超声介导携氧载紫杉醇脂质微泡对乏氧卵巢癌细胞化疗增敏作用研究

发布时间：2018-09-10 13:25

【摘要】：第一部分携氧载紫杉醇脂质微泡的制备及性质检测目的：制备携氧载紫杉醇脂质微泡，检测其性质，并与普通载紫杉醇微泡比较。方法：采用机械振荡法制备携氧载紫杉醇脂质微泡和普通载紫杉醇脂质微泡，测定两种微泡的粒径、粒度分布、电位、包封率、载药量、释氧能力及稳定性。结果：携氧载紫杉醇脂质微泡和普通载紫杉醇脂质微泡的平均粒径分别为(1688.70±107.00)、(2159±144.70)nm，Zeta电位分别为-(10.32±0.31)、-(17.58±0.25)mv，包封率分别为（97.71±1.13）%、(98.63±0.94)%，载药量分别为（22.70±0.82）%、（26.28±0.59）%，携氧载紫杉醇脂质微泡在超声下具有释氧能力。两种微泡于4℃保存2周后微泡数量无明显减少，形态无明显改变，但有聚集现象。结论：成功制备了携氧载紫杉醇微泡，粒度分布较均匀，包封率高，超声介导下具有较好的释氧能力。第二部分卵巢癌乏氧耐药模型细胞株的建立目的：构建乏氧耐药卵巢癌SKOV3细胞模型，为下一步耐药逆转做准备。方法：体外培养卵巢癌SKOV3细胞，加入不同浓度CoCl2培养液作用12、24、48、72h，采用MTT法检测其增殖活性及对紫杉醇的耐药倍数。结果：在CoCl2浓度小于150μmol/L时，对SKOV3细胞生长均无明显抑制作用，而CoCl2浓度大于200μmol/L时，出现明显的细胞生长抑制现象，且其抑制作用与CoCl2呈剂量-时间依赖关系（F=2802.394，，P＜0.05）。不同浓度CoCl2处理SKOV324h，随着CoCl2浓度增加，对紫杉醇的耐药倍数增加（P＜0.05）。结论：CoCl2化学诱导法可成功构建卵巢癌SKOV3细胞的乏氧耐药模型，150μmol/L CoCl2作用24h为合适的构建乏氧耐药SKOV3细胞模型的条件。第三部分超声介导携氧载紫杉醇脂质微泡对乏氧卵巢癌细胞SKOV3化疗增敏的作用研究目的：探讨超声介导携氧载紫杉醇脂质微泡对乏氧卵巢癌SKOV3细胞化疗增敏作用及机制。方法：采用150μmol/L CoCl2乏氧下的对数生长期SKOV3细胞，随机分成7组：（a）PBS组（对照组）、（b）紫杉醇药物组（PTX组）、（c）普通载紫杉醇脂质微泡组（PLMBs组）、（d）携氧载紫杉醇脂质微泡组（OPLMBs组）、（e）紫杉醇药物+超声组（PTX+US组）、（f）普通紫杉醇脂质微泡+超声组（PLMBs+US组）和（g）携氧载紫杉醇脂质微泡+超声组（OPLMBs+US组），分别给予相应处理，24h后光学显微镜观察各组细胞形态， MTT法检测各组细胞的增殖抑制率，Annexin V-FITC/PI双染法检测凋亡情况， PCR检测乏氧诱导因子HIF-1αmRNA和多药耐药基因MDR-1的表达，Western blot检测HIF-1α蛋白和P糖蛋白（P-gp）的表达情况。结果：各处理因素作用24h后，对照组、PLMBs组、OPLMBs组细胞贴壁良好，无明显变化。PTX组见少许圆形透亮细胞，PTX+US组可见较多圆形透亮的死细胞，PLMBs+US组及OPLMBs+US组可见大量圆形透亮的死细胞，其中以OPLMBs+US组更为明显。OPLMBs+US组细胞增殖抑制率及凋亡率分别为（52.80±2.75）%和（32.05±0.34)%，明显高于其他处理组（P＜0.05）。PCR及Westernblot检测结果显示，OPLMBs+US组HIF-1α及多药耐药基因及蛋白表达明显下降，与其他各组相比，差异有统计学意义（P＜0.05）。结论：超声介导携氧载紫杉醇脂质微泡能明显增加紫杉醇对乏氧耐紫杉醇SKOV3细胞（SKOV3/PTXR）的增殖抑制和凋亡诱导作用。其机制可能与下调HIF-1α与MDR-1基因的表达，进而抑制其编码的HIF-1α蛋白及P-gp表达有关。
[Abstract]:Part 1 Preparation and characterization of paclitaxel lipid microbubbles loaded with oxygen
Objective: to prepare paclitaxel loaded lipid microbubbles carrying oxygen and detect its properties and compare with paclitaxel loaded microbubbles.
METHODS: Paclitaxel-loaded lipid microbubbles and paclitaxel-loaded lipid microbubbles were prepared by mechanical oscillation method. The particle size, particle size distribution, potential, entrapment efficiency, drug loading, oxygen-releasing capacity and stability of the two microbubbles were determined.
RESULTS: The average diameters of oxygen-loaded and conventional paclitaxel-loaded lipid microbubbles were (1688.70 65507 The two kinds of microbubbles have no obvious decrease in the number of microbubbles and no obvious change in morphology, but there is aggregation phenomenon.
CONCLUSION: Oxygen-carrying paclitaxel microbubbles were successfully prepared with uniform particle size distribution and high encapsulation efficiency.
The second part is the establishment of ovarian cancer cell line with hypoxia resistance.
Objective: to construct hypoxia resistant ovarian cancer SKOV3 cell model and prepare for the next step of drug resistance reversal.
METHODS: SKOV3 cells were cultured in vitro and treated with different concentrations of CoCl2 for 12,24,48,72 hours. The proliferation activity and drug resistance to paclitaxel were detected by MTT assay.
Results: When the concentration of CoCl2 was less than 150 micromol/L, the growth of SKOV3 cells was not inhibited obviously, but when the concentration of CoCl2 was higher than 200 micromol/L, the growth of SKOV3 cells was inhibited obviously, and the inhibiting effect was dose-time dependent on CoCl2 (F = 2802.394, P < 0.05). Paclitaxel resistance increased (P < 0.05).
CONCLUSION: The hypoxic resistance model of ovarian cancer SKOV3 cells can be successfully constructed by CoCl2 chemical induction method. 150 micromol/L CoCl2 for 24 hours is the suitable condition for constructing the hypoxic resistance SKOV3 cell model.
Part 3 Ultrasound-mediated Sensitization of Oxygen-loaded Paclitaxel Lipid Microbubbles to Hypoxic Ovarian Cancer Cell Line SKOV3
Objective: To investigate the effect and mechanism of ultrasound-mediated oxygen-loaded paclitaxel lipid microbubbles on hypoxic ovarian cancer SKOV3 cells.
METHODS: SKOV3 cells were randomly divided into seven groups: (a) PBS group (control group), (b) paclitaxel-loaded lipid microbubbles group (PTX group), (c) paclitaxel-loaded lipid microbubbles group (PLMBs group), (d) oxygen-loaded paclitaxel lipid microbubbles group (OPLMBs group), (e) paclitaxel drug + ultrasound group (PTX + US group), (f) paclitaxel lipid. Microbubbles + ultrasound group (PLMBs + US group) and oxygen-loaded paclitaxel lipid microbubbles + ultrasound group (OPLMBs + US group) were given corresponding treatment respectively. Cell morphology of each group was observed by optical microscope 24 hours later. Cell proliferation inhibition rate was detected by MTT assay, apoptosis was detected by Annexin V-FITC/PI double staining, HIF-1 alpha mRNA and multidrug detection by PCR. The expression of drug resistance gene MDR-1 was detected by Western blot, and the expression of HIF-1 alpha protein and P glycoprotein (P-gp) was detected.
Results: After 24 hours of treatment, the cells adhered well in the control group, PLMBs group and OPLMBs group, and there was no obvious change. There were a few round bright cells in the PTX group and many round bright dead cells in the PTX+US group. A large number of round bright dead cells were observed in the PLMBs+US group and OPLMBs+US group, especially in the OPLMBs+US group. The inhibiting rate of reproduction and the rate of apoptosis were (52.80+2.75)% and (32.05+0.34)% respectively, which were significantly higher than those in other treatment groups (P < 0.05). The results of PCR and Western blot showed that the expression of HIF-1a and multidrug resistance gene and protein in OPLMBs+US group were significantly lower than those in other groups (P < 0.05).
Conclusion: Ultrasound-mediated paclitaxel-loaded lipid microbubbles can significantly increase the proliferation inhibition and apoptosis induction of paclitaxel-loaded SKOV3 cells (SKOV3/PTXR). The mechanism may be related to the down-regulation of the expression of HIV-1a and MDR-1 genes, thereby inhibiting the expression of HIF-1a protein and P-gp encoded by paclitaxel.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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