神经胶质瘤细胞致瘤性相关基因、抗肿瘤药物以及健康人群认知功能的研究
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摘要
神经胶质瘤是一种恶性的颅内肿瘤,虽然目前在手术和放化疗方面有很大进展,但病人的预后依然很差。寻找特异性靶向神经胶质瘤的药物具有迫切性和临床应用价值。在本研究中,我们首先研究了神经胶质瘤细胞株C6的抗药性与致瘤性的关系。结果发现,只有Nestin阳性的C6细胞才能在SD大鼠脑内形成肿瘤,而Nestin阴性的细胞则没有致瘤能力,这提示Nestin可能与神经胶质瘤细胞的致瘤能力有密切联系。为了更好的研究神经胶质瘤的生物学特性,我们建立了一株人神经胶质瘤细胞系GBM-HSF。在这株细胞中,干细胞相关基因Nestin、 Klf4、Oct4、Sox2、Nanog,神经元标记分子β-tubulin Ⅲ和Map2以及肿瘤迁移相关基因CD26的表达均为阳性。同时,GBM-HSF细胞在条件培养基中可成球生长。流式细胞术的结果表明GBM-HSF细胞中存在对Hoechst33342有极强外排能力的SP细胞。这些结果都提示GBM-HSF细胞中有肿瘤干细胞的存在。该细胞系是研究神经胶质瘤以及肿瘤干细胞的一个非常好的工具。
随后,我们集中研究了三种不同类型药物：氟代柠檬酸,褪黑素和尼克酸对神经胶质瘤的抑制和杀伤作用。结果发现,这三种药物在一定的浓度范围都可显著抑制和杀伤神经胶质瘤细胞。氟代柠檬酸通过抑制三羧酸循环来抑制小鼠神经胶质瘤细胞G422的增殖；褪黑素可显著抑制C6细胞的增殖,并且提高干细胞相关基因Nestin、Sox2和Bmi-1的转录水平；尼克酸则可通过持续提高细胞的内钙水平来诱导人神经胶质瘤细胞U251的凋亡,而同样浓度的尼克酸对神经元则无杀伤作用。这提示这三种药物对神经胶质瘤可能均有一定的治疗作用。
在这三个药物中,尼克酸的选择性较好,因为其对神经元没有杀伤作用。而且尼克酸只有在酸性条件下才对U251细胞具有杀伤作用。肿瘤因其极高的代谢水平,其微环境通常都呈酸性,这就为尼克酸提供了一个天然的选择靶点。
此外,我们对健康人群的大脑认知功能也做了相应研究。在对工作记忆和选择性注意的研究中,我们发现大脑可同时调节视觉工作记忆和听觉选择性注意,并且视觉工作记忆的负荷会对这一调节作用产生一定的影响。我们的结果表明大脑可整合处理来自不同感官通道的信息并以此来指导大脑对认知功能的执行。因为先前的研究表明神经胶质瘤会对人认知功能产生影响,我们的研究结果和方法对研究神经胶质瘤可能具有一定的指导意义。
Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Despite recent advances in neurosurgery, radiation therapy and chemotherapy to treat GBM, the prognosis of this cancer is extremely poor. In the present study, the role of Nestin in the tumorogenesis of glioma cells was investigated. The results showed that, in C6glioma cells, only Nestin positive cells would initiate tumors in SD rat brain. This suggested that Nestin might play an important role in the ability of tumor initiation of glioma cells.
To further study the biological characters of GBM, A new human glioma cell line designated GBM-HSF was established. Stem cell related genes, Nestin、Klf4、 Oct4、Sox2、Nanog; neuronal markers β-tubulin III and Map2; and the metastasis related gene CD26were all positively transcribed in GBM-HSF cells. GBM-HSF cells grew and formed tumor spheres in conditioned cultured medium. Side population (SP) cells, which had a strong ability to efflux the fluorescent dye Hoechst33342, were also detected in the cell line. These results suggested the existence of cancer stem cells (CSC) in this cell line, which was demonstrated to be a good tool to research the biological characters of GBM.
Then we focus on the effects of fluorocitrate (FC), melatonin and nicotinic acid (NA) on glioma cells were tested in order to find effective and specific drugs to GBM. All three drugs were shown to significantly inhibit proliferation and to induce apoptosis of glioma cells. FC suppressed the proliferation of G422glioma cells through inhibiting the tricarboxylic acid cycle of the cells; Melatonin significantly inhibit the growth of C6glioma cells and increase the mRNA levels of stem cell related genes Nestin、Bmi-1and Sox2; NA induced the apoptosis of U251cells, but not neurons, through disrupting intracellular calcium homeostasis. These results indicate that all three drugs might have therapeutic effects on GBM.
NA was the most specific among the three drugs, because it had no effect on the survival of neurons. Furthermore, NA induced apoptosis of U251cells only under the acidic conditions. Importantly, the microenvironment of tumors is also acidic due to the high metabolic rate of cancer growth, which may provide a natural target condition for NA.
In an additional study, cognitive functions were investigated in healthy controls. The modulation of irrelevant auditory stimuli by the brain during a visual working memory (WM) task was evaluated. Visual WM was found to affect the processing of irrelevant auditory stimuli. Visual WM load was also found to play an important role in this process. These results suggested that the brain could integrate information from different sensory channels in order to guide the execution of cognitive functions. These findings and methodology may have implications for GBM research as previous studies have identified gliomas to influence the cognitive functions.
随后,我们集中研究了三种不同类型药物：氟代柠檬酸,褪黑素和尼克酸对神经胶质瘤的抑制和杀伤作用。结果发现,这三种药物在一定的浓度范围都可显著抑制和杀伤神经胶质瘤细胞。氟代柠檬酸通过抑制三羧酸循环来抑制小鼠神经胶质瘤细胞G422的增殖；褪黑素可显著抑制C6细胞的增殖,并且提高干细胞相关基因Nestin、Sox2和Bmi-1的转录水平；尼克酸则可通过持续提高细胞的内钙水平来诱导人神经胶质瘤细胞U251的凋亡,而同样浓度的尼克酸对神经元则无杀伤作用。这提示这三种药物对神经胶质瘤可能均有一定的治疗作用。
在这三个药物中,尼克酸的选择性较好,因为其对神经元没有杀伤作用。而且尼克酸只有在酸性条件下才对U251细胞具有杀伤作用。肿瘤因其极高的代谢水平,其微环境通常都呈酸性,这就为尼克酸提供了一个天然的选择靶点。
此外,我们对健康人群的大脑认知功能也做了相应研究。在对工作记忆和选择性注意的研究中,我们发现大脑可同时调节视觉工作记忆和听觉选择性注意,并且视觉工作记忆的负荷会对这一调节作用产生一定的影响。我们的结果表明大脑可整合处理来自不同感官通道的信息并以此来指导大脑对认知功能的执行。因为先前的研究表明神经胶质瘤会对人认知功能产生影响,我们的研究结果和方法对研究神经胶质瘤可能具有一定的指导意义。
Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Despite recent advances in neurosurgery, radiation therapy and chemotherapy to treat GBM, the prognosis of this cancer is extremely poor. In the present study, the role of Nestin in the tumorogenesis of glioma cells was investigated. The results showed that, in C6glioma cells, only Nestin positive cells would initiate tumors in SD rat brain. This suggested that Nestin might play an important role in the ability of tumor initiation of glioma cells.
To further study the biological characters of GBM, A new human glioma cell line designated GBM-HSF was established. Stem cell related genes, Nestin、Klf4、 Oct4、Sox2、Nanog; neuronal markers β-tubulin III and Map2; and the metastasis related gene CD26were all positively transcribed in GBM-HSF cells. GBM-HSF cells grew and formed tumor spheres in conditioned cultured medium. Side population (SP) cells, which had a strong ability to efflux the fluorescent dye Hoechst33342, were also detected in the cell line. These results suggested the existence of cancer stem cells (CSC) in this cell line, which was demonstrated to be a good tool to research the biological characters of GBM.
Then we focus on the effects of fluorocitrate (FC), melatonin and nicotinic acid (NA) on glioma cells were tested in order to find effective and specific drugs to GBM. All three drugs were shown to significantly inhibit proliferation and to induce apoptosis of glioma cells. FC suppressed the proliferation of G422glioma cells through inhibiting the tricarboxylic acid cycle of the cells; Melatonin significantly inhibit the growth of C6glioma cells and increase the mRNA levels of stem cell related genes Nestin、Bmi-1and Sox2; NA induced the apoptosis of U251cells, but not neurons, through disrupting intracellular calcium homeostasis. These results indicate that all three drugs might have therapeutic effects on GBM.
NA was the most specific among the three drugs, because it had no effect on the survival of neurons. Furthermore, NA induced apoptosis of U251cells only under the acidic conditions. Importantly, the microenvironment of tumors is also acidic due to the high metabolic rate of cancer growth, which may provide a natural target condition for NA.
In an additional study, cognitive functions were investigated in healthy controls. The modulation of irrelevant auditory stimuli by the brain during a visual working memory (WM) task was evaluated. Visual WM was found to affect the processing of irrelevant auditory stimuli. Visual WM load was also found to play an important role in this process. These results suggested that the brain could integrate information from different sensory channels in order to guide the execution of cognitive functions. These findings and methodology may have implications for GBM research as previous studies have identified gliomas to influence the cognitive functions.
引文
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