【作者】 王娜；

【导师】 熊正爱；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2008， 硕士

【摘要】 第一部分紫杉醇对宫颈癌HeLa细胞抑制浓度及超声破坏微泡的辐照参数选择目的:探索紫杉醇对宫颈癌HeLa细胞株的适宜抑制浓度及超声破坏微泡的适宜辐照参数。方法:体外培养宫颈癌细胞,采用MTT比色法检测不同剂量紫杉醇对宫颈癌HeLa细胞的增殖抑制作用和不同辐照时间超声破坏空白微泡对HeLa细胞的急性毒作用,并以此确定适宜的紫杉醇剂量以及超声辐照参数。结果:1.当紫杉醇浓度为10^-9、10^-8、10^-7、10^-6、10^-5、10^-4mol/L时,细胞在72h的增殖抑制率分别为（32.37±1.12）%、（42.97±2.18）%、（51.45±0.86）%、（62.48±0.95）%、（69.81±0.57）%、（76.06±2.4）%。当紫杉醇浓度为10-9mol/L时,24h、48h、72h的增殖抑制率分别为（11.52±1.08）%、（24.6±0.35）%、（32.37±1.12）%。其增殖抑制作用有明显的时间效应和剂量效应关系。当声强为0.5W/cm²,辐照时间20S即可破坏微泡。2.声强固定在0.5W/cm2,时间10S、20S、30S,破坏微泡后即检测细胞的存活率为（92.84±0.66）%、（85.69±2.67）%、（76.61±2.74）%。结论:紫杉醇对宫颈癌HeLa细胞有增殖抑制作用。适宜参数下超声破坏空白微泡对宫颈癌HeLa细胞的急性毒作用影响不大。适宜的超声辐照参数为0.5W/cm²、20S,紫杉醇剂量为10^-7mol/L。第二部分超声破坏载紫杉醇微泡对宫颈癌HeLa细胞株增殖、凋亡的影响研究目的:观察超声破坏载紫杉醇微泡对宫颈癌HeLa细胞株的增殖、凋亡以及细胞周期改变的影响。方法: 1.体外培养宫颈癌细胞,将细胞分为5组,即紫杉醇组、紫杉醇联合超声辐照组、载紫杉醇微泡联合超声辐照组、载紫杉醇微泡组及空白对照组,MTT比色法检测不同观察时间点各组细胞的增殖抑制情况2.透射电镜观察各组细胞的超微结构变化。3.FCM分析各组细胞凋亡诱导及细胞周期的改变。结果:1.超声破坏载紫杉醇微泡组、紫杉醇组及超声联合紫杉醇组均能明显抑制宫颈癌HeLa细胞的增殖,超声破坏紫杉醇微泡组的作用更为明显,三组之间比较,差异具有显著意义（P<0.01）,而相同浓度的载紫杉醇微泡组对HeLa细胞的增殖影响不显著。2.透射电镜观察到超声联合紫杉醇微泡组可见凋亡小体,超声联合紫杉醇可见凋亡细胞,紫杉醇组观察到早期凋亡细胞,空白组和单纯微泡组细胞形态较为正常。3.FCM定量分析结果表明各组细胞的凋亡率分别为:空白组（2.18±0.11）%、单纯紫杉醇微泡组（5.21±0.34）%、单纯紫杉醇组（11.77±0.54）%、紫杉醇联合超声组（15.69±0.43）%、紫杉醇微泡联合超声组（27.5±0.59）%。超声破坏载紫杉醇微泡组、紫杉醇组及紫杉醇联合超声组均表现为G2/M阻滞,进入S期和G0/G1期细胞减少。各组细胞的阻滞率分别为:空白组（9.7±1.81）%、单纯紫杉醇微泡组（14.05±0.8）%、单纯紫杉醇组（22.04±1.51）%、紫杉醇联合超声组（33.28±2.92）%、紫杉醇微泡联合超声组（68.28±2.62）%。结论:超声破坏紫杉醇微泡不仅能显著抑制宫颈癌HeLa细胞增殖,还有显著诱导其凋亡的作用,细胞被阻滞于G2/M期。更多还原

【Abstract】 PARTⅠTHE CONCENTRATION OF PACLITAXEL IN CERVICAL CANCER HELA CELLS IN INHIBITORY EFFECTS AND MICROBUBBLES SECT ULTRASONIC RADIATION DAMAGE PARAMETERSObjective: To study the paclitaxel concentration of cervical cancer cells HeLa in inhibitory effects and ultrasonic radiation parameters of destructing microbubbles.Methods: Cell strain was cultured in vitro. The inhibitory effects of different dosages of paclitaxel on cells proliferation and the acute toxic effects of different irradiation time of ultrasound combined blank microbubbles group on cells was detected by MTT assay.Results:1.The proliferation inhibition ratio of HeLa cells in the 72nd hour were determined as （32.37±1.12）%,（42.97±2.18）%,（51.45±0.86）%,（62.48±0.95）%,（69.81±0.57）%,and（76.06±2.4）% after paclitaxel treatment with different concentrations of 10^-9,10^-8,10^-7,10^-6,10^-5 and 10-4mol/L, respectively. And the inhibition ratio of HeLa cells in the 24th hour, 48th hour and 72nd hour were respectively （11.52±1.08）%,（24.6±0.35）%,（32.37±1.12）%when the palictaxel concentration was fixed in 10-9mol/L. The inhibition effect was positively related with the time and concentration of drug treatment, which showed an obvious dose-response time relationship.2. PLM could be destructed with an acoustic intensity of 0.5W/cm2 and the irradiation time of 20 S. When the acoustic intensity was fixed in 0.5W/cm2, and the time of ultrasound radiation on PLM were 10 S, 20 S and 30 S, the survival rates of cells were determined respectively as （92.84±0.66）%,（85.69±2.67）% and （76.61±2.74）%.Conclusion: paclitaxel could inhibit HeLa cells in vitro. There is a little acute toxic effects on HeLa cells using ultrasound combined blank microbubbles with suitable ultrasonic irradiation parameters. Suitable ultrasonic irradiation parameters is 0.5W/cm2, 20 S, and paclitaxel dosage is 10-7mol/L. PARTⅡTHE EFFECT OF PROLIFERATION INHIBITION AND APOPTOSIS INDUCTION ON CERVICAL CANCER HELA USING PACLITAXEL-CARRYING ULTRASOUND MICROBUBBLESobjective: To study the effects of ultrasound combined paclitaxel-carrying liposome microbubbles（PLM） method on HeLa cell proliferation, apoptosis and changes of cell cycle. Methods: 1.HeLa cell strain was cultured in vitro, and divided into 5 groups: paclitaxel group,paclitaxel plus ultrasound irradiation group,paclitaxel- carrying microbubbles plus ultrasound irradiation group ,paclitaxel-carrying microbubbles group and control group. Proliferation inhibition of cervical cancer HeLa cell lines in different groups were observed at different time points by MTT assay. 2. Apoptosis induction of HeLa cell lines in different groups were observed by transmission electron microscope （TEM）. 3. The apoptosis of cells and the changes of cell cycle were determined by flow cytometry（FCM）.Results:1.Ultrasound combined PLM, paclitaxel alone and ultrasound combined PLM all had significant inhibition effects on HeLa cell proliferation. However, among the three groups, the effects of ultrasound combined PLM group was the strongest （P<0.01）. And PLM alone with the concentration equal with the first group had no significant effects on cells.2. Apoptotic bodies were observed by TEM in the ultrasound combined PLM group, apoptotic cells were observed in the ultrasound combined paclitaxel group, and the early apoptotic cells were observed in the paclitaxel alone group. Cell shape in the contrast group and the PLM alone group were both normal.3.The apoptosis rates of cells determined by FCM were （2.18±0.11）% in the contrast group, （ 5.21±0.34 ） % in the PLM alone group, （11.77±0.54）% in paclitaxel alone group, （15.69±0.43）% in the ultrasound combined paclitaxel group and（27.5±0.59）% in the ultrasound combined PLM group, respectively. With the treatment of ultrasound combined PLM, paclitaxel alone and ultrasound combined PLM, the HeLa cells were all arrested in G2/M phase , and with a concurrent decrease in the proportion of S and G0/G1 phase cells .The arreste rates of cells in G2/M phase determined by FCM were （9.7±1.81）% in the contrast group, （14.05±0.8）% in the PLM alone group, （22.04±1.51）% in paclitaxel alone group, （33.28±2.92）% in the ultrasound combined paclitaxel group and （68.28±2.62）% in the ultrasound combined PLM group, respectively.Conclusion: Cervical cancer HeLa cells with paclitaxel-carrying microbubbles have remarkable proliferation inhibition after ultrasound irradiation. HeLa cells is induced aptosis and all arrested in G2/M phase as well .更多还原