【摘要】：目的 癌胚抗原分子(CEA)在多種腫瘤細(xì)胞中過(guò)表達(dá),作為腫瘤標(biāo)志物被廣泛用于評(píng)價(jià)結(jié)直腸癌、乳腺癌、肺癌等腫瘤的預(yù)后。報(bào)告基因顯像技術(shù)的發(fā)展為評(píng)估腫瘤治療療效以及腫瘤癌基因的表達(dá)提供了優(yōu)越的可行性。如何在活體水平對(duì)癌基因的表達(dá)進(jìn)行有效、實(shí)時(shí)且無(wú)創(chuàng)性的監(jiān)測(cè)是目前評(píng)估腫瘤治療效果而亟需解決的難點(diǎn)。本實(shí)驗(yàn)?zāi)康臄M構(gòu)建含有靶向CEA序列的反式剪接型I型內(nèi)含子核酶(Rib53)為載體核心介導(dǎo)的熒光素酶-核素(Fluc-HSVl-tk)雙報(bào)告基因顯像系統(tǒng),實(shí)現(xiàn)在活體水平直接、靶向、實(shí)時(shí)監(jiān)測(cè)靶基因表達(dá),進(jìn)而達(dá)到評(píng)價(jià)腫瘤療效與預(yù)后的目的。 方法 利用基因工程技術(shù)構(gòu)建靶向癌胚抗原(CEA)的反式剪接型I型內(nèi)含子核酶及核酶-熒光素酶-胸苷激酶(Rib53-Fluc-tk)報(bào)告質(zhì)粒；轉(zhuǎn)染Rib53-Fluc-tk于MCF-7乳腺腫瘤細(xì)胞系及共轉(zhuǎn)pCDNA3.1-CEA及Rib53-Fluc-tk質(zhì)粒于293T細(xì)胞中,分別用雙報(bào)告熒光素酶、逆轉(zhuǎn)錄PCR、Real-time PCR、免疫細(xì)胞熒光、細(xì)胞生物發(fā)光等技術(shù)檢測(cè)核酶對(duì)靶基因的剪接產(chǎn)物以及報(bào)告基因的表達(dá)；Iodogen固相氧化法標(biāo)記氟-阿糖呋喃基尿嘧啶(FAU)后進(jìn)行細(xì)胞攝取動(dòng)力學(xué)實(shí)驗(yàn)。共轉(zhuǎn)染Rib53-Fluc-tk及CEA質(zhì)粒于293T細(xì)胞中,24小時(shí)后將細(xì)胞接種于裸鼠皮下行活體小動(dòng)物生物發(fā)光顯像,驗(yàn)證該雙報(bào)告基因顯像體系的可行性及最佳顯像條件。數(shù)據(jù)分析采用獨(dú)立樣本t檢驗(yàn)、單因素方差分析,P0.05具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.以核酶為核心的雙報(bào)告載體Rib53-Fluc-tk質(zhì)粒的構(gòu)建 四膜蟲(chóng)基因組PCR獲得Rib DNA,大小為516bp,產(chǎn)物大小與預(yù)期一致；將Rib及Fluc序列克隆至pcDNA3.1質(zhì)粒形成Rib3-Fluc報(bào)告質(zhì)粒,瓊脂糖凝膠電泳鑒定酶切條帶大小正確,條帶大小約為2200bp,送檢測(cè)序結(jié)果正確。PCR獲得Rib53-Fluc-tk條帶,大小為3267bp,送檢測(cè)序結(jié)果正確。 2.核酶剪接產(chǎn)物的相關(guān)檢測(cè) 體外培養(yǎng)MCF-7、SK-OV-3以及Hela三種細(xì)胞系,在MCF-7細(xì)胞中檢測(cè)到CEA的表達(dá),而在SK-OV-3及Hela細(xì)胞中均未檢測(cè)到CEA的表達(dá)。 Rib53-Fluc-tk質(zhì)粒轉(zhuǎn)染MCF-7細(xì)胞、SK-OV-3以及Hela細(xì)胞中,在MCF-7細(xì)胞系中檢測(cè)到熒光素酶的表達(dá),比值可達(dá)0.639±0.099,隨著Rib53-Fluc-tk質(zhì)量的增加而比值增加(P=0.006)。而另外兩組細(xì)胞系中熒光素酶的表達(dá)較低,熒光素酶值分別為0.023±0.002(P=0.003),0.033±0.003(P=0.004)。 共轉(zhuǎn)染CEA及Rib53-Fluc-tk1μg于293T細(xì)胞中,當(dāng)CEA質(zhì)量分別為0μg、0.2μg、 lug時(shí),熒光素酶比值分別為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)。 將MCF-7及SK-OV-3細(xì)胞轉(zhuǎn)染Rib53-Fluc-tk后提取RNA,逆轉(zhuǎn)錄PCR提取剪切產(chǎn)物,MCF-7泳道可見(jiàn)一條帶,大小一致,SK-OV-3泳道未見(jiàn)條帶。 Real-time PCR檢測(cè)到MCF-7細(xì)胞轉(zhuǎn)染Rib53-Fluc-tk后CEA的相對(duì)含量表達(dá)減低,而僅轉(zhuǎn)染空載pCDNA3.1的MCF-7細(xì)胞的CEA含量卻無(wú)明顯減少(P=0.019,P=0.003)。 將Rib53-Fluc-Tk轉(zhuǎn)染MCF-7細(xì)胞48小時(shí)后行細(xì)胞免疫熒光,可見(jiàn)細(xì)胞漿內(nèi)熒光較均勻分布。 3.131I-FIAU的合成 采用Iodogen固相氧化法進(jìn)行FAU的131I標(biāo)記,標(biāo)記產(chǎn)物經(jīng)C-18小柱純化。131I-FIAU的標(biāo)記率為64.02±4.79%(n=3),放化純?yōu)?5.96±1.07%(n=3),標(biāo)記產(chǎn)物在人血清24小時(shí)的穩(wěn)定性為95.74±0.86%(n=3)。 4.131I-FIAU的細(xì)胞攝取 在293T細(xì)胞中單獨(dú)轉(zhuǎn)染Rib53-Fluc-tk質(zhì)粒,至4.5h時(shí)細(xì)胞攝取率僅達(dá)到0.310±0.01%(n=4)；在293T細(xì)胞中共轉(zhuǎn)染pCDNA3.1-CEA、Rib53-Fluc-tk質(zhì)粒,隨著時(shí)間的增加,293T細(xì)胞對(duì)131I-FIAU的攝取逐漸增加,至4.5h時(shí)攝取率達(dá)到1.40±0.06%(n=4),P0.01；在MCF-7細(xì)胞中轉(zhuǎn)染Rib53-Fluc-tk質(zhì)粒,隨著時(shí)間的增加細(xì)胞攝取率增加不明顯,在2.5h達(dá)到最高,最高僅達(dá)0.64±0.04%(n=4)。 5.細(xì)胞生物發(fā)光檢測(cè) 將如下質(zhì)粒分別轉(zhuǎn)染293T細(xì)胞：陽(yáng)性參照組pDC316-Fluc-Egfp-Tk1μg;實(shí)驗(yàn)組CEA1μg+Rib53-Fluc-Tk1μg;陰性參照組：Rib53-Fluc-Tk1μg,轉(zhuǎn)染48小時(shí)后加入熒光素酶底物熒光素-D行生物發(fā)光檢測(cè),陽(yáng)性參照組可探測(cè)到較強(qiáng)生物發(fā)光信號(hào),實(shí)驗(yàn)組可探測(cè)生物發(fā)光信號(hào),陰性參照組未探測(cè)明顯生物發(fā)光信號(hào)。 6.裸鼠生物發(fā)光顯像 準(zhǔn)備3-4周齡裸鼠9只,隨機(jī)分成3組,每組3只。將如下質(zhì)粒分別轉(zhuǎn)染293T細(xì)胞：陽(yáng)性參照組pDC316-Fluc-Egfp-Tk5μg;實(shí)驗(yàn)組CEA5μg+Rib53-Fluc-Tk5μg;陰性參照組：Rib53-Fluc-Tk5μg。轉(zhuǎn)染24小時(shí)后收集細(xì)胞,將這三組細(xì)胞分別皮下接種于以上3組裸鼠的右下肢,每組注射3只。行生物發(fā)光現(xiàn)象,陽(yáng)性參照組裸鼠中細(xì)胞注射部位未探測(cè)明顯生物發(fā)光信號(hào)；實(shí)驗(yàn)組未探及生物發(fā)光信號(hào)；陰性參照組未探及生物發(fā)光信號(hào),與預(yù)期一致。結(jié)論 本實(shí)驗(yàn)成功構(gòu)建了以I型內(nèi)含子核酶為核心介導(dǎo)的靶向CEA的雙報(bào)告基因體系。核酶載體能選擇性剪接CEA,形成了腫瘤靶向分子與報(bào)告基因的融合蛋白表達(dá),從而在細(xì)胞水平上成功地對(duì)CEA mRNA進(jìn)行了生物發(fā)光以及核素雙報(bào)告基因的顯像。我們的研究為核酶介導(dǎo)報(bào)告基因顯像提供了體外實(shí)驗(yàn)的前期基礎(chǔ)。 正是因?yàn)楹嗣柑禺愋缘募艚幼饔?報(bào)告基因才能特異性地反映靶向分子的表達(dá)的部位及含量。通過(guò)形成CEA及報(bào)告基因的融合產(chǎn)物,解決了報(bào)告基因系統(tǒng)與靶基因結(jié)合率低、顯像特異性不理想的缺點(diǎn)。如若能提高核酶的剪切效率實(shí)現(xiàn)小動(dòng)物活體顯像的靈敏度,便可以用于監(jiān)測(cè)siRNA、放化療的療效,具有較廣闊的臨床價(jià)值。 目的 正電子發(fā)射斷層掃描(PET)已被廣泛應(yīng)用于腫瘤的診斷、療效評(píng)估、腫瘤復(fù)發(fā)及組織壞死的鑒別診斷。[11C]甲硫氨酸PET顯像是目前最常用的氨基酸PET顯像方法。藥物動(dòng)力學(xué)主要是利用動(dòng)力學(xué)原理與數(shù)學(xué)模型來(lái)描述藥物在體內(nèi)的吸收、分布、代謝與排出情形,其研究方法是利用藥物濃度與時(shí)間的關(guān)系,選擇合適的藥物動(dòng)力學(xué)模型(pharmacokinetic model),計(jì)算出藥物動(dòng)力學(xué)參數(shù)來(lái)反映藥物在體內(nèi)的變化。新近發(fā)表的[11C]甲硫氨酸PET顯像文獻(xiàn)展示了[11C]甲硫氨酸在正常人體及膠質(zhì)瘤患者的生物學(xué)分布,然而目前對(duì)于甲硫氨酸在膠質(zhì)瘤與黑色素瘤相關(guān)模型動(dòng)力學(xué)分析的研究比較有限。因此本次實(shí)驗(yàn)研究是擬在裸大鼠膠質(zhì)瘤以及裸小鼠黑色素腦腫瘤活體對(duì)[11C]甲硫氨酸PET進(jìn)行模型動(dòng)力學(xué)分析,研究相關(guān)動(dòng)力學(xué)參數(shù)以及尋找最佳的數(shù)學(xué)模型來(lái)定量分析[11C]甲硫氨酸在腫瘤的生物分布以及穿透血腦屏障的能力,以便將來(lái)耶魯PET中心在受試人體應(yīng)用[11C]甲硫氨酸PET顯像進(jìn)行模型動(dòng)力學(xué)分析。 方法 分別在裸大鼠(nude rats,n=4)及裸小鼠(nude mice,n=2)腦部立體定位注射腦膠質(zhì)瘤細(xì)胞U87MG以及人黑色素瘤細(xì)胞YUMAC,分別構(gòu)建裸大鼠膠質(zhì)瘤腫瘤模型以及裸小鼠黑色素瘤腦腫瘤模型。 [11C]標(biāo)記甲硫氨酸,并行高效液相層析儀(HPLC)檢測(cè)[11C]甲硫氨酸放化純。 立體定位注射三周后每只裸大鼠分別注射18.5～37MBq [11C]甲硫氨酸,裸小鼠分別注射1.85～3.7MBq[11C]|甲硫氨酸行動(dòng)態(tài)PET顯像。在PET圖像上勾畫(huà)左心室及腦腫瘤等感興趣區(qū)獲得輸入功能曲線、時(shí)間活度曲線。通過(guò)軟件計(jì)算獲得腦腫瘤總分布體積(VD),分別用二腔室模型(one-tissue compartment model、三腔室模型(two-tissue compartment model)、Patlak模型以及多線性(MA1model)模型來(lái)計(jì)算并獲得相應(yīng)的參數(shù)K1,k2,k3, k4和Ki,VD。 結(jié)果 裸大鼠腦腫瘤模型中二室模型更好地?cái)M合[11C]甲硫氨酸在腦膠質(zhì)瘤的生物分布,在裸大鼠腦腫瘤模型中VT為3.894±0.149ml/cc/min(n=4),K1為0.157±0.014ml/cc/min(n=4),k2為0.041±0.004/min(n=4);在裸小鼠黑色素腦腫瘤模型三室模型較二室模型能更好地?cái)M合,VT為1.536±0.030ml/cc/min(n=2),K1為0.175±0.046ml/cc/min (n=2),k2為0.115±0.032/min(n=2),k3為0.407±0.095/min(n=2),k4為0.567±0.139/min(n=2)。Patlak模型在裸大鼠及裸小鼠均能較好地?cái)M合,ki值分別為0.043±0.002ml/cc(n=4),0.019±0.0001ml/cc(n=2)。 結(jié)論 裸大鼠中二室模型能較好地?cái)M合顯像劑在腦組織的生物分布,VT及K1能較好地反映顯像劑在腦組織的分布提及以及穿透血腦屏障的能力,并且K1能夠反映[11C].甲硫氨酸隨腦血流進(jìn)入血腦屏障的速率,k2則反映了[11C]甲硫氨酸隨之被動(dòng)擴(kuò)散排出大腦的速率。裸小鼠中三室模型能更好地描述顯像劑在腦黑色素瘤的分布。ki反映腦組織對(duì)顯像劑由血漿至腫瘤組織不可逆的攝取。藥物動(dòng)力學(xué)參數(shù)較標(biāo)準(zhǔn)化攝取(SUV)等能更靈敏、準(zhǔn)確地反映[11C]甲硫氨酸在體內(nèi)的生物分布,同時(shí)在將來(lái)腫瘤放化療療效評(píng)估時(shí),可通過(guò)計(jì)算以上藥物動(dòng)力學(xué)參數(shù)來(lái)反應(yīng)腫瘤組織[11C]甲硫氨酸分布的變化,從而更敏感地評(píng)價(jià)藥物療效。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R730.44

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