本文選題：適配體 + 循環(huán)腫瘤細(xì)胞　； 參考：《南京醫(yī)科大學(xué)》2016年博士論文

【摘要】：循環(huán)腫瘤細(xì)胞是腫瘤轉(zhuǎn)移復(fù)發(fā)的關(guān)鍵,腫瘤的早期發(fā)現(xiàn)和早期診斷有助于患者及時(shí)接受有效治療,從而降低病死率,改善患者預(yù)后。循環(huán)腫瘤細(xì)胞的檢測(cè)作為一種實(shí)時(shí)"液體活檢"手段為腫瘤疾病的診斷、評(píng)估預(yù)后生存、指導(dǎo)個(gè)體化治療等提供重要價(jià)值。循環(huán)腫瘤細(xì)胞是外周血中的稀有細(xì)胞,對(duì)檢測(cè)方法的敏感性和特異性要求極高。目前的循環(huán)腫瘤細(xì)胞檢測(cè)方法均有檢出率低、敏感性差、特異性不足、價(jià)格成本高等缺陷,鑒于循環(huán)腫瘤細(xì)胞的重要臨床意義,尋找更好的檢測(cè)方法顯得十分迫切。近年來(lái)納米基底法成為檢測(cè)循環(huán)腫瘤細(xì)胞發(fā)展最為迅速的工具。本研究中我們采用腐蝕氣體的新方法,通過(guò)反應(yīng)離子刻蝕法(RIE)干法刻蝕硅硼玻璃,獲得均勻的納米結(jié)構(gòu)的基底。特異性適配體已用于靶向藥物遞送,雙特異性的適配體能否提高腫瘤細(xì)胞的分離效率尚不明確。抗EpCAM適配體(AEA)和抗前列腺特異膜抗原(PSMA)適配體(APA)為常用的適配體,實(shí)驗(yàn)以三種人前列腺癌細(xì)胞 LNCaP(EpCAM+/PSMA+)、PC3(EpCAM+/PSMA-)、Ramos(EpCAM-/PSMA-)為研究對(duì)象,通過(guò)在制備的納米基底上加入AEA和APA適配體以期提高其對(duì)腫瘤細(xì)胞的分離捕獲效率。目的通過(guò)干法刻蝕硅硼玻璃獲得納米結(jié)構(gòu)的基底,聯(lián)合腫瘤細(xì)胞的特異性適配體,建立一種新型的循環(huán)腫瘤細(xì)胞分離的方法。方法利用氧化物蝕刻機(jī)產(chǎn)生的C3F8和C4F8氣體干性刻蝕硅硼玻璃,獲得大小均勻的具有納米結(jié)構(gòu)的基底并通過(guò)原子力顯微鏡對(duì)其進(jìn)行表征鑒定;計(jì)數(shù)一定數(shù)量的4腫瘤細(xì)胞(PC3、MCF-7、HepG2、SW620),用培養(yǎng)液混勻后加入到制備成功的納米基底上,計(jì)算細(xì)胞貼附率;DNA模板滾環(huán)復(fù)制制備APA和AEA適配體并采用凝膠電泳驗(yàn)證;化學(xué)反應(yīng)法將APA和AEA適配體固定到納米基底;將Cy3標(biāo)記的AEA,APA和對(duì)照DNA,分別直接和3種前列腺癌細(xì)胞孵育,通過(guò)Cy3的熒光強(qiáng)度來(lái)計(jì)算適配體與細(xì)胞的結(jié)合水平;納米基底捕獲腫瘤細(xì)胞實(shí)驗(yàn)分為4組:AEA+細(xì)胞組,APA+細(xì)胞組,AEA/APA+細(xì)胞組,以及對(duì)照DNA+細(xì)胞組,培養(yǎng)LNCaP、PC3、Ramos三種前列腺細(xì)胞,分別加入以上四組,DAPI.染色后,使用高內(nèi)涵篩選系統(tǒng)的顯微鏡計(jì)數(shù)計(jì)算納米基底對(duì)三種細(xì)胞的捕獲水平;將AEA和APA二種適配體混合后進(jìn)行凝膠電泳檢測(cè)適配體之間有無(wú)二聚體的產(chǎn)生;采用AEA化的納米基底捕獲模擬腫瘤患者外周血實(shí)驗(yàn),檢測(cè)三種高表達(dá)EpCam的腫瘤細(xì)胞(人肝癌HepG2細(xì)胞、人乳腺癌MCF7細(xì)胞以及人腸癌細(xì)胞SW620細(xì)胞),在高內(nèi)涵篩選系統(tǒng)高通量顯微鏡下全片觀察計(jì)數(shù)異常細(xì)胞,計(jì)算檢出率。CTCs判定標(biāo)準(zhǔn):細(xì)胞呈圓形、橢圓形或長(zhǎng)橢圓形,邊緣清楚完整;免疫熒光染色陽(yáng)性細(xì)胞呈CK18+/DAPI+/CD45-。結(jié)果原子力顯微鏡顯示我們成功的制備了直徑大小在126.8nm-412.9nm之間的納米基底,其中5min、100伏條件下制備的納米基底直徑374.3nm為最佳適宜捕獲腫瘤細(xì)胞的直徑;納米基底對(duì)4種腫瘤細(xì)胞的半小時(shí)貼附率均在93%以上;凝膠電泳結(jié)果顯示經(jīng)過(guò)30min滾環(huán)復(fù)制(RCA)反應(yīng)我們成功擴(kuò)增出APA和AEA適配體;適配體與3種腫瘤細(xì)胞結(jié)合實(shí)驗(yàn)顯示LNCaP細(xì)胞熒光強(qiáng)度最高,其中AEA適配體結(jié)合的熒光強(qiáng)度高于APA適配體,PC3細(xì)胞只檢測(cè)到AEA組的熒光信號(hào),作為對(duì)照的Ramos細(xì)胞和DNA與適配體共孵育后均未檢測(cè)到明顯的熒光信號(hào);細(xì)胞捕獲實(shí)驗(yàn)結(jié)果顯示結(jié)合AEA和APA適配體的納米基底對(duì)LNCaP細(xì)胞的捕獲率分別為85%±5.3%和68%±7.5%,結(jié)合AEA適配體的納米基底對(duì)PC3細(xì)胞的捕獲率分別為76.2%±8.5%,APA組捕獲率僅為1.2%±0.4%;對(duì)照組連接AEA或APA的納米基底對(duì)Ramos細(xì)胞捕獲水平均低于2%;連接有雙適配體AEA/APA的納米基底對(duì)LNCaP和PC3細(xì)胞的捕獲率為8.7%±2.3%和4.5%±1.8%;進(jìn)一步使用了不同比例混合的AEA/APA(1:3,1:1和3:1)來(lái)檢測(cè)對(duì)PC3細(xì)胞的捕獲水平,結(jié)果顯示PC3細(xì)胞捕獲率分別為0.5%、6.7%和42.1%;凝膠電泳結(jié)果顯示在約120-nt的位置出現(xiàn)新的弱條帶;模擬腫瘤患者外周血實(shí)驗(yàn)表明,連接AEA適配的納米基底對(duì)MCF7、HepG2、SW620三組細(xì)胞的檢出率分別為:84%±5.78%、73.6%±5.41%、70%±6.28%,三種細(xì)胞的平均檢出率為75.87%。結(jié)論采用腐蝕性氣體干法蝕刻硼硅玻璃可以快速可靠的制備納米結(jié)構(gòu)直徑均一性良好、面積較大的納米基底,通過(guò)對(duì)反應(yīng)條件的改變可將納米基底的直徑控制在126.8nm-412.9nm之間,其中370nm與細(xì)胞基質(zhì)的大小一致,是最佳適宜捕獲腫瘤細(xì)胞的直徑。特異性適配體與納米基底連接后,單適配體能夠提高納米基底對(duì)腫瘤細(xì)胞的捕獲水平,但雙特異性適配體則導(dǎo)致細(xì)胞捕獲水平明顯下降,原因可能是由于適配體之間形成了異二聚體影響了對(duì)細(xì)胞的捕獲;模擬腫瘤患者外周血實(shí)驗(yàn)證實(shí)納米基底結(jié)合特異性的單適配體對(duì)循環(huán)腫瘤細(xì)胞具有高檢出率。適配體之間形成的異二聚體可能會(huì)削弱多種特異性適配體在檢測(cè)和分子的分離中應(yīng)用,這提示在多適配體的應(yīng)用過(guò)程種需要通過(guò)一定的方法進(jìn)行嚴(yán)格的排除異二聚體的產(chǎn)生。該方法制備的玻璃納米基底具備高效、可靠、面積大(約80ccm2)的優(yōu)勢(shì)。納米基底的快速制備及表面包被處理,繞過(guò)CTCs分離環(huán)節(jié),利用納米基底捕獲CTCs后直接進(jìn)行檢測(cè),避免了分離過(guò)程中的CTC損耗。納米基底的方法不僅能檢測(cè)CTCs數(shù)量,還能夠分析CTCs的腫瘤相關(guān)分子表達(dá)情況,應(yīng)用于臨床后將為腫瘤的診斷、療效監(jiān)控和預(yù)后判斷提供更準(zhǔn)確的信息。放射治療是不可切除的原發(fā)性肝癌重要治療手段之一,但放射性肝損傷及放射乏氧抗拒影響肝癌放療效果。通過(guò)放射增敏的方法可以提高肝癌放療的療效。去唾液酸糖蛋白受體(ASGPR)主要表達(dá)于哺乳動(dòng)物肝實(shí)質(zhì)細(xì)胞表面,是介導(dǎo)細(xì)胞內(nèi)吞的常用靶點(diǎn)。納米金(GNPs)在多個(gè)體內(nèi)外實(shí)驗(yàn)中被證實(shí)有良好的放射增敏效應(yīng),在前期體外實(shí)驗(yàn)中我們合成了攜帶ASPGR特異性配體--半乳糖(GAL)以及聚乙二醇(PEG)修飾的納米金(GNPs)復(fù)合物GAL-PEG-GNPs,證實(shí)了 GAL-PEG-GNPs對(duì)ASGPR陽(yáng)性肝癌細(xì)胞HepG2具有放射增敏效應(yīng)并初步探討其增敏機(jī)制,本研究通過(guò)建立ASGPR陽(yáng)性肝癌裸鼠模型,在體內(nèi)水平研究GAL-PEG-GNPs的靶向性、器官毒性、組織分布、放射增敏。目的建立ASGPR陽(yáng)性肝癌裸鼠模型,研究GAL-PEG-GNPs在裸鼠體內(nèi)的藥物代謝、器官分布、放射增敏效果,為肝癌的放療增敏提供理論依據(jù)。方法培養(yǎng)高表達(dá)ASGPR的HepG2細(xì)胞,建立Balb/c肝癌皮下移植瘤裸鼠模型;通過(guò)尾靜脈途徑將GNPs和GAL-PEG-GNPs注射到裸鼠體內(nèi),抽血裸鼠靜脈血,ICP-MS 在不同時(shí)間點(diǎn)(5min、30min、1h、2h、3h、4h、8h、12h、24h、48h)檢測(cè)納米材料的藥代動(dòng)力學(xué);處死裸鼠后分離心、肝、脾、肺、腎、腫瘤組織及肌肉,通過(guò)ICP-MS檢測(cè)Au在不同時(shí)間點(diǎn)(30min、16h、24h)的臟器組織分布情況;模型建立后,將荷瘤裸鼠固定于特制的盒子里,對(duì)腫瘤局部采取6MeVX線照射(2.5Gy/次,每3天1次,共8次,總劑量為20Gy),通過(guò)裸鼠的體重、移植瘤的體積變化、最終瘤重來(lái)觀察納米材料的放射增敏性。結(jié)果裸鼠在接種HepG2細(xì)胞懸液后3-4天后在右前腋下形成可測(cè)量的瘤塊,從細(xì)胞接種后第5天測(cè)量腫瘤體積,第22天時(shí)所有腫瘤體積達(dá)到60mm3-340mm3。通過(guò)裸鼠尾靜脈注射納米材料后,二種納米材料在血液代謝的半衰期分別為t1/2GNPs=1.007 h,t1/2GAL-PEG-GNPs=3.406 h;組織器官分布檢測(cè)顯示 GNPS 和GAL-PEG-GNPs注射到裸鼠體內(nèi)后大部分都被肝臟和脾臟攝取,GAL-PEG-GNPs組在腫瘤組織內(nèi)Au含量明顯高于GNPs組,3個(gè)時(shí)間點(diǎn)差異均具有統(tǒng)計(jì)學(xué)意義。裸鼠放射增敏實(shí)驗(yàn)發(fā)現(xiàn),單純放射組腫瘤體積在治療開(kāi)始后增加速度明顯大于注射聯(lián)合放射的二組,至放療結(jié)束時(shí)GAL-PEG-GNPs聯(lián)合放療組將腫瘤體積控制到最小,明顯小于單純射線組和GNPs聯(lián)合放療組的(p0.05)。三組裸鼠體重在放療開(kāi)始后均穩(wěn)步增長(zhǎng),GAL-PEG-GNPs聯(lián)合放療組裸鼠生長(zhǎng)最好,體重最大,但較普通放射組及GNPs聯(lián)合放療組無(wú)明顯差別(p0.05)。治療第22天GAL-PEG-GNPs聯(lián)合放療組的腫瘤體積抑制率為82.22%,而GNPs聯(lián)合放療組為47.24%,組間比較差距具有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論本研究在體內(nèi)水平證實(shí)了 GAL-PEG-GNPs具有針對(duì)ASGPR陽(yáng)性肝癌裸鼠模型的靶向性和低毒性,GAL-PEG-GNPs較普通GNPs更能有效的提高腫瘤體積抑制率,改善放療效果,實(shí)驗(yàn)結(jié)果為肝癌的放療增敏及肝癌靶向性藥物載體的應(yīng)用提供了理論依據(jù)。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.4

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