纤维连接蛋白在卵巢癌多细胞团簇中的作用和顺铂对团簇的影响

The Function of Fibronectin in the Formation of Ovarian Cancer Multicellular Aggregates and the Effect of Cisplatin on Aggregates

目的:探讨纤维连接蛋白(FN)对卵巢癌多细胞团簇(MCA)的影响和涉及的整合素受体的调节作用,以及顺铂(DDP)对MCA的影响。方法:用液体重叠技术培养SKOV3和OVCAR-3两种卵巢癌细胞株,获得MCA后用不同浓度的FN进行干预,观察FN对MCA的作用并研究DDP对两种MCA的影响;运用流式细胞术和RT-PCR检测整合素受体α3β1、α4β1、α5β1的表达情况。裸鼠建立人卵巢癌腹水瘤模型后用FN及DDP进行干预,分析两者对裸鼠体内肿瘤和腹水形成的影响,进一步证实MCA形成的相关因素。结果:(1)培养SKOV3和OVCAR-3两种MCA的最佳FN浓度分别为10μg/ml和20μg/ml。(2) SKOV3的MCA和单层细胞DDP的半数抑制浓度(IC50)为20.29μmol/L和29.80μmol/L,OVCAR-3的MCA和单层细胞DDP的IC50为33.11μmol/L和41.69μmol/L; DDP处理前两种卵巢癌细胞株的单层细胞和MCA各细胞周期差异均有统计学意义(P<0.05),DDP作用后MCA凋亡率均明显增加(P<0.05)。(3)流式细胞术、RT-PCR检测显示两种细胞主要表达α4β1(P<0.05),MCA组各整合素均高于单层细胞组(P<0.05)。加入FN后,各整合素表达量均明显增加(P<0.05)。(4)裸鼠干预结果显示,FN组的肿瘤总数和腹水量均超过0.9%氯化纳液组和DDP组(P<0.05),而DDP能有效抑制肿瘤生长和腹水形成(P<0.05)。结论:体外实验说明FN对卵巢癌MCA的形成有明显的促进作用,两种细胞株表面均有整合素受体α3β1、α4β1、α5β1的表达,以α4β1为主,FN可促进它们表达。动物实验证实FN对体内种植的肿瘤细胞也具有明显的促生长作用,而DDP能有效抑制肿瘤生长和腹水形成。

Objective:To investigate the role of fibronectin (FN)on ovarian cancer multicellular aggregate (MCA),the regulation effect of integrin receptors involved and the effect of cisplatin (DDP)on MCA.Methods: MCA of two ovarian cancer cell lines,SKOV3 and OVCAR-3,was obtained by liquid overlay technique.The effect of FN on MCA was investigated by treatment with different concentrations of FN.The effect of DDP on these two different MCA was also determined.The expressions of integrin receptors α3β1 ,α4β1 and α5β1 in two kinds of cells were detected by flow cytometry and RT-PCR.The effects of FN and DDP on tumorous and ascetic formation were analyzed by treatment of nude mice ascites model with FN and DDP.The related factors of MCA formation were further confirmed.Results:①The optimum FN concentrations for the culture of SKOV3 and OVCAR-3 MCAs were 10 μg/ml and 20 μg/ml,respectively.②The IC50 of monolayer DDP and MCA for SKOV3 were 20.29 μmol/L and 29.80 μmol/L,while those for OVCAR-3 were 33.11 μmol/L and 41.69 μmol/L,respectively. There were significant differences between monolayer cell group and MCA group in all cell cycles of both cell lines before DDP treatment (P<0.05).Significant increase of apoptosis of both MCA were detected after treatment with DDP(P<0.05).③Flow cytometry and RT-PCR detection showed that the mainly expressed integrin receptor was α4β1 in both cell lines(P<0.05).The expression of each integrin in MCA group was significantly higher than that in the monolayer cell group(P<0.05).The expression of integrin receptors increased significantly after adding FN(P<0.05).④The nude mice model showed that the total tumor numbers and ascites volume in FN group were more than those in saline group and DDP group significantly(P<0.05).It was also showed that DDP could effectively inhibit tumor growth and ascites formation (P<0.05).Conclusions:These studies showed that FN could promote the formation of ovarian cancer MCA in vitro.The expression of integrin receptor α3β1 ,α4β1 and α5β1 was detected in both cells lines with α4β1 as the main factor.FN could promote the expression of those receptors.Ovarian tumor and ascites model in nude mice showed that FN could significantly facilitate the growth of tumor cells in vivo and DDP could effectively inhibit tumor growth and ascites formation.