硼替佐米联合5-氟尿嘧啶对Hela细胞的抑制作用及其机制的研究

发布时间：2018-06-17 00:19

  本文选题：硼替佐米 + 蛋白酶体抑制剂　； 参考：《苏州大学》2014年硕士论文

【摘要】：目的：宫颈癌为大家所熟知,也是目前所知的妇科最为常见的恶性肿瘤,每年全球都约有二十几万的妇女以为此而去世,宫颈癌对妇女的身心健康和生存品质都有着严重的负面影响,给许多家庭带来了巨大的经济负担。宫颈癌一般的治疗手段主要是以手术和放化疗,这些手段可以使一部分病患得到根治或着相对的缓解病情,但大多数的病患经过手术或者放化疗措施后的远期预后并非特别理想,且部分人群对化疗又不是很敏感,甚至产生抵抗,故预后不良。因此为了全人类的健康及生存环境,迫切需要寻找对宫颈癌较为有效的方法从而大大改善宫颈癌患者的预后。硼替佐米(Bortezomib)是一种细胞内蛋白酶体抑制剂的抑制剂,人体细胞内的蛋白酶体的糜蛋白酶样活性和胰蛋白酶样活性这两者都可以受到Bortezomib的抑制作用。5-FU脱氧核苷酸,5-FU核苷等代谢产物可以由5-氟尿嘧啶(5-FU)在人体细胞内转化而成,这些物质由于和DNA及蛋白质的合成所需底物相似,从而影响或干扰细胞内正常的代谢过程,故5-FU作为一种通过影响细胞代谢而抗肿瘤的药物,在临床恶性肿瘤的治疗方面已被广泛地采用,本文主要探讨的是Bortezomib这一血液肿瘤常用的化疗药物和常见的临床肿瘤化疗药物5-氟尿嘧啶(5-FU)对于Hela细胞的作用是怎样的,并浅表分析其作用机制。 方法：将人的宫颈腺癌Hela细胞培养于培养瓶中,呈单层贴壁样生长,培养基为含10%新生牛血清的DMEM-H(高糖)完全培养基,并将培养瓶放置于拥有饱和的湿度、温度为37摄氏度、含5%二氧化碳环境适合人类细胞生存的培养箱中进行传代培养。分为空白对照组,单纯Bortezomib组,单纯5-FU组及联合组(Bortezomib和5-FU联合应用组)4大组。并通过MTT法来测定各个组别的细胞抑制率情况。采用RT-PCR法测定不同组别中hela细胞的Bcl-2、Survivn、IGF-1及HIF-lamRNA的表达；PI标记流式细胞术(FCM)分析不同组别的细胞周期、AnnexinV/PI双染法法测定不同组别的凋亡的变化。 结果：(1)MTT检测法的检测结果显示,Bortezomib及5-FU单独使用时均可抑制Hela细胞的增殖,且抑制作用与作用的时间及浓度呈正相关,两者联合作用后抑制增殖作用更显著(P0.05);(2)RT-PCR结果显示Bortezomib及5-FU均可使BCL-2、 IGF mRNA及Survivn mRNA的表达下降,且与药物浓度呈正比,并且联合化疗组较单独化疗组的抑制作用更为显著(P0.05); Bortezomib及5-FU对HIF1αmRNA表达无抑制作用,但两者联合应用后其表达量较对照组显著降低(P0.05)。(3)细胞周期结果显示Bortezomib及5-FU可影响Hela细胞的周期比例发生变化,使其处于S期的细胞比例减少,联合化疗组较单独化疗组更加显著(P0.05)。(4)凋亡结果显示Bortezomib和5-FU单独使用时均可引诱导Hela细胞发生凋亡,联合化疗组诱导凋亡的作用较单独化疗组效果更加显著(P0.05)。 结论：本实验结果表明,Bortezomib联合5-FU应用可以明显的增加人宫颈腺癌Hela细胞的凋亡率,而降低Hela细胞的增殖率。
[Abstract]:Objective: cervical cancer is well known and is also known as the most common malignant tumor of gynecology. Every year, more than 20 million women all over the world have died. Cervical cancer has a serious negative impact on the physical and mental health and quality of life of women. It has brought great economic burden to many families. The general treatment of cervical cancer. The treatment means mainly by operation and radiotherapy and chemotherapy, these methods can make a part of the patients get a radical cure or relative relief of the condition, but the long-term prognosis of most patients after surgery or chemotherapy is not particularly ideal, and some people are not very sensitive to chemotherapy and even produce resistance, so the prognosis is bad. The health and living environment of all human beings urgently need to find a more effective method for cervical cancer to greatly improve the prognosis of cervical cancer patients. Borteg Zomi (Bortezomib) is an inhibitor of intracellular proteasome inhibitors, both the chymotrypsin like activity and trypsin like activity of the proteasome in the human body The metabolites such as.5-FU deoxynucleotides, 5-FU nucleosides, and 5-FU nucleosides can be transformed by 5- fluorouracil (5-FU) in human cells, which affect or interfere with normal metabolic processes in cells due to the similar substrates required for the synthesis of DNA and protein, so 5-FU acts as a way of affecting cell metabolism. Antitumor drugs have been widely used in the treatment of clinical malignant tumors. This article mainly discusses how the chemotherapeutic drugs commonly used in the Bortezomib blood tumor and the common clinical tumor chemotherapeutic drugs, 5- fluorouracil (5-FU), have the effect on the Hela cells.
Methods: human cervical adenocarcinoma Hela cells were cultured in a culture bottle with monolayer adherent growth. The culture medium was a DMEM-H (high sugar) complete medium containing 10% newborn bovine serum, and the culture bottle was placed in the incubator with saturated humidity, temperature of 37 degrees Celsius, and 5% carbon dioxide in the incubator suitable for human cell survival. It was divided into 4 groups: blank control group, simple Bortezomib group, simple 5-FU group and combined group (Bortezomib and 5-FU combined application group). The cell inhibition rate of each group was measured by MTT method. The expression of Bcl-2, Survivn, IGF-1 and HIF-lamRNA of HeLa cells in different groups was measured by RT-PCR; PI labeled flow cytometry was divided into two groups. The cell cycle of different groups was analyzed, and the changes of apoptosis in different groups were determined by AnnexinV/PI double staining.
Results: (1) the results of MTT detection showed that the proliferation of Hela cells could be inhibited when Bortezomib and 5-FU were used alone, and the inhibition effect was positively correlated with the time and concentration of the action, and the inhibition of proliferation was more significant (P0.05). (2) RT-PCR knots showed that Bortezomib and 5-FU could make BCL-2, IGF mRNA and Survivn. The expression decreased and was proportional to the drug concentration, and the inhibitory effect of the combined chemotherapy group was more significant than that of the chemotherapy group (P0.05); Bortezomib and 5-FU had no inhibitory effect on the expression of HIF1 alpha mRNA, but the expression of the two groups was significantly lower than that of the control group (P0.05). (3) the cell cycle results showed that Bortezomib and 5-FU could affect Hela fine. The proportion of cell cycle was changed, the proportion of cells in S phase decreased, and the combined chemotherapy group was more significant than that in the chemotherapy group (P0.05). (4) apoptosis results showed that Bortezomib and 5-FU could induce apoptosis of Hela cells, and the effect of combined chemotherapy group to induce apoptosis was more significant than that of chemotherapy group (P0.05).
Conclusion: the results show that Bortezomib combined with 5-FU can significantly increase the apoptosis rate of human cervical adenocarcinoma Hela cells, and decrease the proliferation rate of Hela cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
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本文编号：2028702