【作者】 王娜；

【导师】 吴小华；

【作者基本信息】 河北医科大学 ， 妇产科学， 2013， 硕士

【摘要】 上皮性卵巢癌（epithelial ovarian cancer, EOC）在妇科恶性肿瘤中死亡率居于首位，由于缺乏早期诊断方法和预防筛查措施，约75%的患者发现时已属于晚期。目前卵巢癌的治疗采用手术联合化疗的综合治疗手段，尽管治疗手段的进步使卵巢癌患者的治疗取得了成效，但仍有60-80%的患者死于该病，最主要的原因就是术后高复发率及化疗耐药，因此，迫切需要一种新的治疗手段用于卵巢癌的临床治疗。表皮生长因子受体（Epidermal growth factor receptor, EGFR）信号通路引起了学者们的广泛关注。有证据显示，许多肿瘤中存在EGFR的过表达和异常活化，其中包括卵巢癌[1-2]，EGFR活化后通过激活PI3K/Akt及Ras/Raf/MEK/ERK等在内的下游信号通路参与肿瘤的增殖、凋亡、血管生成、粘附、侵袭、转移等过程[3]，另外，研究表明EGFR信号通路可能介导化疗耐药的形成。吉非替尼（Iressa）是一种EGFR小分子酪氨酸激酶抑制剂，通过与ATP竞争受体胞内区结合位点抑制EGFR的自身磷酸化过程。已有报道应用吉非替尼阻断EGFR信号通路的活化，可以增加化疗药物的敏感性。因此将EGFR信号通路作为药物作用靶点为逆转卵巢癌耐药提供了可能。目的：通过抑制EGFR信号通路的活化，观察其对顺铂耐药卵巢癌细胞株SKOV3/DDP增殖、凋亡的影响，及逆转顺铂耐药的效果，初步探讨EGFR信号通路的活化与顺铂耐药的关系。方法：1Western blot检测SKOV3及SKOV3/DDP细胞中EGFR、p-EGFR（活化的EGFR）蛋白的基础表达状态。2Western blot检测顺铂对SKOV3及SKOV3/DDP细胞中EGFR、p-EGFR蛋白表达的影响。3MTT法检测不同浓度的顺铂（0.625、1.25、2.5、5、10、20μg/ml）对SKOV3及SKOV3/DDP细胞增殖的影响，获得半数抑制浓度（IC50），并计算耐药指数。4MTT法检测不同浓度的吉非替尼（0.01、0.1、1、10、100μM）对SKOV3及SKOV3/DDP细胞增殖的影响。5MTT法检测1μM吉非替尼联合不同浓度的顺铂（0.625、1.25、2.5、5、10、20μg/ml）对SKOV3/DDP细胞增殖的影响，获得联用后顺铂的IC50，并计算耐药逆转倍数。6流式细胞术检测吉非替尼、顺铂、吉非替尼联合顺铂对卵巢癌细胞凋亡的影响。7Western blot检测吉非替尼、顺铂、吉非替尼联合顺铂对SKOV3/DDP细胞EGFR、p-EGFR及下游信号通路Akt、p-Akt（活化的Akt）、ERK、p-ERK（活化的ERK）蛋白表达的影响。8统计学方法：采用SPSS13.0统计软件进行数据统计，计量资料以均数±标准差表示，方差齐性检验，三组以上采用方差分析，组间差异采用LSD法，两组数据比较采用t检验，以α﹦0.05为检验水准，当P＜0.05时，差异有统计学意义；当P＞0.05时，差异无统计学意义。结果：1Western blot检测SKOV3及SKOV3/DDP细胞的EGFR、p-EGFR蛋白表达结果：SKOV3及SKOV3/DDP细胞中均存在EGFR及p-EGFR蛋白的表达，EGFR蛋白在亲本株与耐药株的表达量无显著差别（P>0.05），SKOV3/DDP细胞中p-EGFR蛋白的表达显著高于SKOV3细胞的表达，差异有统计学意义（P <0.05）。2Western blot检测顺铂对SKOV3及SKOV3/DDP细胞的EGFR、p-EGFR蛋白表达结果：顺铂处理前后，SKOV3及SKOV3/DDP细胞中EGFR蛋白的表达量无显著差别（P>0.05），顺铂处理后的SKOV3及SKOV3/DDP细胞中p-EGFR蛋白的表达量相比于顺铂未处理组的细胞明显增高，差异有统计学意义（P <0.05）。3MTT法检测顺铂对SKOV3及SKOV3/DDP细胞增殖影响的结果：顺铂对SKOV3细胞的IC50为3.74±0.03，SKOV3/DDP细胞的IC50为17.66±0.58，耐药指数为4.72。4MTT法检测吉非替尼对SKOV3及SKOV3/DDP细胞增殖影响的结果：吉非替尼可以抑制SKOV3及SKOV3/DDP细胞的生长，这种抑制作用呈剂量依赖性，当浓度为1μM时，抑制作用明显增加。同一浓度下吉非替尼对SKOV3细胞的抑制率高于SKOV3/DDP细胞的抑制率，差异有统计学意义（P <0.05）。5MTT法检测吉非替尼联合顺铂对SKOV3/DDP细胞增殖影响的结果：与单独应用顺铂相比，1μM的吉非替尼联合顺铂可增加对SKOV3/DDP细胞的抑制作用，联合作用后顺铂的IC50为7.42±0.15，逆转耐药倍数为2.38。6流式结果：在SKOV3/DDP细胞中，吉非替尼、顺铂、吉非替尼+顺铂的凋亡率分别为(7.52±0.63)%、(8.13±0.91)%、(17.33±0.84)%，均高于对照组（未加药物处理组）的凋亡率(0.28±0.12）%，差异有统计学意义（P <0.05），且联合组的凋亡率均高于单一用药组（P <0.05），但仍低于顺铂作用于SKOV3细胞诱导的凋亡率（23.03±1.23）%（P <0.05）。7Western blot检测不同处理组对SKOV3/DDP细胞EGFR、p-EGFR、 Akt、p-Akt、ERK、p-ERK蛋白表达的影响结果：与对照组相比，顺铂、吉非替尼，顺铂联合吉非替尼处理SKOV3/DDP细胞，EGFR、Akt及ERK蛋白表达量无显著差别（P>0.05）。顺铂处理后，p-EGFR及p-Akt、p-ERK蛋白的表达量增加，与对照组相比差异有统计学意义（P<0.05）。吉非替尼处理后，EGFR及Akt、ERK的活性降低，与对照组相比差异有统计学意义（P<0.05）。吉非替尼与顺铂联合处理后，p-EGFR及p-Akt、p-ERK蛋白的表达量显著减少，与单用顺铂组相比差异有统计学意义（P<0.05）。结论：1SKOV3/DDP细胞中p-EGFR蛋白的表达量显著高于SKOV3细胞中的表达，提示EGFR信号通路的异常活化可能参与了卵巢癌化疗耐药的过程。2顺铂可以诱导EGFR信号通路的活化，降低顺铂对卵巢癌的细胞毒作用，应用EGFR酪氨酸激酶抑制剂吉非替尼可以协同顺铂增加SKOV3/DDP细胞的敏感性，部分逆转顺铂耐药，这可能与吉非替尼抑制顺铂诱导的EGFR信号通路的激活及Akt，ERK的活性有关。更多还原

【Abstract】 Epithelial ovarian cancer (EOC) is one of the most frequent maliganttumors in the female population. Because of a lack of early diagnosis andpreventive screening measures, approximately70%of all patients withovarian cancer are diagnosed at an advanced stage. The current managementof patients with advanced disease involves optimal surgical debulkingfollowed by chemotherapy. Although this treatment is highly effective,60-80%of women still die of this disease. The main reasons for poorprognosis are a high recurrence rate and resisitance to chemotherapeutics.Therefore, the development new therapy is critical for treatment of ovariancancer patients.Epidermal growth factor receptor (EGFR) signaling pathway causeswidespread concern of scholars. Accumulating evidence suggests that highexpression and constitutional activation of EGFR can be detected in mostcancer including ovarian cancer. EGFR signaling pathway is involved in manycellular process including cell proliferation, apoptosis, angiogenesis, adhesion,invasion and migration via the activation of PI3K/Akt and Ras/Raf/MEK/ERKdownstream signaling pathway. In addition, EGFR may mediatechemoresistance. Gefitinib (Iressa) is an EGFR-tyrosine kenase inhibitor(EGFR-TKI) that competitively inhibits binding of ATP at the ATP site onEGFR, and evidence suggests gefitinib can cause chemosensization in humantumors. Thus EGFR is a promising target for reversing the chemoresistance inovarian cancer.Objective:To investigate the relationship between activity of epidermal growthfactor receptor and the cisplatin resistance in ovarian cancer SKOV3/DDP cells.Methods:1The baseline expression of epidermal growth factor receptor (EGFR) andphosphorylated epidermal growth factor receptor (p-EGFR) protein in SKOV3and SKOV3/DDP cells were analyzed by Western blot.2The expression of EGFR, p-EGFR protein of SKOV3and SKOV3/DDPcells treated with cisplatin were analyzed by Western blot.3The cell proliferation of SKOV3and SKOV3/DDP cells treated withcisplatin at increasing concentrations (0.625,1.25,2.5,5,10,20μg/ml) weremeasured by MTT assay respectively, and the resistance index was calculated.4The cell proliferation of SKOV3and SKOV3/DDP cells treated withgefitinib at increasing concentrations (0.01,0.1,1,10,100μM) were measuredby MTT assay respectively.5The cell proliferation of SKOV3/DDP cells treated with gefitinib (1μM) andcisplatin (0.625,1.25,2.5,5,10,20μg/ml) combined were measured by MTTassay.6The apoptosis was evaluated by flow cytometry with gefitinib and cisplatinalone or combined in ovarian cancer cells.7The expression of EGFR, p-EGFR, Akt, p-Akt, ERK, p-ERK protein ofSKOV3/DDP cells with gefitinib and cisplatin alone or combined wereanalyzed by Western blot.8Statistical methods: Data were evaluated using SPSS13.0statistical software,and data were expressed as mean±standard deviation. The significancedifference was determined using analysis of variance, LSD method and the ttest. P <0.05was considered significant.Results:1Western blot analysis showed that the p-EGFR expression was significantlyenhanced in SKOV3/DDP compared with SKOV3cells (P＜0.05). However,there was no significant difference between the EGFR expression ofSKOV3/DDP and SKOV3cells (P>0.05).2Western blot analysis showed that compared with untreated cells, the p-EGFR expression was significantly enhanced with cisplatin treated inSKOV3and SKOV3/DDP cells (P＜0.05).3The inhibition effects of cisplatin on the SKOV3and SKOV3/DDP cellswere determined by MTT assays, yielding IC50value of3.74±0.03μg/ml forthe SKOV3cells and IC50value of17.66±0.58μg/ml for the SKOV3/DDPcells. The resistance index was4.72.4MTT assays showed that gefitinib could inhibit the growth of the SKOV3and SKOV3/DDP cells in dose-dependent manner. The inhibition rate ofSKOV3cells, induced by gefitinib, was significantly higher than that ofSKOV3/DDP cells and there was significant difference (P＜0.05).5MTT assays showed that the combined treatment of gefitinib and cisplatinsignificantly enhanced the antiproliferative effect compared with single-agenttreatment and there was significant difference (P＜0.05). The IC50value ofcombination of cispaltin with gefitinib to SKOV3/DDP cells was7.42±0.15,and the drug resistance reversal fold was2.38.6The Flow cytometry showed that the apoptosis rate of cisplatin+gefitinib,was significantly higher than that of cispaltin or gefitnib alone (P＜0.05), andthere was no significant difference between the apoptosis rate of cisplatin andgefitinib in SKOV3/DDP cells (P＞0.05). Compared cisplatin with cisplatin+gefitinib, the rate of apoptosis was significant difference in SKOV3/DDP cells(P <0.05) and compared the apoptosis rate of the SKOV3cells induced bycisplatin with that of the SKOV3/DDP cells, there was significant difference(P <0.05).7Western blot analysis showed gefitnib treatment could inhibit EGFR anddownstream signaling pathways Akt, ERK activation. The cisplatin treatmentupregulated the levels of p-EGFR, p-Akt, p-ERK protein expression. Whengefitinib was administered with cisplatin, their expressions weredownregulated.Conclusions:1p-EGFR is significantly enhanced in the SKOV3/DDP cells compared withthe SKOV3cells, proving the activation of EGFR signaling pathway may be involved in chemoresistance in SKOV3/DDP cells.2Cisplatin can activate EGFR signalling pathway, which decreases thechemotherapy sensitivity of ovarian cancer SKOV3/DDP cells. Gefitinib isable to block the activity of EGFR, Akt, ERK，and sensitise SKOV3/DDPcells to chemotherapy. Inhibiting EGFR activation may reverse the cisplatinresistance in ovarian cancer cells.更多还原