载MTX靶向纳米微泡超声造影剂增效HIFU消融对滋养细胞作用的实验研究

发布时间：2018-04-03 22:18

本文选题：甲氨蝶呤　切入点：靶向纳米微泡　出处：《重庆医科大学》2014年博士论文

【摘要】：第一部分载MTX靶向纳米微泡的制备和性能检测目的制备一种包载甲氨蝶呤（MTX）的聚乳酸-羟基乙酸（PLGA）靶向（人类白细胞抗原-G单克隆抗体，， mAbHLA-G）纳米微泡（mAbHLA-G/MTX/PLGA），检测其物理和声学性质。方法采用双乳化法，碳二亚胺连接法和真空冷冻干燥技术制备mAbHLA-G/MTX/PLGA，观察其表形结构，测量粒径电位，包封率和载药量；高强度聚焦超声（HIFU）和普通超声的促发后检测mAbHLA-G/MTX/PLGA纳米微泡中药物的释放情况；并在不同的浓度梯度（200mg/ml，100mg/ml，50mg/ml，25mg/ml，10mg/ml）和时间梯度（0h，2h，4h，8h，16h，24h，48h）观察其超声显影效果。结果mAbHLA-G/MTX/PLGA纳米微泡溶于双蒸水后呈淡黄色混悬液，光镜及扫描电镜观察其形态规则，呈球形，大小较均匀，表面有孔稍欠光滑，分散度好。透射电镜负染后可见MTX均匀分布在纳米微泡中心。马尔文激光测量仪检测出mAbHLA-G/MTX/PLGA平均粒径为（477.6±119.7）nm，分散指数0.171，Zeta电位为（-5.62±5.36）mV。高效液相色谱法检测其包封率为44.11±1.27%，载药量为4.41±0.13%(w/w)。HIFU激发后3h mAbHLA-G/MTX/PLGA纳米微泡药物累计释放率达到50%，72h后药物累计释放率超过80%；而普通超声促发后72h mAbHLA-G/MTX/PLGA纳米微泡药物累计释放率仅为47.8%。体外超声显影实验显示mAbHLA-G/MTX/PLGA纳米微泡能产生较强的超声回声信号，并随着浓度的变化而变化，且制备的mAbHLA-G/MTX/PLGA超声造影剂性能稳定，在制备完成后24h内各时间点超声显像效果没有统计学差异。结论成功制备了载MTX靶向纳米微泡（mAbHLA-G/MTX/PLGANBs）,其形态规则，大小均匀，分散好。与普通超声相比，HIFU更有效的促进了纳米微泡内药物的释放。mAbHLA-G/MTX/PLGA超声造影剂在体外能增强超声成像，且稳定性好，具有良好的声学性能，是一种具有良好应用前景的多功能超声造影剂。第二部分载MTX靶向纳米微泡联合高强度聚焦超声对滋养细胞作用的体外实验研究目的检测人绒毛膜癌细胞株JEG-3细胞HLA-G蛋白的表达和定位，探讨mAbHLA-G/MTX/PLGA纳米微泡体外寻靶能力和被细胞吞噬的情况。探讨mAbHLA-G/MTX/PLGA纳米微泡联合高强度聚焦超声靶向破裂对人绒毛膜癌JEG-3细胞株细胞周期，细胞增殖和侵袭，及诱导凋亡和侵袭相关蛋白表达的影响，为体内实验提供依据。方法体外培养人绒毛膜癌JEG-3细胞株，免疫荧光法检查JEG-3细胞HLA-G蛋白的表达和定位。制备DiI标记的mAbHLA-G/MTX/PLGA纳米微泡（DiI-mAbHLA-G/MTX/PLGANBs），与JEG-3细胞共孵育，观察纳米微泡的体外寻靶能力和被细胞吞噬的情况，实验分组：（1）mAbHLA-G/MTX/PLGA NBs组，（2）mAbHLA-G预处理后+等量的mAbHLA-G/MTX/PLGA NBs组，（3）MTX/PLGA NBs组，共孵育后DAPI染核，采用激光共聚焦显微镜观察纳米微泡的分布情况。 mAbHLA-G/MTX/PLGA NBs联合HIFU体外治疗实验分为12个组：（1）PBS组，（2）空白PLGA NBs组，（3）MTX/PLGANBs组，（4）MTX组，（5）mAbHLA-G/PLGA NBs组，（6）mAbHLA-G/MTX/PLGA NBs组，（7）HIFU+PBS组,（8）HIFU+空白PLGA NBs组,（9）HIFU+MTX/PLGANBs组（,10）HIFU+MTX组，（11）HIFU+mAbHLA-G/PLGANBs组，（12）HIFU+mAbHLA-G/MTX/PLGA NBs组。通过流式细胞仪检测各组细胞凋亡和细胞周期的变化，Western blotting检测凋亡和侵袭相关蛋白Bax，Bcl-2，Caspase3，MMP2，TIMP-2的表达和荧光定量PCR从基因水平检测Caspase3，MMP2的表达情况。结果HLA-G在JEG-3细胞中呈高表达，主要定位在细胞膜表面。体外靶向性验证实验表明，mAbHLA-G/MTX/PLGA纳米微泡能与高表达HLA-G蛋白的JEG-3细胞特异性结合，增加纳米微泡在细胞的聚集和被细胞吞噬。在体外治疗实验中，与其他组相比， HIFU+mAbHLA-G/MTX/PLGA NBs组JEG-3细胞凋亡率最高，细胞明显阻滞在S期。Bax和Caspase3的表达在HIFU+mAbHLA-G/MTX/PLGA NBs组明显高于其他组， Bcl-2蛋白表达低于其他组。 HIFU+mAbHLA-G/MTX/PLGA NBs组细胞侵袭能力明显减弱，MMP2的表达在所有组中最低，TIMP-2的表达明显高于其余各组。结论mAbHLA-G/MTX/PLGA纳米微泡靶向性好，能够改变靶区域周围声环境，增强超声的空化效应和机械效应，同时超声又能促进纳米微泡中化疗药物的释放。HIFU联合mAbHLA-G/MTX/PLGANBs很大程度的抑制了JEC-3细胞的增殖和侵袭，其作用机制可能是通过影响凋亡和侵袭相关蛋白的表达来完成的。第三部分载MTX靶向纳米微泡增效HIFU消融对滋养细胞作用的体内实验研究目的验证HLA-G在JEG-3细胞裸鼠皮下移植瘤模型瘤组织内的表达和定位。评价mAbHLA-G/MTX/PLGA纳米微泡在体内的超声显像效果和体内靶向性。研究mAbHLA-G/MTX/PLGA纳米微泡协同HIFU消融技术对JEG-3细胞裸鼠皮下移植瘤模型的治疗效果和远期生存情况的分析。方法143只BALB/C雌性裸鼠，JEG-3细胞以1~2×106浓度皮下注射于裸鼠背部，构建裸鼠皮下移植瘤模型，肿瘤直径约0.8~1.0cm时可用于后续实验。取瘤组织块石蜡包埋切片，采用免疫组化法检测HLA-G的表达和定位。经尾静脉注射mAbHLA-G/MTX/PLGA纳米微泡，在注射前和注射后（0h和24h）分别观察瘤组织区域的超声显像情况。经尾静脉注射DiI-mAbHLA-G/MTX/PLGA纳米微泡，采用小动物活体荧光成像技术观察不同时间点（3h，24h，48h）靶向载药纳米微泡在荷瘤鼠体内的分布情况，并在不同时间点取瘤组织块冰冻切片观察纳米微泡的分布。体内评价mAbHLA-G/MTX/PLGA纳米微泡增效HIFU消融的效果，120只荷瘤鼠随机分为12组（n=10）：（1）NS组，（2）空白PLGANBs组，（3）MTX/PLGA NBs组，（4）MTX组，（5）mAbHLA-G/PLGANBs组，（6）mAbHLA-G/MTX/PLGA NBs组，（7）HIFU+NS组,（8）HIFU+空白PLGA NBs组,（9）HIFU+MTX/PLGA NBs组（,10）HIFU+MTX组，（11） HIFU+mAbHLA-G/PLGA NBs组，（12） HIFU+mAbHLA-G/MTX/PLGA NBs组。HIFU消融后立即观察各组（7-12组）中瘤组织的灰度变化和取瘤组织测量消融体积。12组在处理完成后24h取瘤组织做HE染色，TUNEL检测凋亡，PCNA评价肿瘤细胞增殖抑制情况。每组余下5只荷瘤鼠用于观察肿瘤的生长情况和进行生存分析。结果HLA-G在JEG-3细胞皮下移植瘤组织中高表达，定位于细胞膜和细胞浆中。mAbHLA-G/MTX/PLGA超声造影剂在体内能稳定显影，并能特异性的到达靶组织，提高靶区域纳米微泡的聚集浓度和停留时间，增加化疗药物在靶区域的释放浓度。mAbHLA-G/MTX/PLGA纳米微泡能明显增强HIFU消融，HIFU+mAbHLA-G/MTX/PLGA NBs组消融后灰度变化显著，瘤组织消融体积明显大于其他组。 HIFU联合mAbHLA-G/MTX/PLGA NBs有效抑制了肿瘤细胞的增殖，促进细胞凋亡，延长荷瘤鼠的生存周期（中位生存期=58天）。结论mAbHLA-G/MTX/PLGA通过改变靶区域声环境，增强HIFU的消融效果，同时HIFU的热效应提高了mAbHLA-G/MTX/PLGA纳米微泡中化疗药物的释放，协同靶向杀灭了残余瘤细胞，抑制了肿瘤的生长和转移。mAbHLA-G/MTX/PLGA超声造影剂联合高强度聚焦超声为临床胎盘植入和滋养细胞疾病的治疗开辟了一条全新的道路。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R445.1;R737.33

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