构建Ⅰ型内含子核酶介导癌胚抗原双报告基因显像体系的实验研究
发布时间：2018-09-01 15:37
【摘要】：目的 癌胚抗原分子(CEA)在多种肿瘤细胞中过表达,作为肿瘤标志物被广泛用于评价结直肠癌、乳腺癌、肺癌等肿瘤的预后。报告基因显像技术的发展为评估肿瘤治疗疗效以及肿瘤癌基因的表达提供了优越的可行性。如何在活体水平对癌基因的表达进行有效、实时且无创性的监测是目前评估肿瘤治疗效果而亟需解决的难点。本实验目的拟构建含有靶向CEA序列的反式剪接型I型内含子核酶(Rib53)为载体核心介导的荧光素酶-核素(Fluc-HSVl-tk)双报告基因显像系统,实现在活体水平直接、靶向、实时监测靶基因表达,进而达到评价肿瘤疗效与预后的目的。 方法 利用基因工程技术构建靶向癌胚抗原(CEA)的反式剪接型I型内含子核酶及核酶-荧光素酶-胸苷激酶(Rib53-Fluc-tk)报告质粒；转染Rib53-Fluc-tk于MCF-7乳腺肿瘤细胞系及共转pCDNA3.1-CEA及Rib53-Fluc-tk质粒于293T细胞中,分别用双报告荧光素酶、逆转录PCR、Real-time PCR、免疫细胞荧光、细胞生物发光等技术检测核酶对靶基因的剪接产物以及报告基因的表达；Iodogen固相氧化法标记氟-阿糖呋喃基尿嘧啶(FAU)后进行细胞摄取动力学实验。共转染Rib53-Fluc-tk及CEA质粒于293T细胞中,24小时后将细胞接种于裸鼠皮下行活体小动物生物发光显像,验证该双报告基因显像体系的可行性及最佳显像条件。数据分析采用独立样本t检验、单因素方差分析,P0.05具有统计学意义。 结果 1.以核酶为核心的双报告载体Rib53-Fluc-tk质粒的构建 四膜虫基因组PCR获得Rib DNA,大小为516bp,产物大小与预期一致；将Rib及Fluc序列克隆至pcDNA3.1质粒形成Rib3-Fluc报告质粒,琼脂糖凝胶电泳鉴定酶切条带大小正确,条带大小约为2200bp,送检测序结果正确。PCR获得Rib53-Fluc-tk条带,大小为3267bp,送检测序结果正确。 2.核酶剪接产物的相关检测 体外培养MCF-7、SK-OV-3以及Hela三种细胞系,在MCF-7细胞中检测到CEA的表达,而在SK-OV-3及Hela细胞中均未检测到CEA的表达。 Rib53-Fluc-tk质粒转染MCF-7细胞、SK-OV-3以及Hela细胞中,在MCF-7细胞系中检测到荧光素酶的表达,比值可达0.639±0.099,随着Rib53-Fluc-tk质量的增加而比值增加(P=0.006)。而另外两组细胞系中荧光素酶的表达较低,荧光素酶值分别为0.023±0.002(P=0.003),0.033±0.003(P=0.004)。 共转染CEA及Rib53-Fluc-tk1μg于293T细胞中,当CEA质量分别为0μg、0.2μg、 lug时,荧光素酶比值分别为0.0794-0.010；0.145±0.033(P=0.192)；0.653+0.130(P=0.048)：3.237±0.09-(P=0.001)。 将MCF-7及SK-OV-3细胞转染Rib53-Fluc-tk后提取RNA,逆转录PCR提取剪切产物,MCF-7泳道可见一条带,大小一致,SK-OV-3泳道未见条带。 Real-time PCR检测到MCF-7细胞转染Rib53-Fluc-tk后CEA的相对含量表达减低,而仅转染空载pCDNA3.1的MCF-7细胞的CEA含量却无明显减少(P=0.019,P=0.003)。 将Rib53-Fluc-Tk转染MCF-7细胞48小时后行细胞免疫荧光,可见细胞浆内荧光较均匀分布。 3.131I-FIAU的合成 采用Iodogen固相氧化法进行FAU的131I标记,标记产物经C-18小柱纯化。131I-FIAU的标记率为64.02±4.79%(n=3),放化纯为95.96±1.07%(n=3),标记产物在人血清24小时的稳定性为95.74±0.86%(n=3)。 4.131I-FIAU的细胞摄取 在293T细胞中单独转染Rib53-Fluc-tk质粒,至4.5h时细胞摄取率仅达到0.310±0.01%(n=4)；在293T细胞中共转染pCDNA3.1-CEA、Rib53-Fluc-tk质粒,随着时间的增加,293T细胞对131I-FIAU的摄取逐渐增加,至4.5h时摄取率达到1.40±0.06%(n=4),P0.01；在MCF-7细胞中转染Rib53-Fluc-tk质粒,随着时间的增加细胞摄取率增加不明显,在2.5h达到最高,最高仅达0.64±0.04%(n=4)。 5.细胞生物发光检测 将如下质粒分别转染293T细胞：阳性参照组pDC316-Fluc-Egfp-Tk1μg;实验组CEA1μg+Rib53-Fluc-Tk1μg;阴性参照组：Rib53-Fluc-Tk1μg,转染48小时后加入荧光素酶底物荧光素-D行生物发光检测,阳性参照组可探测到较强生物发光信号,实验组可探测生物发光信号,阴性参照组未探测明显生物发光信号。 6.裸鼠生物发光显像 准备3-4周龄裸鼠9只,随机分成3组,每组3只。将如下质粒分别转染293T细胞：阳性参照组pDC316-Fluc-Egfp-Tk5μg;实验组CEA5μg+Rib53-Fluc-Tk5μg;阴性参照组：Rib53-Fluc-Tk5μg。转染24小时后收集细胞,将这三组细胞分别皮下接种于以上3组裸鼠的右下肢,每组注射3只。行生物发光现象,阳性参照组裸鼠中细胞注射部位未探测明显生物发光信号；实验组未探及生物发光信号；阴性参照组未探及生物发光信号,与预期一致。结论 本实验成功构建了以I型内含子核酶为核心介导的靶向CEA的双报告基因体系。核酶载体能选择性剪接CEA,形成了肿瘤靶向分子与报告基因的融合蛋白表达,从而在细胞水平上成功地对CEA mRNA进行了生物发光以及核素双报告基因的显像。我们的研究为核酶介导报告基因显像提供了体外实验的前期基础。 正是因为核酶特异性的剪接作用,报告基因才能特异性地反映靶向分子的表达的部位及含量。通过形成CEA及报告基因的融合产物,解决了报告基因系统与靶基因结合率低、显像特异性不理想的缺点。如若能提高核酶的剪切效率实现小动物活体显像的灵敏度,便可以用于监测siRNA、放化疗的疗效,具有较广阔的临床价值。 目的 正电子发射断层扫描(PET)已被广泛应用于肿瘤的诊断、疗效评估、肿瘤复发及组织坏死的鉴别诊断。[11C]甲硫氨酸PET显像是目前最常用的氨基酸PET显像方法。药物动力学主要是利用动力学原理与数学模型来描述药物在体内的吸收、分布、代谢与排出情形,其研究方法是利用药物浓度与时间的关系,选择合适的药物动力学模型(pharmacokinetic model),计算出药物动力学参数来反映药物在体内的变化。新近发表的[11C]甲硫氨酸PET显像文献展示了[11C]甲硫氨酸在正常人体及胶质瘤患者的生物学分布,然而目前对于甲硫氨酸在胶质瘤与黑色素瘤相关模型动力学分析的研究比较有限。因此本次实验研究是拟在裸大鼠胶质瘤以及裸小鼠黑色素脑肿瘤活体对[11C]甲硫氨酸PET进行模型动力学分析,研究相关动力学参数以及寻找最佳的数学模型来定量分析[11C]甲硫氨酸在肿瘤的生物分布以及穿透血脑屏障的能力,以便将来耶鲁PET中心在受试人体应用[11C]甲硫氨酸PET显像进行模型动力学分析。 方法 分别在裸大鼠(nude rats,n=4)及裸小鼠(nude mice,n=2)脑部立体定位注射脑胶质瘤细胞U87MG以及人黑色素瘤细胞YUMAC,分别构建裸大鼠胶质瘤肿瘤模型以及裸小鼠黑色素瘤脑肿瘤模型。 [11C]标记甲硫氨酸,并行高效液相层析仪(HPLC)检测[11C]甲硫氨酸放化纯。 立体定位注射三周后每只裸大鼠分别注射18.5～37MBq [11C]甲硫氨酸,裸小鼠分别注射1.85～3.7MBq[11C]|甲硫氨酸行动态PET显像。在PET图像上勾画左心室及脑肿瘤等感兴趣区获得输入功能曲线、时间活度曲线。通过软件计算获得脑肿瘤总分布体积(VD),分别用二腔室模型(one-tissue compartment model、三腔室模型(two-tissue compartment model)、Patlak模型以及多线性(MA1model)模型来计算并获得相应的参数K1,k2,k3, k4和Ki,VD。 结果 裸大鼠脑肿瘤模型中二室模型更好地拟合[11C]甲硫氨酸在脑胶质瘤的生物分布,在裸大鼠脑肿瘤模型中VT为3.894±0.149ml/cc/min(n=4),K1为0.157±0.014ml/cc/min(n=4),k2为0.041±0.004/min(n=4);在裸小鼠黑色素脑肿瘤模型三室模型较二室模型能更好地拟合,VT为1.536±0.030ml/cc/min(n=2),K1为0.175±0.046ml/cc/min (n=2),k2为0.115±0.032/min(n=2),k3为0.407±0.095/min(n=2),k4为0.567±0.139/min(n=2)。Patlak模型在裸大鼠及裸小鼠均能较好地拟合,ki值分别为0.043±0.002ml/cc(n=4),0.019±0.0001ml/cc(n=2)。 结论 裸大鼠中二室模型能较好地拟合显像剂在脑组织的生物分布,VT及K1能较好地反映显像剂在脑组织的分布提及以及穿透血脑屏障的能力,并且K1能够反映[11C].甲硫氨酸随脑血流进入血脑屏障的速率,k2则反映了[11C]甲硫氨酸随之被动扩散排出大脑的速率。裸小鼠中三室模型能更好地描述显像剂在脑黑色素瘤的分布。ki反映脑组织对显像剂由血浆至肿瘤组织不可逆的摄取。药物动力学参数较标准化摄取(SUV)等能更灵敏、准确地反映[11C]甲硫氨酸在体内的生物分布,同时在将来肿瘤放化疗疗效评估时,可通过计算以上药物动力学参数来反应肿瘤组织[11C]甲硫氨酸分布的变化,从而更敏感地评价药物疗效。
[Abstract]:objective
Carcinoembryonic antigen molecule (CEA) is overexpressed in a variety of tumor cells, and is widely used as a tumor marker to evaluate the prognosis of colorectal cancer, breast cancer, lung cancer and other tumors. The purpose of this study is to construct a fluorescein-HSVl-tk dual-reporter gene imaging system with trans-spliced ribozyme type I (Rib53) as the carrier core to achieve in vivo water. Direct, targeted, real-time monitoring of target gene expression, and then achieve the purpose of evaluating tumor efficacy and prognosis.
Method
Trans-spliced ribozyme type I and ribozyme-fluorescein-thymidine kinase (Rib53-Fluc-tk) reporter plasmids targeting carcinoembryonic antigen (CEA) were constructed by genetic engineering technology; Rib53-Fluc-tk was transfected into MCF-7 breast cancer cell line and co-transfected pCDNA3.1-CEA and Rib53-Fluc-tk plasmids into 293T cells, respectively, using double-reporting luciferase and reverse-reporting luciferase. Transcriptional PCR, Real-time PCR, immunocytofluorescence and cell bioluminescence were used to detect the splicing products of ribozyme and the expression of reporter gene; the uptake kinetics of fluoro-arbofuran-uracil (FAU) labeled by Iodogen solid-phase oxidation was studied. Rib53-Fluc-tk and CEA plasmids were co-transfected into 293T cells 24 hours later. Cells were inoculated into nude mice subcutaneously for bioluminescence imaging in vivo to verify the feasibility and optimal imaging conditions of the double reporter gene imaging system.
Result
1. construction of Rib53-Fluc-tk plasmid with ribozyme as core dual report vector
Rib DNA was obtained from tetrahydrohymena genome by PCR with a size of 516 bp, and the product size was the same as expected. Rib and Fluc sequences were cloned into pcDNA3.1 plasmid to form Rib3-Fluc reporter plasmid. The RIBBON size was correct by agarose gel electrophoresis, and the ribbon size was about 2200 bp. Rib53-Fluc-tk was obtained by PCR with a size of 3267 bp. Sending test results are correct.
2. detection of ribozyme splicing products
CEA expression was detected in MCF-7, SK-OV-3 and Hela cells in vitro, but not in SK-OV-3 and Hela cells.
The expression of luciferase was detected in MCF-7 cells, SK-OV-3 cells and Hela cells transfected with Rib53-Fluc-tk plasmid. The ratio of luciferase expression in MCF-7 cells was 0.639 (+ 0.099) and increased with the increase of Rib53-Fluc-tk mass (P = 0.006). The expression of luciferase in the other two cell lines was lower, and the luciferase value was 0.023 (+ 0.002) (P = 0.003) respectively. 0.033 + 0.003 (P=0.004).
The luciferase ratios were 0.0794-0.010, 0.145+0.033 (P=0.192), 0.653+0.130 (P=0.048):3.237+0.09-(P=0.001) when CEA and Rib53-Fluc-tk1 were 0, 0.2 UG and lug, respectively.
After transfection of MCF-7 and SK-OV-3 cells into Rib53-Fluc-tk, RNA was extracted and the splicing products were extracted by reverse transcription polymerase chain reaction (RT-PCR). A band was found in the MCF-7 swimming lane, and no band was found in the SK-OV-3 swimming lane.
Real-time PCR showed that the relative expression of CEA in MCF-7 cells transfected with Rib53-Fluc-tk was decreased, while that in MCF-7 cells transfected with pCDNA3.1 did not decrease significantly (P=0.019, P=0.003).
Immunofluorescence of MCF-7 cells transfected with Rib53-Fluc-Tk for 48 hours showed that the fluorescence was uniformly distributed in the cytoplasm.
Synthesis of 3.131I-FIAU
Iodogen solid-phase oxidation method was used to label FAU with 131I. The labeling rate of 131I-FIAU was 64.02 (?) 4.79% (n = 3) and the radiochemical purity was 95.96 (?) 1.07% (n = 3), and the labeling product was 95.74 (?) 0.86% (n = 3) in human serum for 24 hours.
Cellular uptake of 4.131I-FIAU
Rib53-Fluc-tk plasmid was transfected into 293T cells separately, and the uptake rate of Rib53-Fluc-tk was only 0.310+0.01%(n=4) at 4.5 H. pCDNA3.1-CEA and Rib53-Fluc-tk plasmid were co-transfected into 293T cells. The uptake rate of 131I-FIAU increased gradually with the increase of time, and reached 1.40+0.06%(n=4) and P 0.01 at 4.5 H. The uptake rate of Rib53-Fluc-tk plasmid increased slightly with the increase of time, reaching the highest level at 2.5 h, reaching only 0.64 (+ 0.04%).
5. cell bioluminescence detection
293T cells were transfected with the following plasmids: pDC316-Fluc-Egfp-Tk1 UG in the positive control group, CEA1 UG + Rib53-Fluc-Tk1 UG in the experimental group, and Rib53-Fluc-Tk1 UG in the negative control group. After 48 hours of transfection, fluorescein-D substrate was added to the positive control group for bioluminescence detection, and strong bioluminescence signals were detected in the experimental group. No obvious bioluminescence signal was detected in the negative reference group.
6. bioluminescence imaging of nude mice
Nine nude mice aged 3-4 weeks were prepared and randomly divided into three groups, three in each group. 293T cells were transfected with the following plasmids: positive control group pDC316-Fluc-Egfp-Tk5 ug; experimental group CEA 5 UG + Rib53-Fluc-Tk5 ug; negative control group: Rib53-Fluc-Tk5 ug. After 24 hours of transfection, the cells were collected and subcutaneously inoculated into the right lower part of the above three groups of nude mice. Limbs were injected with 3 mice in each group. Bioluminescence was detected in the injection site of the cells in the positive control group, no obvious bioluminescence signal was detected in the experimental group, and no bioluminescence signal was detected in the negative control group.
In this study, we successfully constructed a dual reporter gene system targeting CEA mediated by intron ribozyme type I. The ribozyme vector can selectively splice CEA and form the fusion protein expression of tumor-targeting molecule and reporter gene. Our research provides a preliminary basis for in vitro experiments for ribozyme mediated reporter gene imaging.
It is precisely because of the ribozyme-specific splicing that the reporter gene can specifically reflect the location and content of the target molecule expression. By forming the fusion products of CEA and reporter gene, the defects of low binding rate and poor imaging specificity between the reporter gene system and the target gene can be solved. The sensitivity of in vivo imaging can be used to monitor the efficacy of siRNA, radiotherapy and chemotherapy, and has a broad clinical value.
objective
Positron emission tomography (PET) has been widely used in tumor diagnosis, therapeutic evaluation, differential diagnosis of tumor recurrence and tissue necrosis. Metabolism and excretion are studied by choosing the appropriate pharmacokinetic model and calculating the pharmacokinetic parameters to reflect the changes of drugs in vivo according to the relationship between drug concentration and time. Biological distribution of tumor patients, however, the current study on methionine in glioma and melanoma-related model dynamics analysis is relatively limited. Therefore, this experiment is intended to study the kinetics of [11C] methionine PET in nude rat glioma and nude mouse melanoma in vivo. Number and search for the best mathematical model to quantitatively analyze the biological distribution of [11C] methionine in tumors and its ability to penetrate the blood-brain barrier, so as to apply [11C] methionine PET imaging in the future in Yale PET Center for model dynamics analysis.
Method
Glioma cell U87MG and human melanoma cell YUMAC were injected into the brain of nude rats (n=4) and nude mice (n=2) to construct glioma model in nude rats and melanoma model in nude mice.
[11C] was labeled with methionine, and [11C] methionine radiochemical purity was detected by parallel high performance liquid chromatography (HPLC).
Three weeks after stereotactic injection, each nude rat was injected with 18.5-37 MBq [11C] methionine, and the nude mice were injected with 1.85-3.7 MBq [11C] | methionine dynamic PET imaging respectively. The input function curves and time activity curves were obtained by drawing the left ventricle and brain tumor regions of interest on the PET images. Volume (VD) was calculated by one-tissue compartment model, two-tissue compartment model, Patlak model and MA1 model respectively, and the corresponding parameters K1, k2, k3, K4 and Ki, VD were obtained.
Result
The two-compartment model fitted the biological distribution of [11C] methionine better in the brain tumor model of nude rats. The VT of the three-compartment model was 3.894 [0.149ml / cc / min (n = 4), K1 was 0.157 [0.014ml / cc / min (n = 4) and K2 was 0.041 [0.004]/ min (n = 4); the three-compartment model of the melanoma model of nude mice was better than the two-compartment model. Patlak model could be well fitted in both nude rats and nude mice. Ki values of Patlak model were 0.043 [0.002 ml / cc / cc / cc / cc / min (n = 4.019 [0.019 [0.019 [0.019 [0.019 [0 0 0 0 0.019 [0.019] 1] 1 [0 [0.019 [0.019 [0] 1]]. Patlakmodel in nunude rats and nununude micewere 0.043 3 [0.043 [0.043 [0.002 [0.002 ml / cc / ml]]], K 2 cc (n = 2).
conclusion
The two-compartment model of nude rats can well fit the biological distribution of imaging agent in brain tissue, VT and K1 can better reflect the distribution of imaging agent in brain tissue and the ability to penetrate blood-brain barrier, and K1 can reflect [11C]. The rate of methionine entering blood-brain barrier with cerebral blood flow, K2 reflects the passive expansion of [11C] methionine. Ki reflects irreversible uptake of imaging agents from plasma to tumor tissue. Pharmacokinetic parameters are more sensitive than standardized uptake (SUV) and accurately reflect the biological distribution of [11C] methionine in the body. In the future, these pharmacokinetic parameters can be calculated to reflect the changes of [11C] methionine distribution in tumor tissues, so as to evaluate the therapeutic effect more sensitively.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R730.44
本文编号：2217624
[Abstract]:objective
Carcinoembryonic antigen molecule (CEA) is overexpressed in a variety of tumor cells, and is widely used as a tumor marker to evaluate the prognosis of colorectal cancer, breast cancer, lung cancer and other tumors. The purpose of this study is to construct a fluorescein-HSVl-tk dual-reporter gene imaging system with trans-spliced ribozyme type I (Rib53) as the carrier core to achieve in vivo water. Direct, targeted, real-time monitoring of target gene expression, and then achieve the purpose of evaluating tumor efficacy and prognosis.
Method
Trans-spliced ribozyme type I and ribozyme-fluorescein-thymidine kinase (Rib53-Fluc-tk) reporter plasmids targeting carcinoembryonic antigen (CEA) were constructed by genetic engineering technology; Rib53-Fluc-tk was transfected into MCF-7 breast cancer cell line and co-transfected pCDNA3.1-CEA and Rib53-Fluc-tk plasmids into 293T cells, respectively, using double-reporting luciferase and reverse-reporting luciferase. Transcriptional PCR, Real-time PCR, immunocytofluorescence and cell bioluminescence were used to detect the splicing products of ribozyme and the expression of reporter gene; the uptake kinetics of fluoro-arbofuran-uracil (FAU) labeled by Iodogen solid-phase oxidation was studied. Rib53-Fluc-tk and CEA plasmids were co-transfected into 293T cells 24 hours later. Cells were inoculated into nude mice subcutaneously for bioluminescence imaging in vivo to verify the feasibility and optimal imaging conditions of the double reporter gene imaging system.
Result
1. construction of Rib53-Fluc-tk plasmid with ribozyme as core dual report vector
Rib DNA was obtained from tetrahydrohymena genome by PCR with a size of 516 bp, and the product size was the same as expected. Rib and Fluc sequences were cloned into pcDNA3.1 plasmid to form Rib3-Fluc reporter plasmid. The RIBBON size was correct by agarose gel electrophoresis, and the ribbon size was about 2200 bp. Rib53-Fluc-tk was obtained by PCR with a size of 3267 bp. Sending test results are correct.
2. detection of ribozyme splicing products
CEA expression was detected in MCF-7, SK-OV-3 and Hela cells in vitro, but not in SK-OV-3 and Hela cells.
The expression of luciferase was detected in MCF-7 cells, SK-OV-3 cells and Hela cells transfected with Rib53-Fluc-tk plasmid. The ratio of luciferase expression in MCF-7 cells was 0.639 (+ 0.099) and increased with the increase of Rib53-Fluc-tk mass (P = 0.006). The expression of luciferase in the other two cell lines was lower, and the luciferase value was 0.023 (+ 0.002) (P = 0.003) respectively. 0.033 + 0.003 (P=0.004).
The luciferase ratios were 0.0794-0.010, 0.145+0.033 (P=0.192), 0.653+0.130 (P=0.048):3.237+0.09-(P=0.001) when CEA and Rib53-Fluc-tk1 were 0, 0.2 UG and lug, respectively.
After transfection of MCF-7 and SK-OV-3 cells into Rib53-Fluc-tk, RNA was extracted and the splicing products were extracted by reverse transcription polymerase chain reaction (RT-PCR). A band was found in the MCF-7 swimming lane, and no band was found in the SK-OV-3 swimming lane.
Real-time PCR showed that the relative expression of CEA in MCF-7 cells transfected with Rib53-Fluc-tk was decreased, while that in MCF-7 cells transfected with pCDNA3.1 did not decrease significantly (P=0.019, P=0.003).
Immunofluorescence of MCF-7 cells transfected with Rib53-Fluc-Tk for 48 hours showed that the fluorescence was uniformly distributed in the cytoplasm.
Synthesis of 3.131I-FIAU
Iodogen solid-phase oxidation method was used to label FAU with 131I. The labeling rate of 131I-FIAU was 64.02 (?) 4.79% (n = 3) and the radiochemical purity was 95.96 (?) 1.07% (n = 3), and the labeling product was 95.74 (?) 0.86% (n = 3) in human serum for 24 hours.
Cellular uptake of 4.131I-FIAU
Rib53-Fluc-tk plasmid was transfected into 293T cells separately, and the uptake rate of Rib53-Fluc-tk was only 0.310+0.01%(n=4) at 4.5 H. pCDNA3.1-CEA and Rib53-Fluc-tk plasmid were co-transfected into 293T cells. The uptake rate of 131I-FIAU increased gradually with the increase of time, and reached 1.40+0.06%(n=4) and P 0.01 at 4.5 H. The uptake rate of Rib53-Fluc-tk plasmid increased slightly with the increase of time, reaching the highest level at 2.5 h, reaching only 0.64 (+ 0.04%).
5. cell bioluminescence detection
293T cells were transfected with the following plasmids: pDC316-Fluc-Egfp-Tk1 UG in the positive control group, CEA1 UG + Rib53-Fluc-Tk1 UG in the experimental group, and Rib53-Fluc-Tk1 UG in the negative control group. After 48 hours of transfection, fluorescein-D substrate was added to the positive control group for bioluminescence detection, and strong bioluminescence signals were detected in the experimental group. No obvious bioluminescence signal was detected in the negative reference group.
6. bioluminescence imaging of nude mice
Nine nude mice aged 3-4 weeks were prepared and randomly divided into three groups, three in each group. 293T cells were transfected with the following plasmids: positive control group pDC316-Fluc-Egfp-Tk5 ug; experimental group CEA 5 UG + Rib53-Fluc-Tk5 ug; negative control group: Rib53-Fluc-Tk5 ug. After 24 hours of transfection, the cells were collected and subcutaneously inoculated into the right lower part of the above three groups of nude mice. Limbs were injected with 3 mice in each group. Bioluminescence was detected in the injection site of the cells in the positive control group, no obvious bioluminescence signal was detected in the experimental group, and no bioluminescence signal was detected in the negative control group.
In this study, we successfully constructed a dual reporter gene system targeting CEA mediated by intron ribozyme type I. The ribozyme vector can selectively splice CEA and form the fusion protein expression of tumor-targeting molecule and reporter gene. Our research provides a preliminary basis for in vitro experiments for ribozyme mediated reporter gene imaging.
It is precisely because of the ribozyme-specific splicing that the reporter gene can specifically reflect the location and content of the target molecule expression. By forming the fusion products of CEA and reporter gene, the defects of low binding rate and poor imaging specificity between the reporter gene system and the target gene can be solved. The sensitivity of in vivo imaging can be used to monitor the efficacy of siRNA, radiotherapy and chemotherapy, and has a broad clinical value.
objective
Positron emission tomography (PET) has been widely used in tumor diagnosis, therapeutic evaluation, differential diagnosis of tumor recurrence and tissue necrosis. Metabolism and excretion are studied by choosing the appropriate pharmacokinetic model and calculating the pharmacokinetic parameters to reflect the changes of drugs in vivo according to the relationship between drug concentration and time. Biological distribution of tumor patients, however, the current study on methionine in glioma and melanoma-related model dynamics analysis is relatively limited. Therefore, this experiment is intended to study the kinetics of [11C] methionine PET in nude rat glioma and nude mouse melanoma in vivo. Number and search for the best mathematical model to quantitatively analyze the biological distribution of [11C] methionine in tumors and its ability to penetrate the blood-brain barrier, so as to apply [11C] methionine PET imaging in the future in Yale PET Center for model dynamics analysis.
Method
Glioma cell U87MG and human melanoma cell YUMAC were injected into the brain of nude rats (n=4) and nude mice (n=2) to construct glioma model in nude rats and melanoma model in nude mice.
[11C] was labeled with methionine, and [11C] methionine radiochemical purity was detected by parallel high performance liquid chromatography (HPLC).
Three weeks after stereotactic injection, each nude rat was injected with 18.5-37 MBq [11C] methionine, and the nude mice were injected with 1.85-3.7 MBq [11C] | methionine dynamic PET imaging respectively. The input function curves and time activity curves were obtained by drawing the left ventricle and brain tumor regions of interest on the PET images. Volume (VD) was calculated by one-tissue compartment model, two-tissue compartment model, Patlak model and MA1 model respectively, and the corresponding parameters K1, k2, k3, K4 and Ki, VD were obtained.
Result
The two-compartment model fitted the biological distribution of [11C] methionine better in the brain tumor model of nude rats. The VT of the three-compartment model was 3.894 [0.149ml / cc / min (n = 4), K1 was 0.157 [0.014ml / cc / min (n = 4) and K2 was 0.041 [0.004]/ min (n = 4); the three-compartment model of the melanoma model of nude mice was better than the two-compartment model. Patlak model could be well fitted in both nude rats and nude mice. Ki values of Patlak model were 0.043 [0.002 ml / cc / cc / cc / cc / min (n = 4.019 [0.019 [0.019 [0.019 [0.019 [0 0 0 0 0.019 [0.019] 1] 1 [0 [0.019 [0.019 [0] 1]]. Patlakmodel in nunude rats and nununude micewere 0.043 3 [0.043 [0.043 [0.002 [0.002 ml / cc / ml]]], K 2 cc (n = 2).
conclusion
The two-compartment model of nude rats can well fit the biological distribution of imaging agent in brain tissue, VT and K1 can better reflect the distribution of imaging agent in brain tissue and the ability to penetrate blood-brain barrier, and K1 can reflect [11C]. The rate of methionine entering blood-brain barrier with cerebral blood flow, K2 reflects the passive expansion of [11C] methionine. Ki reflects irreversible uptake of imaging agents from plasma to tumor tissue. Pharmacokinetic parameters are more sensitive than standardized uptake (SUV) and accurately reflect the biological distribution of [11C] methionine in the body. In the future, these pharmacokinetic parameters can be calculated to reflect the changes of [11C] methionine distribution in tumor tissues, so as to evaluate the therapeutic effect more sensitively.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R730.44
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