本文選題：胃癌診斷 + 分子影像�。� 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【背景】胃癌是嚴(yán)重威脅人類生命健康的惡性腫瘤之一,其高病死率與發(fā)現(xiàn)時病變已處于晚期密切相關(guān),早期診斷是降低胃癌病死率的關(guān)鍵環(huán)節(jié)和有效手段,但現(xiàn)有臨床診斷手段在診斷靈敏度與特異性上均不能滿足早診的需求。MG7Ab是胃癌特異性單克隆抗體,靶向特異性強(qiáng)、靈敏度高,具有作為胃癌早期診斷分子標(biāo)志物的應(yīng)用前景。分子影像作為新興的成像手段,具有可在活體連續(xù)動態(tài)觀察特定分子改變的特點(diǎn),逐漸成為基礎(chǔ)研究中不可或缺的手段,也極具臨床應(yīng)用前景,其中多模態(tài)成像可以使不同的成像模態(tài)互補(bǔ),得到更為精準(zhǔn)和信息量更大的影像,為診斷提供有力證據(jù)�！灸康摹恳晕赴┨禺愋詥慰寺】贵wMG7Ab為靶向分子,制備系列可用于胃癌診斷成像的分子影像探針:MG7Ab-Cy5.5、64Cu-NODAGA-MG7Ab以及MG7Ab-ICG等,開展體外和在體成像研究,探索并優(yōu)化成像條件�！痉椒ā�1.胃癌特異性單克隆抗體MG7Ab的制備與功能驗證:通過雜交瘤-腹水抗體制備系統(tǒng)制備抗體,辛酸-飽和硫酸銨沉淀法及后續(xù)免疫學(xué)手段純化MG7Ab,通過Western Blot與免疫熒光染色實(shí)驗,驗證此次所制備抗體的胃癌特異性與親和力。2.MG7Ab核素顯像探針的制備與表征:通過化學(xué)耦聯(lián)法,將螯合劑NODAGA-NHS ester和p-NCS-benzyl-NODAGA與MG7Ab耦聯(lián),使用64Cu Cl2進(jìn)行放射性標(biāo)記,利用PD-10凝膠柱收集純化放射性探針,并計算放射性標(biāo)記效率;通過PBS與FBS孵育,檢驗探針的溶液穩(wěn)定性。3.MG7Ab-ICG探針的制備與表征:通過不同化學(xué)修飾將ICG與MG7Ab耦聯(lián),利用紫外-可見光吸收光譜對探針表征;通過PBS孵育,檢測探針的溶液穩(wěn)定性;利用濃度梯度成像,檢測探針的光聲成像性質(zhì)。4.體外細(xì)胞學(xué)成像研究:在細(xì)胞水平,通過細(xì)胞攝取實(shí)驗分別檢測各探針的胃癌細(xì)胞結(jié)合,利用激光共聚焦顯微鏡明確探針的結(jié)合模式,通過競爭性抑制實(shí)驗驗證探針結(jié)合的特異性。5.在體成像研究建立荷胃癌細(xì)胞的裸鼠模型,分別使用IVIS活體成像、小動物PET/CT成像、光聲成像以及近紅外成像內(nèi)鏡等系統(tǒng),通過不同時間點(diǎn)的成像探索最佳成像時間,通過競爭性抑制明確腫瘤成像的特異性,通過生物學(xué)分布研究探針在體內(nèi)的代謝情況�！窘Y(jié)果】1.胃癌特異性單克隆抗體MG7Ab的制備與功能:成功通過雜交瘤-腹水抗體制備體系,獲得一批含高濃度MG7Ab的腹水,經(jīng)進(jìn)一步純化,共獲得干粉態(tài)MG7Ab 8.67 mg。選用5種胃癌細(xì)胞株和永生化胃粘膜上皮細(xì)胞進(jìn)行Western Blot實(shí)驗,結(jié)果顯示以本次制備的MG7Ab為一抗,在胃癌細(xì)胞SGC-7901、KATOIII和MKN-28三株細(xì)胞株中出現(xiàn)了明顯的130 k Da顯色條帶,胃癌細(xì)胞AGS中有弱陽性條帶出現(xiàn),而胃癌細(xì)胞MKN-45和永生化胃粘膜上皮細(xì)胞GES中,在相應(yīng)位置無明顯條帶出現(xiàn);免疫熒光實(shí)驗顯示,MG7Ab與胃癌SGC-7901和MKN-28細(xì)胞株結(jié)合均主要位于細(xì)胞膜部位,另在細(xì)胞漿中可見微弱染色,而在陰性對照GES細(xì)胞株中,未見胞膜及胞質(zhì)有明確染色。2.MG7Ab-Cy5.5探針制備與體外、在體成像:通過耦聯(lián)反應(yīng),成功將Cy5.5-NHS ester與MG7Ab耦聯(lián),平均每個抗體上聯(lián)有2.5個熒光分子。細(xì)胞攝取實(shí)驗顯示,MG7Ab-Cy5.5探針可以特異性結(jié)合胃癌細(xì)胞SGC-7901和MKN-28,而不與永生化胃粘膜上皮細(xì)胞GES結(jié)合;激光共聚焦顯微鏡成像表明探針與胃癌細(xì)胞結(jié)合主要定位在胞膜,并有少量胞質(zhì)分布;荷瘤裸鼠活體近紅外成像顯示探針可特異性聚集在腫瘤部位;生物學(xué)分布提示探針能特異性聚集于腫瘤部位,并主要經(jīng)肝臟代謝。3.64Cu-NODAGA-MG7Ab探針制備與體外、在體成像:在不同反應(yīng)條件下成功將螯合劑NODAGA-NHS ester和p-NCS-benzyl-NODAGA與MG7Ab耦聯(lián),制備了兩種核素探針前體;在溫和反應(yīng)條件下實(shí)現(xiàn)了64Cu核素標(biāo)記,經(jīng)PD-10分離純化,測得兩種探針的標(biāo)記效率分別為85.7%和80.9%;PBS與FBS孵育后,兩種探針均有較好的溶液穩(wěn)定性,在孵育240 min后,兩種探針的完整度分別為96.37%、94.60%(PBS中)和95.39%、94.75%(40%FBS中);細(xì)胞攝取實(shí)驗表明兩種探針均能特異性結(jié)合胃癌細(xì)胞SGC-7901,孵育120 min,探針1的攝取率為13.93±0.45%ID,探針2的攝取率為10.62±0.07%ID,而加入MG7Ab競爭性抑制后,細(xì)胞攝取率明顯下降,在孵育60 min時,探針1的攝取率為12.29±0.27%ID,而加入MG7Ab后,攝取率下降至3.85±0.21%ID;探針2在孵育60 min時攝取率為10.39±0.04%ID,而加入MG7Ab競爭后,攝取率下降至3.26±0.04%ID;細(xì)胞親和力測定得出探針的解離常數(shù)KD=1.15±0.17μM;在體成像顯示探針1較探針2有更好的代謝行為和更佳的成像對比度,注射后24 h,兩種探針在裸鼠腫瘤攝取量分別為2.27±0.54%ID/g和0.25±0.13%ID/g;生物學(xué)分布研究顯示,MG7Ab可競爭性抑制探針在腫瘤部位的攝取,在注射后24 h,探針組腫瘤攝取為3.44±0.29%ID/g,而競爭抑制組僅為1.01±0.08%ID/g,進(jìn)一步驗證了探針結(jié)合的特異性。4.MG7Ab-ICG探針制備與體外、在體成像:通過共價耦聯(lián)和非共價耦聯(lián)兩種方式制備了MG7Ab-ICG探針,紫外-可見光譜吸收顯示,共價耦聯(lián)探針中,ICG/MG7Ab為5.4,而非共價耦聯(lián)探針為12.3;光聲成像顯示探針光聲信號與探針濃度具有良好的線性關(guān)系,相關(guān)性系數(shù)R2=0.9984;體外穩(wěn)定性檢測顯示,共價耦聯(lián)探針的穩(wěn)定性優(yōu)于非共價耦聯(lián)探針,其在孵育12 h、24 h和48 h的完整度分別為79.6%、75.9%以及72.2%,而非共價耦聯(lián)探針為31.7%、26.0%以及25.2%,但最終ICG/MG7Ab在兩種探針均在3-4區(qū)間;細(xì)胞結(jié)合實(shí)驗顯示,孵育2 h,探針組細(xì)胞信號強(qiáng)度為1.37*106±1.93*105 p/s/cm2/sr,競爭組為1.04*106±1.05*105 p/s/cm2/sr,陰性對照組為1.07*106±1.28*105 p/s/cm2/sr,提示探針可特異性與胃癌細(xì)胞結(jié)合;在體光聲成像顯示MG7Ab-ICG探針可顯著增強(qiáng)腫瘤區(qū)域信號強(qiáng)度,注射2 h,探針組信號為667.3%±102.2%,競爭抑制組為339.8±41.3%,信號強(qiáng)度差別約2倍;近紅外內(nèi)鏡可在探針注射15 min區(qū)別腫瘤部位的特異性成像信號與非特異成像信號�！窘Y(jié)論】成功制備、純化了一批具有胃癌特異性的單克隆抗體MG7Ab,以其為靶向核心制備了系列可用于胃癌診斷成像的分子影像探針:MG7Ab-Cy5.5、64Cu-NODAGA-MG7Ab以及MG7Ab-ICG等,通過開展體外和在體成像實(shí)驗,驗證了探針成像的腫瘤特異性,探索并優(yōu)化了成像條件,為提高胃癌診斷的準(zhǔn)確率和特異性提供新的分子影像學(xué)支持。
[Abstract]:[background] gastric cancer is one of the malignant tumors that seriously threaten human life and health. The high mortality rate is closely related to the disease in the late stage. Early diagnosis is the key link and effective means to reduce the mortality of gastric cancer. However, the diagnostic sensitivity and specificity of the existing diagnostic methods can not meet the needs of early diagnosis.MG7Ab. It is a specific monoclonal antibody for gastric cancer with high specificity and high sensitivity. It has the potential to be used as a molecular marker for early diagnosis of gastric cancer. As a new imaging means, molecular imaging has the characteristics of continuous dynamic observation of specific molecules in living bodies, and has gradually become an indispensable means in basic research, and it is also highly clinical. With the prospect, multi-modal imaging can make different imaging modalities complementary, get more accurate and more information, and provide strong evidence for diagnosis. [Objective] to prepare a series of molecular imaging probes for gastric cancer diagnosis imaging with the specific monoclonal antibody MG7Ab of gastric cancer as the target molecule, and to prepare a series of molecular imaging probes for diagnosis of gastric cancer: MG7Ab-Cy5.5,64Cu-NODAGA-MG7Ab And MG7Ab-ICG and so on, carry out in vitro and in vivo imaging research, explore and optimize imaging conditions. [Methods] preparation and functional verification of 1. gastric cancer specific monoclonal antibody MG7Ab: preparation of antibodies by hybridoma ascites antibody preparation, octane saturated ammonium sulfate precipitation and sequel immunological methods to purify MG7Ab by Western Blot and immunity The immunofluorescence staining experiments were conducted to verify the preparation and characterization of the.2.MG7Ab nuclear imaging probe for the specificity and affinity of the prepared antibodies. By coupling the chelating agent NODAGA-NHS ester and p-NCS-benzyl-NODAGA and MG7Ab by chemical coupling, the radioactivity markers were labeled with 64Cu Cl2, and the radioactive probe was collected and purified by the PD-10 gel column, and the radioprobe was collected and purified by the PD-10 gel column. The efficiency of radioactivity labeling was calculated; the preparation and characterization of the solution stability.3.MG7Ab-ICG probe of the probe by PBS and FBS were prepared and characterized: the probe was characterized by the coupling of ICG with MG7Ab by different chemical modifications and the UV visible light absorption spectrum was used to characterize the probe; the solution stability of the probe was detected by PBS incubation; the probe was detected by the concentration gradient imaging. .4. in vitro cytological imaging study of photoacoustic imaging: at the cell level, cell uptake was detected by cell uptake experiments, and the binding mode of gastric cancer cells was detected by the laser confocal microscopy, and the specific.5. of the probe was verified by competitive inhibition experiments to establish nude mice bearing gastric cancer cells in vivo. Model, using IVIS living body imaging, small animal PET/CT imaging, photoacoustic imaging and near infrared imaging endoscopy, the optimal imaging time was explored by imaging at different time points, the specificity of tumor imaging was determined by competitive inhibition, and the metabolic status of the probe in the body was studied by biological distribution. [results] 1. gastric cancer was specific. The preparation and function of sex monoclonal antibody MG7Ab: successfully using hybridoma ascites antibody preparation system to obtain a batch of ascites containing high concentration of MG7Ab. After further purification, a total of 5 kinds of gastric cancer cell lines and immortalized gastric mucosa epithelial cells were selected for Western Blot experiment of dry powder MG7Ab 8.67 mg.. The results showed that the MG7Ab was prepared by this method. For one resistance, there were obvious 130 K Da bands in the three cells of gastric cancer cells SGC-7901, KATOIII and MKN-28, and there were weak positive bands in gastric cancer cell AGS, while MKN-45 in gastric cancer cells and GES of immortalized gastric mucosa epithelial cells had no obvious bands in the corresponding position, and the immunofluorescence experiment showed MG7Ab and SGC-7901 and M of gastric cancer. The combination of KN-28 cell lines is mainly located in the membrane part of the cell, and the weak staining is visible in the cytoplasm. In the negative control GES cell line, there is no clear staining.2.MG7Ab-Cy5.5 probe in cell membrane and cytoplasm, and in vivo imaging: by coupling reaction, Cy5.5-NHS ester is coupled to MG7Ab, and the average of each antibody is 2.5. The cell uptake experiments showed that the MG7Ab-Cy5.5 probe could specifically bind the gastric cancer cells SGC-7901 and MKN-28, but not with the immortalized gastric epithelial cells GES. The laser confocal microscope imaging showed that the probe and gastric cancer cells were located mainly in the cell membrane, with a small amount of cytoplasm distribution, and the near infrared imaging of the tumor bearing nude mice was near infrared imaging. It shows that the probe can be specifically clustered at the tumor site. Biological distribution suggests that the probe can be specifically clustered at the tumor site and is mainly prepared and in vitro by the liver metabolism.3.64Cu-NODAGA-MG7Ab probe. In vivo imaging, two kinds of nuclei are prepared by coupling the chelating agent NODAGA-NHS ester and p-NCS-benzyl-NODAGA with MG7Ab under different reaction conditions. 64Cu nuclide labelling was realized under mild reaction conditions. The labeling efficiency of the two probes was 85.7% and 80.9% respectively by PD-10 separation and purification. After incubating with PBS and FBS, the two probes had better solution stability. After incubating 240 min, the integrity of two probes was 96.37%, 94.60% (PBS) and 95.39%, 94.75% (40%). In FBS), the cell uptake experiments showed that the two probes were able to specifically bind the gastric cancer cell SGC-7901 and incubate 120 min, the uptake rate of probe 1 was 13.93 + 0.45%ID, and the uptake rate of probe 2 was 10.62 + 0.07%ID, and the uptake rate of cells decreased significantly after the competitive inhibition of MG7Ab, and the uptake rate of probe 1 was 12.29 + 0.27%ID when incubating 60 min. After entering MG7Ab, the uptake rate decreased to 3.85 + 0.21%ID, and the uptake rate of probe 2 at 60 min was 10.39 + 0.04%ID, and the uptake rate decreased to 3.26 + 0.04%ID after MG7Ab competition, and the dissociation constant of the probe was determined by KD=1.15 + 0.17 mu M, and in vivo imaging probe 1 was better than the probe 2 for better metabolic behavior and better imaging pair. The uptake of the two probes in nude mice was 2.27 + 0.54%ID/g and 0.25 + 0.13%ID/g, respectively. The biological distribution study showed that the uptake of the MG7Ab competitive inhibition probe at the tumor site was 24 h after the injection, and the tumor uptake was 3.44 + 0.29%ID/g in the probe group, while the competition inhibition group was only 1.01 + 0.08%ID/g, which was further verified. The probe binding specific.4.MG7Ab-ICG probe was prepared and in vitro, in vivo imaging: MG7Ab-ICG probes were prepared by covalent coupling and non covalent coupling two methods. UV visible absorption showed that ICG/MG7Ab was 5.4 in covalent coupling probe and 12.3 for non covalent coupling probe; photoacoustic imaging showed the probe photoacoustic signal and probe concentration. With good linear relationship and correlation coefficient R2=0.9984, the stability test in vitro showed that the stability of covalent coupling probe was better than non covalent coupling probe, and the integrity of 12 h, 24 h and 48 h were 79.6%, 75.9% and 72.2% respectively, while the non covalent coupling probes were 31.7%, 26% and 25.2%, but finally ICG/MG7Ab was in two probes. In the 3-4 interval, the cell binding experiment showed that the cell signal intensity of the incubated 2 h was 1.37*106 + 1.93*105 p/s/cm2/sr, the competition group was 1.04*106 + 1.05*105 p/s/cm2/sr, and the negative control group was 1.07*106 + 1.28*105 p/s/cm2/sr, suggesting that the probe was specific to the gastric cancer cells, and the bulk photoacoustic imaging showed that the MG7Ab-ICG probe could be significantly enhanced. The signal intensity of the tumor was 2 h, the probe group signal was 667.3% + 102.2%, the competition inhibition group was 339.8 + 41.3% and the signal intensity difference was about 2 times. The specific imaging signal and the nonspecific imaging signal could be distinguished by the near infrared endoscopy with the probe injection of 15 min. A series of molecular imaging probes, such as MG7Ab-Cy5.5,64Cu-NODAGA-MG7Ab and MG7Ab-ICG, were prepared by cloning antibody MG7Ab, which were used as the target core for the diagnosis of gastric cancer. In vitro and in vivo imaging experiments, the specificity of the probe imaging was verified. The imaging conditions were explored and optimized to improve the accuracy of diagnosis of gastric cancer and the accuracy of the diagnosis of gastric cancer. Specificity provides new molecular imaging support.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.2
，

本文編號：2088105