| 1. The preparation and testing of Docetaxel loaded CD105 labelled lipid microbubbles.Objective: To preparation a CD105 labelled and docetaxel loaded lipid microbubble.Methods: 1. The Docetaxel Loaded Lipid Microbubble(DLLM) preparation: using modified thin-film hydration method. 2. The Docetaxel loaded Lipid Microbubbles(DLLM) and CD105 were linked by biotin-streptavidin system to obtain CD105 targeted and docetaxel loaded lipid microbubbles(CD105-DLLM). 3. The characteristics of CD105 labeled and docetaxel loaded lipid microbubbles: The shape of microbubbles was observed under electron microscope. The diameter of microbubbles was measured by laser particle size analyzer. 4. The encapsulation rate and drug loading rate of CD105-DLLM was examined by reversed-phase High Performance Liquid Chromatography(HPLC) method. 5. In vitro drug release test was also determined by HPLC too. 6. A preliminary study stability of CD105-DLLM had tested the conditions of 4℃ and 25℃ on 1d, 3d, 5d,10d.Results: 1. The scanning electron microscope(SEM) showed CD105-DLLM were in round shape, good dispersion, less obvious adhesion, uniform particle size distribution. The average particle size was 3220±1570 nm. 2. The HPLC measured encapsulation rate was 80-86.5%, drug loading rate was 18.5-23%. 3. Stability experiment results showed 4℃ for an optimum temperature, microbubble should be fresh prepared before use. 4. The drug release test showed CD105-DLLM would release drug within 48 h by ultrasound destruction, then the drug would soon reach a release rate of 100%.Conclusion: In this part of the experiment, we obtained CD105 labelled and Docetaxel loaded lipid microbubbles with good stability, satisfied encapsulation rate and drug loading capacity. Which also could quick release under ultrasound destruction.2. Targeting ability and proliferation inhibition test on human liver cancer cells MHCC–H in CD105-DLLM+UTMD group.Objective: To observe CD105 labeled docetaxel loaded liposomes microbubble(CD105-DLLM+ UTMD) target ability on human liver cancer cells MHCC- H in vitro and in vivo experiments. Through in vitro(cells) and in vivo(animals) experiment, CD105-DLLM+ UTMD method had proliferation inhibition effect human liver cancer cell MHCC- H.Methods: In vitro cultivation MHCC-H liver cancer cell lines. Cell culture conditions: 37 ℃ and 5% CO2, saturated humidity, DMEM containing 10% fetal bovine serum-HG training day in liquid, 90% fusion in batches. In cell growth state stability experiment, divided into four groups Control, DOC, DLLM, CD105-DLLM+ UTMD. To determine as following: 1. Test the targeting ability of CD105-DLLM in vitro experiment: using Cy3-PEG-Biotin labeling with CD105-DLLM, which can make the microbubble dye with red fluorescence. Selection of logarithmic phase MHCC-H cells, after that, we put it in 6 orifice to grow after dyeing, and observed under laser confocal microscope(LSCM) IPP7.0 analysis software for analysis. 2. Test the cell vitality, proliferation inhibition in vitro experiment. CCK-8 method was used to detect different processing methods on human liver cancer cell MHCC-H proliferation inhibition, including:(1) Through theCCK 8 method determination of different processing methods on MHCC-H the vitality of four groups.(2) Determination of different processing methods on MHCC-H liver cancer cell proliferation. 3. Target experiment in tumor-burdened nude mice in vivo: To establish a subcutaneous xenograft model of human liver cancer cell in nude mice. Though the mice tail vein injection of Cy-3 in two groups: CD105-DLLM after UTMD and Cy-3 marking the DLLM. Put nude mice after anesthesia in small animals living imaging system, detection with Cy-3 dyes.(2) To observe the survival time influence on different treatment for a tumor-burdened nude mice.(3) To observe tumor growth volume in different groups on tumor-burdened nude mice via B-Mode ultrasonic imaging and small animal in vivo imaging system.Results: 1. In vitro cell vitality results show CD105-DLLM+ UTMD group after 4h the cell vitality of inhibitory effect was significantly higher than other groups(P < 0.01). 2. The cell proliferation inhibition experiment results show that the CD105-DLLM+ UTMD inhibition of cell proliferation in various time points were significantly different than other groups(P < 0.01). 3. In immunofluorescence experiments, CD105-DLLM in vitro was good targeting property. 4. A tumor-burdened nude mice in vivo experiment:(1) HE staining and shown MHCC-H a tumor-burdened nude mice model is set up;(2) Animals find target determination results: after the small animals living fluorescence imaging findings: CD105-DLLM group can be detected in the tumor site of red fluorescence, and the Control group was unable to detected, which confirmed CD105-DLLM also has a good targeting ability in vivo.(3) After that, we compared the different survival curve between two groups of tumor-burdened nude mice. CD105-DLLM+UTMD group of nude mice than in the control group, significantly prolong survival time.(4) In ultrasonic two-dimensional graphic system, CD105-DLLM+UTMD nude mice tumor volume were reduced obviously.Conclusion: 1. CD105-DLLM+UTMD reduced cell vitality of MHCC-H. 2. CD105-DLLM+UTMD group in MHCC-H cells were proliferation inhibition. 3. CD105-DLLM+UTMD had targeting ability in vitro and in vivo experiments.3. The mechanism of inhibition and apoptosis-promoting effect of CD105-DLLM with UTMD in human liver cancer cell MHCC-H.Objective: To discuss the mechanism of inhibition and apoptosis-promoting effect of CD105-DLLM with UTMD in MHCC-H. By test of cell cycle, cell apoptosis and apoptosis related proteins.Methods: The logarithmic phase MHCC-H cells divided into four groups: unloaded microbubble(CON), docetaxel alone(DOC), docetaxel loaded lipid microbubble(DLLM), CD105 loaded and docetaxel loaded lipid microbubbles with ultrasound destruction(CD105-DLLM+UTMD). Various test were as follow: 1.Flow cytometry is used to test the cell cycle and early apoptosis. 2. MHCC-H cell apoptosis rate were also observed with TUNEL detection, data analyzed by Cell Quest software. 3. Use western blotting to evaluate the apoptosis related proteins Bax, Bcl 2, Cyto-c, P53, and Caspase 3 expression. 4. To observe Caspase-3 activation and translocation in living cells by laser confocal microscopy.Results: 1. Flow cytometry tests showed CD105-DLLM+UTMD group inhibited the liver cancer cell from proliferation by significantly inhibit the split phase cell proportion. At the same time CD105-DLLM+UTMD significantly increased the rate of apoptosis MHCC-H. 2. In situ TUNEL apoptosis detection also shows that CD105-DLLM+UTMD could caused liver cancer cell apoptosis. 3. Western-blotting showed apoptosis related protein Cyt-c, Bax/Bcl-2 and of P53 in CD105-DLLM+UTMD group were significantly different than other groups(P < 0.01). We also observed content of active Caspase-3 was more than tenfold increased in CD105-DLLM+UTMD group. 4. By LSCM observation of living cells, the content of Caspase-3 in five hours can be seen in the cell nucleus. But there were no distinct changes in the cytoplasm and cell morphology. After five hours cytoplasm gradually appear Caspase-3 activation and apoptotic bodies. At 10 hours after most activated Caspase-3 were transferred to the membrane surface.Conclusion: CD105 DLLM+UTMD inhibited the liver cancer cell from proliferation by reduce the split phase cell proportion. Also it promoted apoptosis by increasing the proteinCyt-c, Bax/Bcl-2, and of P53 expression and caused Caspase-3 activation and translocation.Summary: This experiment prepared a CD105 antibody labelled docetaxel lipid microbubble. This microbubble has good stability, the encapsulation rate and drug loadings were higher, also can quick release by UTMD mediated. Through cell vitality, determination of the level of cell proliferation found: CD105-DLLM+UTMD significantly lower UTMD MHCC-H cell vitality, at the same time of MHCC-H cells with proliferation inhibition; Through the target test both in vitro and in vivo, we verified that UTMD mediated CD105-DLLM induced apoptosis in human cancer cell MHCC-H. UTMD mediated CD105-DLLM to induce MHCC-H liver cell apoptosis, Cyto-c and Caspase 3 plays an important role in the process of apoptosis. Western blotting test shown in UTMD mediated CD105-DLLM to induce MHCC-H liver cell apoptosis, Cyto-c and Caspase-3 plays an important role in the process of cell apoptosis; UTMD mediated CD105-DLLM caused Caspase3 activation and translocation. |
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