Survivin表达对HepG2细胞高LET射线辐射敏感性的影响

<正>Survivin是1997年发现的凋亡抑制蛋白家族(IAPs)的成员,它特异性的表达于大多数的恶性肿瘤细胞中,而在正常组织中检测不到,具有组织特异性。以前的研究表

Survivin expressions in human hepatoma HepG2 cells exposed to ionizing radiation of different LET

Survivin is a member of the inhibitors of apoptosis (IAP) protein family that interferes with post-mitochondrial events including activation of caspases. To examine the regulation of survivin expression in response to irradiation with different linear energy transfer (LET), human hepatoma HepG2 cells were irradiated in vitro with X-rays and carbon ions. Cellular sensitivities to low- and high-LET radiation were determined by colony formation. Survivin expression at mRNA and protein level were measured with RT-PCR and Western blot analyses, respectively. Radiation-induced cell cycle arrest and apoptosis were investigated with flow cytometry. We found that low-LET X-rays induced dose-dependent increases in survivin expression. After exposure to high-LET carbon ions, survivin expression gradually increased from 0 to 4 Gy, and then declined at 6 Gy. More pronounced survivin expression, stronger G(2)/M phase arrest was observed after exposure to carbon ions in comparison with X-rays at doses from 0 to 4 Gy. These observations indicate that there is a differential survivin expression in response to different LET radiations and the cycle arrest mechanism may be associated with it. In addition, our data on induction of apoptosis are compatible with the assumption that survivin expression induced by low-LET X-rays radiation may play a critical role in inhibiting apoptosis. However, after irradiation with ions, an anti-apoptotic function of survivin is not evident, possibly because of the serious damage produced by densely ionizing radiation.

抑制survivin表达增强人肝癌HepG2细胞对高线性能量转移射线的辐射敏感性；Inhibiting Survivin Expression Increases The Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Radiation

探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.

Survivin表达在高LET射线诱导细胞凋亡中作用机理的初步研究；A Preliminary Study on Action Mechanisms of Survivin Expression in Cell Apoptosis Induced by High-LET Radiation

先前的研究表明,肿瘤细胞中survivin的高表达与细胞对高传能线密度(LET)射线的辐射抗性相关。研究了survivin表达在高LET射线诱导的细胞凋亡中的作用,发现抑制survivin表达对高LETC离子辐射诱导的Bcl-2和Bax表达没有明显的影响。在高LET射线辐照中,survivin可能通过抑制caspase-3和-9活性的途径,抑制了细胞凋亡。

Inhibiting Survivin Expression Increases The Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Radiation

The influence of survivin expression on the radiosensitivity of tumor cells to high linear energy transfer (LET) radiation is investigated. Survivin-specific short-interfering RNA (siRNA) oligonucleotides were synthesized based on the survivin sequence provided by GenBank. Human hepatoma HepG2 cells were transfected with survivin-specific siRNA to inhibit its expressions. It was found that the transfection with surviving-specific siRNA increased the levels of G2/M arrest and the apoptotic rates induced by radiation in HepG2 cells. After exposure to high-LET carbon ions, a reduced clonogenic survival effect was observed in the cells treated with siRNA. These results show that survivin plays a key role in mediating the radioresistance of cells to high-LET radiation.

survivin表达对重离子诱导人肝癌HepG2细胞生物学效应的影响

放射治疗是肿瘤三大治疗手段之一（手术治疗、放疗、化疗），如何提高肿瘤细胞的放射敏感性一直是科研人员关注的研究方向。电离辐射导致细胞死亡的主要方式是细胞凋亡，然而肿瘤细胞内往往细胞凋亡信号通路异常，降低了治疗效果。其中细胞内高水平表达的细胞凋亡抑制蛋白（Inhibitor of Apoptosis Protein，IAP）抑制了caspase分子的活性，而caspase分子正是细胞凋亡的执行分子。因此科学家们通过各种手段尤其是RNA干涉的方法以抑制肿瘤细胞内细胞凋亡抑制蛋白的表达及蛋白活性来达到提高肿瘤治疗效果的目的。 Survivin是凋亡抑制蛋白家族的一员，该蛋白在大多数恶性肿瘤中高表达，而在正常组织中检测不到，因此具有组织特异性。Survivin参与肿瘤细胞分化并抑制肿瘤细胞凋亡，它的高表达被证明与很多恶性肿瘤对放射治疗中产生的辐射抗性相关。本文主要研究了不同LET射线辐照下人肝癌HepG2细胞 survivin的表达及其表达对重离子诱导的生物学效应的影响。首先，我们使用不同LET的碳离子束和X射线辐照HepG2细胞，采用标准克隆形成法确定其辐射敏感性，利用流式细胞技术（FCM）检测辐射后细胞周期分布，RT-PCR和western blotting检测survivin的表达。结果显示，人肝HepG2癌细胞经不同LET射线照射，survivin表达是不同的。与低LET的X射线相比，高LET碳离子诱导的细胞损伤和周期阻滞更明显，从而诱导了更强烈的survivin表达。 接着，根据Genbank提供的survivin序列，合成特异性survivin-siRNA寡核苷酸，转染HepG2细胞，抑制survivin的表达。我们发现siRNA转染后诱导了细胞G2/M期阻滞，增加了自发性和辐射诱导的细胞凋亡。在碳离子辐照后，siRNA细胞克隆存活率明显下降。这些结果显示survivin表达是细胞产生高LET辐射抗性的关键因素。最后，我们初步探讨了在细胞凋亡过程中，survivin基因的作用机制。发现抑制survivin表达，对离子束辐射诱导的Bcl-2和Bax表达没有明显的影响。Survivin表达直接抑制了caspase-3和-9的活性，从而抑制了细胞凋亡。以上的实验结果表明：不同LET射线辐照细胞后survivin出现差异表达，与X射线相比，高LET重离子诱导的HepG2细胞中survivin表达更明显，以survivin为靶基因的siRNA技术应用于HepG2细胞，可以极大提高该细胞对重离子辐射的敏感性。本论文研究为临床应用重离子束治疗癌症提供了非常有用的基础数据，同时也为重离子束放射治疗联合基因治疗提供了新的思路

Studies on alkaloids binding to GC-rich human survivin promoter DNA using positive and negative ion electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding of 13 alkaloids to two GC-rich DNA duplexes which are critical sequences in human survivin promoter. Negative ion ESI-MS was first applied to screen the binding of the alkaloids to the duplexes. Six alkaloids (including berberine, jatrorrhizine, palmatine, reserpine, berbamine, and tetrandrine) show complexation with the target DNA sequences. Relative binding affinities were estimated from the negative ion ESI data, and the alkaloids show a binding preference to the duplex with higher GC content. Positive ion ESI mass spectra of the complexes were also recorded and compared with those obtained in negative ion mode.