卵巢癌化疗耐药相关分子机制的探讨
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摘要
研究背景与目的
细胞在长期的进化过程中产生的监控纺锤体形态、染色体着丝点与微管的连接及其产生的张力,确保姐妹染色单体精确分离的质控机制,即为纺锤体检查点。纺锤体检查点是细胞周期的最后关卡,是近年来细胞分子生物学的研究热点。早在上世纪90年代初,研究人员就在酵母体内确定了6种执行纺锤体检查点功能所必需的基因：Bub1、Bub2、Bub3、Mad1、Mad2及Mad3,而蛋白激酶Bub1是纺锤体检查点的平台蛋白。截至目前,有关纺锤体检查点的功能尚存在较多争议,而其精细调节机制更有待于深入探讨。紫杉醇作为一线化疗药物在实体瘤包括乳腺癌及卵巢癌等恶性肿瘤的治疗方面取得了巨大成功,但临床上耐药病例仍频繁发生。众所周知,紫杉醇是一种微管稳定剂,通过抑制微管解聚进而阻滞细胞周期杀伤肿瘤,而有研究提示这种杀伤作用必须有赖于具有完整功能的纺锤体检查点。虽然突变在实体瘤中并不常见,但纺锤体检查点功能低下却时有发生。Bub1是否直接影响纺锤体功能并进而导致肿瘤的化疗耐药,国内尚未见诸报道。siRNA在干扰基因表达的效率和时长均显不足,而本研究通过稳定转染pEGFP-Bub1-shRNA质粒,使干扰效率大为增强,且其具有更高的稳定性,为深入探讨卵巢癌细胞紫杉醇化疗耐药的精细机制提供有力工具。
方法
一、Bub1免疫组织化学染色；
二、构建人pEGFP-Bub1-shRNA质粒,随后以G418进行稳定筛选；
三、应用不同浓度梯度紫杉醇作用卵巢癌细胞,选择后续实验浓度；
四、采用MTT法和流氏细胞术检测紫杉醇作用各组细胞的存活率、凋亡率以及细胞周期；
五.Hochest 33342染色观察细胞分裂及凋亡情况。
结果
在一定剂量紫杉醇作用后,A2780及SKOV3细胞均呈现生长受抑及凋亡增加。细胞首先出现体积缩小,连接消失,与周围的细胞脱离,然后是细胞质密度增加,核质浓缩,核膜核仁破碎。凋亡细胞周期主要阻滞在G2／M期。在干扰Bub1的表达后,A2780及SKOV3细胞的药物敏感性显著降低,G2／M期比例亦随之下降。
结论
一定剂量的紫杉醇可使人卵巢癌A2780及SKOV3细胞发生生长受抑、凋亡及G2／M期阻滞。转染pEGFP-Bub1-shRNA可导致卵巢癌细胞紫杉醇敏感性降低及G2／M期细胞逃逸。
研究背景及目的
卵巢癌是妇科肿瘤死亡的首要原因,化疗是其除手术以外最为重要的治疗方式。然而,在临床上,耐药病例频繁发生。因此,新的化疗方法可能有助于改善患者的预后。紫杉醇作为卵巢癌化疗的一线药物,在实体瘤的治疗方面取得了巨大成功,但对其耐药的患者并不少见’。既往研究表明,在卵巢癌细胞紫杉醇化疗耐药的过程中,包括B2在内的许多分子靶标涉及其中,为此,我们进行相关实验来探究其内在机制。作为B家族的一员,B2被认为是一种重要的抗凋亡基因,在包括卵巢癌的众多肿瘤发生发展的过程中发挥了重要作用。纺锤体检查点是监控纺锤体,确保姐妹染色单体精确分离的一套质控机制,而Bub1是其重要组分。有研究表明Bub1是纺锤体检查点发挥功能所必需的2,而紫杉醇作用的发挥有赖于功能完整的纺锤体检查点。我们通过构建过表达B2基因及分别上调和下调Bub1基因的细胞模型,进而探究纺锤体检查点人卵巢癌A2780细胞化疗过程中Bub1与A/B通路蛋白的相互作用关系。
方法
本研究试图通过设计相关实验探索B2基因与纺锤体检查点平台蛋白Bubl之间的相互作用关系。为此,我们运用脂质体转染的方法将野生型B2及pEGFP-Bub1-shRNA质粒导入人卵巢癌A2780细胞,此后以G418稳定筛选并扩大培养；pcs2-Bub1正义全长质粒瞬时转染。此后,在以紫杉醇,AⅠ及BⅠ等药物进行处理后,我们运用半定量PCR及western blot检测各组细胞中Bub1、B2及p-B的含量,流式细胞术及Hoechst33342染色检测细胞凋亡率及周期变化；免疫共沉淀验证与Bubl结合之蛋白。
结果
结果表明,重组B2质粒及pcs2-Bub1正义全长质粒分别能有效增强B2和Bub1mRNA及蛋白水平的表达。在特定时间点,中等剂量紫杉醇作用后,过表达B2的细胞不仅凋亡率明显降低,G2／M期阻滞细胞比例亦明显降低,Hoechst33342染色的结果也为后者提供佐证。有趣的是,我们发现在上调B2表达并以一定浓度的紫杉醇处理,纺锤体检查点平台蛋白Bub1的表达水平较对照组明显降低,而在那些下调Bub1表达的细胞,在以中等剂量紫杉醇处理后的特定时间点,p-B呈现持续活化的状态；在以紫杉醇处理后,Bub1和p-B呈现蛋白与蛋白结合的状态,以上结果初步证明,纺锤体检查点蛋白Bub1与B在卵巢癌细胞化疗过程中可能存在相互作用。
结论
Bub1可能与B之间存在密切联系,本研究拓展了对于细胞周期调控和凋亡机制的认识,可能为恶性肿瘤的化疗提供新的分子靶标。
研究背景与目的
恶性肿瘤有其独特的,难以预期的生物学行为,对人类的健康造成了极大危害,因此,需要更好的分子生物技术来破解谜题,而蛋白质组学就是一个很好的选择。这项技术可能有助于我们了解恶性肿瘤的始动因素及其预后。截至目前,最为常用的大规模蛋白筛选技术就是结合图像和质谱分析的二维电泳技术。运用这项技术,我们将获得大量有价值的有关于蛋白状态及翻译后水平改变的信息。
方法
一、将处于对数生长期的A2780、A2780/DDP、A2780/Taxol、C13K及OV2008细胞均匀接种于10 cm培养皿中,24小时后以无血清1640培养基处理C13K及OV2008细胞过夜,以使其同步化,其后提取细胞总蛋白；
二、采用二维电泳法筛选各配对细胞表达差异蛋白；
三、MALDI-TOF-MS质谱分析鉴定选取蛋白质。
结果
C13K细胞SDS-PAGE胶中共有502±30个点被检测出,OV2008细胞SDS-PAGE胶中共有430±21个点被检测出。选取两块胶中表达差异明显的20个蛋白点进行后续MALDI-TOF MS质谱分析。其他细胞株的蛋白图谱分析工作尚在进行中。
结论
作为高通量筛选差异蛋白的有效技术手段,二维电泳技术可被用于筛选新的卵巢癌细胞化疗耐药的相关调控基因。
Background and Objective
The spindle checkpoint, known as a surveillance mechanism, is crucial for ensuring the chromosome segregation process. Past studies indicated that the spindle checkpoint controls the cell cycle. The well-characterized components of the spindle checkpoint include Bub1, Bub2, Bub3, Mps1, Mad1, Mad2 and Mad3. The family of Mad (mitotic arrest deficient) and Bub (budding uninhibited by benzimidazole) proteins were identified by yeast mutagenesis screens for mutants unable to survive a temporary exposure to microtubule toxin. Bub1 is the paltform of spindle checkpoint. There are many controversial issues on the spindle checkpoint. The innner mechanisms are yet waiting to be explored. Paclitaxel succeeded in the treatment of solid tumors, including breast cancer, ovarian cancer and so on. Paclitaxel is a microtube stable reagent, which bloking cell cycle to kill the tumor. Some studies demonsteated that the killing effects depend on a functional spindle checkpoint. The mutant is rare, but the of spindle checkpoint often occour. There are no reports on the Bub1-mediated chemoresistance at home. Both of the efficiency and time length of siRNA is not enough. The pEGFPBub1-shRNA plasmid we used can obviously enhance the silent efficiency, supply powerful tool to probe the mechanisms of paclitaxel-resistance in ovarian cancer cells.
Methods
1. The immunohistochemistry staining of Bub 1;
2. The construction of full-length and pEGFP-Bub1-shRNA plasmids;
3. Apply paclitaxel with different concentration gradient to affect tumour cells, choose the concentration when the rate of apoptosis and G2/M was highest and without obvious apoptosis;
4. Apply MTT and FACS method to test the apoptosis and cell cycle of tumour cells;
5. Apply Hoechst33342 staining method to test the cell mitosis and apoptosis.
Results
After the treatment of certain paclitaxel, the rate of proliferation inhibition and apoptosis increased. The volume of apoptosis cell shrunk, the links dissolved that isolate from cells around, and following that, the density of cytoplasm increased and the nucleoplasm to condense. The cell cycle of apoptotic cells mainly in G2/M phase. The drug-sensitivity of A2780 and SKOV3 reduced after transfected with pEGFP-Bubl-shRNA plasmid.
Conclusion
Both A2780 and SKOV3 cells could be induced into apoptosis by a certain dose paclitaxel. The cell apoptosis and arrest induced by paclitaxel could be reversed by transfecting with pEGFP-Bubl-shRNA plasmid.
Background and Objective
Recent evidence has suggested that B2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and controlling the cell cycle. In addition, some scholars suggested that the PTX sensitivity dependent on a functional spindle assembly checkpoint. Bub1, as a platform protein, considered as an important component of spindle checkpoint. In the present study, we investigated the role of the B2/Bub1 cross-talking in apoptosis and cell cycle when exposure to PTX.
Methods
In order to explore the relationship between the Bubl and A/B pathway, recombinant expression plasmid WT-B2 and pEGFP-Bub1-shRNA were transfected into A2780 cells by lipofectamine2000, and then detected the expression level of Bub1、B2及p-B gene by using RT-PCR and western blot. Cell apoptosis and cycle were measured by flow cytometry and Hoechst33342 after treatment with PTX, AI and BI. In addition, we used CO-IP to find the ralated protein combined with Bibl.
Results
Our data showed that up-regulation of B2 contributed ovarian cancer cells to override PTX-induced G2/M arrest, and inhibited Bubl expression. The expression level of P-B is higher in pEGFP-Bubl-shRNA/A2780 cells than control cells. The result of CO-IP indicated that Bub1 protein may combine with p-B. Our findings will aid in understanding the molecular mechanism of PTX-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.
Conclusion
There are intimate relationships between Bub1 and B. This study has expanded the knowledge on regulation of cell cycle and apoptotic mechanisms, and may supply new targets.
Background and Objective
Malignant tumors have almost individually unpredictable behavior and aggressiveness, therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of malignant tumors, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications.
Methods
1. extracted total protein.
2. Screened differential expression protein between C13K, OV2008, A2780, A2780/DDP,A2780/Taxol cells through two-dimensional gel electrophoresis proteomics technology. Repeated the experiment three times and picked out the special points for the follow-up MALDI-TOF-MS analysis.
Results
A total of 502±30 points were detected in C13K cells, while there were 430±21 points detected in OV2008 cells through two-dimensional gel electrophoresis. Selected 20 significantly differential expression protein spots for the follow-up MALDI-TOF MS analysis.
Conclusion
As one of effective technique for isolating the different expression protein, two-dimensional gel electrophoresis can be successfully used for screening new chemoresistance-associated genes.
细胞在长期的进化过程中产生的监控纺锤体形态、染色体着丝点与微管的连接及其产生的张力,确保姐妹染色单体精确分离的质控机制,即为纺锤体检查点。纺锤体检查点是细胞周期的最后关卡,是近年来细胞分子生物学的研究热点。早在上世纪90年代初,研究人员就在酵母体内确定了6种执行纺锤体检查点功能所必需的基因：Bub1、Bub2、Bub3、Mad1、Mad2及Mad3,而蛋白激酶Bub1是纺锤体检查点的平台蛋白。截至目前,有关纺锤体检查点的功能尚存在较多争议,而其精细调节机制更有待于深入探讨。紫杉醇作为一线化疗药物在实体瘤包括乳腺癌及卵巢癌等恶性肿瘤的治疗方面取得了巨大成功,但临床上耐药病例仍频繁发生。众所周知,紫杉醇是一种微管稳定剂,通过抑制微管解聚进而阻滞细胞周期杀伤肿瘤,而有研究提示这种杀伤作用必须有赖于具有完整功能的纺锤体检查点。虽然突变在实体瘤中并不常见,但纺锤体检查点功能低下却时有发生。Bub1是否直接影响纺锤体功能并进而导致肿瘤的化疗耐药,国内尚未见诸报道。siRNA在干扰基因表达的效率和时长均显不足,而本研究通过稳定转染pEGFP-Bub1-shRNA质粒,使干扰效率大为增强,且其具有更高的稳定性,为深入探讨卵巢癌细胞紫杉醇化疗耐药的精细机制提供有力工具。
方法
一、Bub1免疫组织化学染色；
二、构建人pEGFP-Bub1-shRNA质粒,随后以G418进行稳定筛选；
三、应用不同浓度梯度紫杉醇作用卵巢癌细胞,选择后续实验浓度；
四、采用MTT法和流氏细胞术检测紫杉醇作用各组细胞的存活率、凋亡率以及细胞周期；
五.Hochest 33342染色观察细胞分裂及凋亡情况。
结果
在一定剂量紫杉醇作用后,A2780及SKOV3细胞均呈现生长受抑及凋亡增加。细胞首先出现体积缩小,连接消失,与周围的细胞脱离,然后是细胞质密度增加,核质浓缩,核膜核仁破碎。凋亡细胞周期主要阻滞在G2／M期。在干扰Bub1的表达后,A2780及SKOV3细胞的药物敏感性显著降低,G2／M期比例亦随之下降。
结论
一定剂量的紫杉醇可使人卵巢癌A2780及SKOV3细胞发生生长受抑、凋亡及G2／M期阻滞。转染pEGFP-Bub1-shRNA可导致卵巢癌细胞紫杉醇敏感性降低及G2／M期细胞逃逸。
研究背景及目的
卵巢癌是妇科肿瘤死亡的首要原因,化疗是其除手术以外最为重要的治疗方式。然而,在临床上,耐药病例频繁发生。因此,新的化疗方法可能有助于改善患者的预后。紫杉醇作为卵巢癌化疗的一线药物,在实体瘤的治疗方面取得了巨大成功,但对其耐药的患者并不少见’。既往研究表明,在卵巢癌细胞紫杉醇化疗耐药的过程中,包括B2在内的许多分子靶标涉及其中,为此,我们进行相关实验来探究其内在机制。作为B家族的一员,B2被认为是一种重要的抗凋亡基因,在包括卵巢癌的众多肿瘤发生发展的过程中发挥了重要作用。纺锤体检查点是监控纺锤体,确保姐妹染色单体精确分离的一套质控机制,而Bub1是其重要组分。有研究表明Bub1是纺锤体检查点发挥功能所必需的2,而紫杉醇作用的发挥有赖于功能完整的纺锤体检查点。我们通过构建过表达B2基因及分别上调和下调Bub1基因的细胞模型,进而探究纺锤体检查点人卵巢癌A2780细胞化疗过程中Bub1与A/B通路蛋白的相互作用关系。
方法
本研究试图通过设计相关实验探索B2基因与纺锤体检查点平台蛋白Bubl之间的相互作用关系。为此,我们运用脂质体转染的方法将野生型B2及pEGFP-Bub1-shRNA质粒导入人卵巢癌A2780细胞,此后以G418稳定筛选并扩大培养；pcs2-Bub1正义全长质粒瞬时转染。此后,在以紫杉醇,AⅠ及BⅠ等药物进行处理后,我们运用半定量PCR及western blot检测各组细胞中Bub1、B2及p-B的含量,流式细胞术及Hoechst33342染色检测细胞凋亡率及周期变化；免疫共沉淀验证与Bubl结合之蛋白。
结果
结果表明,重组B2质粒及pcs2-Bub1正义全长质粒分别能有效增强B2和Bub1mRNA及蛋白水平的表达。在特定时间点,中等剂量紫杉醇作用后,过表达B2的细胞不仅凋亡率明显降低,G2／M期阻滞细胞比例亦明显降低,Hoechst33342染色的结果也为后者提供佐证。有趣的是,我们发现在上调B2表达并以一定浓度的紫杉醇处理,纺锤体检查点平台蛋白Bub1的表达水平较对照组明显降低,而在那些下调Bub1表达的细胞,在以中等剂量紫杉醇处理后的特定时间点,p-B呈现持续活化的状态；在以紫杉醇处理后,Bub1和p-B呈现蛋白与蛋白结合的状态,以上结果初步证明,纺锤体检查点蛋白Bub1与B在卵巢癌细胞化疗过程中可能存在相互作用。
结论
Bub1可能与B之间存在密切联系,本研究拓展了对于细胞周期调控和凋亡机制的认识,可能为恶性肿瘤的化疗提供新的分子靶标。
研究背景与目的
恶性肿瘤有其独特的,难以预期的生物学行为,对人类的健康造成了极大危害,因此,需要更好的分子生物技术来破解谜题,而蛋白质组学就是一个很好的选择。这项技术可能有助于我们了解恶性肿瘤的始动因素及其预后。截至目前,最为常用的大规模蛋白筛选技术就是结合图像和质谱分析的二维电泳技术。运用这项技术,我们将获得大量有价值的有关于蛋白状态及翻译后水平改变的信息。
方法
一、将处于对数生长期的A2780、A2780/DDP、A2780/Taxol、C13K及OV2008细胞均匀接种于10 cm培养皿中,24小时后以无血清1640培养基处理C13K及OV2008细胞过夜,以使其同步化,其后提取细胞总蛋白；
二、采用二维电泳法筛选各配对细胞表达差异蛋白；
三、MALDI-TOF-MS质谱分析鉴定选取蛋白质。
结果
C13K细胞SDS-PAGE胶中共有502±30个点被检测出,OV2008细胞SDS-PAGE胶中共有430±21个点被检测出。选取两块胶中表达差异明显的20个蛋白点进行后续MALDI-TOF MS质谱分析。其他细胞株的蛋白图谱分析工作尚在进行中。
结论
作为高通量筛选差异蛋白的有效技术手段,二维电泳技术可被用于筛选新的卵巢癌细胞化疗耐药的相关调控基因。
Background and Objective
The spindle checkpoint, known as a surveillance mechanism, is crucial for ensuring the chromosome segregation process. Past studies indicated that the spindle checkpoint controls the cell cycle. The well-characterized components of the spindle checkpoint include Bub1, Bub2, Bub3, Mps1, Mad1, Mad2 and Mad3. The family of Mad (mitotic arrest deficient) and Bub (budding uninhibited by benzimidazole) proteins were identified by yeast mutagenesis screens for mutants unable to survive a temporary exposure to microtubule toxin. Bub1 is the paltform of spindle checkpoint. There are many controversial issues on the spindle checkpoint. The innner mechanisms are yet waiting to be explored. Paclitaxel succeeded in the treatment of solid tumors, including breast cancer, ovarian cancer and so on. Paclitaxel is a microtube stable reagent, which bloking cell cycle to kill the tumor. Some studies demonsteated that the killing effects depend on a functional spindle checkpoint. The mutant is rare, but the of spindle checkpoint often occour. There are no reports on the Bub1-mediated chemoresistance at home. Both of the efficiency and time length of siRNA is not enough. The pEGFPBub1-shRNA plasmid we used can obviously enhance the silent efficiency, supply powerful tool to probe the mechanisms of paclitaxel-resistance in ovarian cancer cells.
Methods
1. The immunohistochemistry staining of Bub 1;
2. The construction of full-length and pEGFP-Bub1-shRNA plasmids;
3. Apply paclitaxel with different concentration gradient to affect tumour cells, choose the concentration when the rate of apoptosis and G2/M was highest and without obvious apoptosis;
4. Apply MTT and FACS method to test the apoptosis and cell cycle of tumour cells;
5. Apply Hoechst33342 staining method to test the cell mitosis and apoptosis.
Results
After the treatment of certain paclitaxel, the rate of proliferation inhibition and apoptosis increased. The volume of apoptosis cell shrunk, the links dissolved that isolate from cells around, and following that, the density of cytoplasm increased and the nucleoplasm to condense. The cell cycle of apoptotic cells mainly in G2/M phase. The drug-sensitivity of A2780 and SKOV3 reduced after transfected with pEGFP-Bubl-shRNA plasmid.
Conclusion
Both A2780 and SKOV3 cells could be induced into apoptosis by a certain dose paclitaxel. The cell apoptosis and arrest induced by paclitaxel could be reversed by transfecting with pEGFP-Bubl-shRNA plasmid.
Background and Objective
Recent evidence has suggested that B2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and controlling the cell cycle. In addition, some scholars suggested that the PTX sensitivity dependent on a functional spindle assembly checkpoint. Bub1, as a platform protein, considered as an important component of spindle checkpoint. In the present study, we investigated the role of the B2/Bub1 cross-talking in apoptosis and cell cycle when exposure to PTX.
Methods
In order to explore the relationship between the Bubl and A/B pathway, recombinant expression plasmid WT-B2 and pEGFP-Bub1-shRNA were transfected into A2780 cells by lipofectamine2000, and then detected the expression level of Bub1、B2及p-B gene by using RT-PCR and western blot. Cell apoptosis and cycle were measured by flow cytometry and Hoechst33342 after treatment with PTX, AI and BI. In addition, we used CO-IP to find the ralated protein combined with Bibl.
Results
Our data showed that up-regulation of B2 contributed ovarian cancer cells to override PTX-induced G2/M arrest, and inhibited Bubl expression. The expression level of P-B is higher in pEGFP-Bubl-shRNA/A2780 cells than control cells. The result of CO-IP indicated that Bub1 protein may combine with p-B. Our findings will aid in understanding the molecular mechanism of PTX-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.
Conclusion
There are intimate relationships between Bub1 and B. This study has expanded the knowledge on regulation of cell cycle and apoptotic mechanisms, and may supply new targets.
Background and Objective
Malignant tumors have almost individually unpredictable behavior and aggressiveness, therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of malignant tumors, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications.
Methods
1. extracted total protein.
2. Screened differential expression protein between C13K, OV2008, A2780, A2780/DDP,A2780/Taxol cells through two-dimensional gel electrophoresis proteomics technology. Repeated the experiment three times and picked out the special points for the follow-up MALDI-TOF-MS analysis.
Results
A total of 502±30 points were detected in C13K cells, while there were 430±21 points detected in OV2008 cells through two-dimensional gel electrophoresis. Selected 20 significantly differential expression protein spots for the follow-up MALDI-TOF MS analysis.
Conclusion
As one of effective technique for isolating the different expression protein, two-dimensional gel electrophoresis can be successfully used for screening new chemoresistance-associated genes.
引文
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