YKL-40分子对卵巢癌SKOV-3细胞体外迁移能力和化疗敏感性的影响

发布时间：2018-03-20 12:00

本文选题：卵巢癌　切入点：YKL-40　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：卵巢癌是妇科三大常见恶性肿瘤之一,上皮性卵巢癌(EOC)为主要病理类型(85%以上),死亡率高居女性肿瘤相关性死亡之首。上皮性卵巢癌恶性程度高,早期症状隐匿,且极易发生侵袭转移,致使70%的患者在确诊时即已出现盆腹腔器官的侵袭播散和(或)远处脏器的转移。目前,最大程度的外科减瘤术和术后铂类、紫杉醇的联合化疗是上皮性卵巢癌的主要治疗手段,并可使大部分的患者获得缓解。但仍有75%-80%的晚期患者在治疗后复发,并在之后进行的重复循环的化疗过程中发生获得性耐药,出现对铂类等一线化疗方案药物敏感性的降低。上皮性卵巢癌的高侵袭转移特性以及耐药的出现,是阻碍患者预后、导致患者高死亡率的重要原因,严重威胁了女性的健康。因此,探讨并阐明影响卵巢癌转移和化学耐药的参与分子和机制具有重要意义。YKL-40,又被称作壳质酶3样1(CHI3L1),是一种分泌型糖蛋白,在乳腺癌、肺癌、脑胶质瘤等多种恶性肿瘤中高表达。研究表明,YKL-40可通过多种机制参与恶性脑胶质瘤、子宫内膜癌等肿瘤细胞的侵袭转移过程。另有研究证实,YKL-40分子可通过AKT/ERK通路介导耐替莫唑胺(TMZ)的脑胶质瘤细胞株(TMZ-R U87)对替莫唑胺的耐药。然而,YKL-40是否可影响EOC细胞的迁移能力及对铂类药物的敏感性,还尚不明确。本研究选用经典的EOC细胞系SKOV-3细胞作为研究对象,共分三个部分,分别从体外验证YKL-40分子对上皮性卵巢癌细胞的迁移能力以及铂类药物敏感性的影响,并探讨相关调控因子。第一部分 YKL-40在卵巢癌SKOV-3细胞的表达[目的]检测EOC细胞SKOV-3中YKL-40分子的表达。[方法]选用胶质母细胞瘤U87细胞作为阳性对照,采用RT-PCR、ELISA、Western Blot试验,分别从mRNA和蛋白水平检测SKOV-3细胞中YKL-40的表达水平。[结果]在阳性对照U87细胞中,RT-PCR及ELISA可检测到YKL-40的显著表达,其琼脂糖凝胶电泳可见YKL-40 mRNA扩增产物,细胞上清分泌水平的表达量为(18.41±1.69)ng/mL;而在SKOV-3细胞,无论mRNA水平还是分泌蛋白水平,均未检测到YKL-40的表达。[结论]YKL-40在SKOV-3细胞无明显表达。第二部分 YKL-40对卵巢癌SKOV-3细胞体外迁移能力的影响[目的]探讨YKL-40对EOC细胞SKOV-3体外迁移能力的影响及相关分子机制。[方法]根据第一部分检测结果,构建YKL-40过表达慢病毒,采用慢病毒感染的方法构造过表达YKL-40的SKOV-3细胞株LV-SKOV-3细胞,和空载体阴性对照细胞株 NC-SKOV-3,qRT-PCR、ELISA、Western Blot 验证 YKL-40过表达情况;Transwell试验检测YKL-40过表达对SKOV-3迁移能力的影响;特异性抑制剂阻滞过表达细胞中p38 MAPK通路,检测细胞迁移能力的改变。[结果]1.成功构建YKL-40过表达细胞株LV-SKOV-3及空载体阴性对照细胞株NC-SKOV-3。qRT-PCR、ELISA、Western Blot 检测到 LV-SKOV-3 细胞 YKL-40表达显著增强,其mRNA的相对表达量为NC-SKOV-3细胞的9.89倍,蛋白分泌量达(32.35±1.38)ng/ml,为 NC-SKOV-3 细胞的 17.43 倍。2.LV-SKOV-3细胞的迁移数量平均为(255.75士 12.75)个/视野,NC-SKOV-3细胞为(119.25±13.25)个/视野。LV-SKOV-3细胞的迁移数量上调为空载体阴性对照细胞的1.14倍。3.抑制p38 MAPK通路,可部分下调LV-SKOV-3的迁移能力。Transwell试验结果示,抑制剂组LV-SKOV-3细胞的迁移数量较DMSO对照组减少,为对照组的 63.30%。[结论]YKL-40分子可显著增强上皮性卵巢癌SKOV-3细胞的迁移能力,且这一作用可能是通过p38 MAPK通路介导的。第三部分 YKL-40对卵巢癌SKOV-3细胞化疗药物敏感性的影响[目的]探讨YKL-40对卵巢癌SKOV-3细胞铂类药物敏感性的影响及相关分子机制。[方法]Western Blot试验检测LV-SKOV-3细胞中NF-κB分子的表达;NF-κB 特异性抑制剂、ELISA、Western Blot 检测 NF-κB 对 SKOV-3 细胞 YKL-40蛋白表达的调控;分别采用不同浓度(1μg/ml、2.5μg/ml、5μg/ml、1.0μg/ml)的顺铂(DDP)处理YKL-40过表达LV-SKOV-3细胞和空载体阴性对照NC-SKOV-3细胞48h,CCK-8试验检测细胞的抑制率;抑制剂Bay11-7082特异性抑制LV-SKOV-3细胞中的NF-κB,CCK-8试验再次检测细胞LV-SKOV-3对DDP敏感性的变化。[结果]1.Western Blot结果显示,LV-SKOV-3细胞中NF-κB蛋白表达水平较阴性对照NC-SKOV-3细胞增高。2.抑制 NF-κB 分子后,ELISA、Western Blot 检测到 LV-SKOV-3 细胞 YKL-40的表达下降,其细胞上清分泌水平为(25.40±3.30)ng/ml,是DMSO对照组的0.64倍。其细胞YKL-40蛋白的相对表达量为DMSO组的0.68倍。3.经不同浓度DDP处理后,CCK-8检测均表现为细胞LV-SKOV-3的抑制率较NC-SKOV-3降低。这说明,YKL-40过表达可降低DDP对卵巢癌细胞SKOV-3的杀伤力。4.抑制NF-κB后,在各浓度梯度DDP作用下,抑制剂组LV-SKOV-3细胞的抑制率较DMSO组增高,但仍低于NC-SKOV-3细胞组。这说明,抑制NF-κB可部分恢复顺铂对SKOV-3细胞的杀伤力。[结论]1.NF-κB可调控上皮性卵巢癌SKOV-3细胞中YKL-40的表达。2.YKL-40可降低上皮性卵巢癌SKOV-3细胞铂类药物的敏感性,且这一过程部分受到NF-κB因子的调控。
[Abstract]:Ovarian cancer is one of the three most common gynecologic malignant tumor, epithelial ovarian cancer (EOC) is the main pathological type (more than 85%), the highest mortality of tumor related death in women with epithelial ovarian cancer. The first high degree of malignancy, early symptoms and occult, prone to invasion and metastasis, resulting in 70% of the patients at the time of diagnosis is occurred in the pelvic organ invasion and dissemination and (or) distant metastasis of organs. At present, the greatest degree of surgical cytoreductive surgery and postoperative chemotherapy of paclitaxel combined with platinum, is the main method for the treatment of epithelial ovarian cancer, and can make the most of the patients in remission. But there is still a relapse in patients with advanced 75%-80% after treatment, the acquired resistance occurred repeatedly during chemotherapy and after in reduced to platinum as first-line chemotherapy drug sensitivity. High invasive characteristics and the emergence of multidrug resistance in epithelial ovarian cancer, is An important cause of hindering the prognosis of the patients, patients with high mortality, a serious threat to women's health. Therefore, to explore and illustrate the effects of molecules involved in ovarian cancer metastasis and chemical resistance and mechanism of.YKL-40 has an important significance, also known as chitinase 3 1 (CHI3L1), is a secreted glycoprotein in breast cancer, lung cancer and, high expression of brain glioma and other malignant tumors. Studies show that YKL-40 may be involved in malignant glioma through a variety of mechanisms, endometrial cancer and other tumor cell invasion and metastasis. Another study confirmed that YKL-40 molecules can guide resistance to temozolomide via the AKT/ERK pathway (TMZ) of brain glioma cells strain (TMZ-R U87) resistance to temozolomide. However, whether YKL-40 can influence the migration ability of EOC cells and the sensitivity to platinum drugs, also is not clear. This research used the EOC cell line SKOV-3 classic as the research object, Is divided into three parts, respectively from the in vitro validation of YKL-40 molecules on epithelial ovarian cancer cell migration and the effects of platinum drug sensitivity, and to explore the related regulatory factors. The first part of YKL-40 in the expression of SKOV-3 [Objective] to detect EOC cells of ovarian cancer SKOV-3 cells in YKL-40 molecule expression. Methods with glioblastoma U87 cells were used as positive control, using RT-PCR, ELISA, Western Blot test, the expression level of SKOV-3 cells. The results from the detection of mRNA and protein levels in YKL-40 in the positive control U87 cells, RT-PCR and ELISA can detect the expression of YKL-40 significantly, the agarose gel electrophoresis showed YKL-40 mRNA amplification, expression the supernatant secretion was (18.41 + 1.69) ng/mL; while in SKOV-3 cells, whether mRNA or protein level was not detected. Conclusion the expression of YKL-40]YKL-40 in SKOV-3 cells No significant cell expression. In the second part, the influence of YKL-40 on migration of ovarian cancer SKOV-3 cells in vitro [Objective] to explore the method. The effect of YKL-40 on the migration ability of EOC cells SKOV-3 in vitro and its molecular mechanism. According to the first part of the detection result, construct YKL-40 lentivirus expressing, using lentivirus infection constructed over expression SKOV-3 cell line LV-SKOV-3 cell YKL-40, empty vector and negative control cell lines NC-SKOV-3, qRT-PCR, ELISA, Western Blot confirmed the YKL-40 over expression; Transwell test to detect the effect of overexpression of YKL-40 on the migration ability of SKOV-3; the specific inhibitor of p38 pathway in cell block MAPK expression changes. Results the ability to detect cell migration]1. successfully constructed YKL-40 the expression of LV-SKOV-3 cells and empty vector control cells NC-SKOV-3.qRT-PCR, ELISA, Western Blot LV-SKOV-3 was detected in YKL-40 cells The expression was significantly enhanced and the relative expression amount of mRNA was 9.89 times higher than that of NC-SKOV-3 cells, protein secretion of (32.35 + 1.38) ng/ml, the number of NC-SKOV-3 cell migration of.2.LV-SKOV-3 cells was 17.43 times the average (255.75 + 12.75) / field, NC-SKOV-3 cells (119.25 + 13.25) / field in.LV-SKOV-3 cells the number of up to 1.14 times the migration of.3. empty vector negative control cells inhibition of p38 MAPK pathway, the migration ability of.Transwell test of down-regulation of LV-SKOV-3 in migration inhibitor group in LV-SKOV-3 cells compared with DMSO control group, the control group 63.30%.[conclusion]YKL-40 can significantly enhance the migration of epithelial ovarian cancer SKOV-3 cells. And this effect may be mediated by p38 MAPK pathway. In the third part, the influence of YKL-40 on sensitivity of ovarian cancer SKOV-3 cells [Objective] to investigate the chemotherapy of ovarian YKL-40 The expression of NF- kappa B. Molecular cancer SKOV-3 cells influence the sensitivity of platinum and related molecular mechanism of]Western Blot test in LV-SKOV-3 cells; NF- kappa B inhibitor, ELISA, Blot detection of NF- regulation of Western kappa B on the expression of SKOV-3 protein in YKL-40 cells; were collected with different concentrations (1 g/ml, 2.5 g/ml, 5 g/ml, 1 g/ml) and cisplatin (DDP) treatment of YKL-40 cells overexpressing LV-SKOV-3 and empty vector negative control NC-SKOV-3 cell 48h, CCK-8 test cell inhibition rate; inhibitor Bay11-7082 specific inhibition of LV-SKOV-3 cells in NF- kappa B, CCK-8 test results. LV-SKOV-3 cells again change the sensitivity to DDP the results show]1.Western Blot, LV-SKOV-3 cells expression of NF- kappa B protein levels compared with negative control NC-SKOV-3 cells increased.2. inhibition of NF- kappa B molecules, ELISA, Western Blot detected LV-SKOV-3 cell YKL- 40 drop in expression, its secretion level of cell supernatant (25.40 + 3.30) ng/ml, DMSO is 0.64 times higher than the control group. The cell YKL-40 expression of the protein is 0.68 times.3. in group DMSO were treated with different concentrations of DDP, CCK-8 detection showed inhibitory rate of LV-SKOV-3 cells is lower than NC-SKOV-3. This shows that YKL-40, over expression of DDP can be reduced on ovarian cancer cells SKOV-3.4. inhibition of NF- kappa B after the destruction, in the different concentration of DDP, the inhibition rate of LV-SKOV-3 cells was higher than that of DMSO group, but still lower than NC-SKOV-3 cell group. This shows that the inhibition of NF- kappa B partially restored cisplatin on SKOV-3 cell killing. Conclusion]1.NF- kappa B can express.2.YKL-40 YKL-40 regulation of epithelial ovarian cancer cell SKOV-3 can reduce the sensitivity of epithelial ovarian cancer cell SKOV-3 platinum drugs, and this process is regulated by NF- factor kappa B.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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