本文選題：結(jié)核分枝桿菌 + 雜交瘤�。� 參考：《復(fù)旦大學(xué)》2013年博士論文

【摘要】：第一部分 碘與熒光標(biāo)記抗ESAT-6單克隆抗體靶向探針的制備及免疫活性評(píng)價(jià)目的 制備抗結(jié)核分枝桿菌ESAT-6蛋白的單克隆抗體,以碘原子和熒光標(biāo)記單克隆抗體研制CT和熒光靶向探針,并評(píng)價(jià)兩種探針的免疫活性。 方法 1.以重組大腸桿菌表達(dá)純化的ESAT-6蛋白為免疫原免疫小鼠,采用淋巴細(xì)胞雜交瘤技術(shù),獲得了4株針對(duì)結(jié)核分枝桿菌ESAT-6抗原的單克隆抗體雜交瘤細(xì)胞株,進(jìn)行小鼠腹水制備、純化,并測(cè)定抗體純度和效價(jià)。 2.采用Iodogen法,以放射性碘Na125I示蹤非放射性碘Na127I標(biāo)記抗ESAT-6單克隆抗體,紙層析法測(cè)定放化純度和穩(wěn)定性。 3.將抗ESAT-6單克隆抗體偶聯(lián)兩種熒光基團(tuán)IR783和羅丹明制備熒光探針,SDS-PAGE凝膠電泳確定熒光探針表征。 4.間接ELSIA法測(cè)定碘原子與熒光標(biāo)記探針的抗體效價(jià),評(píng)價(jià)標(biāo)記后探針免疫活性。 結(jié)果 1.4株單克隆抗體的腹水效價(jià)達(dá)到1：128000,純化后抗體純度高于90%,抗體亞型均為IgGl/k型。ELISA結(jié)果顯示制備的單克隆抗體與結(jié)核分枝桿菌ESAT-6抗原可發(fā)生特異反應(yīng),而不與非相關(guān)性蛋白His-85b發(fā)生反應(yīng)。 2.純化后的碘原子標(biāo)記抗ESAT-6抗體的放化純度大于98%。穩(wěn)定性試驗(yàn)測(cè)定結(jié)果顯示,放射性碘標(biāo)記抗ESAT-6抗體在體外4℃條件下存放一周后放化純度大于95%。 3.熒光探針每個(gè)抗體分子上標(biāo)記2個(gè)IR783和1.5個(gè)羅丹明。采用不同色調(diào)分析方式進(jìn)行處理,顯示探針無雜條帶。 4.標(biāo)記后單克隆抗體仍能與ESAT-6抗原發(fā)生特異性反應(yīng),抗體效價(jià)大于1：128000,碘原子和熒光標(biāo)記探針具有較強(qiáng)免疫活性。 結(jié)論 1.制備了高純度、高效價(jià)抗結(jié)核分枝桿菌ESAT-6單克隆抗體,可與ESAT-6蛋白產(chǎn)生特異性反應(yīng)。 2.碘原子標(biāo)記抗ESAT-6單克隆抗體具有較好的體外穩(wěn)定性；熒光探針純度優(yōu)良。碘與熒光標(biāo)記抗ESAT-6單克隆抗體能與ESAT-6抗原特異性結(jié)合,較好保持其免疫活性。為進(jìn)一步應(yīng)用于動(dòng)物體內(nèi)成像實(shí)驗(yàn)奠定了基礎(chǔ)。 第二部分 小鼠肺結(jié)核模型建立及ESAT-6抗原在小鼠肺結(jié)核組織中的表達(dá) 目的 探討滴鼻途徑建立C57BL/6J小鼠肺部結(jié)核分枝桿菌感染模型的可行性,分析ESAT-6抗原在小鼠肺結(jié)核組織中的表達(dá)和意義。 方法 1. C57BL/6J小鼠30只,分為感染組24只,對(duì)照組6只。結(jié)核分枝桿菌H37Rv標(biāo)準(zhǔn)株滴鼻法接種感染組小鼠,對(duì)照組小鼠給予同等劑量生理鹽水。 2.感染后4周、8周、12周,Micro-CT掃描小鼠肺部評(píng)價(jià)肺部病灶變化,并與HE染色及抗酸染色病理對(duì)照分析,小鼠肺組織勻漿培養(yǎng)計(jì)算肺部結(jié)核分枝桿菌菌落數(shù)。 3.應(yīng)用免疫組化及免疫熒光染色法檢測(cè)ESAT-6抗原在小鼠肺結(jié)核組織中的表達(dá)。 結(jié)果 1.感染后4周、8周、12周,小鼠肺組織勻漿培養(yǎng)結(jié)核分枝桿菌菌落計(jì)數(shù)在106CFU左右,呈輕度上升趨勢(shì)；小鼠肺部Micro-CT掃描顯示肺部病灶陽性率分別為50.0%、83.3%、100%,肺部病灶體積增加與時(shí)間遞增相關(guān)(P0.05)；3個(gè)監(jiān)測(cè)時(shí)間點(diǎn)小鼠肺部病理學(xué)檢查陽性率分別為83.3%、100%、100%。感染后8周、12周兩個(gè)時(shí)間點(diǎn)小鼠肺部Micro-CT掃描陽性率及病理陽性率相似,病理改變均以增殖性實(shí)變和肉芽腫為主,干酪樣壞死少見。感染后4周、8周、12周,抗酸桿菌染色均可見病變區(qū)散在或大量結(jié)核分枝桿菌聚集。 2.免疫組化結(jié)果顯示感染后4周、8周、12周可見在小鼠肺結(jié)核滲出及實(shí)變區(qū)域片狀褐色陽性物質(zhì)沉積,在結(jié)核分枝桿菌及巨噬細(xì)胞分布區(qū)域見條狀、顆粒狀、結(jié)節(jié)狀褐色陽性物質(zhì)沉積。免疫熒光染色在實(shí)變區(qū)及肉芽腫區(qū)域熒光信號(hào)明顯增強(qiáng)。 結(jié)論 1. Micro-CT動(dòng)態(tài)觀察與病理學(xué)對(duì)照分析表明經(jīng)滴鼻感染途徑成功建立了小鼠肺結(jié)核感染模型。 2.感染后8周是成像實(shí)驗(yàn)?zāi)Ｐ徒⒌睦硐霑r(shí)間點(diǎn)。. 3.免疫組化及免疫熒光觀察表明ESAT-6抗原在小鼠肺結(jié)核組織中的表達(dá)明顯增強(qiáng),為進(jìn)一步研究單克隆抗體探針靶向診斷小鼠肺結(jié)核提供了依據(jù)。 第三部分 小鼠肺結(jié)核Micro-CT和近紅外熒光成像的實(shí)驗(yàn)研究 目的 探討抗ESAT-6單克隆抗體靶向探針Micro-CT和近紅外熒光成像診斷小鼠肺結(jié)核的可行性。 方法 1.24只C57BL/6J小鼠分為CT組和熒光組,每組12只。CT組和熒光組又分為實(shí)驗(yàn)組和對(duì)照組,實(shí)驗(yàn)組各9只小鼠,對(duì)照組各3只小鼠。小鼠經(jīng)滴鼻法感染結(jié)核后8周用于成像實(shí)驗(yàn)。實(shí)驗(yàn)組經(jīng)尾靜脈注射碘和熒光標(biāo)記單克隆抗體探針,對(duì)照組注射等量碘和熒光標(biāo)記非特異IgG抗體。 2.CT組6只小鼠注射探針前、注射探針后6h、24h、48h活體Micro-CT動(dòng)態(tài)成像,測(cè)量不同時(shí)間點(diǎn)肺部病灶CT值并計(jì)算平均值；對(duì)照組小鼠相同時(shí)間點(diǎn)觀察。CT組3只小鼠在注射探針24h后,放射性活度儀檢測(cè)肺部病灶及周圍正常組織放射性碘分布。 3.熒光組9只小鼠注射熒光探針后24h,取小鼠肺、心、肝、脾、腎、肌肉組織離體近紅外熒光成像。測(cè)量肺部病灶、正常肺組織、背景噪聲信號(hào)強(qiáng)度(SI),計(jì)算對(duì)比噪聲比(CNR), CNR=(SI病灶-SI正常肺)／SI背景噪聲。肺組織冰凍切片熒光顯微鏡觀察并與HE染色對(duì)照分析,評(píng)價(jià)熒光探針到達(dá)結(jié)核病灶的能力。對(duì)照組小鼠相同方法觀察。 結(jié)果 1.注射碘標(biāo)記抗ESAT-6靶向探針后,Micro-CT小鼠肺部動(dòng)態(tài)增強(qiáng)顯示肺部結(jié)核病灶強(qiáng)化,且24h時(shí)CT值最高。對(duì)照組小鼠肺部病灶未見強(qiáng)化。放射性活度儀檢測(cè)實(shí)驗(yàn)組小鼠肺部病灶放射性碘分布高于正常肺組織(P0.05)。 2.離體近紅外熒光成像顯示實(shí)驗(yàn)組小鼠肺部結(jié)核病灶區(qū)域熒光信號(hào)明顯增強(qiáng),對(duì)照組結(jié)核病灶區(qū)域微弱熒光或無熒光信號(hào)。單克隆抗體誘導(dǎo)的肺部病灶信號(hào)明顯高于同型對(duì)照抗體,對(duì)比噪聲比分別為60.33±3.44(n=9)和10.49±2.03(n＝3),兩者存在統(tǒng)計(jì)學(xué)差異(P0.05)。熒光顯微鏡顯示熒光積聚區(qū)域與HE染色肺結(jié)核組織部位一致。 結(jié)論 1.活體Micro-CT成像顯示碘原子標(biāo)記探針能夠靶向性聚集于結(jié)核病灶,放射性碘體內(nèi)生物學(xué)分布與活體成像一致。 2.離體近紅外熒光成像及熒光顯微鏡觀察證明了抗ESAT-6單克隆抗體熒光靶向探針可特異性到達(dá)小鼠肺結(jié)核組織。 3.應(yīng)用以上兩種抗ESAT-6單克隆抗體探針可靶向、特異成像小鼠肺結(jié)核,為進(jìn)一步多模式成像診斷肺結(jié)核奠定了基礎(chǔ)。
[Abstract]:Part one
Preparation of iodine and fluorescent labeled anti ESAT-6 monoclonal antibody targeting probes and evaluation of their immune activity
The monoclonal antibodies against Mycobacterium tuberculosis ESAT-6 protein were prepared. The CT and fluorescent target probe were developed with iodine atom and fluorescence labeled monoclonal antibody, and the immunological activity of the two probes was evaluated.
Method
1. the ESAT-6 protein expressed and purified by recombinant Escherichia coli was immunized with immunogenic mice. By using lymphocyte hybridoma technique, 4 monoclonal antibody hybridoma cell lines were obtained for ESAT-6 antigen of Mycobacterium tuberculosis. The mouse ascites was prepared, purified, and the antibody purity and titer were measured.
2. using Iodogen method, radioiodine Na125I was used to trace the non radioactive iodine Na127I labeled monoclonal antibody against ESAT-6. The radiochemical purity and stability were determined by paper chromatography.
3. the monoclonal antibody against ESAT-6 was coupled with two fluorescent groups IR783 and Luo Danming to prepare fluorescent probes. SDS-PAGE gel electrophoresis was used to determine the fluorescence probe characterization.
4. indirect ELSIA method was used to determine the antibody titer of iodine atom and fluorescent labeled probe, and the immune activity of labeled probe was evaluated.
Result
The ascitic titer of 1.4 monoclonal antibodies reached 1:128000, and the purity of the purified antibody was higher than 90%. The antibody subtypes were all IgGl/k.ELISA results showed that the monoclonal antibody was specific to the ESAT-6 antigen of Mycobacterium tuberculosis and did not react against the unrelated protein His-85b.
The purity of anti ESAT-6 antibody after 2. purified iodine was greater than that of 98%. stability test, and the radioiodine labeled anti ESAT-6 antibody was stored for one week at 4 centigrade, and the purity was more than 95%.
3. fluorescent probes labeled 2 IR783 and 1.5 rhodamine on each antibody molecule. Different hue analysis methods were used to show that there were no heterozygous bands in the probe.
After 4. labeling, the monoclonal antibody can still react with ESAT-6 antigen specifically, the titer of the antibody is greater than 1:128000, and the iodine atom and the fluorescent labeled probe have strong immune activity.
conclusion
1. a highly purified and highly effective anti Mycobacterium tuberculosis ESAT-6 monoclonal antibody was prepared, which could react specifically with ESAT-6 protein.
2. iodide labeled anti ESAT-6 monoclonal antibody has good in vitro stability, and the purity of fluorescence probe is excellent. Iodine and fluorescent labeling anti ESAT-6 monoclonal antibody can be specifically combined with ESAT-6 antigen, and better maintain its immune activity. It lays a foundation for further application of imaging experiments in animal body.
The second part
Establishment of a mouse model of pulmonary tuberculosis and expression of ESAT-6 antigen in mice with pulmonary tuberculosis
objective
To explore the feasibility of establishing a model of Mycobacterium tuberculosis infection in C57BL/6J mice by means of nasal drip, and to analyze the expression and significance of ESAT-6 antigen in the tuberculosis of mice.
Method
1. C57BL/6J mice were divided into infection group (24 mice) and control group (6 rats). The H37Rv standard strain of Mycobacterium tuberculosis was inoculated with nosed mice in infection group, and the same dose of saline was given to the control group.
2. after 4 weeks of infection, 8 weeks and 12 weeks, the lung lesions were evaluated by Micro-CT scan in the lungs, and the lung tissue homogenate culture was used to calculate the number of Mycobacterium tuberculosis in the lung tissue of mice.
3. immunohistochemistry and immunofluorescence staining were used to detect the expression of ESAT-6 antigen in mouse pulmonary tuberculosis.
Result
1. after 4 weeks of infection, 8 weeks and 12 weeks, the colony count of Mycobacterium tuberculosis in lung tissue homogenate in mice was about 106CFU, showing a slight increase. The positive rates of lung lesions were 50%, 83.3%, 100% in lung Micro-CT scan in mice, respectively, and the increase of lung lesion volume was associated with time increasing (P0.05), and 3 monitoring time points of lung pathology in mice. The positive rates of the examination were 83.3%, 100%, 8 weeks after 100%. infection and two time at 12 weeks in 12 weeks, and the pathological positive rates were similar. The pathological changes were mainly proliferative and granulomatous, and the caseous necrosis was rare. 4 weeks, 8 weeks and 12 weeks after infection, the pathological area was scattered in the lesion area or a large number of tuberculosis branches. Bacteria gather.
2. the results of immunofluorescence showed that 4 weeks, 8 weeks, and 12 weeks after infection, the nodular Brown positive substance in the tubercular exudation and real region of the mice was deposited. In the distribution area of Mycobacterium tuberculosis and macrophage, a strip, granular and nodular Brown positive substance was deposited. The fluorescence staining of immunofluorescence in the real and granulomatous regions was significantly increased. Strong.
conclusion
1. Micro-CT dynamic observation and pathology analysis showed that a mouse model of pulmonary tuberculosis infection was successfully established through nasal infection.
2. the 8 week after infection is the ideal time to establish the imaging experimental model.
3. immunofluorescence and immunofluorescence showed that the expression of ESAT-6 antigen was obviously enhanced in the tuberculosis tissues of mice, which provided a basis for the further study of the monoclonal antibody probe targeted to the diagnosis of tuberculosis in mice.
The third part
Experimental study on Micro-CT and near infrared fluorescence imaging of pulmonary tuberculosis in mice
objective
Objective to investigate the feasibility of using ESAT-6 monoclonal antibody targeted probe Micro-CT and near infrared fluorescence imaging in the diagnosis of pulmonary tuberculosis in mice.
Method
1.24 C57BL/6J mice were divided into CT group and fluorescence group, each group of 12.CT groups and fluorescent groups were divided into experimental group and control group, 9 mice in the experimental group and 3 mice in the control group. The mice were infected by the nose drip method for 8 weeks after tuberculosis and were used for imaging experiments. The experimental group was injected with iodine and fluorescence labeled monoclonal antibody probes by the tail vein, and the control group was injected with equal amount of iodine. Non specific IgG antibodies were labeled with fluorescence and fluorescence.
Before injection of probe into 6 mice in group 2.CT, the 6h, 24h, 48h living body Micro-CT dynamic imaging after injection of probe was used to measure the CT value of lung lesions at different time points and to calculate the average value. In the control group, 3 mice in the.CT group were observed at the same time point, and the radioactivity meter was used to detect the radioactive iodine distribution in the lung lesions and surrounding normal tissues after the injection probe 24h.
3. fluorescent group 9 mice were injected with 24h after injection of fluorescent probe. The lung, heart, liver, spleen, kidney, and muscle tissue were taken near infrared fluorescence imaging in mice. The lung lesions, normal lung tissues, background noise signal intensity (SI) were measured, contrast noise ratio (CNR), CNR= (SI lesion -SI normal lung) / SI background noise. HE staining was used to evaluate the ability of fluorescent probes to reach tuberculosis foci.
Result
After 1. injection of iodine labeled anti ESAT-6 target probe, the lung dynamic enhancement showed that the pulmonary tuberculosis focus was enhanced in the Micro-CT mice, and the CT value was the highest at 24h. The lung lesions in the control group did not strengthen. The radioiodine distribution in the lung lesions of the mice was higher than that of the normal lung tissue (P0.05).
2. in vitro near infrared fluorescence imaging showed that the fluorescence signal of the pulmonary tuberculosis focus in the experimental group was obviously enhanced and the tuberculosis focus area of the control group was weak fluorescent or no fluorescence signal. The lung focal signal induced by the monoclonal antibody was significantly higher than that of the same type control antibody. The contrast noise ratio was 60.33 + 3.44 (n=9) and 10.49 + 2.03 (n = 3), respectively. There was statistical difference (P0.05). Fluorescence microscopy showed that the area of fluorescence accumulation was consistent with that of HE stained tuberculosis tissue.
conclusion
1. in vivo Micro-CT imaging showed that the iodine labeled probe could target aggregation in the tuberculosis foci. The biodistribution of radioiodine in vivo coincide with in vivo imaging.
2. in vitro near infrared fluorescence imaging and fluorescence microscopy confirmed that the fluorescent targeting probe against ESAT-6 monoclonal antibody could specifically reach the pulmonary tuberculosis tissue in mice.
3. the above two anti ESAT-6 monoclonal antibody probes can be targeted and specifically imaging pulmonary tuberculosis in mice, thus laying the foundation for further multi-mode imaging diagnosis of pulmonary tuberculosis.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R52;R816.4

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