L-SS-Luc R、p CMV-Luc L-SS-Luc R共轉(zhuǎn)染PC-3細(xì)胞和293T細(xì)胞,進(jìn)行體內(nèi)基因重組介導(dǎo)的細(xì)胞光學(xué)報(bào)告基因成像,實(shí)現(xiàn)并驗(yàn)證CRISPR/Cas9基因編輯系統(tǒng)用于分子成像的可行性。結(jié)果:1、含組成型啟動(dòng)子的Luc基因分別經(jīng)腺病毒、陽離子脂質(zhì)體Lipofectamine2000、陽離子聚合物PEI處理后,腫瘤細(xì)胞后均可發(fā)出熒光,其中以腺病毒轉(zhuǎn)染方式成像效果最佳。轉(zhuǎn)染試劑及腺病毒載體轉(zhuǎn)染后的腫瘤細(xì)胞注射小鼠體內(nèi)后均可實(shí)現(xiàn)細(xì)胞示蹤;以腺病毒載體Ad.p CMV-Luc感染前列腺癌種植瘤小鼠后,腫瘤區(qū)域可見較強(qiáng)熒光,所示熒光區(qū)域與腫瘤范圍無相關(guān)性。2、Ad.p DD3-Tf R-Luc轉(zhuǎn)染不同細(xì)胞后,僅前列腺癌細(xì)胞發(fā)出熒光,而正常細(xì)胞、及其他腫瘤細(xì)胞無熒光顯示;WB結(jié)果顯示僅前列腺癌細(xì)胞出現(xiàn)Tf R蛋白過表達(dá)。Ad.p DD3-Tf R-Luc介導(dǎo)的活體光學(xué)成像顯示小鼠體內(nèi)僅腫瘤部位發(fā)出熒光,而瘤周組織及全身其他器官均無熒光顯示,腫瘤邊界顯示清楚,其熒光范圍與與腫瘤增長(zhǎng)保持一致。3、經(jīng)測(cè)序驗(yàn)證,本實(shí)驗(yàn)設(shè)計(jì)的CRISPR/Cas9載體質(zhì)粒p U6-g RNA(SS)-pβactin-Cas9和類Luc基因載體質(zhì)粒p CMV-Luc L-SS-Luc R及p Sur-Luc L-SS-Luc R構(gòu)建成功。由p U6-g RNA(SS)-pβactin-Cas9質(zhì)粒和組成型啟動(dòng)子調(diào)控的p CMV-Luc L-SS-Luc R質(zhì)粒共轉(zhuǎn)染后,293T及PC-3細(xì)胞均有熒光顯示,且成像效果較好。由p U6-g RNA(SS)-pβactin-Cas9質(zhì)粒和組織特異性啟動(dòng)子調(diào)控的p Sur-Luc L-SS-Luc R質(zhì)粒共轉(zhuǎn)染后,PC-3細(xì)胞有熒光顯示。結(jié)論:1、腺病毒、陽離子脂質(zhì)體、陽離子聚合物均可實(shí)現(xiàn)腫瘤細(xì)胞的Luc基因成像,其中在細(xì)胞層面上腺病毒載體的轉(zhuǎn)染效率較高,同時(shí),腺病毒介導(dǎo)的光學(xué)基因成像可獲得較好的活體光學(xué)分子成像結(jié)果,但受組成型啟動(dòng)子調(diào)控的基因成像無法實(shí)現(xiàn)腫瘤靶向成像,因此該類Luc基因探針在分子成像中的應(yīng)用主要局限于細(xì)胞示蹤與分子生物學(xué)實(shí)驗(yàn)研究領(lǐng)域。2、基于腺病毒載體Ad.p DD3-Tf R-Luc的成像研究成功驗(yàn)證了由組織特異性啟動(dòng)子介導(dǎo)的基因成像在細(xì)胞及活體層面腫瘤靶向成像中的應(yīng)用,表明利用體外重組技術(shù),借助組織特異性啟動(dòng)子可為腫瘤靶向性報(bào)告基因成像提供了一個(gè)有效的生物“開關(guān)”。3、本研究成功構(gòu)建了一種基于CRISPR/Cas9系統(tǒng)的新型光學(xué)基因成像系統(tǒng),實(shí)現(xiàn)了由CRISPR/Cas9在體基因重組技術(shù)介導(dǎo)的正常及腫瘤細(xì)胞的靶向光學(xué)基因成像,從而將目前國(guó)際前沿的CRISPR/Cas9在體基因重組技術(shù)創(chuàng)新性地融入到分子影像研究中,為未來實(shí)現(xiàn)精準(zhǔn)分子成像、檢測(cè)基因水平的生物學(xué)行為以及實(shí)現(xiàn)活體、“多靶點(diǎn)”、“多因素”成像為目的的新型基因分子成像研究提供了一種全新的、基于在體基因編輯技術(shù)的基因成像方案。
[Abstract]:OBJECTIVE: To construct Luc optical gene probe regulated by constitutive and tissue-specific promoters by in vitro gene recombination technology, and to compare the characteristics, differences and technical limitations of optical molecular imaging of non-targeted and targeted tumors. The system was designed and implemented to perform in vivo gene editing-mediated optical gene molecular imaging of inactivated Luc genes, which laid the foundation for the application of in vivo gene targeting imaging strategy designed in this study to target optical gene imaging under multi-target conditions in vivo. Methods: 1. Different transfection reagents and adenoviruses were used as delivery agents. Carrier, after in vitro recombinant gene P CMV-Luc was transfected into prostate cancer cells for optical imaging, to explore the imaging effect of non-targeted optical gene cells; after the transfected cells were further cultured and injected into mice subcutaneously, optical imaging was completed to evaluate the effect of in vivo cell tracing, and a prostate cancer mouse model was constructed to inject the tumor into mice. Adenoviral vector Ad.p CMV-luc was injected directly into the tumor to perform in vivo optical imaging. G method to detect the expression of Tf R after gene probe treatment and evaluate the characteristics of Tf R-mediated cell-targeted optical imaging; 3. To construct a mouse model of prostate cancer, the tissue-specific expression probe Ad.pD3-Tf R-Luc was injected into the tumor for optical imaging to analyze the fluorescence intensity and range of the image, and then to evaluate the targeting-based imaging. In vivo tumor targeting imaging with gene probes, the advantages and disadvantages of in vitro gene recombination for gene imaging were discussed by comparing the results of the first and second parts of imaging. 4. survivin promoter sequence, stop sequence (SS) sequence and G RNA (SS) sequence targeting SS sequence were designed and synthesized. CRISPR/Cas9 was constructed using plasmid PX459 and P SG-target as templates. The plasmid P U6-g RNA (SS) -pbeta actin-Cas9, P U6-g RNA (N) -pbetactin-Cas9, P U6-g RNA (N) -pbetactin-Cas9 and inactivated Luc gene plasmplasmid P CMV-Luc L-SS-SS-Luc R, P Sur-Luc L-SS-SS-Luc L-SS-SS-Luc R, P Sur-Luc L-SS-SS-SS-SS-Luc L-SS-Luc R; 5, the plasmplasmid P U6-g RNA (SS) -pbetactin-pbetactin-Cas9, P U6-g RNA (N) -pbetactin-pbetactin-Cas9, P U6-p U6-g RNA (N) -Cells and 293T cells, proceeding Results: 1. Luc gene containing constitutive promoter was treated with adenovirus, Lipofectamine 2000 and PEI respectively, and the tumor cells could emit fluorescence. Adenovirus transfection was the best method for imaging. The tumor cells transfected with adenovirus vector and adenovirus vector could be traced in vivo. The tumor region showed strong fluorescence after adenovirus vector Ad.p CMV-Luc was used to infect prostate cancer implant tumor mice, and the fluorescence region showed no correlation with tumor range. 2, Ad.p DD After 3-Tf R-Luc was transfected into different cells, only prostate cancer cells emitted fluorescence, while normal cells and other tumor cells showed no fluorescence. WB results showed that only prostate cancer cells showed Tf R protein overexpression. Ad.p DD3-Tf R-Luc-mediated in vivo optical imaging showed that only tumor sites emitted fluorescence in mice, while the surrounding tissues and the whole body emitted fluorescence. No fluorescence was detected in other organs, and the tumor boundary was clearly displayed. The fluorescence range was consistent with the tumor growth. 3. The designed plasmids of CRISPR/Cas9 vector p U6-g RNA (SS) -pBeactin-Cas9 and Luc-like gene vector p CMV-Luc L-SS-Luc R and P Sur-Luc L-SS-Luc R were successfully constructed by sequencing. After co-transfection of P CMV-Luc L-SS-Luc R plasmid and P CMV-Luc L-SS-Luc R plasmid regulated by tissue-specific promoter, 293T and PC-3 cells showed fluorescence and good imaging effect. After co-transfection of P Sur-Luc L-SS-Luc R plasmid regulated by P U6-g RNA (SS) -pbeta actin-Cas9 plasmid and tissue-specific promoter, PC-3 cells showed fluorescence. Both liposome and cationic polymer can achieve Luc gene imaging of tumor cells, and adenovirus vectors have higher transfection efficiency at the cellular level. At the same time, optical gene imaging mediated by adenovirus can obtain better in vivo optical molecular imaging results, but gene imaging regulated by constitutive promoters can not achieve tumor targeting synthesis. Therefore, the application of Luc gene probes in molecular imaging is mainly limited to the field of cell tracing and molecular biology experiments. 2. Imaging studies based on adenovirus vector Ad.p DD3-Tf R-Luc have successfully demonstrated the application of tissue-specific promoter-mediated gene imaging in cell and in vivo tumor targeting imaging. In this study, a novel optical gene imaging system based on CRISPR/Cas9 system was successfully constructed, and the normal and swelling induced by CRISPR/Cas9 in vivo gene recombination technology were realized. Targeted optical gene imaging of tumor cells will innovatively incorporate the current international frontier RISPR/Cas9 in vivo gene recombination technology into molecular imaging research, in order to achieve accurate molecular imaging in the future, detect the biological behavior of gene level and achieve in vivo, "multi-target" and "multi-factor" imaging for the purpose of new gene components. Sub imaging research provides a new technology of gene imaging based on in vivo gene editing technology.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R730.4

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