【摘要】：目的 卵巢上皮性癌是常見(jiàn)的女性惡性腫瘤之一，在女性生殖系統(tǒng)惡性腫瘤中，其死亡率也是最高的。卵巢癌患者的早期癥狀不典型，目前尚無(wú)有效的早期篩查和特異性診斷方法，超過(guò)70%的患者已是中晚期。目前標(biāo)準(zhǔn)治療方案為理想的腫瘤減滅術(shù)和以鉑類(lèi)為基礎(chǔ)的化療，但近年來(lái)卵巢癌患者五年生存率僅為30%-40%左右[42]。尋找卵巢癌有效的基因治療的分子靶點(diǎn)是卵巢癌目前亟待解決的臨床問(wèn)題。 鈣周期蛋白(Calcyclin，Cacy)屬于S100蛋白家族。文獻(xiàn)報(bào)道，Calcyclin在胰腺癌、肺癌、乳腺癌、卵巢癌、肝癌、結(jié)直腸癌等多種類(lèi)型的惡性腫瘤中均出現(xiàn)表達(dá)明顯增加[3,27-32]。有研究證實(shí)Calcyclin與上皮性癌發(fā)生發(fā)展相關(guān)[36,37]。還有研究表明Calcyclin高表達(dá)與胰腺癌、骨肉瘤、胃癌、肺腺癌、黑色素瘤等多種惡性腫瘤的轉(zhuǎn)移和不良預(yù)后相關(guān)[28,38-40]。這些研究均提示，Calcyclin可能是腫瘤基因治療的良好候選靶點(diǎn)。 目前Calcyclin蛋白在卵巢上皮性癌中的表達(dá)及其臨床意義尚不清楚。本研究旨在從組織水平、血清水平統(tǒng)計(jì)分析Calcyclin蛋白表達(dá)與卵巢癌的相關(guān)性，并初步探討Calcyclin作為腫瘤基因治療的可行性。 方法 （1）本研究首先用免疫組化技術(shù)檢測(cè)60例卵巢癌病理組織標(biāo)本中Calcyclin的蛋白表達(dá)情況，統(tǒng)計(jì)分析Calcyclin與卵巢癌的發(fā)生發(fā)展的關(guān)系，探討Calcyclin與卵巢上皮性癌臨床病理因素之間的相關(guān)性； （2）用ELISA法檢測(cè)正常血清、良性卵巢腫瘤和卵巢癌患者Calcyclin血清濃度，統(tǒng)計(jì)分析Calcyclin血清濃度與卵巢癌的相關(guān)性，初步驗(yàn)證Calcyclin作為檢測(cè)卵巢癌病情發(fā)展的分子標(biāo)志物的可行性； （3）我們利用RNA干擾技術(shù)特異性降低Calcyclin表達(dá)水平后，觀察卵巢癌細(xì)胞細(xì)胞周期、侵襲能力、順鉑敏感性等生物學(xué)行為的變化，探討Calcyclin作為卵巢癌靶向治療候選基因的可行性。 結(jié)果 （1）免疫組化檢測(cè)卵巢癌組織中Calcyclin表達(dá)情況，結(jié)果表明低分化的卵巢癌患者中Calcyclin高表達(dá)比例（55.0%）顯著高于高分化的卵巢癌患者中Calcyclin高表達(dá)比例（15.4%），有統(tǒng)計(jì)學(xué)差異(P＜0.05)。Calcyclin表達(dá)水平與手術(shù)分期有關(guān)，隨著手術(shù)分期的增加，Calcyclin表達(dá)水平也隨之明顯上升，有統(tǒng)計(jì)學(xué)差異(P＜0.05)。在出現(xiàn)腹水和淋巴結(jié)轉(zhuǎn)移的卵巢癌患者中，Calcyclin高表達(dá)的比例明顯增加，有統(tǒng)計(jì)學(xué)意義(P＜0.001)。 （2）ELISA法檢測(cè)Calcyclin血清濃度，結(jié)果表明卵巢癌患者Calcyclin血清平均濃度為1242.47+178.17pg/ml，顯著高于正常血清（489.73+37.86pg/ml）和良性卵巢腫瘤（700.75+43.14pg/ml），差異有統(tǒng)計(jì)學(xué)意義（P0.01）。然后我們統(tǒng)計(jì)分析了不同手術(shù)分期和細(xì)胞學(xué)分級(jí)的卵巢癌患者Calcyclin血清濃度。結(jié)果顯示Ⅲ期、Ⅳ期卵巢癌患者Calcyclin血清濃度分別為644.79+67.89pg/ml和1087.44+104.66pg/ml，顯著高于Ⅰ期、Ⅱ期卵巢癌患者（359.35+37.82pg/ml和499.44+42.56pg/ml），差異有統(tǒng)計(jì)學(xué)意義（P0.001）。同時(shí)我們還發(fā)現(xiàn)低分化的卵巢癌患者Calcyclin血清濃度顯著升高，為1320.08+102.59pg/ml，與高分化（573.58+37.28pg/ml）和中分化（832.91+75.62pg/ml）相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 （3）利用RNA干擾技術(shù)特異性降低Calcyclin表達(dá)水平后，細(xì)胞周期檢測(cè)結(jié)果表明，Calcyclin表達(dá)降低后A2780細(xì)胞出現(xiàn)了G0/G1期比例升高，，S期比例降低。轉(zhuǎn)染6.25、12.5nmol/LCalcyclinsiRNA后A2780細(xì)胞增殖指數(shù)分別為39.4和31.0，較空白對(duì)照組和陰性對(duì)照siRNA組顯著降低。 （4）Transwell試驗(yàn)結(jié)果表明，CalcyclinsiRNA分別轉(zhuǎn)染A2780細(xì)胞后穿膜細(xì)胞數(shù)目顯著減少，有統(tǒng)計(jì)學(xué)意義（p＜0.05）。這一結(jié)果提示隨著Calcyclin表達(dá)降低，A2780細(xì)胞侵襲能力也隨之明顯降低。 （5）MTT法檢測(cè)CalcyclinsiRNA轉(zhuǎn)染后A2780細(xì)胞對(duì)順鉑敏感性的影響，結(jié)果顯示CalcyclinsiRNA引起順鉑對(duì)A2780細(xì)胞的抑制率增加，即一定程度提高了A2780細(xì)胞對(duì)順鉑的敏感性。對(duì)順鉑半數(shù)致死劑量（IC50）的計(jì)算結(jié)果顯示，轉(zhuǎn)染6.25、12.5nmol/LCalcyclinsiRNA的A2780細(xì)胞對(duì)順鉑IC50值分別為13.42μM和11.32μM，和陰性對(duì)照siRNA組IC50值（18.12μM）均顯著降低。 結(jié)論 （1）免疫組化結(jié)果顯示Calcyclin表達(dá)水平與卵巢癌細(xì)胞分化程度、手術(shù)分期、腹水和淋巴結(jié)轉(zhuǎn)移有關(guān)，提示Calcyclin表達(dá)和與卵巢癌的發(fā)生發(fā)展、侵襲轉(zhuǎn)移和不良預(yù)后密切相關(guān)，有可能成為臨床評(píng)價(jià)病情進(jìn)展和隨訪的參考指標(biāo)。 （2）Calcyclin的血清濃度在卵巢癌患者中顯著升高，并與手術(shù)分期和細(xì)胞學(xué)分級(jí)相關(guān)，提示Calcyclin的血清濃度與卵巢癌病情發(fā)展相關(guān)。但本研究由于時(shí)間有限，病例樣本量較小，Calcyclin能否成為監(jiān)測(cè)腫瘤病情發(fā)展的分子標(biāo)志物還需要進(jìn)一步大樣本研究驗(yàn)證。 （3）CalcyclinsiRNA轉(zhuǎn)染卵巢癌A2780細(xì)胞后，引起A2780細(xì)胞G0/G1期阻滯，侵襲能力降低，同時(shí)對(duì)順鉑的敏感性有所增強(qiáng)。Calcyclin蛋白高表達(dá)對(duì)卵巢癌A2780細(xì)胞的惡性生物學(xué)行為有促進(jìn)作用，有可能成為卵巢癌靶向治療的新候選靶標(biāo)。
[Abstract]:objective
Ovarian epithelial carcinoma is one of the most common malignant tumors in women, and its mortality rate is the highest among the malignant tumors of the female reproductive system. The early symptoms of ovarian cancer patients are atypical, there is no effective early screening and specific diagnosis method, more than 70% of the patients are in the middle and advanced stage. In recent years, the 5-year survival rate of ovarian cancer patients is only about 30%-40%[42]. It is an urgent clinical problem to find effective gene therapy for ovarian cancer.
Calcyclin (Cacy) belongs to the S100 protein family. The expression of Calcyclin in pancreatic cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, colorectal cancer and other types of malignant tumors was significantly increased [3,27-32]. Overexpression is associated with metastasis and poor prognosis in pancreatic cancer, osteosarcoma, gastric cancer, lung adenocarcinoma, melanoma and other malignant tumors [28,38-40].
At present, the expression of Calcyclin protein in ovarian epithelial carcinoma and its clinical significance are still unclear. The purpose of this study is to analyze the correlation between the expression of Calcyclin protein and ovarian cancer at tissue level and serum level, and to explore the feasibility of Calcyclin as a gene therapy for ovarian cancer.
Method
(1) Immunohistochemical technique was used to detect the expression of Calcyclin in 60 ovarian cancer specimens. The relationship between Calcyclin and the development of ovarian cancer was analyzed statistically.
(2) Serum Calcyclin levels in normal serum, benign ovarian tumors and ovarian cancer patients were detected by ELISA, and the correlation between serum Calcyclin levels and ovarian cancer was statistically analyzed.
(3) After specific reduction of Calcyclin expression by RNA interference, we observed the changes of cell cycle, invasiveness and cisplatin sensitivity of ovarian cancer cells, and explored the feasibility of Calcyclin as a candidate gene for targeted therapy of ovarian cancer.
Result
(1) The expression of Calcyclin in ovarian cancer tissues was detected by immunohistochemistry. The results showed that the expression of Calcyclin in poorly differentiated ovarian cancer patients (55.0%) was significantly higher than that in well differentiated ovarian cancer patients (15.4%). The expression of Calcyclin was related to the stage of operation (P < 0.05). The expression of Calcyclin increased significantly with the increase of lymph node metastasis and ascites (P < 0.05). In ovarian cancer patients with ascites and lymph node metastasis, the expression of Calcyclin increased significantly (P < 0.001).
(2) Calcyclin serum concentration was detected by ELISA. The results showed that the average serum concentration of Calcyclin in ovarian cancer patients was 1242.47+178.17 pg/ml, which was significantly higher than that in normal serum (489.73+37.86 pg/ml) and benign ovarian tumor (700.75+43.14 pg/ml), and the difference was statistically significant (P 0.01). The results showed that the serum concentration of Calcyclin in patients with stage III and stage IV ovarian cancer was 644.79+67.89pg/ml and 1087.44+104.66 pg/ml, respectively, which was significantly higher than that in patients with stage I and stage II ovarian cancer (359.35+37.82 pg/ml and 499.44+42.56 pg/ml), and the difference was statistically significant (P 0.001). The serum concentration of Calcyclin in cancer patients was 1320.08+102.59 pg/ml, which was significantly higher than that in well-differentiated (573.58+37.28 pg/ml) and moderately differentiated (832.91+75.62 pg/ml) patients (P 0.05).
(3) Cell cycle analysis showed that the proportion of G0/G1 phase in A2780 cells increased and that of S phase decreased after specific reduction of Calcyclin expression by RNA interference technique. Group decreased significantly.
(4) Transwell assay showed that the number of transfected A2780 cells significantly decreased (p < 0.05). The results suggested that the invasive ability of A2780 cells decreased with the decrease of Calcyclin expression.
(5) MTT assay showed that the inhibition rate of cisplatin on A2780 cells was increased after transfection of CalcyclinsiRNA, that is to say, the sensitivity of A2780 cells to cisplatin was increased to a certain extent. The IC50 values of 2780 cells to cisplatin were 13.42 and 11.32 mu M, respectively. The IC50 values (18.12 mu M) of the negative control siRNA group were significantly decreased.
conclusion
(1) Immunohistochemistry showed that the expression of Calcyclin was correlated with the differentiation of ovarian cancer cells, surgical stage, ascites and lymph node metastasis, suggesting that the expression of Calcyclin was closely related to the occurrence and development of ovarian cancer, invasion and metastasis and poor prognosis, which might be a reference index for clinical evaluation of disease progression and follow-up.
(2) The serum concentration of Calcyclin was significantly increased in ovarian cancer patients and correlated with surgical stage and cytological grade, suggesting that the serum concentration of Calcyclin was associated with the development of ovarian cancer. Validation of large sample studies.
(3) After transfection of CalcyclinsiRNA into ovarian cancer A2780 cells, the G0/G1 phase arrest of A2780 cells was induced, and the invasiveness of A2780 cells was decreased, and the sensitivity to cisplatin was enhanced. The high expression of Calcyclin protein could promote the malignant biological behavior of ovarian cancer A2780 cells and might be a new candidate target for targeted therapy of ovarian cancer.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.31

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