本文選題：甲氨蝶呤　切入點(diǎn)：靶向納米微泡　出處：《重慶醫(yī)科大學(xué)》2014年博士論文

【摘要】：第一部分載MTX靶向納米微泡的制備和性能檢測(cè) 目的制備一種包載甲氨蝶呤（MTX）的聚乳酸-羥基乙酸（PLGA）靶向（人類白細(xì)胞抗原-G單克隆抗體，， mAbHLA-G）納米微泡（mAbHLA-G/MTX/PLGA），檢測(cè)其物理和聲學(xué)性質(zhì)。 方法采用雙乳化法，碳二亞胺連接法和真空冷凍干燥技術(shù)制備mAbHLA-G/MTX/PLGA，觀察其表形結(jié)構(gòu)，測(cè)量粒徑電位，包封率和載藥量；高強(qiáng)度聚焦超聲（HIFU）和普通超聲的促發(fā)后檢測(cè)mAbHLA-G/MTX/PLGA納米微泡中藥物的釋放情況；并在不同的濃度梯度（200mg/ml，100mg/ml，50mg/ml，25mg/ml，10mg/ml）和時(shí)間梯度（0h，2h，4h，8h，16h，24h，48h）觀察其超聲顯影效果。 結(jié)果mAbHLA-G/MTX/PLGA納米微泡溶于雙蒸水后呈淡黃色混懸液，光鏡及掃描電鏡觀察其形態(tài)規(guī)則，呈球形，大小較均勻，表面有孔稍欠光滑，分散度好。透射電鏡負(fù)染后可見MTX均勻分布在納米微泡中心。馬爾文激光測(cè)量?jī)x檢測(cè)出mAbHLA-G/MTX/PLGA平均粒徑為（477.6±119.7）nm，分散指數(shù)0.171，Zeta電位為（-5.62±5.36）mV。高效液相色譜法檢測(cè)其包封率為44.11±1.27%，載藥量為4.41±0.13%(w/w)。HIFU激發(fā)后3h mAbHLA-G/MTX/PLGA納米微泡藥物累計(jì)釋放率達(dá)到50%，72h后藥物累計(jì)釋放率超過80%；而普通超聲促發(fā)后72h mAbHLA-G/MTX/PLGA納米微泡藥物累計(jì)釋放率僅為47.8%。體外超聲顯影實(shí)驗(yàn)顯示mAbHLA-G/MTX/PLGA納米微泡能產(chǎn)生較強(qiáng)的超聲回聲信號(hào)，并隨著濃度的變化而變化，且制備的mAbHLA-G/MTX/PLGA超聲造影劑性能穩(wěn)定，在制備完成后24h內(nèi)各時(shí)間點(diǎn)超聲顯像效果沒有統(tǒng)計(jì)學(xué)差異。 結(jié)論成功制備了載MTX靶向納米微泡（mAbHLA-G/MTX/PLGANBs）,其形態(tài)規(guī)則，大小均勻，分散好。與普通超聲相比，HIFU更有效的促進(jìn)了納米微泡內(nèi)藥物的釋放。mAbHLA-G/MTX/PLGA超聲造影劑在體外能增強(qiáng)超聲成像，且穩(wěn)定性好，具有良好的聲學(xué)性能，是一種具有良好應(yīng)用前景的多功能超聲造影劑。 第二部分載MTX靶向納米微泡聯(lián)合高強(qiáng)度聚焦超聲對(duì)滋養(yǎng)細(xì)胞作用的體外實(shí)驗(yàn)研究 目的檢測(cè)人絨毛膜癌細(xì)胞株JEG-3細(xì)胞HLA-G蛋白的表達(dá)和定位，探討mAbHLA-G/MTX/PLGA納米微泡體外尋靶能力和被細(xì)胞吞噬的情況。探討mAbHLA-G/MTX/PLGA納米微泡聯(lián)合高強(qiáng)度聚焦超聲靶向破裂對(duì)人絨毛膜癌JEG-3細(xì)胞株細(xì)胞周期，細(xì)胞增殖和侵襲，及誘導(dǎo)凋亡和侵襲相關(guān)蛋白表達(dá)的影響，為體內(nèi)實(shí)驗(yàn)提供依據(jù)。 方法體外培養(yǎng)人絨毛膜癌JEG-3細(xì)胞株，免疫熒光法檢查JEG-3細(xì)胞HLA-G蛋白的表達(dá)和定位。制備DiI標(biāo)記的mAbHLA-G/MTX/PLGA納米微泡（DiI-mAbHLA-G/MTX/PLGANBs），與JEG-3細(xì)胞共孵育，觀察納米微泡的體外尋靶能力和被細(xì)胞吞噬的情況，實(shí)驗(yàn)分組：（1）mAbHLA-G/MTX/PLGA NBs組，（2）mAbHLA-G預(yù)處理后+等量的mAbHLA-G/MTX/PLGA NBs組，（3）MTX/PLGA NBs組，共孵育后DAPI染核，采用激光共聚焦顯微鏡觀察納米微泡的分布情況。 mAbHLA-G/MTX/PLGA NBs聯(lián)合HIFU體外治療實(shí)驗(yàn)分為12個(gè)組：（1）PBS組，（2）空白PLGA NBs組，（3）MTX/PLGANBs組，（4）MTX組，（5）mAbHLA-G/PLGA NBs組，（6）mAbHLA-G/MTX/PLGA NBs組，（7）HIFU+PBS組,（8）HIFU+空白PLGA NBs組,（9）HIFU+MTX/PLGANBs組（,10）HIFU+MTX組，（11）HIFU+mAbHLA-G/PLGANBs組，（12）HIFU+mAbHLA-G/MTX/PLGA NBs組。通過流式細(xì)胞儀檢測(cè)各組細(xì)胞凋亡和細(xì)胞周期的變化，Western blotting檢測(cè)凋亡和侵襲相關(guān)蛋白Bax，Bcl-2，Caspase3，MMP2，TIMP-2的表達(dá)和熒光定量PCR從基因水平檢測(cè)Caspase3，MMP2的表達(dá)情況。 結(jié)果HLA-G在JEG-3細(xì)胞中呈高表達(dá)，主要定位在細(xì)胞膜表面。體外靶向性驗(yàn)證實(shí)驗(yàn)表明，mAbHLA-G/MTX/PLGA納米微泡能與高表達(dá)HLA-G蛋白的JEG-3細(xì)胞特異性結(jié)合，增加納米微泡在細(xì)胞的聚集和被細(xì)胞吞噬。在體外治療實(shí)驗(yàn)中，與其他組相比， HIFU+mAbHLA-G/MTX/PLGA NBs組JEG-3細(xì)胞凋亡率最高，細(xì)胞明顯阻滯在S期。Bax和Caspase3的表達(dá)在HIFU+mAbHLA-G/MTX/PLGA NBs組明顯高于其他組， Bcl-2蛋白表達(dá)低于其他組。 HIFU+mAbHLA-G/MTX/PLGA NBs組細(xì)胞侵襲能力明顯減弱，MMP2的表達(dá)在所有組中最低，TIMP-2的表達(dá)明顯高于其余各組。 結(jié)論mAbHLA-G/MTX/PLGA納米微泡靶向性好，能夠改變靶區(qū)域周圍聲環(huán)境，增強(qiáng)超聲的空化效應(yīng)和機(jī)械效應(yīng)，同時(shí)超聲又能促進(jìn)納米微泡中化療藥物的釋放。HIFU聯(lián)合mAbHLA-G/MTX/PLGANBs很大程度的抑制了JEC-3細(xì)胞的增殖和侵襲，其作用機(jī)制可能是通過影響凋亡和侵襲相關(guān)蛋白的表達(dá)來完成的。 第三部分載MTX靶向納米微泡增效HIFU消融對(duì)滋養(yǎng)細(xì)胞作用的體內(nèi)實(shí)驗(yàn)研究 目的驗(yàn)證HLA-G在JEG-3細(xì)胞裸鼠皮下移植瘤模型瘤組織內(nèi)的表達(dá)和定位。評(píng)價(jià)mAbHLA-G/MTX/PLGA納米微泡在體內(nèi)的超聲顯像效果和體內(nèi)靶向性。研究mAbHLA-G/MTX/PLGA納米微泡協(xié)同HIFU消融技術(shù)對(duì)JEG-3細(xì)胞裸鼠皮下移植瘤模型的治療效果和遠(yuǎn)期生存情況的分析。 方法143只BALB/C雌性裸鼠，JEG-3細(xì)胞以1~2×106濃度皮下注射于裸鼠背部，構(gòu)建裸鼠皮下移植瘤模型，腫瘤直徑約0.8~1.0cm時(shí)可用于后續(xù)實(shí)驗(yàn)。取瘤組織塊石蠟包埋切片，采用免疫組化法檢測(cè)HLA-G的表達(dá)和定位。經(jīng)尾靜脈注射mAbHLA-G/MTX/PLGA納米微泡，在注射前和注射后（0h和24h）分別觀察瘤組織區(qū)域的超聲顯像情況。經(jīng)尾靜脈注射DiI-mAbHLA-G/MTX/PLGA納米微泡，采用小動(dòng)物活體熒光成像技術(shù)觀察不同時(shí)間點(diǎn)（3h，24h，48h）靶向載藥納米微泡在荷瘤鼠體內(nèi)的分布情況，并在不同時(shí)間點(diǎn)取瘤組織塊冰凍切片觀察納米微泡的分布。體內(nèi)評(píng)價(jià)mAbHLA-G/MTX/PLGA納米微泡增效HIFU消融的效果，120只荷瘤鼠隨機(jī)分為12組（n=10）：（1）NS組，（2）空白PLGANBs組，（3）MTX/PLGA NBs組，（4）MTX組，（5）mAbHLA-G/PLGANBs組，（6）mAbHLA-G/MTX/PLGA NBs組，（7）HIFU+NS組,（8）HIFU+空白PLGA NBs組,（9）HIFU+MTX/PLGA NBs組（,10）HIFU+MTX組，（11） HIFU+mAbHLA-G/PLGA NBs組，（12） HIFU+mAbHLA-G/MTX/PLGA NBs組。HIFU消融后立即觀察各組（7-12組）中瘤組織的灰度變化和取瘤組織測(cè)量消融體積。12組在處理完成后24h取瘤組織做HE染色，TUNEL檢測(cè)凋亡，PCNA評(píng)價(jià)腫瘤細(xì)胞增殖抑制情況。每組余下5只荷瘤鼠用于觀察腫瘤的生長(zhǎng)情況和進(jìn)行生存分析。 結(jié)果HLA-G在JEG-3細(xì)胞皮下移植瘤組織中高表達(dá)，定位于細(xì)胞膜和細(xì)胞漿中。mAbHLA-G/MTX/PLGA超聲造影劑在體內(nèi)能穩(wěn)定顯影，并能特異性的到達(dá)靶組織，提高靶區(qū)域納米微泡的聚集濃度和停留時(shí)間，增加化療藥物在靶區(qū)域的釋放濃度。mAbHLA-G/MTX/PLGA納米微泡能明顯增強(qiáng)HIFU消融，HIFU+mAbHLA-G/MTX/PLGA NBs組消融后灰度變化顯著，瘤組織消融體積明顯大于其他組。 HIFU聯(lián)合mAbHLA-G/MTX/PLGA NBs有效抑制了腫瘤細(xì)胞的增殖，促進(jìn)細(xì)胞凋亡，延長(zhǎng)荷瘤鼠的生存周期（中位生存期=58天）。 結(jié)論mAbHLA-G/MTX/PLGA通過改變靶區(qū)域聲環(huán)境，增強(qiáng)HIFU的消融效果，同時(shí)HIFU的熱效應(yīng)提高了mAbHLA-G/MTX/PLGA納米微泡中化療藥物的釋放，協(xié)同靶向殺滅了殘余瘤細(xì)胞，抑制了腫瘤的生長(zhǎng)和轉(zhuǎn)移。mAbHLA-G/MTX/PLGA超聲造影劑聯(lián)合高強(qiáng)度聚焦超聲為臨床胎盤植入和滋養(yǎng)細(xì)胞疾病的治療開辟了一條全新的道路。
[Abstract]:Preparation and performance detection of MTX targeted nano microbubbles in the first part
Objective to prepare a poly (lactic acid glycolic acid) (PLGA) targeted human leukocyte antigen -G monoclonal antibody (mAbHLA-G) nano microbubble (mAbHLA-G/MTX/PLGA) loaded with methotrexate (MTX), and to detect its physical and acoustic properties.
Methods using double emulsion method, two carbon imine connection method and vacuum freeze drying technology for preparing mAbHLA-G/MTX/PLGA, observe the morphological structure, measurement of particle size and zeta potential, encapsulation efficiency and drug loading; high intensity focused ultrasound (HIFU) to promote the release after detection of mAbHLA-G/MTX/PLGA nano microbubble in medicine and conventional ultrasound; and in different concentration gradient (200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml, 10mg/ml) and time gradient (0h, 2h, 4h, 8h, 16h, 24h, 48h) to observe the effect of ultrasound imaging.
The results of mAbHLA-G/MTX/PLGA nano microbubble dissolved in double distilled water after the pale yellow suspension, light microscope and scanning electron microscope to observe the morphological rules, spherical shape, uniform size, surface pore slightly less smooth, well dispersion. TEM negative staining showed uniform distribution of MTX in nano microbubble Malvin laser measuring instrument center. The detection of mAbHLA-G/MTX/PLGA average particle size (477.6 + 119.7) nm, the polydispersity index of 0.171, Zeta potential (-5.62 + 5.36) mV. HPLC method for the determination of the entrapment efficiency was 44.11 + 1.27%, the drug loading was 4.41 + 0.13% (w/w).HIFU 3H mAbHLA-G/MTX/PLGA after excitation of nano microbubble accumulated drug release rate 50%, after 72h accumulated drug release rate of more than 80%; while the ordinary ultrasonic trigger after 72h mAbHLA-G/MTX/PLGA nano microbubble drug cumulative release rate was only 47.8%. in vitro ultrasound imaging experiments showed that mAbHLA-G/MTX/PLGA nano microbubble can produce strong ultrasound Echo signal changes with the concentration. The prepared mAbHLA-G/MTX/PLGA ultrasound contrast agent has stable performance. After the completion of the preparation, there is no statistical difference in the effect of 24h in each time point.
Conclusion the successful preparation of MTX loaded targeted microbubble contrast agent (mAbHLA-G/MTX/PLGANBs), the regular shape, uniform size, good dispersion. Compared with conventional ultrasound, HIFU is more effective to promote the release of.MAbHLA-G/MTX/PLGA nano microbubble ultrasound contrast agent in medicine can enhance ultrasound imaging in vitro, and good stability, with good acoustic performance that is a kind of multifunctional ultrasound contrast agent has good application prospect.
Experimental study on the effect of MTX targeting nano microbubbles combined with high intensity focused ultrasound on trophoblast in second parts
Objective to detect the expression and localization of human choriocarcinoma cell line JEG-3 cells HLA-G protein, explore the mAbHLA-G/MTX/PLGA nano microbubble targeting and phagocytosis. The study of mAbHLA-G/MTX/PLGA nano microbubble combined with high intensity focused ultrasound targeted rupture of cell cycle in human choriocarcinoma JEG-3 cell line, cell proliferation and invasion, and the effect of induced expression and invasion apoptosis related protein, provide the basis for in vivo experiments.
Human choriocarcinoma cell line JEG-3 cultured in vitro, the expression and localization of immunofluorescence examination of JEG-3 cells HLA-G protein. MAbHLA-G/MTX/PLGA nano preparation of DiI labeled microbubbles (DiI-mAbHLA-G/MTX/PLGANBs), and JEG-3 cells were incubated in vitro, observe the nano microbubble targeting ability and phagocytosis, experimental groups: (1 mAbHLA-G/MTX/PLGA) NBs group, (2) mAbHLA-G/MTX/PLGA + NBs group with mAbHLA-G pretreatment, MTX/PLGA (3) NBs group, after incubation of DAPI nuclear staining and distribution by laser confocal microscopy nano microbubbles.
MAbHLA-G/MTX/PLGA NBs combined with HIFU treatment in vitro experiment was divided into 12 groups: (1) PBS (2) PLGA group, blank NBs group, MTX/PLGANBs group (3), (4) MTX group, (5) mAbHLA-G/PLGA NBs (6) mAbHLA-G/MTX/PLGA group, NBs group, HIFU+PBS group (7), (8) HIFU+ blank PLGA NBs group (9), HIFU+MTX/PLGANBs group (group HIFU+MTX, 10), (11) HIFU+mAbHLA-G/PLGANBs (12) HIFU+mAbHLA-G/MTX/PLGA group, NBs group. The changes were detected by flow cytometry and cell apoptosis and cell cycle, invasion and related protein Bax, Western blotting Bcl-2 Caspase3 MMP2, apoptosis, and expression of TIMP-2. Fluorescence quantitative PCR detection of Caspase3 from the gene level, the expression of MMP2.
The high expression of HLA-G in JEG-3 cells was mainly localized in the cell membrane. In vitro targeting experiment shows that mAbHLA-G/MTX/PLGA nano microbubble can be combined with JEG-3 cell specific HLA-G protein expression, increase of nano microbubble in cell aggregation and phagocytosis in vitro. The experimental treatment, compared with the other HIFU+mAbHLA-G/MTX/PLGA group, NBs group the apoptosis rate of JEG-3 cells was the highest, the expression of S and.Bax block in Caspase3 HIFU+mAbHLA-G/MTX/PLGA in NBs group was significantly higher than other groups, the expression of Bcl-2 protein was lower than that of the other group. The invasion ability of HIFU+ mAbHLA-G/MTX/PLGA cells in NBs group significantly decreased the expression of MMP2 in all groups in the lowest, the expression of TIMP-2 was significantly higher than that of other groups.
Conclusion mAbHLA-G/MTX/PLGA nano microbubble targeting, can change the sound environment around the target area, enhance the cavitation effect and mechanical effect of ultrasound, and ultrasound can promote the release of.HIFU and mAbHLA-G/MTX/PLGANBs in nano microbubble chemotherapy significantly inhibited the proliferation and invasion of JEC-3 cells, the mechanism may be accomplished by expression the effect of apoptosis and invasion associated protein.
Experimental study on the effect of MTX targeting nano microbubbles on the effect of HIFU ablation on trophoblastic cells in the third part
Objective to validate the expression and localization of HLA-G in tumor tissue of JEG-3 xenografts in nude mice model. The evaluation of mAbHLA-G/MTX/PLGA nano microbubbles in ultrasound imaging in vivo and in vivo targeting. Analysis of mAbHLA-G/MTX/PLGA nano microbubble treatment effect of ablation technology in cooperative HIFU transplantation tumor model of JEG-3 cell in nude mice skin down and long-term survival.
Methods 143 female BALB/C nude mice, JEG-3 cells in a concentration of 106 1~2 * subcutaneously in the back of nude mice, construct the subcutaneous tumor model in nude mice, tumor diameter of about 0.8~1.0cm can be used for subsequent experiments. The tumor tissue was embedded in paraffin, expression and localization were detected by immunohistochemistry HLA-G. After intravenous injection of mAbHLA-G/MTX/PLGA nanoparticles after the injection of microbubbles, before and after the injection (0h and 24h) were used to observe the ultrasound imaging of tumor tissue. The situation of regional intravenous injection of DiI-mAbHLA-G/MTX/PLGA nano microbubble, using small animal in vivo fluorescence imaging was observed at different time points (3H, 24h, 48h) targeting drug loaded microbubbles in distribution of tumor in vivo, and tumor tissue frozen sections of nano bubble distribution at different time points. In vivo evaluation of mAbHLA-G/MTX/PLGA nano microbubble enhancing effect of HIFU ablation, 120 mice were randomly divided into 12 groups (n=10) (1): NS group, blank group PLGANBs (2), (3) MTX/PLGA (4) NBs group, MTX group, mAbHLA-G/PLGANBs group (5), (6) mAbHLA-G/MTX/PLGA (7) NBs group, HIFU+NS group, PLGA NBs (8) HIFU+ blank group (9), HIFU+MTX/PLGA (group NBs, 10) HIFU+MTX group, (11) HIFU+mAbHLA-G/PLGA (12) NBs group, HIFU+mAbHLA-G/MTX/PLGA NBs group.HIFU were observed immediately after ablation (Group 7-12) measurement in gray change tumor tissue and tumor tissue ablation volume in.12 group after the treatment of 24h tumor specimens were stained by HE and TUNEL for the detection of apoptosis, inhibition of proliferation of PCNA tumor each group of cells. The remaining 5 mice to observe the growth of tumors and survival analysis.
Results the expression of HLA-G in JEG-3 cells in subcutaneous tumor, stable in vivo in the developing orientation of ultrasound contrast agent.MAbHLA-G/MTX/PLGA in cell membrane and cytoplasm, and can reach the target tissue specificity, improve the target region of nano microbubble aggregation concentration and residence time, increase the concentration of.MAbHLA-G/MTX/PLGA in the release of drug nano target region the microbubbles can enhance HIFU ablation intensity changes after ablation of HIFU+mAbHLA-G/MTX/PLGA NBs group, the tumor tissue ablation volume was significantly higher than other groups. HIFU combined with mAbHLA-G/MTX/PLGA NBs can effectively inhibit tumor cell proliferation, promote cell apoptosis, prolong the mice survival period (median survival period of =58 days).
Conclusion mAbHLA-G/MTX/PLGA can change the target regional acoustic environment, enhance the ablation effect of HIFU, HIFU and improve the thermal effect of mAbHLA-G/MTX/PLGA nano microbubble in drug release, cooperative target and kill residual tumor cells, treatment inhibited the growth and metastasis of.MAbHLA-G/ ultrasound contrast agent MTX/PLGA combined with high intensity focused ultrasound for clinical implantation and placenta trophoblastic disease has opened up a new road.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R445.1;R737.33

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2 鄧?guó)P蓮;姜振東;鄒建中;李銳;申俊玲;孫立群;;高強(qiáng)度聚焦超聲治療子宮肌瘤臨床并發(fā)癥分析[J];臨床超聲醫(yī)學(xué)雜志;2010年05期

3 ;Complications of high intensity focused ultrasound in patients with recurrent and metastatic abdominal tumors[J];World Journal of Gastroenterology;2007年19期

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