一种利用适配体修饰纳米基底检测循环肿瘤细胞方法的建立

发布时间：2018-05-10 07:17

本文选题：适配体 + 循环肿瘤细胞　；参考：《南京医科大学》2016年博士论文

【摘要】：循环肿瘤细胞是肿瘤转移复发的关键,肿瘤的早期发现和早期诊断有助于患者及时接受有效治疗,从而降低病死率,改善患者预后。循环肿瘤细胞的检测作为一种实时"液体活检"手段为肿瘤疾病的诊断、评估预后生存、指导个体化治疗等提供重要价值。循环肿瘤细胞是外周血中的稀有细胞,对检测方法的敏感性和特异性要求极高。目前的循环肿瘤细胞检测方法均有检出率低、敏感性差、特异性不足、价格成本高等缺陷,鉴于循环肿瘤细胞的重要临床意义,寻找更好的检测方法显得十分迫切。近年来纳米基底法成为检测循环肿瘤细胞发展最为迅速的工具。本研究中我们采用腐蚀气体的新方法,通过反应离子刻蚀法(RIE)干法刻蚀硅硼玻璃,获得均匀的纳米结构的基底。特异性适配体已用于靶向药物递送,双特异性的适配体能否提高肿瘤细胞的分离效率尚不明确。抗EpCAM适配体(AEA)和抗前列腺特异膜抗原(PSMA)适配体(APA)为常用的适配体,实验以三种人前列腺癌细胞 LNCaP(EpCAM+/PSMA+)、PC3(EpCAM+/PSMA-)、Ramos(EpCAM-/PSMA-)为研究对象,通过在制备的纳米基底上加入AEA和APA适配体以期提高其对肿瘤细胞的分离捕获效率。目的通过干法刻蚀硅硼玻璃获得纳米结构的基底,联合肿瘤细胞的特异性适配体,建立一种新型的循环肿瘤细胞分离的方法。方法利用氧化物蚀刻机产生的C3F8和C4F8气体干性刻蚀硅硼玻璃,获得大小均匀的具有纳米结构的基底并通过原子力显微镜对其进行表征鉴定;计数一定数量的4肿瘤细胞(PC3、MCF-7、HepG2、SW620),用培养液混匀后加入到制备成功的纳米基底上,计算细胞贴附率;DNA模板滚环复制制备APA和AEA适配体并采用凝胶电泳验证;化学反应法将APA和AEA适配体固定到纳米基底;将Cy3标记的AEA,APA和对照DNA,分别直接和3种前列腺癌细胞孵育,通过Cy3的荧光强度来计算适配体与细胞的结合水平;纳米基底捕获肿瘤细胞实验分为4组:AEA+细胞组,APA+细胞组,AEA/APA+细胞组,以及对照DNA+细胞组,培养LNCaP、PC3、Ramos三种前列腺细胞,分别加入以上四组,DAPI.染色后,使用高内涵筛选系统的显微镜计数计算纳米基底对三种细胞的捕获水平;将AEA和APA二种适配体混合后进行凝胶电泳检测适配体之间有无二聚体的产生;采用AEA化的纳米基底捕获模拟肿瘤患者外周血实验,检测三种高表达EpCam的肿瘤细胞(人肝癌HepG2细胞、人乳腺癌MCF7细胞以及人肠癌细胞SW620细胞),在高内涵筛选系统高通量显微镜下全片观察计数异常细胞,计算检出率。CTCs判定标准:细胞呈圆形、椭圆形或长椭圆形,边缘清楚完整;免疫荧光染色阳性细胞呈CK18+/DAPI+/CD45-。结果原子力显微镜显示我们成功的制备了直径大小在126.8nm-412.9nm之间的纳米基底,其中5min、100伏条件下制备的纳米基底直径374.3nm为最佳适宜捕获肿瘤细胞的直径;纳米基底对4种肿瘤细胞的半小时贴附率均在93%以上;凝胶电泳结果显示经过30min滚环复制(RCA)反应我们成功扩增出APA和AEA适配体;适配体与3种肿瘤细胞结合实验显示LNCaP细胞荧光强度最高,其中AEA适配体结合的荧光强度高于APA适配体,PC3细胞只检测到AEA组的荧光信号,作为对照的Ramos细胞和DNA与适配体共孵育后均未检测到明显的荧光信号;细胞捕获实验结果显示结合AEA和APA适配体的纳米基底对LNCaP细胞的捕获率分别为85%±5.3%和68%±7.5%,结合AEA适配体的纳米基底对PC3细胞的捕获率分别为76.2%±8.5%,APA组捕获率仅为1.2%±0.4%;对照组连接AEA或APA的纳米基底对Ramos细胞捕获水平均低于2%;连接有双适配体AEA/APA的纳米基底对LNCaP和PC3细胞的捕获率为8.7%±2.3%和4.5%±1.8%;进一步使用了不同比例混合的AEA/APA(1:3,1:1和3:1)来检测对PC3细胞的捕获水平,结果显示PC3细胞捕获率分别为0.5%、6.7%和42.1%;凝胶电泳结果显示在约120-nt的位置出现新的弱条带;模拟肿瘤患者外周血实验表明,连接AEA适配的纳米基底对MCF7、HepG2、SW620三组细胞的检出率分别为:84%±5.78%、73.6%±5.41%、70%±6.28%,三种细胞的平均检出率为75.87%。结论采用腐蚀性气体干法蚀刻硼硅玻璃可以快速可靠的制备纳米结构直径均一性良好、面积较大的纳米基底,通过对反应条件的改变可将纳米基底的直径控制在126.8nm-412.9nm之间,其中370nm与细胞基质的大小一致,是最佳适宜捕获肿瘤细胞的直径。特异性适配体与纳米基底连接后,单适配体能够提高纳米基底对肿瘤细胞的捕获水平,但双特异性适配体则导致细胞捕获水平明显下降,原因可能是由于适配体之间形成了异二聚体影响了对细胞的捕获;模拟肿瘤患者外周血实验证实纳米基底结合特异性的单适配体对循环肿瘤细胞具有高检出率。适配体之间形成的异二聚体可能会削弱多种特异性适配体在检测和分子的分离中应用,这提示在多适配体的应用过程种需要通过一定的方法进行严格的排除异二聚体的产生。该方法制备的玻璃纳米基底具备高效、可靠、面积大(约80ccm2)的优势。纳米基底的快速制备及表面包被处理,绕过CTCs分离环节,利用纳米基底捕获CTCs后直接进行检测,避免了分离过程中的CTC损耗。纳米基底的方法不仅能检测CTCs数量,还能够分析CTCs的肿瘤相关分子表达情况,应用于临床后将为肿瘤的诊断、疗效监控和预后判断提供更准确的信息。放射治疗是不可切除的原发性肝癌重要治疗手段之一,但放射性肝损伤及放射乏氧抗拒影响肝癌放疗效果。通过放射增敏的方法可以提高肝癌放疗的疗效。去唾液酸糖蛋白受体(ASGPR)主要表达于哺乳动物肝实质细胞表面,是介导细胞内吞的常用靶点。纳米金(GNPs)在多个体内外实验中被证实有良好的放射增敏效应,在前期体外实验中我们合成了携带ASPGR特异性配体--半乳糖(GAL)以及聚乙二醇(PEG)修饰的纳米金(GNPs)复合物GAL-PEG-GNPs,证实了 GAL-PEG-GNPs对ASGPR阳性肝癌细胞HepG2具有放射增敏效应并初步探讨其增敏机制,本研究通过建立ASGPR阳性肝癌裸鼠模型,在体内水平研究GAL-PEG-GNPs的靶向性、器官毒性、组织分布、放射增敏。目的建立ASGPR阳性肝癌裸鼠模型,研究GAL-PEG-GNPs在裸鼠体内的药物代谢、器官分布、放射增敏效果,为肝癌的放疗增敏提供理论依据。方法培养高表达ASGPR的HepG2细胞,建立Balb/c肝癌皮下移植瘤裸鼠模型;通过尾静脉途径将GNPs和GAL-PEG-GNPs注射到裸鼠体内,抽血裸鼠静脉血,ICP-MS 在不同时间点(5min、30min、1h、2h、3h、4h、8h、12h、24h、48h)检测纳米材料的药代动力学;处死裸鼠后分离心、肝、脾、肺、肾、肿瘤组织及肌肉,通过ICP-MS检测Au在不同时间点(30min、16h、24h)的脏器组织分布情况;模型建立后,将荷瘤裸鼠固定于特制的盒子里,对肿瘤局部采取6MeVX线照射(2.5Gy/次,每3天1次,共8次,总剂量为20Gy),通过裸鼠的体重、移植瘤的体积变化、最终瘤重来观察纳米材料的放射增敏性。结果裸鼠在接种HepG2细胞悬液后3-4天后在右前腋下形成可测量的瘤块,从细胞接种后第5天测量肿瘤体积,第22天时所有肿瘤体积达到60mm3-340mm3。通过裸鼠尾静脉注射纳米材料后,二种纳米材料在血液代谢的半衰期分别为t1/2GNPs=1.007 h,t1/2GAL-PEG-GNPs=3.406 h;组织器官分布检测显示 GNPS 和GAL-PEG-GNPs注射到裸鼠体内后大部分都被肝脏和脾脏摄取,GAL-PEG-GNPs组在肿瘤组织内Au含量明显高于GNPs组,3个时间点差异均具有统计学意义。裸鼠放射增敏实验发现,单纯放射组肿瘤体积在治疗开始后增加速度明显大于注射联合放射的二组,至放疗结束时GAL-PEG-GNPs联合放疗组将肿瘤体积控制到最小,明显小于单纯射线组和GNPs联合放疗组的(p0.05)。三组裸鼠体重在放疗开始后均稳步增长,GAL-PEG-GNPs联合放疗组裸鼠生长最好,体重最大,但较普通放射组及GNPs联合放疗组无明显差别(p0.05)。治疗第22天GAL-PEG-GNPs联合放疗组的肿瘤体积抑制率为82.22%,而GNPs联合放疗组为47.24%,组间比较差距具有统计学意义(p0.05)。结论本研究在体内水平证实了 GAL-PEG-GNPs具有针对ASGPR阳性肝癌裸鼠模型的靶向性和低毒性,GAL-PEG-GNPs较普通GNPs更能有效的提高肿瘤体积抑制率,改善放疗效果,实验结果为肝癌的放疗增敏及肝癌靶向性药物载体的应用提供了理论依据。
[Abstract]:Circulating tumor cells are the key to the recurrence of tumor metastasis. Early detection and early diagnosis of tumor can help the patient to receive effective treatment in time, thus reducing the mortality and improving the prognosis of the patients. The circulating tumor cells are rare cells in peripheral blood, and the sensitivity and specificity of the detection methods are very high. The current detection methods of circulating tumor cells have low detection rate, poor sensitivity, lack of specificity, high price and high cost, and look for better detection in view of the important clinical significance of circulating tumor cells. The method appears to be very urgent. In recent years, nano substrate method has become the most rapid tool for detecting circulating tumor cells. In this study, we used a new method of corrosion gas to etch silicon boron glass by reactive ion etching (RIE) method to obtain the base of homogeneous nanostructure. It is not clear whether specific aptamers can improve the separation efficiency of tumor cells. Anti EpCAM aptamer (AEA) and anti prostatic specific membrane antigen (PSMA) aptamer (APA) are commonly used aptamers. The experiment is based on three human prostate cancer cells LNCaP (EpCAM+/PSMA+), PC3 (EpCAM+ /PSMA-), Ramos (EpCAM-/PSMA-), through the preparation of the preparation. Mickey and APA aptamers are added to improve the separation and capture efficiency of the tumor cells. Objective to establish a new method for the separation of tumor cells by using the dry etching silicon boron glass to obtain the substrate of the nanostructure and the specific aptamers of the tumor cells. The formula of C3F8 and C4F8 produced by the oxide etch machine by square method. Gas dry etching of silicon boron glass to obtain a homogeneous substrate with nano structure and characterization by atomic force microscopy; count a certain number of 4 tumor cells (PC3, MCF-7, HepG2, SW620), and then add the culture solution to the prepared nanoscale Mickey base and calculate the cell attachment rate; DNA template rolling ring replication preparation APA and AEA aptamers were verified by gel electrophoresis; the chemical reaction method immobilized APA and AEA aptamers to the nanoscale substrate; Cy3 labeled AEA, APA and control DNA were incubated directly with 3 kinds of prostate cancer cells, respectively, and the binding level of the aptamers and cells was calculated by the fluorescence intensity of Cy3, and the nano substrate to capture tumor cells was divided into 4 groups: AE A+ cell group, APA+ cell group, AEA/APA+ cell group, and control DNA+ cell group, and culture LNCaP, PC3, Ramos three kinds of prostate cells, respectively added to the above four groups. After DAPI. staining, the capture level of nanoscale substrate to three cells was calculated using the microscope count of high intension screening system, and the gel electricity was mixed with AEA and APA two aptamers. The formation of two polymer was found between the aptamers, and the peripheral blood test of the simulated tumor patients was captured by AEA nano substrate. Three kinds of tumor cells with high expression of EpCam (human liver cancer HepG2 cells, human breast cancer MCF7 cells and human colon cancer cells SW620 cells) were detected and counted under the high flux high flux microscope. Abnormal cells, detection rate.CTCs criteria: cells were round, oval or long oval, and the edges were clear and complete; immunofluorescence staining positive cells showed CK18+/DAPI+/CD45-. results atomic force microscopy showed that we successfully prepared the nanoscale substrate between the diameter and the size of 126.8nm-412.9nm, which was prepared under the condition of 5min, 100 volt. The diameter of the nano base diameter 374.3nm is the best suitable for the capture of tumor cells; the half hour adherent rate of the nano substrate to 4 tumor cells is more than 93%. The gel electrophoresis results showed that the APA and AEA aptamers were successfully amplified by 30min rolling ring replication (RCA), and the combination of aptamers and 3 tumor cells showed the fluorescence of LNCaP cells. The intensity of the AEA aptamer binding was higher than that of the APA aptamer, and the PC3 cells only detected the fluorescence signal in the AEA group. As the control Ramos cells and the DNA and the aptamers, the fluorescence signals were not detected. The results of the cell capture experiment showed the capture of the LNCaP cells with the nano base of the AEA and APA aptamers. The rates were 85% + 5.3% and 68% + 7.5% respectively. The capture rate of PC3 cells with AEA aptamers was 76.2% + 8.5% respectively, and the capture rate of APA group was only 1.2% + 0.4%, and the control group with AEA or APA nanoscale substrate to Ramos cells was less than 2%, and the capture rate of the nano base to LNCaP and PC3 cells connected with the BIS aptamer AEA/ APA was 8.7% + 2.3% and 4.5% + 1.8%; using different proportions of AEA/APA (1:3,1:1 and 3:1) to detect the capture level of PC3 cells. The results showed that the capture rates of PC3 cells were 0.5%, 6.7% and 42.1%, respectively. The gel electrophoresis results showed a new weak strip in the location of about 120-nt; the peripheral blood experiment of the simulated tumor patients showed that the connection of AEA was suitable. The detection rates of the MCF7, HepG2, SW620 three groups were 84% + 5.78%, 73.6% + 5.41% and 70% + 6.28%, respectively. The average detection rate of three cells was 75.87%. conclusion that etching borosilicate glass by corrosive gas dry etching could quickly and reliably prepare nanostructures with good diameter homogenization and large area of nano substrates. The diameter of nanoscale substrate can be controlled in the diameter of 126.8nm-412.9nm, in which the size of 370nm is in accordance with the size of the cell matrix. It is the best fit to capture the diameter of the tumor cells. After the specific aptamers are connected with the nano substrate, the mono aptamer can improve the capture level of the nano base to the tumor cells. The cell capture level was significantly reduced because of the formation of the hetero two polymer between the aptamers and the influence of the cell capture. The peripheral blood test of the simulated tumor patients confirmed the high detection rate of the circulating tumor cells with the nano substrate binding to the specific monosaptams. The hetero two polymer formed between the aptamers may weaken a lot. The application of specific aptamers in detection and molecular separation suggests that the application process of polyaptamers should be strictly excluded from the production of isoamomers through a certain method. The prepared glass nano substrates have the advantages of high efficiency, reliability, large area (about 80ccm2), rapid preparation of nano substrates and the surface of the surface of bread. Treatment, bypassing the CTCs separation link, using the nano substrate to capture CTCs directly after the detection, to avoid the CTC loss during the separation process. The nano base method can not only detect the number of CTCs, but also analyze the expression of the tumor related molecules of CTCs. After clinical application, it will be more accurate for the diagnosis of swollen tumors, the monitoring of curative effect and the prognosis of the prognosis. Radiation therapy is one of the important treatments for non resectable primary liver cancer, but radiation-induced liver injury and hypoxic resistance affect the effect of radiotherapy on liver cancer. The effect of radiation sensitization can improve the therapeutic effect of liver cancer. ASGPR is mainly expressed on the surface of mammalian liver parenchyma cells. The common target of cell endocytosis. Gold nanoparticles (GNPs) have been proved to have a good radiosensitization effect in a number of experiments and internal and external experiments. In the early in vitro experiments, we synthesized the nano gold (GNPs) complex GAL-PEG-GNPs modified by ASPGR specific ligands, galactose (GAL) and polyethylene glycol (PEG), which confirmed that GAL-PEG-GNPs is positive for ASGPR. Hepatoma cell HepG2 has radiosensitization effect and its sensitizing mechanism is preliminarily discussed. In this study, the target, organ toxicity, tissue distribution and radiosensitivity of GAL-PEG-GNPs were studied in vivo by establishing a nude mouse model of ASGPR positive liver cancer. Objective to establish a nude mouse model of ASGPR positive liver cancer, and to study the drug metabolism of GAL-PEG-GNPs in nude mice. The distribution of organs and the effect of radiation sensitization provide a theoretical basis for the enhancement of radiation sensitization of liver cancer. Methods the HepG2 cells with high expression of ASGPR were cultured, and the nude mice model of Balb/c liver cancer subcutaneous transplanted tumor was established. GNPs and GAL-PEG-GNPs were injected into nude mice through the tail vein pathway, and the venous blood of nude mice was pumped. ICP-MS at different time points (5min, 30min, 1H, 2h, 3h, 4h, 8) H, 12h, 24h, 48h) detects the pharmacokinetics of nanomaterials; after the death of nude mice, the heart, liver, spleen, lung, kidney, tumor tissue and muscles are isolated and the tissues and tissues of Au at different time points (30min, 16h, 24h) are detected by ICP-MS; after the model is established, the tumor bearing nude mice are fixed in a special box, and 6MeV X ray irradiation (2.5Gy/ times) is taken to the tumor locally. 1 times every 3 days, a total of 8 times, the total dose of 20Gy), the body weight of the nude mice, the volume change of the transplanted tumor, and the final tumor weight to observe the radiosensitivity of the nanomaterials. Results the nude mice formed a measurable tumor in the right anterior axillary 3-4 days after the inoculation of the nude mice, and measured the tumor volume fifth days after the inoculation of the cells, and all the tumor bodies at twenty-second days. After the nanomaterials were injected into the tail vein of the nude mice, the half-life of the two nanomaterials in the blood was t1/2GNPs=1.007 h and t1/2GAL-PEG-GNPs=3.406 h, respectively. The tissue and organ distribution detection showed that most of the GNPS and GAL-PEG-GNPs were injected into the nude mice by the liver and spleen, and the GAL-PEG-GNPs group was in the tumor. The content of Au in the tissue was significantly higher than that in the GNPs group, and the difference between the 3 time points was statistically significant. The radiation sensitization test in nude mice showed that the increase of tumor volume in the simple radiation group was obviously greater than that of the two groups of the injection combined radiation, and the volume of the GAL-PEG-GNPs combined with the radiotherapy group at the end of the radiotherapy was minimal, obviously less than that of the radiotherapy group. The weight of the three groups of nude mice increased steadily after the radiotherapy, and the three group of nude mice increased steadily after the radiotherapy. The GAL-PEG-GNPs combined with radiotherapy group had the best growth and the maximum weight, but there was no significant difference between the normal radiation group and the GNPs combined with the radiotherapy group (P0.05). The tumor volume inhibition rate of the twenty-second day GAL-PEG-GNPs combined radiotherapy group was 82.22%. The GNPs combined with radiotherapy group was 47.24%, and the comparative gap between the groups was statistically significant (P0.05). Conclusion the study in vivo confirmed the targeting and low toxicity of GAL-PEG-GNPs in the nude mice model of ASGPR positive liver cancer. GAL-PEG-GNPs was more effective to improve the tumor volume inhibition rate and improve the effect of radiotherapy, and the experimental results were improved. It provides a theoretical basis for the radiosensitization of liver cancer and the application of targeted drug carriers for liver cancer.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.4

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