促性腺激素釋放激素激動劑以及聯(lián)合順鉑對卵巢癌裸鼠移植瘤化療效果的影響及分子機(jī)制的研究

1、促性腺激素釋放激素激動劑以及聯(lián)合順鉑對卵巢癌裸鼠移植瘤化療效果的影響及分子機(jī)制的研究         11-05-21 16:12:00     編輯：studa20                   作者：翁慧男, 王沂峰,林瓊燕  【摘要】  目的 探討單獨應(yīng)用促

2、性腺激素釋放激素激動劑(gonadotropin-releasing hormone agonist,GnRHa)及與順鉑聯(lián)合應(yīng)用對卵巢癌裸鼠移植瘤化療效果的影響及其分子機(jī)制。方法 將24只裸鼠腹腔種植低分化卵巢漿液性囊腺癌細(xì)胞株OVCAR-3，成瘤后隨機(jī)分為4組，即對照組(NS)、順鉑組、GnRHa組(GnRHa+NS)、聯(lián)合用藥組(GnRHa+順鉑)。剝?nèi)∧[瘤組織稱重，透射電鏡檢測各組腫瘤組織中細(xì)胞凋亡情況，免疫組織化學(xué)法檢測腫瘤組織中ki67、NFkb及caspase-3表達(dá)水平。結(jié)果 對照組中未見到明顯凋亡細(xì)胞及凋亡小體;順鉑組以核邊集固縮的早期凋亡細(xì)胞為主;GnRHa+NS組可見凋亡

3、細(xì)胞及壞死溶解細(xì)胞;聯(lián)合用藥組以中晚期凋亡細(xì)胞為主。腫瘤重量及ki67評分：對照組分別為(0.149±0.072)g、(9.533±2.380);順鉑組(0.034±0.010)g、(6.283±0.491);GnRHa組(0.116±0.039)g、(8.733±0.531);聯(lián)合用藥組(0.026±0.024)g、(7.70±0.745)。統(tǒng)計結(jié)果，順鉑組和聯(lián)合用藥組腫瘤重量及ki67分別與對照組相比，差異有顯著性(P<0.05);而GnRHa組與對照組腫瘤重量及ki67比較、順鉑組和聯(lián)合用藥組腫瘤重量及

4、ki67比較差異均無顯著性(P>0.05)。順鉑組與聯(lián)合用藥組腫瘤重量及ki67均明顯低于其余兩組，差異有顯著性(P<0.05);與對照組比較，順鉑組、GnRHa組、聯(lián)合用藥組NFkb表達(dá)減弱，caspase-3表達(dá)增強，差異均有顯著性(P<0.05)。聯(lián)合用藥組NFkb表達(dá)量比順鉑組減少，差異具有顯著性(P<0.05)。結(jié)論 單獨應(yīng)用GnRHa不影響腫瘤細(xì)胞的增殖;聯(lián)合應(yīng)用GnRHa及順鉑不影響順鉑對卵巢癌細(xì)胞的抗增殖作用;單獨應(yīng)用GnRHa、順鉑及GnRHa與順鉑聯(lián)合應(yīng)用均能誘導(dǎo)卵巢癌裸鼠移植瘤腫瘤細(xì)胞的凋亡。下調(diào)NFkb表達(dá)、上調(diào)caspase-3表達(dá)可能為誘導(dǎo)腫

5、瘤細(xì)胞凋亡的分子機(jī)制之一。 【關(guān)鍵詞】  卵巢腫瘤;促性腺激素釋放激素激動劑;順鉑;細(xì)胞凋亡Abstract objective To explore the anti-proliferation and apoptosis inducing effects of GnRHa ,cisplatin or both on the ovarian cancer model of athymic mice and their related molecular mechanisms.Methods Twenty-four nu/nu athymic mice were injected i

6、ntraperitoneally with human ovarian serous cystadenocarcinoma cells of the line OVCAR-3 and then randomly divided into 4 groups:control group (n=6,used as controls),CDDP group (n=6,receiving NS 0.2ml after inoculation, CDDP 4 weeks after 2 weeks of the injection of NS),GnRHa group(n=6,receiving GnRH

7、a + NS) and GnRHa + CDDP group(n=6,receiving GnRHa + CDDP). At the end of the experiment,mice were killed,and the tumor tissue was excised and histochemical analysis.Transmissino electron microscopy detect the apoptosis of the tumor tissue . The protein levels of ki67,NFkb and caspase-3 in the tumor

8、 tissue was detected by immunohistochemistry.Results There was no apoptosis cell or apoptosis body in control group.The viable apoptotic cells were observed in the cisplatin group.Apotosis cells and necrosis dissolve cells were seen in GnRH a+NS group.In combined group there were mid and non-viable

9、apoptotic cells. The immunohistochemistryscore of ki67 and the weight of the ovarian cancer of the CDDP group and the combination group were lower than the control group and the GnRHa group,but there were no significantly difference between the CDDP group and the combination group(P>0.05).Compare

10、d with the control group,the protein level of NFkb was decreased while caspase-3 expression was increased in the other three groups(P>0.05).The level of NFkb in combined group was lower than the group of cisplatin,which had significantly difference (P<0.05).Conclusion GnRHa combined with CDDP

11、in the nu/nu athymic mice ovarian cancer model do not influence the anti-proliferation of CDDP to the ovarian cancer.GnRHa ,cisplatin or both of them could also induce the apoptosis through up-regulating caspase-3 and down-regulating expression of NFkb on the ovarian cancer model of athymic mice,whi

12、ch probably one of its molecular mechanisms.Key words ovarian cancer;gonadotropin-releasing hormone agonist;cisplatin;anti-proliferation apoptosis卵巢癌目前仍為女性惡性腫瘤死亡率最高的疾病，且發(fā)病年齡日趨年輕化。傳統(tǒng)手術(shù)治療、放化療及生物治療等治療措施療效有限，不良反應(yīng)大，5年生存率徘徊在30%左右。目前有報道在腫瘤化療過程中，使用GnRHa有卵巢功能保護(hù)的作用。而順鉑是卵巢癌標(biāo)準(zhǔn)化療方案的用藥之一。目前尚未有研究聯(lián)合使用GnRHa及順鉑對腫瘤細(xì)胞化

13、療的相關(guān)影響。在體外實驗方面，有研究證實GnRHa能直接抑制卵巢癌細(xì)胞增殖1，并呈時間劑量依賴性2，3。而在凋亡方面，GnRHa是否能誘導(dǎo)腫瘤細(xì)胞凋亡，與順鉑聯(lián)合用藥是否有協(xié)同作用尚未清楚。本研究擬建立卵巢癌裸鼠移植瘤模型，探討體內(nèi)單獨應(yīng)用GnRHa、GnRHa與順鉑聯(lián)合應(yīng)用對卵巢癌裸鼠移植瘤模型腫瘤細(xì)胞凋亡及增殖的影響及可能的分子機(jī)制。現(xiàn)報道如下。1 材料與方法1.1 細(xì)胞株和裸鼠 人卵巢漿液性乳頭狀囊腺癌細(xì)胞株OVCAR-3購自中國科學(xué)院細(xì)胞庫，46周齡成年雌性BALB/c-nu裸鼠24只，體重1416g。購自廣東省醫(yī)學(xué)實驗動物研究中心動物許可證號：SYXK(粵)2005-0058，中山大

14、學(xué)眼科中心SPF實驗動物部飼養(yǎng)，恒溫(25±2)，恒濕45%50%，飼養(yǎng)于無菌實驗室的凈化空氣層流架中。順鉑(CDDP)為齊魯制藥廠產(chǎn)品;醋酸曲普瑞林(達(dá)菲林)為博福-益普生(天津)制藥有限公司產(chǎn)品;高糖型DMEM培養(yǎng)基、胰蛋白酶、胎牛血清為美國GIBCO公司產(chǎn)品;免疫組化試劑(北京中杉金橋)一抗為NFkb抗體及caspase-3抗體(廣州杰特偉生物科技有限公司)。選擇即用型免疫組化(IHC)二步法染色試劑盒(北京中杉金橋);透射電鏡型號為日本電子株式會社(JELO100CX)。1.2 細(xì)胞培養(yǎng) 人卵巢癌細(xì)胞株OVCAR-3在高糖型DMEM培養(yǎng)基中培養(yǎng)至對數(shù)生長期，0.25%胰蛋白酶

15、消防立管化3min，終止反應(yīng)，制備成單細(xì)胞懸液，細(xì)胞計數(shù)，調(diào)整細(xì)胞密度為2×107/ml。1.3 動物接種及實驗分組 在SPF環(huán)境超凈工作臺內(nèi)，以75%酒精消毒裸鼠腹部皮膚，腹腔接種0.2ml細(xì)胞懸液，觀察動物成瘤情況，接種后第10天可觀察到裸鼠腹部稍膨隆，活動減少，腹腔進(jìn)針處可見米粒樣大小結(jié)節(jié)。腹腔腫瘤原代培養(yǎng)細(xì)胞形態(tài)與OVCAR-3細(xì)胞形態(tài)一致。遂將24只裸鼠隨機(jī)分為4組每組6只：即對照組腹腔注射生理鹽水(NS)0.2ml/w×5w、順鉑組(第一天腹腔注射NS 0.2ml,2周后腹腔注射順鉑5mg/w×4w)、GnRHa+NS組(GnRHa 0.3mg皮下注射，2周后NS 0.2ml/w腹腔注射×4w)、聯(lián)合用藥組(GnRHa 0.3mg皮下注射，2周后順鉑腹腔注射5mg/w×4w)。1.4 觀察指標(biāo)1.5 統(tǒng)計學(xué)處理 數(shù)據(jù)以(x±s)表示，運用SPSS