基因体外重组与在体编辑介导的肿瘤靶向光学分子成像

发布时间：2018-08-23 21:39

【摘要】：目的:利用体外基因重组技术,构建组成型及组织特异型启动子调控的Luc光学基因分子探针,对比研究无靶向及靶向肿瘤光学分子成像的特点、区别及各自技术局限;根据CRISPR/Cas9基因编辑系统原理,借助该系统的精准靶向性及在体基因编辑特点,设计并实现利用该系统对失活Luc基因进行在体基因编辑介导的光学基因分子成像,为将本研究设计的在体基因靶向成像策略用于活体内多靶点条件下的靶向光学基因成像奠定基础。方法:1、以不同转染试剂及腺病毒为递送载体,将体外重组基因p CMV-Luc转染前列腺癌肿瘤细胞后进行光学成像,探讨非靶向光学基因细胞成像效果;将上述转染后的细胞继续培养后注入小鼠皮下,完成光学成像以评价活体细胞示踪效果,同时构建前列腺癌小鼠模型,以瘤内注射的方式向其肿瘤内直接注射腺病毒载体Ad.p CMV-luc后进行活体光学成像;2、利用含前列腺癌组织特异性启动子的体外重组基因p DD3-Tf R-Luc的腺病毒载体感染正常细胞、前列腺癌细胞及其他肿瘤细胞,完成细胞光学成像,并用Western-blotting方法检测基因探针处理后Tf R的表达水平,评估该类基因介导的细胞靶向光学成像特点;3、构建前列腺癌小鼠模型,向其肿瘤内注射组织特异性表达探针Ad.p DD3-Tf R-Luc后进行光学成像,分析图像荧光强度及范围的变化,进而评估基于靶向基因探针的活体内肿瘤靶向成像效果,对比第一、二部分成像结果讨论体外基因重组用于基因成像的优势与不足;4、设计并合成survivin启动子序列、stop sequence(SS)序列及靶向SS序列的g RNA(SS)序列;以质粒PX459和p SG-target为模板构建CRISPR/Cas9系统的载体质粒p U6-g RNA(SS)-pβactin-Cas9、p U6-g RNA(N)-pβactin-Cas9和失活Luc基因质粒p CMV-Luc L-SS-Luc R、p Sur-Luc L-SS-Luc R;5、将质粒p U6-g RNA(SS)-pβactin-Cas9、p U6-g RNA(N)-pβactin-Cas9分别与p Sur-Luc L-SS-Luc R、p CMV-Luc L-SS-Luc R共转染PC-3细胞和293T细胞,进行体内基因重组介导的细胞光学报告基因成像,实现并验证CRISPR/Cas9基因编辑系统用于分子成像的可行性。结果:1、含组成型启动子的Luc基因分别经腺病毒、阳离子脂质体Lipofectamine2000、阳离子聚合物PEI处理后,肿瘤细胞后均可发出荧光,其中以腺病毒转染方式成像效果最佳。转染试剂及腺病毒载体转染后的肿瘤细胞注射小鼠体内后均可实现细胞示踪;以腺病毒载体Ad.p CMV-Luc感染前列腺癌种植瘤小鼠后,肿瘤区域可见较强荧光,所示荧光区域与肿瘤范围无相关性。2、Ad.p DD3-Tf R-Luc转染不同细胞后,仅前列腺癌细胞发出荧光,而正常细胞、及其他肿瘤细胞无荧光显示;WB结果显示仅前列腺癌细胞出现Tf R蛋白过表达。Ad.p DD3-Tf R-Luc介导的活体光学成像显示小鼠体内仅肿瘤部位发出荧光,而瘤周组织及全身其他器官均无荧光显示,肿瘤边界显示清楚,其荧光范围与与肿瘤增长保持一致。3、经测序验证,本实验设计的CRISPR/Cas9载体质粒p U6-g RNA(SS)-pβactin-Cas9和类Luc基因载体质粒p CMV-Luc L-SS-Luc R及p Sur-Luc L-SS-Luc R构建成功。由p U6-g RNA(SS)-pβactin-Cas9质粒和组成型启动子调控的p CMV-Luc L-SS-Luc R质粒共转染后,293T及PC-3细胞均有荧光显示,且成像效果较好。由p U6-g RNA(SS)-pβactin-Cas9质粒和组织特异性启动子调控的p Sur-Luc L-SS-Luc R质粒共转染后,PC-3细胞有荧光显示。结论:1、腺病毒、阳离子脂质体、阳离子聚合物均可实现肿瘤细胞的Luc基因成像,其中在细胞层面上腺病毒载体的转染效率较高,同时,腺病毒介导的光学基因成像可获得较好的活体光学分子成像结果,但受组成型启动子调控的基因成像无法实现肿瘤靶向成像,因此该类Luc基因探针在分子成像中的应用主要局限于细胞示踪与分子生物学实验研究领域。2、基于腺病毒载体Ad.p DD3-Tf R-Luc的成像研究成功验证了由组织特异性启动子介导的基因成像在细胞及活体层面肿瘤靶向成像中的应用,表明利用体外重组技术,借助组织特异性启动子可为肿瘤靶向性报告基因成像提供了一个有效的生物“开关”。3、本研究成功构建了一种基于CRISPR/Cas9系统的新型光学基因成像系统,实现了由CRISPR/Cas9在体基因重组技术介导的正常及肿瘤细胞的靶向光学基因成像,从而将目前国际前沿的CRISPR/Cas9在体基因重组技术创新性地融入到分子影像研究中,为未来实现精准分子成像、检测基因水平的生物学行为以及实现活体、“多靶点”、“多因素”成像为目的的新型基因分子成像研究提供了一种全新的、基于在体基因编辑技术的基因成像方案。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.4

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