携氧载ICG纳米粒协同光声动力对卵巢癌SKOV3细胞增殖抑制的实验研究
发布时间：2018-08-05 12:15
【摘要】：第一部分携氧载ICG纳米粒制备及特性检测目的:制备携氧液态氟碳载吲哚菁绿PLGA纳米粒并进行其特性检测方法:采用双乳化-溶剂挥发法(w/o/w)制备携氧液态氟碳载吲哚菁绿PLGA纳米粒(OPI-NPs)、载携氧液态氟碳PLGA纳米粒(OP-NPs)和载吲哚菁绿PLGA纳米粒(I-NPs),光镜下观察形态、Malvern粒径测定仪测定OPI-NPs的粒径大小分布,UV-Vis分光光度计测定ICG的包封率及载药量,光声成像仪观察其光声显影能力,并与OP-NPs+free ICG及I-NPs的光声显影能力进行比较。结果:OPI-NPs为球形,平均粒径大小(275.24±7.28)nm,大小分布范围在80 nm—1000 nm之间,ICG的包封率约55%。OP-NPs+free ICG、I-NPs及OPI-NPs的光声显影强度分别为0.336±0.022,0.744±0.029及1.112±0.072。OPI-NPs及I-NPs的光声显影强度明显强于OP-NPs+free ICG,而OPI-NPs的光声显影强度明显强于I-NPs,差异具有统计学意义(P0.05)。结论:成功制备OPI-NPs纳米粒,粒径相对比较均匀,且具有良好的光声显影能力。第二部分纳米粒激发的适宜激光、超声参数及纳米粒浓度的筛选目的:筛选出可以激发纳米粒相变及其破裂的适宜激光及超声参数及用于光声动力治疗的纳米粒ICG浓度。方法:1、激光参数筛选:将制备好的纳米粒稀释20倍,按照激光强度及辐照时间分组如下:0.5W/cm2,30s、0.5W/cm2,60s、1.0W/cm2,30s,1.0 W/cm2,60s、1.5 W/cm2,30s、1.5 W/cm2,60s、2.0 W/cm2,30s和2.0 W/cm2,60s。分别给予上述不同处理条件后,光镜下观察微球形态变化。2、超声参数筛选:将制备好的纳米粒稀释20倍,激光(2.0W/cm2,30s)辐照后,根据超声强度及辐照时间分组如下:0.44W/cm2,30s、0.44 W/cm2,60s、0.66 W/cm2,30s、0.66 W/cm2,60s、0.88 W/cm2,30s、0.88 W/cm2,60s、1.10 W/cm2,30s和1.10 W/cm2,60s。分别给予相应处理,处理后即刻光镜观察微球破碎情况。3、纳米粒ICG浓度筛选:取对数生长期的SKOV3细胞,根据纳米粒中ICG的浓度分组如下:对照组、0.5μg/ml组、1.0μg/ml组、2.0μg/ml组、4.0μg/ml组和8.0μg/ml组,共6组。分别将纳米粒与细胞共培养24h后,CCK-8检测细胞存活率。4、适宜激光、超声作用下对微球相变及击碎情况观察:,将制备好的纳米粒稀释20倍,分别取等量的纳米粒悬浮液放入凝胶孔中,分别在激光(2.0W/cm2,30s)辐照前后、激光(2.0W/cm2,30s)加超声(0.88W/cm2)辐照30秒及60秒后,诊断超声观察凝胶管回声变化、光镜观察微球形态变化、粒径测定仪测定微球粒径大小分布。5、适宜激光、超声作用下对细胞增殖的影响:取对数生长期的SKOV3细胞,分组如下:对照组、激光组、超声组及激光联合超声组。分别给予相应处理。应用CCK-8检测细胞存活率,观察各组对细胞增殖抑制情况。结果:1、当激光辐照能量达到1.5W/cm2时纳米粒发生明显相变,伴随着辐照时间的延长相变数目进一步增加,当激光能量达到2.0W/cm2时纳米粒相变数目明显增多,但随着时间的延长,相变数目无明显变化。2、当超声辐照能量为0.44W/cm2和0.66W/cm2时相变的纳米粒未见明显变化,伴随着辐照时间的延长仍未见明显的变化;当超声辐照能量达到0.88W/cm2时相变的纳米粒数目明显减少,伴随着辐照能量增加到1.10W/cm2时相变的纳米粒数目减少不明显,但随着辐照时间的延长至60s时,相变的纳米粒数目明显减少。3、被包裹的ICG浓度0.5μg/ml至4μg/ml之间对细胞无明显抑制作用,被包裹的ICG浓度达8μg/ml时细胞增殖明显受到抑制。4、激光激发前后的超声显影明显增强,光镜下观察纳米粒粒径增大,纳米粒平均粒径分别为275.24nm和1533.65nm;激光辐照后再进行超声辐照30s和60s较激光激发后的超声显影明显减弱,纳米粒平均粒径分别为1060.56nm和771.38 nm。5、激光(2.0W/cm2,30s)及超声(0.88W/cm2,60s)对细胞无明显抑制作用。结论:激光(2.0W/cm2,30s)可有效激发纳米粒发生相变,超声(0.88W/cm2,60s)可有效使相变的纳米粒破碎,且此能量下的超声及激光无论单独或联合作用,对SKOV3细胞生长均无影响。纳米粒ICG浓度≤4μg/ml对SKOV3细胞生长无抑制作用,故选择激光(2.0W/cm2,30s)、超声(0.88W/cm2,60s)及纳米粒ICG浓度4μg/ml作为本实验适宜参数用于后续研究中。第三部分携氧载ICG纳米粒协同光声动力对卵巢癌SKOV3细胞增殖抑制及其机制的实验研究目的:观察光动力和声动力联合携氧纳米粒对卵巢癌SKOV3细胞的增殖抑制及其机制。方法:1、不同处理方式对细胞生长抑制情况检测:将对数生长期的SKOV3细胞随机分6组:ICG+L+U组、I-NPs+L+U组、PI-NPs+L+U组、OPI-NPs+L+U组、OPI-NPs+L组和OPI-NPs+U组。分别进行相应处理,24小时后应用CCK-8检测各组细胞存活率;2、不同的纳米粒ICG浓度对细胞生长抑制情况检测:将不同ICG浓度(0.25μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和4.0μg/ml)的纳米粒加入到对数生长期的SKOV3细胞共培养24小时后,分别用激光(2.0W/cm2,30s)及超声(0.88W/cm2,60s)处理,处理后24小时,用CCK-8检测细胞存活率;3、不同方式处理后无细胞体系溶液内活性氧检测:按照ICG终浓度为4μg/ml的各样本加入96孔培养板中,然后各孔中加入浓度为50m M SOSG 0.02ml,按照不同处理方式分组如下:对照组、PBS+L+U组、ICG+L+U组、I-NPs+L+U组、PI-NPs+L+U组、OPI-NPs+L+U组、OPI-NPs+L组和OPI-NPs+U组;处理后20分钟,用酶标仪测各组溶液的荧光强度;4、纳米粒ICG浓度对无细胞体系溶液内活性氧产生情况的影响:将不同ICG浓度(0.25μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和4.0μg/ml)的纳米粒分别加入到96孔板钟,然后再加入浓度为50m M SOSG 0.02ml,用激光(2.0W/cm2,30s)联合超声(0.88W/cm2,60s)处理,处理后20分钟,用酶标仪测溶液的荧光强度;5、不同方式处理后细胞内活性氧检测:将对数生长期的SKOV3细胞分8组:对照组、PBS+L+U组、ICG+L+U组、I-NPs+L+U组、PI-NPs+L+U组、OPI-NPs+L+U组、OPI-NPs+L组和OPI-NPs+U组,分别加入ICG或纳米粒使溶液中的ICG浓度为4μg/ml,与SKOV3细胞共培养24h后,分别加入2μl DCFH-DA(10m M)共培养20分钟,然后用PBS冲洗3次,加入无血清培养液2ml,再予以激光和/或超声进行处理,处理后20分钟分别用流式仪检测活性氧荧光强度及荧光显微镜下观察细胞内活性氧指示剂显影情况。结果:1、PI-NPs+L+U组,OPI-NPs+L+U组和OPI-NPs+L组对细胞的增殖抑制率分别为(30.2±1.291)%,(77.24±0.4634)%和(59.06±2.003)%,明显高于其他各处理组(P0.05);OPI-NPs+L+U组和OPI-NPs+L组对细胞的增殖抑制率明显高于PI-NPs+L+U组,OPI-NPs+L+U组对细胞的增殖抑制率明显高于OPI-NPs+L组,差异均有统计学意义(P0.05)。2、伴随着纳米粒ICG浓度的增大细胞增殖抑制率也随着增加,4μg/ml组达到(72.04±5.273)%,明显高于其他各浓度组,差异均有统计学意义(P0.05)。3、PI-NPs+L+U组,OPI-NPs+L+U组和OPI-NPs+L组无细胞体系溶液中的活性氧指示剂的荧光强度分别为185.3±19.46、440.1±11.47和355.6±31.15明显高于其他各处理组(P0.05);OPI-NPs+L+U组和OPI-NPs+L组的活性氧含量明显高于PI-NPs+L+U组,OPI-NPs+L+U组的活性氧含量明显高于OPI-NPs+L组,差异均有统计学意义(P0.05)。4、伴随着纳米粒浓度增加,细胞外活性氧的产生进一步增加,4μg/ml组达到440.1±11.47,明显高于其他各浓度组,差异均有统计学意义(P0.05)。5、荧光显微镜见PI-NPs+L+U组,OPI-NPs+L+U组和OPI-NPs+L组细胞内均出现明显绿色荧光,,OPI-NPs+L+U组和OPI-NPs+L组的荧光较PI-NPs+L+U组明显;流式仪定量检测细胞内活性氧指示剂荧光强度可见:PI-NPs+L+U组,OPI-NPs+L+U组和OPI-NPs+L组细胞内活性氧指示剂的荧光强度分别为1935±53.14,2939±300.2和2935±295.3,明显高于其他各处理组,OPI-NPs+L+U组和OPI-NPs+L组细胞内活性氧的荧光强度明显高于PI-NPs+L+U组,差异均有统计学意义(P0.05),而OPI-NPs+L+U组细胞内活性氧的荧光强度与OPI-NPs+L组无明显差异(P0.05)。结论:激光联合超声激发携氧纳米粒能够明显抑制卵巢癌SKOV3细胞的增殖,其机制可能与细胞内外活性氧的产生有关。
[Abstract]:The preparation and characterization of oxygen loaded ICG nanoparticles in the first part: preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles and its properties detection methods: the preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles (OPI-NPs), oxygen carrying liquid fluorocarbon PLGA nanoparticles (OP-NPs) and indoles carrying oxygen carrying liquid fluorocarbon indocyanine green (w/o/w) Cyanine green PLGA nanoparticles (I-NPs) were observed under light microscope. The size distribution of OPI-NPs was measured by Malvern particle size analyzer. The encapsulation efficiency and drug loading of ICG were measured by UV-Vis spectrophotometer. The photoacoustic imaging ability of ICG was observed by the photoacoustic imaging instrument. The results were compared with that of OP-NPs+free ICG and I-NPs. The results showed that OPI-NPs was spherical and average. The size of the particle size (275.24 + 7.28) nm, the size distribution range from 80 nm to 1000 nm, the encapsulation efficiency of ICG is about 55%.OP-NPs+free ICG, the photoacoustic intensity of I-NPs and OPI-NPs is 0.336 + 0.022,0.744 + 0.029 and 1.112 + 0.072.OPI-NPs and I-NPs is obviously stronger than that of OP-NPs+free. Better than I-NPs, the difference is statistically significant (P0.05). Conclusion: the successful preparation of OPI-NPs nanoparticles with relatively uniform particle size and good photoacoustic development ability. The suitable laser, ultrasonic parameters and nanoparticle concentration of the second part nanoparticles are screened. The suitable laser can be selected to select the suitable laser to stimulate the phase transition and rupture of nanoparticles. And ultrasonic parameters and the concentration of nanoparticles ICG for photoacoustic power therapy. Method: 1, laser parameters screening: the prepared nanoparticles are diluted 20 times, according to laser intensity and irradiation time as follows: 0.5W/cm2,30s, 0.5W/cm2,60s, 1.0W/cm2,30s, 1 W/cm2,60s, 1.5 W/ cm2,30s, 1.5 W/cm2,60s, 2 W/cm2,30s and 2 W/cm2,60s. respectively given. After the above treatment conditions, the morphology of the microspheres was observed under the light microscope.2, the ultrasonic parameters were screened: the prepared nanoparticles were diluted 20 times. After the laser (2.0W/cm2,30s) irradiation, the ultrasonic intensity and the irradiation time were grouped as follows: 0.44W/cm2,30s, 0.44 W/cm2,60s, 0.66 W/ cm2,30s, 0.66 W/cm2,60s, 0.88 W/cm2,30s, 0.88 W/cm2,60s, 1.10 W/cm2,30s. And 1.10 W/cm2,60s. were treated respectively. After treatment, the microsphere fragmentation was observed by immediate light microscopy (.3) and nanoparticles ICG concentration was screened: the logarithmic growth period SKOV3 cells were selected according to the concentration of ICG in the nanoparticles as follows: the control group, the 0.5 mu g/ml group, the 1 mu g/ml group, the 2 mu g/ml group, the 4 micron g/ml group and 8 micron g/ml group, and 6 groups respectively. The nanoparticles and the group were respectively. After the cell co culture of 24h, CCK-8 was used to detect the cell survival rate.4, suitable for laser. Under ultrasonic action, the microsphere phase transformation and fragmentation were observed: the prepared nanoparticles were diluted 20 times, and the same amount of nanoparticle suspension was put into the gel pores respectively. Before and after the laser (2.0W/cm2,30s) irradiation, the laser (2.0W/cm2,30s) and ultrasonic (0.88W/cm2) irradiated 30 respectively. After second and 60 seconds, the echo changes of gel tube were observed by diagnostic ultrasound. The morphology of microspheres was observed by light microscope. The size distribution of microspheres was measured by particle size measuring instrument.5. The effect of laser and ultrasound on cell proliferation: SKOV3 cells of logarithmic growth period were taken as follows: control group, laser group, ultrasound group and laser combined ultrasound group. The cell survival rate was detected by CCK-8, and the inhibition of cell proliferation was observed. The results were as follows: 1, when the laser irradiation energy reached 1.5W/cm2, the nanoparticles had obvious phase transition, and the number of phase transition was further increased with the prolongation of irradiation time. The number of nanoparticles phase transition was significantly increased when the laser energy reached 2.0W/ cm2, but with the time, the number of phase transition was significantly increased. There is no obvious change in the number of phase changes of.2. When the energy of ultrasound irradiation is 0.44W/cm2 and 0.66W/cm2, the phase transition nanoparticles do not change obviously. There is no obvious change with the prolongation of the irradiation time. When the energy reaches 0.88W/cm2, the number of nanoparticles decreases obviously, with the increase of radiation energy to 1.10W/cm2. The decrease of the number of phase transition nanoparticles was not obvious, but with the prolongation of the irradiation time to 60s, the number of phase transition nanoparticles decreased obviously by.3, and the concentration of the encapsulated ICG between 0.5 and 4 Mu was not obviously inhibited. The proliferation of the encapsulated ICG was 8 mu g/ml and the cell proliferation was obviously inhibited by.4 and the ultrasonic development before and after laser excitation. The size of nanoparticles was increased by light microscopy. The average particle size of nanoparticles was 275.24nm and 1533.65nm, respectively. The ultrasonic development of 30s and 60s after laser irradiation was obviously weakened after laser irradiation. The average particle size of nanoparticles was 1060.56nm and 771.38 nm.5 respectively, and the laser (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s) were used to the cells. Conclusion: laser (2.0W/cm2,30s) can effectively stimulate the phase transition of nanoparticles. Ultrasound (0.88W/cm2,60s) can effectively break the phase of phase transition, and the ultrasound and laser under this energy have no effect on the growth of SKOV3 cells either alone or in combination. The ICG concentration of nanoparticles is less than 4 mu g/ml to the growth of SKOV3 cells. Use, select laser (2.0W/cm2,30s), ultrasound (0.88W/cm2,60s) and nanoparticles ICG concentration of 4 mu g/ml as the appropriate parameters of this experiment in the follow-up study. Third experimental study on the inhibition of the proliferation of ovarian cancer SKOV3 cells with oxygen loaded ICG nanoparticles and the mechanism of the inhibition of proliferation of ovarian cancer cells: the observation of photodynamic and acoustic power combined oxygen carrying nanoparticles Inhibition and mechanism of the proliferation of SKOV3 cells in ovarian cancer cells. Methods: 1, detection of cell growth inhibition by different treatments: SKOV3 cells in logarithmic growth period were randomly divided into 6 groups: ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U group, OPI-NPs+L+U group, OPI-NPs+L group and OPI-NPs+U group. 24 hours after 24 hours, CCK-8 detection was used. Group cell survival rate; 2, different nanoparticles ICG concentration for cell growth inhibition detection: the nanoparticles with different ICG concentrations (0.25 g/ml, 0.5 mu g/ml, 1 mu g/ml, 2 micron g/ml and 4 mu g/ml) were added to the logarithmic growth period SKOV3 cells for 24 hours, and respectively treated with stimulated light (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s), and 24 smaller after treatment. At the time, the cell survival rate was detected by CCK-8. 3, the reactive oxygen species in the cell solution was detected in different ways after different treatments: the samples were added to the 96 hole culture plate according to the final concentration of 4 mu g/ml, and the concentration was 50m M SOSG 0.02ml in each hole. The control group, PBS+L+U, ICG+L+U, I-NPs+L+U, PI-NPs+L were grouped according to the different treatments. Group +U, group OPI-NPs+L+U, group OPI-NPs+L and group OPI-NPs+U; 20 minutes after treatment, the fluorescence intensity of each solution was measured by an enzyme scale. 4, the effect of ICG concentration on the production of reactive oxygen in the cell solution: the nanoparticles with different ICG concentrations (0.25 mu g/ml, 0.5 mu g/ml, 1 micron ml, 2 mu g/ml and 4 micron g/ml) were added to the 96 hole clock, respectively. Then the concentration of 50m M SOSG 0.02ml was added with the laser (2.0W/cm2,30s) combined ultrasound (0.88W/cm2,60s) treatment, after 20 minutes, the fluorescence intensity of the solution was measured by the enzyme scale. 5, the intracellular reactive oxygen species were detected in different ways: the logarithmic growth period SKOV3 cells were divided into 8 groups: control group, PBS+L+U group, ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U. Group OPI-NPs+L+U, group OPI-NPs+L+U, group OPI-NPs+L and OPI-NPs+U were added to ICG or nanoparticles to make ICG concentration in the solution respectively. After co culture 24h with SKOV3 cells, 2 micron DCFH-DA (10m M) were added for 20 minutes respectively. Then 3 times were washed with serum free culture solution and then treated with laser and / or ultrasound for 20 minutes after treatment. The fluorescence intensity of reactive oxygen species was detected by flow meter and the development of active oxygen indicator was observed under the fluorescence microscope. Results: 1, PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group were respectively (30.2 + 1.291)%, (77.24 + 0.4634)% and (59.06 + 2.003)%, respectively, higher than the other treatment groups (P0.05); OPI-NPs+L+U The inhibitory rate of cell proliferation in the group and OPI-NPs+L group was significantly higher than that in the PI-NPs+L+U group. The proliferation inhibition rate of the OPI-NPs+L+U group was significantly higher than that in the OPI-NPs+L group. The difference was statistically significant (P0.05).2, with the increasing of the ICG concentration of the nanoparticles, the proliferation inhibition rate was also increased, and the 4 mu g/ml group reached (72.04 + 5.273)%, obviously higher than the others. The difference of the concentration group was statistically significant (P0.05).3, group PI-NPs+L+U, group OPI-NPs+L+U and OPI-NPs+L group, the fluorescence intensity of active oxygen indicator in no cell solution was 185.3 + 19.46440.1 + 11.47 and 355.6 + 31.15 respectively higher than that of other treatment groups (P0.05), and the active oxygen content in OPI-NPs+L+U and OPI-NPs+L groups was significantly higher than that of the group OPI-NPs+L+U and OPI-NPs+L. In group PI-NPs+L+U, the content of active oxygen in group OPI-NPs+L+U was significantly higher than that in group OPI-NPs+L, and the difference was statistically significant (P0.05).4. With the increase of the concentration of nanoparticles, the production of extracellular reactive oxygen species increased further, the group of 4 micron g/ml reached 440.1 + 11.47, obviously higher than the other concentration groups, the difference was statistically significant (P0.05).5, and the fluorescence microscope was PI -NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group showed obvious green fluorescence, and the fluorescence intensity of OPI-NPs+L+U group and OPI-NPs+L group was more obvious than that of PI-NPs+L+U group. The fluorescence intensity of intracellular reactive oxygen indicator quantitative detection by flow meter was visible: the fluorescence intensity of active oxygen indicator in PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group was respectively. 1935 + 53.142939 + 300.2 and 2935 + 295.3, significantly higher than the other treatment groups, OPI-NPs+L+U group and OPI-NPs+L group intracellular reactive oxygen fluorescence intensity was significantly higher than the PI-NPs+L+U group, the difference was statistically significant (P0.05), but the fluorescence intensity of active oxygen in the OPI-NPs+L+U group was not significantly different from that of the OPI-NPs+L group (P0.05). Conclusion: laser coupling Oxygen-carrying nanoparticles stimulated by ultrasound can inhibit the proliferation of ovarian cancer SKOV3 cells, and the mechanism may be related to the production of reactive oxygen species inside and outside the cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
本文编号：2165769
[Abstract]:The preparation and characterization of oxygen loaded ICG nanoparticles in the first part: preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles and its properties detection methods: the preparation of oxygen carrying liquid fluorocarbon indocyanine green PLGA nanoparticles (OPI-NPs), oxygen carrying liquid fluorocarbon PLGA nanoparticles (OP-NPs) and indoles carrying oxygen carrying liquid fluorocarbon indocyanine green (w/o/w) Cyanine green PLGA nanoparticles (I-NPs) were observed under light microscope. The size distribution of OPI-NPs was measured by Malvern particle size analyzer. The encapsulation efficiency and drug loading of ICG were measured by UV-Vis spectrophotometer. The photoacoustic imaging ability of ICG was observed by the photoacoustic imaging instrument. The results were compared with that of OP-NPs+free ICG and I-NPs. The results showed that OPI-NPs was spherical and average. The size of the particle size (275.24 + 7.28) nm, the size distribution range from 80 nm to 1000 nm, the encapsulation efficiency of ICG is about 55%.OP-NPs+free ICG, the photoacoustic intensity of I-NPs and OPI-NPs is 0.336 + 0.022,0.744 + 0.029 and 1.112 + 0.072.OPI-NPs and I-NPs is obviously stronger than that of OP-NPs+free. Better than I-NPs, the difference is statistically significant (P0.05). Conclusion: the successful preparation of OPI-NPs nanoparticles with relatively uniform particle size and good photoacoustic development ability. The suitable laser, ultrasonic parameters and nanoparticle concentration of the second part nanoparticles are screened. The suitable laser can be selected to select the suitable laser to stimulate the phase transition and rupture of nanoparticles. And ultrasonic parameters and the concentration of nanoparticles ICG for photoacoustic power therapy. Method: 1, laser parameters screening: the prepared nanoparticles are diluted 20 times, according to laser intensity and irradiation time as follows: 0.5W/cm2,30s, 0.5W/cm2,60s, 1.0W/cm2,30s, 1 W/cm2,60s, 1.5 W/ cm2,30s, 1.5 W/cm2,60s, 2 W/cm2,30s and 2 W/cm2,60s. respectively given. After the above treatment conditions, the morphology of the microspheres was observed under the light microscope.2, the ultrasonic parameters were screened: the prepared nanoparticles were diluted 20 times. After the laser (2.0W/cm2,30s) irradiation, the ultrasonic intensity and the irradiation time were grouped as follows: 0.44W/cm2,30s, 0.44 W/cm2,60s, 0.66 W/ cm2,30s, 0.66 W/cm2,60s, 0.88 W/cm2,30s, 0.88 W/cm2,60s, 1.10 W/cm2,30s. And 1.10 W/cm2,60s. were treated respectively. After treatment, the microsphere fragmentation was observed by immediate light microscopy (.3) and nanoparticles ICG concentration was screened: the logarithmic growth period SKOV3 cells were selected according to the concentration of ICG in the nanoparticles as follows: the control group, the 0.5 mu g/ml group, the 1 mu g/ml group, the 2 mu g/ml group, the 4 micron g/ml group and 8 micron g/ml group, and 6 groups respectively. The nanoparticles and the group were respectively. After the cell co culture of 24h, CCK-8 was used to detect the cell survival rate.4, suitable for laser. Under ultrasonic action, the microsphere phase transformation and fragmentation were observed: the prepared nanoparticles were diluted 20 times, and the same amount of nanoparticle suspension was put into the gel pores respectively. Before and after the laser (2.0W/cm2,30s) irradiation, the laser (2.0W/cm2,30s) and ultrasonic (0.88W/cm2) irradiated 30 respectively. After second and 60 seconds, the echo changes of gel tube were observed by diagnostic ultrasound. The morphology of microspheres was observed by light microscope. The size distribution of microspheres was measured by particle size measuring instrument.5. The effect of laser and ultrasound on cell proliferation: SKOV3 cells of logarithmic growth period were taken as follows: control group, laser group, ultrasound group and laser combined ultrasound group. The cell survival rate was detected by CCK-8, and the inhibition of cell proliferation was observed. The results were as follows: 1, when the laser irradiation energy reached 1.5W/cm2, the nanoparticles had obvious phase transition, and the number of phase transition was further increased with the prolongation of irradiation time. The number of nanoparticles phase transition was significantly increased when the laser energy reached 2.0W/ cm2, but with the time, the number of phase transition was significantly increased. There is no obvious change in the number of phase changes of.2. When the energy of ultrasound irradiation is 0.44W/cm2 and 0.66W/cm2, the phase transition nanoparticles do not change obviously. There is no obvious change with the prolongation of the irradiation time. When the energy reaches 0.88W/cm2, the number of nanoparticles decreases obviously, with the increase of radiation energy to 1.10W/cm2. The decrease of the number of phase transition nanoparticles was not obvious, but with the prolongation of the irradiation time to 60s, the number of phase transition nanoparticles decreased obviously by.3, and the concentration of the encapsulated ICG between 0.5 and 4 Mu was not obviously inhibited. The proliferation of the encapsulated ICG was 8 mu g/ml and the cell proliferation was obviously inhibited by.4 and the ultrasonic development before and after laser excitation. The size of nanoparticles was increased by light microscopy. The average particle size of nanoparticles was 275.24nm and 1533.65nm, respectively. The ultrasonic development of 30s and 60s after laser irradiation was obviously weakened after laser irradiation. The average particle size of nanoparticles was 1060.56nm and 771.38 nm.5 respectively, and the laser (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s) were used to the cells. Conclusion: laser (2.0W/cm2,30s) can effectively stimulate the phase transition of nanoparticles. Ultrasound (0.88W/cm2,60s) can effectively break the phase of phase transition, and the ultrasound and laser under this energy have no effect on the growth of SKOV3 cells either alone or in combination. The ICG concentration of nanoparticles is less than 4 mu g/ml to the growth of SKOV3 cells. Use, select laser (2.0W/cm2,30s), ultrasound (0.88W/cm2,60s) and nanoparticles ICG concentration of 4 mu g/ml as the appropriate parameters of this experiment in the follow-up study. Third experimental study on the inhibition of the proliferation of ovarian cancer SKOV3 cells with oxygen loaded ICG nanoparticles and the mechanism of the inhibition of proliferation of ovarian cancer cells: the observation of photodynamic and acoustic power combined oxygen carrying nanoparticles Inhibition and mechanism of the proliferation of SKOV3 cells in ovarian cancer cells. Methods: 1, detection of cell growth inhibition by different treatments: SKOV3 cells in logarithmic growth period were randomly divided into 6 groups: ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U group, OPI-NPs+L+U group, OPI-NPs+L group and OPI-NPs+U group. 24 hours after 24 hours, CCK-8 detection was used. Group cell survival rate; 2, different nanoparticles ICG concentration for cell growth inhibition detection: the nanoparticles with different ICG concentrations (0.25 g/ml, 0.5 mu g/ml, 1 mu g/ml, 2 micron g/ml and 4 mu g/ml) were added to the logarithmic growth period SKOV3 cells for 24 hours, and respectively treated with stimulated light (2.0W/cm2,30s) and ultrasound (0.88W/cm2,60s), and 24 smaller after treatment. At the time, the cell survival rate was detected by CCK-8. 3, the reactive oxygen species in the cell solution was detected in different ways after different treatments: the samples were added to the 96 hole culture plate according to the final concentration of 4 mu g/ml, and the concentration was 50m M SOSG 0.02ml in each hole. The control group, PBS+L+U, ICG+L+U, I-NPs+L+U, PI-NPs+L were grouped according to the different treatments. Group +U, group OPI-NPs+L+U, group OPI-NPs+L and group OPI-NPs+U; 20 minutes after treatment, the fluorescence intensity of each solution was measured by an enzyme scale. 4, the effect of ICG concentration on the production of reactive oxygen in the cell solution: the nanoparticles with different ICG concentrations (0.25 mu g/ml, 0.5 mu g/ml, 1 micron ml, 2 mu g/ml and 4 micron g/ml) were added to the 96 hole clock, respectively. Then the concentration of 50m M SOSG 0.02ml was added with the laser (2.0W/cm2,30s) combined ultrasound (0.88W/cm2,60s) treatment, after 20 minutes, the fluorescence intensity of the solution was measured by the enzyme scale. 5, the intracellular reactive oxygen species were detected in different ways: the logarithmic growth period SKOV3 cells were divided into 8 groups: control group, PBS+L+U group, ICG+L+U group, I-NPs+L+U group, PI-NPs+L+U. Group OPI-NPs+L+U, group OPI-NPs+L+U, group OPI-NPs+L and OPI-NPs+U were added to ICG or nanoparticles to make ICG concentration in the solution respectively. After co culture 24h with SKOV3 cells, 2 micron DCFH-DA (10m M) were added for 20 minutes respectively. Then 3 times were washed with serum free culture solution and then treated with laser and / or ultrasound for 20 minutes after treatment. The fluorescence intensity of reactive oxygen species was detected by flow meter and the development of active oxygen indicator was observed under the fluorescence microscope. Results: 1, PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group were respectively (30.2 + 1.291)%, (77.24 + 0.4634)% and (59.06 + 2.003)%, respectively, higher than the other treatment groups (P0.05); OPI-NPs+L+U The inhibitory rate of cell proliferation in the group and OPI-NPs+L group was significantly higher than that in the PI-NPs+L+U group. The proliferation inhibition rate of the OPI-NPs+L+U group was significantly higher than that in the OPI-NPs+L group. The difference was statistically significant (P0.05).2, with the increasing of the ICG concentration of the nanoparticles, the proliferation inhibition rate was also increased, and the 4 mu g/ml group reached (72.04 + 5.273)%, obviously higher than the others. The difference of the concentration group was statistically significant (P0.05).3, group PI-NPs+L+U, group OPI-NPs+L+U and OPI-NPs+L group, the fluorescence intensity of active oxygen indicator in no cell solution was 185.3 + 19.46440.1 + 11.47 and 355.6 + 31.15 respectively higher than that of other treatment groups (P0.05), and the active oxygen content in OPI-NPs+L+U and OPI-NPs+L groups was significantly higher than that of the group OPI-NPs+L+U and OPI-NPs+L. In group PI-NPs+L+U, the content of active oxygen in group OPI-NPs+L+U was significantly higher than that in group OPI-NPs+L, and the difference was statistically significant (P0.05).4. With the increase of the concentration of nanoparticles, the production of extracellular reactive oxygen species increased further, the group of 4 micron g/ml reached 440.1 + 11.47, obviously higher than the other concentration groups, the difference was statistically significant (P0.05).5, and the fluorescence microscope was PI -NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group showed obvious green fluorescence, and the fluorescence intensity of OPI-NPs+L+U group and OPI-NPs+L group was more obvious than that of PI-NPs+L+U group. The fluorescence intensity of intracellular reactive oxygen indicator quantitative detection by flow meter was visible: the fluorescence intensity of active oxygen indicator in PI-NPs+L+U group, OPI-NPs+L+U group and OPI-NPs+L group was respectively. 1935 + 53.142939 + 300.2 and 2935 + 295.3, significantly higher than the other treatment groups, OPI-NPs+L+U group and OPI-NPs+L group intracellular reactive oxygen fluorescence intensity was significantly higher than the PI-NPs+L+U group, the difference was statistically significant (P0.05), but the fluorescence intensity of active oxygen in the OPI-NPs+L+U group was not significantly different from that of the OPI-NPs+L group (P0.05). Conclusion: laser coupling Oxygen-carrying nanoparticles stimulated by ultrasound can inhibit the proliferation of ovarian cancer SKOV3 cells, and the mechanism may be related to the production of reactive oxygen species inside and outside the cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
【参考文献】
相关期刊论文 前1条
1 杨凯,陈绍维,陈睿,温玉明;隐形顺铂聚乳酸纳米微粒对口腔鳞癌原发灶的靶向性研究[J];华西口腔医学杂志;2005年05期
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