低频低能量超声联合微泡增加人前列腺癌细胞化疗疗效的体外实验及其机制初探

发布时间：2018-02-27 09:13

本文关键词： 低频低能量超声微泡造影剂前列腺癌体外实验化疗机制　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：目的:研究低频低能量超声联合微泡对两种不同的人雄激素非依赖型前列腺癌细胞的化疗增效作用并对其机制进行初步探索。方法:1.流式细胞仪检测Calcein(分子量622.53)、FD4(分子量3,000-5,000)以及FD70(分子量59,000-77,000)三种不同分子量荧光物质进入细胞的阳性率;2.实验分4组:对照组、超声联合微泡组,米托蒽醌组,超声联合微泡+米托蒽醌组。不同分组的DU-145细胞干预后培养24小时,使用MTT和克隆实验检测细胞的增殖情况,使用Transwell小室测定细胞的迁移能力,使用透射电镜观察超声联合微泡对细胞亚显微结构的影响;3.实验分4组:对照组、超声联合微泡组,米托蒽醌组,超声联合微泡+米托蒽醌组。不同分组的PC-3细胞干预后培养24小时,使用MTT和克隆实验进行细胞增殖的检测,并与DU-145细胞结果进行比较,使用流式细胞仪,Hoechst染色检测细胞的凋亡情况,使用透射电镜观察超声联合微泡对细胞凋亡以及亚显微结构的影响,最后使用流式细胞仪检测Calcein(分子量622.53)进入细胞的阳性率。结果:1.该参数的低频低能量超声联合微泡可以增加三种不同分子量物质Calcein、FD4以及FD70进入细胞的百分比,空白对照组与超声联合微泡组的Calcein、FD4以及FD70荧光阳性百分比分别为:9.74±2.55%vs 31.26±3.34%;6.15±0.93%vs 20.57±2.89%;2.80±1.68%vs 12.92±3.54%(P0.05);2.超声联合微泡+米托蒽醌组的DU-145细胞增殖率为50.68%±5.85%,明显小于米托蒽醌组72.28%±4.61%(P0.05);超声联合微泡+米托蒽醌组的DU-145细胞集落数量为18.7±5.5,明显小于米托蒽醌组的45.0±6.0(P0.05);超声联合微泡+米托蒽醌组的DU-145细胞迁移数为2.7±2.5,明显小于米托蒽醌组的DU-145细胞迁移数17.7±7.1(P0.05);透射电镜结果显示低频低能量超声联合微泡组的DU-145细胞的细胞质中出现的自噬泡比空白对照组的多。3.超声联合微泡治疗组的DU-145细胞的吸光度为0.7078±0.0604,与空白对照组的0.7533±0.1275无统计学差异(P0.05);超声联合微泡治疗组的PC-3细胞的吸光度为0.6337±0.0577,明显小于空白对照组的0.7413±0.1252(P0.05);超声联合微泡+米托蒽醌组的PC-3细胞的吸光度为0.3663±0.0493,明显小于米托蒽醌组的0.5295±0.0668(P0.05);超声联合微泡+米托蒽醌组的PC-3细胞的细胞集落数量为11.0±5.0,明显小于米托蒽醌组的41.3±4.5(P0.05);流式细胞仪检测超声联合微泡组的PC-3细胞的凋亡率为9.30%±0.72%,明显高于空白对照组的2.37%±0.71%(P0.05);Hoechst染色检测检测超声联合微泡组的PC-3细胞的凋亡率为8.17±2.77%,明显高于空白对照组的1.80±1.30%(P0.05)。透射电镜结果显示超声联合微泡治疗组的PC-3细胞出现凋亡细胞,同时发现超声联合微泡组的PC-3细胞的细胞质中出现的自噬泡比空白对照组的多。空白对照组与超声联合微泡组的Calcein荧光阳性百分比分别为:7.27±3.20%vs 28.30±4.67%(P0.05)。结论:本实验条件下的低频低能量超声联合微泡可以增加DU-145细胞对Calcein、FD4以及FD70这三种不同分子量物质的通透性,并且可以增加人前列腺癌DU-145细胞以及PC-3细胞的化疗疗效,但二者的机制是不同的,对于DU-145细胞主要是通过增加通透性实现的,对于PC-3细胞是通过增加通透性及诱导凋亡实现的。
[Abstract]:Objective: To study the low-frequency and low-energy ultrasound combined with microbubbles on two different human androgen independent prostate cancer cells to chemotherapy synergistic effect and preliminary exploration on its mechanism. Methods: 1. flow cytometry Calcein (molecular weight 622.53), FD4 (molecular weight 3000-5000) and FD70 (molecular weight 59000-77000). The rate of three different molecular weight fluorescent substances into cells; 2. were divided into 4 groups: control group, ultrasound and microbubble group, mitoxantrone group, ultrasound and microbubble + mitoxantrone group. The prognosis of 24 hours of training in different groups of DU-145 stem cells, proliferation of MTT and cloning assay, migration cells were measured using Transwell cell, using transmission electron microscope to observe the effect of ultrasound mediated microbubble destruction on cell ultrastructure; 3. were divided into 4 groups: control group, ultrasound and microbubble group, mitoxantrone group, ultrasound and microbubble + mitoxantrone Anthraquinone group. Prognosis of 24 hours of training in different groups of PC-3 stem cells, detected by MTT and cloning experiments of cell proliferation, and compared with the results of DU-145 cells, flow cytometry was used to detect the apoptosis of Hoechst cells were examined by transmission electron microscopy, ultrasound combined microbubble on apoptosis and ultrastructure finally, using flow cytometry Calcein (molecular weight 622.53) positive rate into cells. Results: the low frequency ultrasonic energy 1. the parameters of the microbubbles can increase three different molecular weight substances Calcein, FD4 and FD70 into the cell percentage of Calcein control group and ultrasound combined with microbubbles group FD4, FD70 and fluorescence positive percentage were 9.74 + 2.55%vs 31.26 + 3.34%; 6.15 + 0.93%vs 20.57 + 2.89%; 2.80 + 1.68%vs 12.92 + 3.54% (P0.05); 2. ultrasound combined with microbubbles + mitoxantrone group DU-145 The cell proliferation rate was 50.68% + 5.85%, significantly less than the mitoxantrone group 72.28% + 4.61% (P0.05); ultrasound combined with microbubbles + mitoxantrone group DU-145 cell colony number was 18.7 + 5.5, significantly less than the mitoxantrone group 45 + 6 (P0.05); ultrasound combined with microbubbles + DU-145 cell migration mitoxantrone group is 2.7 + 2.5, significantly less than the mitoxantrone group DU-145 cell migration number 17.7 + 7.1 (P0.05); transmission electron microscopy results showed that the absorbance of low frequency and low energy ultrasound combined with microbubbles group in the cytoplasm of DU-145 cells appeared more autophagic vacuoles than the control group.3. ultrasound combined microbubble treatment group of DU-145 cells 0.7078 + 0.0604 and 0.7533 + 0.1275 in the control group had no significant difference (P0.05); the absorbance of ultrasound combined with microbubble treatment group of PC-3 cells was 0.6337 + 0.0577, significantly less than the control group 0.7413 + 0.1252 (P0.05); ultrasound combined with microbubbles + The absorbance of the mitoxantrone group of PC-3 cells was 0.3663 + 0.0493, significantly less than the mitoxantrone group 0.5295 + 0.0668 (P0.05); ultrasound combined with microbubbles + mitoxantrone group PC-3 cell colony number was 11 + 5, significantly less than the mitoxantrone group 41.3 + 4.5 (P0.05); flow cytometry detection of ultrasound combined with micro bubble group apoptosis rate of PC-3 cells was 9.30% + 0.72%, significantly higher than the control group of 2.37% + 0.71% (P0.05); Hoechst staining detected by ultrasound micro bubble group apoptosis rate of PC-3 cells was 8.17 + 2.77%, significantly higher than the control group of 1.80 + 1.30% (P0.05) transmission. The results of SEM showed that ultrasound combined with microbubbles group PC-3 cells apoptosis, also found that ultrasound combined with microbubbles group in the cytoplasm of PC-3 cells and the control group of autophagosomes than the blank. The blank control group and ultrasound combined with microbubbles group Calcein fluorescence The positive percentage was 7.27 + 3.20%vs 28.30 + 4.67% (P0.05). Conclusion: low frequency ultrasound energy under the experimental conditions the microbubbles can increase DU-145 cells to Calcein, FD4 and FD70 of the three different molecular weight substances and can increase the permeability of human prostate cancer DU-145 cells and PC-3 cells in response to chemotherapy. But the different mechanisms of the two, for the DU-145 cell is mainly by increasing the permeability to achieve, by increasing the permeability and inducing apoptosis for PC-3 cells.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

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