本文選題：卵巢癌 + 腫瘤標志物�。� 參考：《東南大學》2015年博士論文

【摘要】：目的：卵巢癌是臨床最常見的婦科腫瘤之一,其死亡率居婦科腫瘤首位。因其早期癥狀隱匿,超過70%的患者就診時已為晚期。血清腫瘤標志物(Tumor marker,TM)檢測在臨床早期診斷卵巢癌方面已廣泛應用,但由于早期腫瘤患者血液中含量極低,并可有多種等非癌癥因素(腹水、炎癥、子宮內(nèi)膜異位癥等)引起TM的異常升高,早期腫瘤的標志物信號便被埋沒在這些噪聲信號中無法鑒別出來。因此本研究通過對超聲生物效應的研究,初探超聲致卵巢癌腫瘤標志物濃度增量的方法,進而分析影響TM增量釋放的超聲參數(shù),建立在細胞、和小鼠移植瘤上超聲放大的方法,探索該方法在卵巢癌早期診斷中的應用。方法：(1)在超聲頻率F=1MHz的條件下(0.1 W/cm2～0.3 W/cm2,1 min～5 min),以電化學發(fā)光法檢測細胞上清中的TM濃度(CA125、CA199),以TM增量和超聲能量水平為主要考察指標,優(yōu)化能使多種標志物增量達到最大的超聲參數(shù)組合,建立超聲能量和標志物增量之間的劑量-效應關(guān)系；并在建立卵巢癌異種移植腫瘤動物模型上驗證超聲放大卵巢癌腫瘤標志物方法的可行性。(2)采用自制1 MHz脈沖超聲發(fā)生器,并采用該設備施加超聲于人卵巢癌上皮細胞(SKOV3),用臺盼藍染色、Edu細胞增殖實驗、MTT增殖抑制實驗、Transwell侵襲實驗檢測超聲對卵巢癌細胞的死亡率、增殖能力和侵襲能力的影響；建立卵巢癌異種移植腫瘤動物模型,用免疫組化對瘤體進行E-鈣粘蛋白、波形蛋白的染色,檢測超聲對腫瘤上皮間質(zhì)轉(zhuǎn)化的影響。(3)以低功率超聲(1MHz)輻射卵巢癌細胞(0.1 W/cm2-0.3 W/cm2,1 min-5 min),以臨床公認的卵巢癌標志物CA125為對象,用熒光定量RT-PCR、Western Blot分別檢測超聲處理前后CA125的相關(guān)基因表達及合成情況。(4)無菌取材人卵巢癌組織標本,原代培養(yǎng)上皮性卵巢癌細胞,測定超聲處理前后的CA125、CA199濃度,探討本方法的臨床應用可行性。結(jié)果：(1)不同功率、不同輻射時間的超聲處理后CA125、CA199兩者濃度均升高(P0.05)。其中CA125濃度隨功率的增加、輻射時間的延長而增加,相對于0.1W/cm2,CA125在0.2 W/cm2和0.3 W/cm2功率照下濃度擴增倍數(shù)分別為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濃度擴增倍數(shù)則不如CA125顯著,分別為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內(nèi)兩組功率條件下CA199濃度擴增倍數(shù)差異不顯著(P0.05)。裸鼠經(jīng)超聲處理后,血清TM濃度隨功率增加而升高(P0.01),血清CA125濃度較對照組提高約1.3倍,CA199濃度提高約2.4倍。CA125濃度的變化與聲處理時間、功率、觀察時間有線性回歸關(guān)系,“最優(yōu),,回歸方程為：夕=34.793+12.889T-15.281 P+13.632 D,功率對CA125濃度的影響最大；CA199濃度的變化與聲處理時間、功率有線性回歸關(guān)系,“最優(yōu)”回歸方程為：y=1.545—0.151 P+0.302D,觀察時間對CA199濃度的影響最大。血清CA125濃度的變化與功率有線性回歸關(guān)系,“最優(yōu)”回歸方程為：y=5.971+2.250 P；血清CA199濃度的變化與功率有線性回歸關(guān)系,“最優(yōu),,回歸方程為：少=1.524+1.191 P。(2)結(jié)合臺盼藍染色結(jié)果顯示,當功率高于0.2 W/cm2時,隨著聲處理時間的延長,細胞瞬時死亡率逐步攀升,且相同處理時間下,0.3 W/cm2功率組死亡率均高于0.2 W/cm2功率組死亡率(P0.05)。超聲對SKOV3細胞的增殖抑制作用結(jié)果表明,當功率≥0.2 W/cm2時,24 h時均表現(xiàn)具有一定的抑制生長作用,其中0.2 W/cm2為2%左右,0.3 W/cm2為3.5%左右,隨觀察時間延長,兩組功率對SKOV3田胞的增殖抑制作用趨近于O。超聲照射后裸鼠移植瘤E-cad表達以弱陽性(+)為主,VIM表達以強陽性(+++)為主,超聲組與對照組間兩種EMT標志物表達水平差異無統(tǒng)計學意義(P0.05)。(3)0.1 W/cm2和0.2 W/cm2功率下,CA125 mRNA水平變化差異較大,0.3W/cm2功率下CA125 mRNA水平均明顯降低。超聲后不同培養(yǎng)時間：在0h、24h時間段中CA125 mRNA變化差異較大,0.1 W/cm2超聲照射4 min后培養(yǎng)24 hCA125 mRNA相對表達上調(diào)最顯著,各組CA125 mRNA 48 h后表達水平均降低(P0.05)。三種功率超聲于卵巢癌細胞1-5 min后：對不同超聲功率組間進行比較發(fā)現(xiàn)：0.1 W/cm2與0.2W/cm2組間CA125蛋白相對表達量無顯著差異性(P=0.2470.05),0.1 W/cm2與0.3 W/cm2組間CA125蛋白相對表達量差異有統(tǒng)計學意義(P=0.010.05),0.2 W/cm2與0.3 W/cm2組間CA125蛋白相對表達量差異有統(tǒng)計學意義(P=0.040.05)；對相同功率超聲照射后培養(yǎng)時項間進行比較發(fā)現(xiàn)：0h與24h組間CA125蛋白相對表達量無顯著差異性(P=0.5360.05),Oh分別與48h、72h組間CA125蛋白相對表達量差異均有統(tǒng)計學意義(P=0.0010.05)。(4)與對照組相比,原代上皮性卵巢癌細胞經(jīng)超聲(0.2 W/cm2,1MHz,3 min)處理后,CA125、CA199濃度分別擴增約14.5和3.8倍。1.結(jié)論：①超聲可在細胞和活體水平使多種卵巢癌TM濃度增加,所建立模型可為卵巢癌預警和早期診斷提供一定依據(jù)。②本文采用的低功率超聲(低于美國食藥監(jiān)規(guī)定)對離體卵巢癌細胞和活體卵巢癌移植瘤進行照射后,并未引起細胞和瘤體顯著性的損傷,證實本文建立的超聲放大TM新方法安全可靠。③本文采用的低功率超聲對離體卵巢癌細胞作用后可以引起CA125蛋白和基因表達水平的變化。④借助超聲放大卵巢癌TM以早期診斷卵巢癌的方法在臨床應用中有一定可行性。
[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.

【學位授予單位】：東南大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.31

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