【作者】 邢军；

【导师】 岳天孚；

【作者基本信息】 天津医科大学 ， 妇产科学（专业学位）， 2011， 博士

【摘要】 目的：观察组蛋白去乙酰化酶抑制剂：SAHA (suberoylanilide hydrox amic acid)联合顺铂或X射线照射后人宫颈癌SiHa细胞生长抑制、细胞凋亡、细胞集落形成率等情况,探讨SAHA(?)寸宫颈癌细胞化疗和放疗增敏的效果。同时检测(?)P21、Bax及Ku70的mRNA和蛋白的表达,探讨其增敏的机制。方法：1、以SAHA0.25、0.5、1、2、4、6μ mol/L和DDP1、2、4、6、8、10μg/mL分成单药组和不同浓度SAHA+DDP联合用药组,另设不加药对照组,作用于体外培养的人宫颈癌SiHa(?)细胞。采用MTT比色法检测各组SiHa细胞的增殖抑制率；流式细胞术检测各组的细胞周期；用AnnexinV-FITC标记药物作用后的细胞,了解其早期凋亡的情况；2、20%IC50SAHA预处理SiHa细胞24h后与不同剂量X射线(0Gy、1Gy、2Gy、4Gy、6Gy、8Gy)联合,利用细胞克隆形成实验检测照射后集落形成率,并计算细胞存活分数；根据单击多靶模型SF=1-(1-e-D/Do。)n拟合生存曲线并计算外推值,平均致死剂量和准阈剂量以及放射增敏比；3、用RT-PCR法和Western blot法分析药物作用及射线照射后宫颈癌细胞中P21、Bax和Ku70的mRNA和蛋白的表达；结果：1、SAHA(?)单药处理细胞时,各组对SiHa细胞的生长抑制率均比对照组升高(P<0.05)；随着给药时间延长,细胞的生长抑制率逐渐升高(P<0.05)。2、SAHA与DDP联合作用SiHa细胞24h,不同剂量联合组对细胞的抑制率均较单用DDP明显(P<0.05)；与1、2、4ug/ml DDP联合应用时,随SAHA剂量的增大抑制率逐渐增高(P<0.05)；SAHA在剂量浓度为1、2、4μ mol/L时,细胞的抑制率随DDP浓度的增加而升高；联用的效果Q值显示SAHA和相对中低剂量的DDP联合用药表现为协同作用,较高剂量的DDP联合用药为单纯相加作用。3、SAHA1、2、4μ mol/L(S组)分别与DDP1、2、4ug/ml(D组)联合(SD组)作用于SiHa细胞,与对照组比较,S组、D组及SD组G0/G1期比例明显增加,而G2/M+S期细胞比例明显减少,PI明显下降(P<0.05)；与S及D组比较,SD组G0/G1期比例明显增加,而G2/M+S期细胞比例明显减少,PI明显下降(P<0.05)；4、各联合组早期凋亡细胞比例均较单药组明显升高(P<0.05)；在2μ g/ml的DDP作用SiHa细胞24h后,细胞凋亡不明显,但经2μ mol/L SAHA预处理后,细胞凋亡率显著增加,且随着SAHA浓度的增加,凋亡率也增加(P<0.05)。5、Bax mRNA及蛋白在S、D组表达比较微弱,与对照组比较无差异(P>0.05),联合作用后BaxmRNA及蛋白的表达显著高于其他各组(P<0.05)；6、P21mRNA及蛋白在各组的表达均上调,以联合组的表达上调显著,明显高于D组和对照组(P<0.05)。7、SAHA联合照射组的细胞存活分数明显低于单独放疗组,随放疗剂量的增高,细胞存活分数逐渐降低(P<0.05)；利用多靶单击数学模型绘制出细胞辐射存活曲线,可见联合组曲线较单纯照射组左移,且较为平直、“肩区”不明显。单独照射组和联合组平均致死剂量Do分别为2.329、1.213,外推数N分别为2.761、4.770,准阈剂量Dq分别为1.721、0.823。放射增敏比为1.92。8、SAHA组BaxmRNA及蛋白的表达与对照组比较差异无统计学意义(P<0.05)，照射组明显高于对照组(P<0.05),SAHA联合照射组明显高于其它各组(P<0.05)；SAHA(?)关合照射组细胞Ku70mRNA及蛋白的表达明显低于照射组(P<0.05)；结论：1、SAHA在体外能够抑制人宫颈癌SiHa (?)细胞的生长,抑制作用呈时间剂量依赖性。2、SAHA联合DDP作用SiHa细胞,在体外可以显著诱导细胞凋亡,并使细胞周期阻滞于GO/G1期,二者具有协同作用。3、SAHA联合DDP作用SiHa细胞,能够显著地上调p21、Bax的mRNA及蛋白表达水平,可见促进细胞凋亡及阻断细胞周期可能是SAHA (?)曾强SiHa细胞对DDP敏感性的机制之一。4、小剂量SAHA在体外能够促进SiHa细胞对X-线照射的敏感性。5小剂量SAHA通过增加放射诱导细胞凋亡基因Bax的表达及下调DNA修复基因Ku70的表达,促进了细胞的凋亡,抑制了细胞的修复,从而实现对SiHa(?)细胞的放射增敏作用。更多还原

【Abstract】 ObjectiveTo observe the sensitizing effects of the HDAC inhibitor suberoylanilide ydroxamic acid (SAHA) combined with DDP or X ray to cervical cancer SiHa cells and the mechanism of sensitization as well through studying the growth inhibition and apoptosis, cell colons formation rate and the result of the radiation sensitive parameters of cerical cancer cells.Methods1. SiHa cells were treated with0.25,0.5,1,2,4and6μmol/L of SAHA, and1.2,4,6,8and10μg/mL of DDP, respectivly. SiHa cells were treated with the vary combination dose of the two drugs. The inhibitory rate of SiHa cells were analyzed with MTT; Cell cycle distribution was detected by flow cytometry; Apoptosis of cells was measured by Annexin V-FITC.2. SiHa cells were incubated with SAHA at the concentration of20%IC50for24h and then were exposed to X-ray irradiation (IR)(0,2,4,6,8Gy). Colonies and surviving fraction (SF) of cells were detected by Clonogenic assay, the SF、Do and sensitizing enhancement ratio (SER) of each groups and the cell survival curve was gained by single-hit multi-target model.3. P21, Bax and Ku70’s mRNA and protein expression levels were investigated by RT-PCR and Western bolt in SiHa cell. Results1. SAHA was capable of inhibiting SiHa cells growth comparing with control group (P<0.05); SAHA appears more inhibiting SiHa cells growth effect attending by the increasing dose and time.2. The combined groups inhibited the cell growth obversely than single drug groups after24hours (P<0.05); Inhibition rate increased gradually with SAHA increasing dose combined with DDP; The effect of SAHA combined With DDP showed that low doses of DDP combination is synergy, while higher doses of DDP combination has simple addition effect.3. When1,2,4umol/L of SAHA (S group) combined with the DDP1,2,4ug/ml (D group) respectively, all the groups appear arresting cell at G1/G0phase, while the G2/M+S period cells proportion markedly reduced. PI reduced compared with control group significantly (P<0.05). GO/G1phase ratio increased significantly in SD group, while the G2/M+S period cells proportion markedly reduced. PI reduced significantly compared with the S group and D group (P<0.05).4. SAHA combing with DDP induced more severe apoptosis than separate groups (P<0.05). Apoptosis in2ug/mL DDP group was not obviously, but after pretreatment by2umol/L SAHA, apoptosis rate increased significantly.5. Compared the expression of Bax mRNA with the control group, there was no statistical significance of D, S group(P>0.05), while the expressions of combination groups were higher then other groups(P<0.05); P21expression in each group is raised significantly, especially in combination groups comparing with D group and control group (P<0.05).6. Bax protein expression between SAHA groups and the control group was not statistically significant (P>0.05). The combination of two drugs increased Bax protein level (P<0.05). P21protein in each group are raised higher than D group and control group, especially in combination groups, significantly (P<0.05).7. Cell survival fraction was obviously lower after treatment with the combination of SAHA and radiation than that of radiation alone (P<0.05). The cell survival curve of combination groups were to the left compared with radiotherapy groups. The front one was relatively flat and the shoulder area is not obvious. Do of the X-ray groups and the combination groups were2.329and1.213respectivly. The Dq were1.721and0.823respectively. The SFR was1.92.8. Expression of Bax mRNA of radiotherapy groups were higher then the control groups(P<0.05). The expressions of combination groups of Ku70mRNA were were lower then than the radiotherapy groups (P<0.05).Conclusions1. SAHA has the synergistic effect with DDP on SiHa cells.2. SAHA can arrest cell cycle and induce apoptosis of SiHa cells with DDP.3. The mechanism might be associated with up-regulating expression of P21and Bax mRNA and protein.4. Low concentrations of SAHA enhanced the radiosensitivity of SiHa cells.6. Up-regulating the expression of Bax and down-regulating the expression of Ku70response to the sensitization enhancement of SAHA to Siha cells.更多还原