超声放大肿瘤标志物及卵巢癌预警和早期诊断新方法的实验研究

发布时间：2018-04-29 12:29

本文选题：卵巢癌 + 肿瘤标志物　；参考：《东南大学》2015年博士论文

【摘要】：目的：卵巢癌是临床最常见的妇科肿瘤之一,其死亡率居妇科肿瘤首位。因其早期症状隐匿,超过70%的患者就诊时已为晚期。血清肿瘤标志物(Tumor marker,TM)检测在临床早期诊断卵巢癌方面已广泛应用,但由于早期肿瘤患者血液中含量极低,并可有多种等非癌症因素(腹水、炎症、子宫内膜异位症等)引起TM的异常升高,早期肿瘤的标志物信号便被埋没在这些噪声信号中无法鉴别出来。因此本研究通过对超声生物效应的研究,初探超声致卵巢癌肿瘤标志物浓度增量的方法,进而分析影响TM增量释放的超声参数,建立在细胞、和小鼠移植瘤上超声放大的方法,探索该方法在卵巢癌早期诊断中的应用。方法：(1)在超声频率F=1MHz的条件下(0.1 W/cm2～0.3 W/cm2,1 min～5 min),以电化学发光法检测细胞上清中的TM浓度(CA125、CA199),以TM增量和超声能量水平为主要考察指标,优化能使多种标志物增量达到最大的超声参数组合,建立超声能量和标志物增量之间的剂量-效应关系；并在建立卵巢癌异种移植肿瘤动物模型上验证超声放大卵巢癌肿瘤标志物方法的可行性。(2)采用自制1 MHz脉冲超声发生器,并采用该设备施加超声于人卵巢癌上皮细胞(SKOV3),用台盼蓝染色、Edu细胞增殖实验、MTT增殖抑制实验、Transwell侵袭实验检测超声对卵巢癌细胞的死亡率、增殖能力和侵袭能力的影响；建立卵巢癌异种移植肿瘤动物模型,用免疫组化对瘤体进行E-钙粘蛋白、波形蛋白的染色,检测超声对肿瘤上皮间质转化的影响。(3)以低功率超声(1MHz)辐射卵巢癌细胞(0.1 W/cm2-0.3 W/cm2,1 min-5 min),以临床公认的卵巢癌标志物CA125为对象,用荧光定量RT-PCR、Western Blot分别检测超声处理前后CA125的相关基因表达及合成情况。(4)无菌取材人卵巢癌组织标本,原代培养上皮性卵巢癌细胞,测定超声处理前后的CA125、CA199浓度,探讨本方法的临床应用可行性。结果：(1)不同功率、不同辐射时间的超声处理后CA125、CA199两者浓度均升高(P0.05)。其中CA125浓度随功率的增加、辐射时间的延长而增加,相对于0.1W/cm2,CA125在0.2 W/cm2和0.3 W/cm2功率照下浓度扩增倍数分别为4.05、10.57、20.22、26.43、39.82倍和19.62、31.24、32.28、44.72、62.59倍(1、2、3、4、5 min)；而CA199浓度扩增倍数则不如CA125显著,分别为1.22、1.37、1.18、1.26、1.2倍和1.16、1.25、1.27、1.5、1.54倍(1、2、3、4、5 min),且3 min内两组功率条件下CA199浓度扩增倍数差异不显著(P0.05)。裸鼠经超声处理后,血清TM浓度随功率增加而升高(P0.01),血清CA125浓度较对照组提高约1.3倍,CA199浓度提高约2.4倍。CA125浓度的变化与声处理时间、功率、观察时间有线性回归关系,“最优,,回归方程为：夕=34.793+12.889T-15.281 P+13.632 D,功率对CA125浓度的影响最大；CA199浓度的变化与声处理时间、功率有线性回归关系,“最优”回归方程为：y=1.545—0.151 P+0.302D,观察时间对CA199浓度的影响最大。血清CA125浓度的变化与功率有线性回归关系,“最优”回归方程为：y=5.971+2.250 P；血清CA199浓度的变化与功率有线性回归关系,“最优,,回归方程为：少=1.524+1.191 P。(2)结合台盼蓝染色结果显示,当功率高于0.2 W/cm2时,随着声处理时间的延长,细胞瞬时死亡率逐步攀升,且相同处理时间下,0.3 W/cm2功率组死亡率均高于0.2 W/cm2功率组死亡率(P0.05)。超声对SKOV3细胞的增殖抑制作用结果表明,当功率≥0.2 W/cm2时,24 h时均表现具有一定的抑制生长作用,其中0.2 W/cm2为2%左右,0.3 W/cm2为3.5%左右,随观察时间延长,两组功率对SKOV3田胞的增殖抑制作用趋近于O。超声照射后裸鼠移植瘤E-cad表达以弱阳性(+)为主,VIM表达以强阳性(+++)为主,超声组与对照组间两种EMT标志物表达水平差异无统计学意义(P0.05)。(3)0.1 W/cm2和0.2 W/cm2功率下,CA125 mRNA水平变化差异较大,0.3W/cm2功率下CA125 mRNA水平均明显降低。超声后不同培养时间：在0h、24h时间段中CA125 mRNA变化差异较大,0.1 W/cm2超声照射4 min后培养24 hCA125 mRNA相对表达上调最显著,各组CA125 mRNA 48 h后表达水平均降低(P0.05)。三种功率超声于卵巢癌细胞1-5 min后：对不同超声功率组间进行比较发现：0.1 W/cm2与0.2W/cm2组间CA125蛋白相对表达量无显著差异性(P=0.2470.05),0.1 W/cm2与0.3 W/cm2组间CA125蛋白相对表达量差异有统计学意义(P=0.010.05),0.2 W/cm2与0.3 W/cm2组间CA125蛋白相对表达量差异有统计学意义(P=0.040.05)；对相同功率超声照射后培养时项间进行比较发现：0h与24h组间CA125蛋白相对表达量无显著差异性(P=0.5360.05),Oh分别与48h、72h组间CA125蛋白相对表达量差异均有统计学意义(P=0.0010.05)。(4)与对照组相比,原代上皮性卵巢癌细胞经超声(0.2 W/cm2,1MHz,3 min)处理后,CA125、CA199浓度分别扩增约14.5和3.8倍。1.结论：①超声可在细胞和活体水平使多种卵巢癌TM浓度增加,所建立模型可为卵巢癌预警和早期诊断提供一定依据。②本文采用的低功率超声(低于美国食药监规定)对离体卵巢癌细胞和活体卵巢癌移植瘤进行照射后,并未引起细胞和瘤体显著性的损伤,证实本文建立的超声放大TM新方法安全可靠。③本文采用的低功率超声对离体卵巢癌细胞作用后可以引起CA125蛋白和基因表达水平的变化。④借助超声放大卵巢癌TM以早期诊断卵巢癌的方法在临床应用中有一定可行性。
[Abstract]:Objective: ovarian cancer is one of the most common gynecologic tumors in clinic, and its mortality rate ranks first in gynecologic tumor. Because of its early symptoms, more than 70% of the patients are late. The detection of serum tumor markers (Tumor marker, TM) has been widely used in the early diagnosis of ovarian cancer, but the blood content of early cancer patients is extremely high. Low, and a variety of non cancer factors (ascites, inflammation, endometriosis, etc.) cause abnormal increase of TM. The signal of early tumor markers can not be identified in these noise signals. Therefore, this study is to explore the method of increasing the concentration of tumor markers in ovarian cancer by studying the biological effects of ultrasound. Furthermore, the ultrasonic parameters affecting the TM increment were analyzed, and the methods of ultrasound amplification on cells and mice transplanted tumor were established to explore the application of this method in the early diagnosis of ovarian cancer. Methods: (1) the concentration of TM (CA1) in cell supernatant (CA1) was detected by electrochemiluminescence (0.1 W/cm2 to 0.3 W/cm2,1 min to 5 min) under the ultrasonic frequency. 25, CA199), using the TM increment and the ultrasonic energy level as the main index to optimize the combination of ultrasonic parameters which can maximize the increment of multiple markers, establish the dose effect relationship between the ultrasonic energy and the increment of the marker, and verify the ultrasonic amplification of the tumor marker of ovarian cancer in the animal model of ovarian cancer xenotransplantation. The feasibility of the method. (2) using the self-made 1 MHz pulse ultrasonic generator, and using the device to apply ultrasound to human ovarian cancer epithelial cells (SKOV3), trypan blue staining, Edu cell proliferation test, MTT proliferation inhibition test, and Transwell invasion test to detect the effect of ultrasound on the mortality, proliferation and invasion of ovarian cancer cells; E- cadherin and vimentin were used to detect the effect of ultrasound on the transformation of epithelial mesenchymal transition of tumor. (3) the ovarian cancer cells (0.1 W/cm2-0.3 W/cm2,1 min-5 min) were radiated by low power ultrasound (1MHz), and the clinically recognized ovarian cancer marker CA125 was used as the target, and fluorescence determination was used. RT-PCR and Western Blot were used to detect the expression and synthesis of CA125 related genes before and after ultrasonic treatment. (4) tissue specimens of ovarian cancer in aseptic human ovarian cancer, primary cultured epithelial ovarian cancer cells, CA125, CA199 concentration before and after ultrasonic treatment, and the feasibility of the clinical application of this method. Results: (1) different power, different radiation time The concentration of CA125 and CA199 increased (P0.05) after ultrasonic treatment, and the concentration of CA125 increased with the increase of power and the prolongation of radiation time. Relative to 0.1W/cm2, the concentration multiplier of CA125 at 0.2 W/cm2 and 0.3 W/cm2 power was 4.05,10.57,20.22,26.43,39.82 times and 19.62,31.24,32.28,44.72,62.59 times (1,2,3,4,5 min), respectively. The multiplier of A199 concentration was less significant than that of CA125, 1.22,1.37,1.18,1.26,1.2 times and 1.16,1.25,1.27,1.5,1.54 times (1,2,3,4,5 min), and there was no significant difference (P0.05) in the multiplier of CA199 concentration in the two groups of 3 min. The serum TM concentration increased with the increase of power (P0.01) in nude mice after ultrasound treatment (P0.01), and the serum CA125 concentration was more than the control. The group increased about 1.3 times, the concentration of CA199 increased about 2.4 times the.CA125 concentration, and there was a linear regression relationship with the time of sound treatment, power and observation time. "Optimal, the regression equation was: the eve =34.793+12.889T-15.281 P+13.632 D, the power had the greatest influence on the CA125 concentration; the variation of CA199 concentration was linear with the sound treatment time and the power had linear regression relationship." The "optimal" regression equation is: y=1.545 - 0.151 P+0.302D, the effect of observation time on CA199 concentration is the greatest. The change of serum CA125 concentration has linear regression relation with power, and the "optimal" regression equation is y=5.971+2.250 P; the change of serum CA199 concentration is linear regression relation with power, "the optimal, regression equation is less =1.524+1." .191 P. (2) combined with trypan blue staining results showed that when the power was higher than 0.2 W/cm2, the cell instantaneous mortality increased gradually with the prolongation of the sound processing time, and the mortality rate of the 0.3 W/cm2 power group was higher than that of the 0.2 W/cm2 Power Group (P0.05) at the same processing time. 0.2 W/cm2, 24 h showed a certain inhibitory effect on growth, of which 0.2 W/cm2 was 2%, 0.3 W/cm2 was about 3.5%, with the prolonged observation time, the inhibitory effect of two groups of power on the proliferation of SKOV3 field was near O. ultrasound irradiation, E-cad expression in nude mice was mainly weak positive (+), VIM expression was strongly positive (+ + +), and ultrasound group was the dominant group. There was no significant difference in the expression level of two EMT markers between the control group and the control group (P0.05). (3) 0.1 W/cm2 and 0.2 W/cm2 power, CA125 mRNA level varied greatly, CA125 mRNA level under 0.3W/cm2 power decreased obviously. After ultrasound, the different incubation time: in 0h, 24h time period, the difference was great, 0.1 ultrasonic irradiation 4 After in, the relative expression of 24 hCA125 mRNA was up to the most significant, and the expression level of CA125 mRNA 48 h was decreased (P0.05). Three kinds of ultrasonic power ultrasound were found after 1-5 min of ovarian cancer cells: compared with the different ultrasonic power groups, there was no significant difference between the 0.1 W/cm2 and 0.2W/cm2 groups of CA125 protein (P=0.2470.05), 0.1 The relative expression of CA125 protein in 0.3 W/cm2 groups was statistically significant (P=0.010.05), and the relative expression of CA125 protein between 0.2 W/cm2 and 0.3 W/cm2 groups was statistically significant (P=0.040.05). The relative expression of CA125 protein between 0h and 24h group was not significantly different (P=0.). 5360.05) the difference of the relative expression of CA125 protein between Oh and 48h, 72h group was statistically significant (P=0.0010.05). (4) compared with the control group, the primary epithelial ovarian cancer cells were treated with ultrasound (0.2 W/cm2,1MHz, 3 min), CA125 and CA199 concentrations were amplified by 14.5 and 3.8 times.1. conclusions, respectively: (1) ultrasound can make a variety of eggs at the level of cell and living body. The concentration of TM in nests is increased, and the established model provides a basis for early diagnosis and early diagnosis of ovarian cancer. 2. The low power ultrasound (below the US food drug regulation) has not caused significant damage to the cell and tumor cells after irradiation of the ovarian cancer cells and living ovarian cancer cells. The new method of TM is safe and reliable. 3. The effect of low power ultrasound on ovarian cancer cells in vitro can cause changes in the level of CA125 protein and gene expression. (4) the method of ultrasonic amplification of ovarian cancer TM for early diagnosis of ovarian cancer is feasible in clinical application.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.31

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