【摘要】：第一部分?jǐn)y氧載紫杉醇脂質(zhì)微泡的制備及性質(zhì)檢測(cè) 目的：制備攜氧載紫杉醇脂質(zhì)微泡，檢測(cè)其性質(zhì)，并與普通載紫杉醇微泡比較。 方法：采用機(jī)械振蕩法制備攜氧載紫杉醇脂質(zhì)微泡和普通載紫杉醇脂質(zhì)微泡，測(cè)定兩種微泡的粒徑、粒度分布、電位、包封率、載藥量、釋氧能力及穩(wěn)定性。 結(jié)果：攜氧載紫杉醇脂質(zhì)微泡和普通載紫杉醇脂質(zhì)微泡的平均粒徑分別為(1688.70±107.00)、(2159±144.70)nm，Zeta電位分別為-(10.32±0.31)、-(17.58±0.25)mv，包封率分別為（97.71±1.13）%、(98.63±0.94)%，載藥量分別為（22.70±0.82）%、（26.28±0.59）%，攜氧載紫杉醇脂質(zhì)微泡在超聲下具有釋氧能力。兩種微泡于4℃保存2周后微泡數(shù)量無(wú)明顯減少，形態(tài)無(wú)明顯改變，但有聚集現(xiàn)象。 結(jié)論：成功制備了攜氧載紫杉醇微泡，粒度分布較均勻，包封率高，超聲介導(dǎo)下具有較好的釋氧能力。 第二部分卵巢癌乏氧耐藥模型細(xì)胞株的建立 目的：構(gòu)建乏氧耐藥卵巢癌SKOV3細(xì)胞模型，為下一步耐藥逆轉(zhuǎn)做準(zhǔn)備。 方法：體外培養(yǎng)卵巢癌SKOV3細(xì)胞，加入不同濃度CoCl2培養(yǎng)液作用12、24、48、72h，采用MTT法檢測(cè)其增殖活性及對(duì)紫杉醇的耐藥倍數(shù)。 結(jié)果：在CoCl2濃度小于150μmol/L時(shí)，對(duì)SKOV3細(xì)胞生長(zhǎng)均無(wú)明顯抑制作用，而CoCl2濃度大于200μmol/L時(shí)，出現(xiàn)明顯的細(xì)胞生長(zhǎng)抑制現(xiàn)象，且其抑制作用與CoCl2呈劑量-時(shí)間依賴關(guān)系（F=2802.394，，P＜0.05）。不同濃度CoCl2處理SKOV324h，隨著CoCl2濃度增加，對(duì)紫杉醇的耐藥倍數(shù)增加（P＜0.05）。 結(jié)論：CoCl2化學(xué)誘導(dǎo)法可成功構(gòu)建卵巢癌SKOV3細(xì)胞的乏氧耐藥模型，150μmol/L CoCl2作用24h為合適的構(gòu)建乏氧耐藥SKOV3細(xì)胞模型的條件。 第三部分超聲介導(dǎo)攜氧載紫杉醇脂質(zhì)微泡對(duì)乏氧卵巢癌細(xì)胞SKOV3化療增敏的作用研究 目的：探討超聲介導(dǎo)攜氧載紫杉醇脂質(zhì)微泡對(duì)乏氧卵巢癌SKOV3細(xì)胞化療增敏作用及機(jī)制。 方法：采用150μmol/L CoCl2乏氧下的對(duì)數(shù)生長(zhǎng)期SKOV3細(xì)胞，隨機(jī)分成7組：（a）PBS組（對(duì)照組）、（b）紫杉醇藥物組（PTX組）、（c）普通載紫杉醇脂質(zhì)微泡組（PLMBs組）、（d）攜氧載紫杉醇脂質(zhì)微泡組（OPLMBs組）、（e）紫杉醇藥物+超聲組（PTX+US組）、（f）普通紫杉醇脂質(zhì)微泡+超聲組（PLMBs+US組）和（g）攜氧載紫杉醇脂質(zhì)微泡+超聲組（OPLMBs+US組），分別給予相應(yīng)處理，24h后光學(xué)顯微鏡觀察各組細(xì)胞形態(tài)， MTT法檢測(cè)各組細(xì)胞的增殖抑制率，Annexin V-FITC/PI雙染法檢測(cè)凋亡情況， PCR檢測(cè)乏氧誘導(dǎo)因子HIF-1αmRNA和多藥耐藥基因MDR-1的表達(dá)，Western blot檢測(cè)HIF-1α蛋白和P糖蛋白（P-gp）的表達(dá)情況。 結(jié)果：各處理因素作用24h后，對(duì)照組、PLMBs組、OPLMBs組細(xì)胞貼壁良好，無(wú)明顯變化。PTX組見(jiàn)少許圓形透亮細(xì)胞，PTX+US組可見(jiàn)較多圓形透亮的死細(xì)胞，PLMBs+US組及OPLMBs+US組可見(jiàn)大量圓形透亮的死細(xì)胞，其中以O(shè)PLMBs+US組更為明顯。OPLMBs+US組細(xì)胞增殖抑制率及凋亡率分別為（52.80±2.75）%和（32.05±0.34)%，明顯高于其他處理組（P＜0.05）。PCR及Westernblot檢測(cè)結(jié)果顯示，OPLMBs+US組HIF-1α及多藥耐藥基因及蛋白表達(dá)明顯下降，與其他各組相比，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論：超聲介導(dǎo)攜氧載紫杉醇脂質(zhì)微泡能明顯增加紫杉醇對(duì)乏氧耐紫杉醇SKOV3細(xì)胞（SKOV3/PTXR）的增殖抑制和凋亡誘導(dǎo)作用。其機(jī)制可能與下調(diào)HIF-1α與MDR-1基因的表達(dá)，進(jìn)而抑制其編碼的HIF-1α蛋白及P-gp表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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