【作者】 顾科；

【导师】 蒋国梁；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 第一部分：索拉非尼联合放射抗人肝细胞癌放射增敏效应的体外及体内实验研究目的：观察索拉非尼联合放射对人肝癌细胞株SK-Hep-1及其裸鼠移植瘤的抑瘤效应,明确索拉非尼与放射是否具有协同抗肝细胞癌效应并对两种方法联合应用的有效方式进行探讨。材料与方法：研究分体外及体内实验。体外实验中：(1)细胞增殖毒性试验。采用CCK-8法,测出索拉非尼作用SK-Hep-1细胞株48小时的半致死浓度(IC50),进而确定索拉非尼用于放射增敏实验的合适浓度。(2)克隆形成实验。分为3组,分别给予单纯放射、索拉非尼作用48小时后同步联合放射、放射后序贯应用索拉非尼作用48小时。根据各剂量点存活分数(SF),经多靶单击数学模型拟合,获得细胞存活曲线,将联合处理组与对照组进行参数D0、SF10%、SF1%比较,得出放射增敏比(SER)。体内实验中：(1)建立人肝癌细胞株SK-Hep-1裸鼠移植瘤模型。(2)荷瘤裸鼠分为3组：单纯放射组、30mg/kg索拉非尼灌胃7天后放射组、100mg/kg索拉非尼灌胃7天后放射组。放射剂量分别为单次0Gy、2Gy、4Gy、6Gy、8Gy、10Gy。观察各组肿瘤的生长延迟时间,比较肿瘤体积生长至原先2倍的生长延迟。经Gompertz模型拟合获得肿瘤的剂量效应曲线,将联合处理组与单纯放射组进行参数比较,计算出剂量修饰因子(DMF)。结果：体外实验中,索拉非尼作用48小时对SK-Hep-1增殖的IC50为9.5625μM,索拉非尼浓度选用1μM,用于联合放射组的克隆形成实验。多靶单击模型拟合得出,索拉非尼同步联合放射的SER(DO)、SER(SF10%)及SER(SF1%)分别为1.1、1.3、1.2。体内实验中,30mg/kg索拉非尼联合放射2Gy、4Gy、6Gy、8Gy后的TGD分别为5.3天、15.1天、19天、20.9天；100mg/kg索拉非尼联合放射2Gy、4Gy、6Gy、8Gy后的TGD分别为10.9天、16.6天、20.3天、22.6天。30mg/kg及100mg/kg索拉非尼联合放射的DMF分别为1.3至1.8,以100mg/kg索拉非尼联合放射更明显。结论：体外及体内实验结果显示,索拉非尼同步联合放射有放射增敏效应。第二部分：索拉非尼同步联合放射抗人肝细胞癌放射增敏机制的实验研究目的：第一部分实验结果表明,索拉非尼同步放射治疗人肝癌细胞株SK-Hep-1有放射增敏作用。本研究探讨索拉非尼放射增敏效应的机制。材料与方法：(1)SK-Hep-1细胞经1μM索拉非尼处理48小时后同步放射OGy、1Gy、2Gy、6Gy,应用Western blot法检测细胞蛋白VEGFR-2.ERK、NF-κB及Ku 70磷酸化及非磷酸化表达,与单纯放射组进行比较。(2)给予SK-Hep-1细胞OGy、1Gy、2Gy、4Gy、6Gy、8Gy放射后,应用ELISA法检测放射后0h、24h、48h、72h不同时期培养液内VEGF含量；并用RT-PCR检测在0Gy、1Gy、2Gy、6Gy、8Gy放射后24h、48h,细胞的VEGF mRNA表达,观察放射对人肝癌细胞分泌VEGF的影响、放射是否可以引起人肝癌细胞株过分泌VEGF。结果：(1)Western blot检测结果：单纯放射后,DNA修复蛋白Ku 70磷酸化水平略增高；1μM索拉非尼处理后,VEGFR-2及Ku 70磷酸化明显抑制；索拉非尼同步联合放射后,VEGFR-2、ERK及Ku 70蛋白的磷酸化均明显受抑制：NF-κB的磷酸化在索拉非尼联合6Gy放射后有轻度抑制。(2)ELISA检测结果：放射后,VEGF浓度整体上高于对照组,且增长幅度强于对照组；在24h及48h时以2Gy放射组显著,在72h时,放射组VEGF浓度均高于对照组,1,2,4,6Gy组增高显著(P<0.05),其中1Gy组最明显,8Gy组不明显(P>0.05)。在放射组中,以1、2、4、6Gy放射后48h-72h培养液中VEGF浓度为最高,放射剂量继续增加并不能引起VEGF的升高；72h时,8Gy组与对照组相比无显著差异,明显低于1、2、4、6Gy组。8Gy高剂量放射诱发VEGF增高的效应并不明显,可能与高剂量放射后细胞死亡增加显著有关。(3)RT-PCR结果SK-Hep-1细胞8Gy放射24h后VEGF mRNA表达增强,48h后更为明显；放射1Gy、2Gy、6Gy后48h,VEGF mRNA表达也明显上升；而对照组(0Gy)在24h及48h时VEGF mRNA表达无明显差异。用FR-2000型图像分析系统对电泳图像进行光密度扫描,计算得到VEGF mRNA的相对转录量(与β-Actin mRNA光密度值的比值),结果显示,VEGF mRNA的表达随放射剂量及放射后时间的延长,有加速升高的趋势,在8Gy放射后最为明显。结果提示,未放射时SK-Hep-1细胞培养液中VEGF浓度随时问延长的升高,是由于培养瓶中VEGF的长期蓄积；而受放射后VEGF的进行性加速升高,与放射诱导的VEGF mRNA表达增强有关。结论：索拉非尼放射增敏机制可能为：放射诱发SK-Hep-1细胞过高分泌VEGF,且通过上调DNA修复蛋白的表达加速DNA损伤修复；索拉非尼同步联合放射后,可通过抑制VEGFR-2、ERK1/2及Ku 70的磷酸化,从而抑制放射后肿瘤细胞DNA损伤的修复,起到放射增敏作用。在体索拉非尼的放射增敏机制可能是：索拉非尼抑制放射后肿瘤细胞DNA损伤的修复；同时,放射可引起人肝癌细胞过度分泌VEGF,增加肝细胞癌(HCC)的增殖；而索拉非尼单独或联合放射均可明显抑制VEGFR-2磷酸化,从而可以减少细胞膜VEGFR-2与胞外VEGF的活性结合,从而在体内可减少肿瘤新生血管形成,延缓或抑制肿瘤的生长。本实验研究结果提示,在临床使用放射治疗HCC时,同步联合应用索拉非尼有可能增强局部疗效,同时减少放射诱发的VEGF升高,降低复发及远地转移的几率第三部分：肝硬化大鼠模型建立及肝硬化自然消退中肝再生的研究目的：我们在对硬化肝脏放射后的肝再生进行实验研究时,需要在肝硬化形成后继续观察120天。为此我们建立一个适合长时期观察的肝硬化动物模型,并对肝硬化自然消退过程中的肝功能及肝再生进行研究,所得到的数据可以作为基础值,更好的理解、解释放射实验的结果。材料与方法：给予112只Wistar大鼠饮用0.03%硫代乙酰胺(TAA)29周,来建立肝硬化动物模型。肝硬化模型建立后,将TAA撤除,继续观察120天,每隔30天处死5只大鼠,获取肝脏和血液样本,观察硬化及肝再生的动态变化。(1)全自动生化仪检测血清ALT、AST、ALP及PA,观察肝功能变化；(2)三色染色观察胶原纤维含量、血清TGF-β1浓度、TGF-β1免疫组化染色表达及肝TGF-β1 mRNA表达、肝羟脯氨酸含量,以此观察肝硬化的动态变化；(3)肝脏指数、H&E染色分析肝细胞有丝分裂指数(MI)、流式细胞术检测肝细胞增殖分数(PI)、PCNA免疫组化染色表达及qRT-PCR检测肝脏PCNA mRNA表达,用于分析肝再生变化；(4)ELISA法检测血清肝再生相关生长因子HGF、VEGF、TGF-α及IL-6。结果：大鼠应用TAA29周后,经病理检测证实形成肝硬化,模型建立,建模率96%。模型建立时,表现为明显肝再生,伴肝功能不全。撤除TAA后,大鼠肝硬化出现白发消退,肝脏胶原纤维减少、羟脯氨酸含量下降、肝脏TGF-β1表达下调。在肝硬化消退时,伴随肝功能的改善。在肝硬化消退120天后,明显肝纤维化仍然可见。撤除TAA后,肝再生显著降低,肝细胞MI、PI、PCNA mRNA表达下降,免疫组化PCNA阳性染色比例下降,此外,血清HGF和VEGF也出现下降。结论：肝硬化大鼠动物模型可以经口服0.03%TAA29周后诱导形成。肝硬化能发生自发消退,消退过程中肝功能改善、肝再生下降。此模型形成的肝硬化可以维持120天以上,适合需长时期观察的研究使用。第四部分：硬化肝脏放射后肝损伤及肝再生的实验研究目的：研究肝硬化大鼠右半肝脏给予亚致死性放射后,是否能产生肝再生、是否能触发未放射的左半肝肝脏出现肝再生材料与方法：将研究分成连续的两个实验。实验一中,肝硬化大鼠分成4组,即对照组(未放射)、5-0Gy、10-0Gy及15-0Gy组,后3组分别给予右半肝单次放射5Gy、10Gy及15Gy,左半肝不放射。将放射前定义为d0,放射后观察120天,每隔30天,分别于d30、d60、d90及d120每组处死5只大鼠,获取血清和肝脏标本进行肝损伤与肝再生评价。实验二的设计依赖于实验一结果。实验一结果显示,右半肝放射5Gy、10Gy、15Gy均能引起放射半肝及未放射半肝发生肝再生,以15Gy为最明显。实验二中,将肝硬化大鼠分成5组,分别为假照对照组(0-0 Gy)、15-0Gy、15-2.5Gy、15-5Gy及15-7.5Gy组,后4组选择15Gy作为初始刺激放射右半肝,并同时分别放射左半肝0Gy、2.5Gy、5Gy及7.5Gy。将放射前定义为d0,放射后观察150天,每隔30天,分别于d30、d60、d90、d120及d150每组处死5只大鼠,获取血清和肝脏标本进行肝损伤与肝再生评价。实验一及实验二的观察指标相同：(1)肝损伤：大鼠体重、血清ALT、AST、ALP及PA；(2)肝再生：肝脏指数、H&E染色分析肝细胞有丝分裂指数(MI)、流式细胞术检测肝细胞增殖分数(PI)、PCNA免疫组化染色表达及qRT-PCR检测肝脏PCNA mRNA表达、ELISA法检测血清肝再生相关生长因子HGF、VEGF、TGF-α及IL-6；(3)血清TGF-β1浓度、TGF-β1免疫组化染色表达及肝TGF-β1 mRNA表达。结果：单纯给予右半肝5Gy、10Gy、15Gy放射,或者给予右半肝15Gy放射同时左半肝放射2.5Gy、5 Gy、7.5Gy,均可引起肝再生,且在左右半肝均发生。肝再生指标中,PCNA免疫组化阳性染色比例、PCNA mRNA表达、PI、MI明显升高,以左半肝略显著；同时血清HGF、VEGF、IL-6浓度均显著升高。放射后肝功能受损,血清ALT、AST、ALP、TGF-β1升高,PA降低,肝组织中TGF-β1表达明显增高,以右半肝显著。随放射剂量及放射肝体积增加,肝再生与肝损伤表现更显著。实验中大鼠死亡率均不高于11.11%,实验使用的放射剂量及范围对肝硬化大鼠致死性不高。结论：硬化肝脏能耐受一定程度的放射,15Gy的亚致死性部分肝脏放射可以在放射肝及未放射肝,引起剂量和体积依赖性肝损伤与肝再生。组织学上,肝损伤在受到更高剂量放射的右半肝更明显,而肝再生在接受较低剂量的左半肝更显著。更多还原

【Abstract】 Part I:Study on radiosensitization of sorafenib in treatment of hepatocellular carcinoma (HCC) in vitro and in vivoObjective:To investigate the radiosensitization of sorafenib in treatment of HCC cell line SK-Hep-1 and its transplanted tumor in nude mice.Material & Methods:The present study was designed into two experiments in vitro and in vivo. For experiment in vitro, cell proliferation toxicity was measured by using CCK-8 method to obtain 50% inhibition concentration (IC50) of sorafenib 48 hours after treating SK-Hep-1 cell line. Clone formation assay was applied to obtain survival fraction (SF) of each treatment, i.e., IR alone as control, IR synchronizing sorafenib 48 hours afer incubing cell line and IR followed by sorafenib. Based on multi-target single-hit model, cell survival curve was gained and theoretical IR doses of D0, SF10% and SF1% could be calculated. Sensitizing enhancement ratios (SERs) of sorafenib combing IR were obtained by compared with those of group control. For experiment in vivo, nude mice transplanted tumor model was established firstly. The mice were feeded vehicle,30mg/kg and 100mg/kg sorafenib respectively for 7 days, and the tumors were irradiated with doses of OGy,2Gy,4Gy,6Gy,8Gy and 10Gy on day 8 repectively. Tumor growth delay (TGD) was assessed. Dose response curve of transplanted tumor was obtained according to Gompertz model. Given growing to double of initial tumor volume before treatment, TGD of combination group was compared with that of IR alone group, then dose modify fraction (DMF) was calculated to evaluate synergetic anti-tumor effect of sorafenib and IR.Results:Cell proliferation toxicity assay presented IC50 of sorafenib 48 hours after treating SK-Hep-1 cell line was 9.5625μM and the concentration of 1μM, less than IC10, was used in clone formation experiment. According to multi-target single-hit model, cell survival curve of treatment with sorafenib combined with IR concurrently and theoretical IR doses of D0, SF10% and SF1% were gained. SER (DO)、SER(SF10%)and SER(SF1%) were 1.1138、1.2749、1.2017 repectively, which were more than 1 and radiosensitizing effect of sorafenib combined with IR concurrently was confirmed. In vivo, after irradiated with 2Gy、4Gy、6Gy、8Gy concurrently combined with 30mg/kg sorafenib, TGD were 5.3 days,15.1 days,19 days and 20.9 days, respectively. As to IR combined with 100mg/kg sorafenib, TGD were 10.9 days,16.6 days,20.3 days and 22.6 days, respectively. DMFs of 30mg/kg and 100mg/kg sorafenib combined IR were between 1.3 and 1.8. The stronger radiosensitization was shown in 100mg/kg sorafenib combined with IR.Conclusion:Sorafenib combined with IR concurrently presented radiosensitation in vitro and in vivo.Part II:The mechanism of radiosensitization of sorafinib in treatment of hepatocellular carcinoma (HCC)Objective:The previous data had shown the radiosensitization of sorafenib in treatment of HCC cell line. This study is to investigate the underlying mechanism.Material & Methods:The methods of Western blotting, enzyme linked immunosorbent assay (ELISA) and RT-PCR were used to investigate molecular mechanism of radiosensitizing effect. SK-Hep-1 cell line was treated with 1μM sorafenib for 48 hours concurrently combined with IR of OGy, 1Gy,2Gy and 6Gy respectively. Then the cells were collected, from which the total protein was extracted. The expressions of VEGFR-2, ERK, NF-κB and Ku 70 were assessed with or without phosphorylation. ELISA was applied to measure concentration of VEGF in medium 0 hour,24 hours,48 hours and 72 hours after SK-Hep-1 being irradiated with single dose of OGy, 1Gy,2Gy,4Gy,6Gy and 8Gy respectively. Otherwise, expression of VEGF mRNA of these cells 24 hours and 48 hours after treated with IR of OGy, 1Gy, 2Gy,6Gy,8Gy was assayed also.Results:(1) The data of Western blotting showed the up-regulation of phosphorylation in DNA repair protein, Ku 70 with IR alone. Phosphorylations in VEGFR-2 and Ku 70 were inhibited obviously by treatment with 1μM sorafenib alone. The treatment of sorafenib combined with IR inhibited the expressions of phosphorylated VEGFR-2, ERK and Ku 70. (2) The results of ELISA assay presented that medium concentration of VEGF increased after IR higher than that of control group. The significant increase presented 24 hours and 48 hours after IR of 2 Gy. The concentrations of VEGF at 72 hours after IR of 1Gy,2Gy,4Gy and 6Gy were significantly higher than those of control group (P<0.05), the highest value after 1 Gy IR, while the difference was not notable after 8Gy IR (P>0.05). As a whole, concentration of VEGF rose with the increase of IR dose, but decreased with high IR dose of 8Gy, of which the reason was the decline of survival cells after 8Gy IR probably. (3) The data of RT-PCR showed expression of VEGF mRNA of Sk-hep-1 was enhanced 24 hours, especially 48 hours after IR. The increase of VEGF mRNA expression presented 48 hours after IR of 1Gy,2Gy,6Gy also. The expressions of VEGF mRNA in control group showed no significant difference between 24 hours and 48 hours after sham IR. VEGF relative transcription ratio was calculated with image analysis system compared with expression ofβ-Actin mRNA, indicating that expression of VEGF mRNA upregulated accelerated parallel to the increase of dose of IR and time after IR, most evident with 8Gy IR. The data implied that the concentration of medium VEGF after IR increased owing to IR-induced enhancement of VEGF mRNA expression.Conclusion:IR could stimulate VEGF secretion of SK-Hep-1 cell line and induce upregulation of DNA repair protein to accelerate repair of DNA damage. Sorafenib combined with IR was capable of inhibiting phosphorylations of VEGFR-2, ERK and Ku 70. These mechanisms resulted in radiosensitization of sorafenib.PartⅢ:A natural process of cirrhosis resolution and deceleration of liver regeneration after thioacetamide withdrawal in a rat modelObjective:To establish a useful rat model with cirrhosis and evaluate liver injury and liver regeneration during cirrhosis resolution.Material & Methods:In a radiobiological study of partial liver irradiation on thioacetamide (TAA) induced cirrhotic liver in rat, the observation time was 120 days after TAA withdrawal. The natural process was recorded, focusing on cirrhosis and regeneration as the baseline to understand and interpret outcome. Cirrhosis in rats was induced by orally drinking 0.03% TAA water for 29 weeks. After establishment of cirrhosis model, the rats were observed for 120 days upon TAA withdrawal to investigate the dynamic changes of liver cirrhosis and regeneration. The endpoints were:(1). Histological change. (2). Liver functions. (3). Cirrhosis:Trichrome stain serum and in situ transforming growth factor beta-1 (TGF-β1), hydroxyproline content of liver. (4). Liver regeneration:liver index; hepatocyte mitotic index (MI); hepatocyte proliferation index (PI) by flow cytometry; PCNA labeling index (LI) by IHC and expression of PCNA mRNA. (5). Growth factors:serum HGF, VEGF, TGF-a and IL-6.Results:The cirrhosis model was established after 29 weeks. And upon TAA withdrawal, the cirrhosis presented resolution. Gradual improvement in liver functions was noted with decreasing ALT, AST and ALP, and increasing PA during cirrhosis resolution. The resolution of cirrhosis was evident by histological improvement with attenuation of collagen fiber and hydroxyproline and decrease of TGF-β1 IHC index, but cirrhosis was still existed on 120 days after TAA withdrawal. Significant deceleration of liver regeneration was demonstrated with TAA withdrawal, evidenced by decrease of hepatocyte MI and PI, reduced expression of PCNA mRNA and PCNA LI, and decreases of serum HGF and VEGF.Conclusion:Cirrhosis was induced by drinking 0.03% TAA water for 29 weeks in rats. Upon TAA withdrawal it was revealed that hepatic cirrhosis continuously resolved, but still existed up to 120 days, and liver regeneration significantly decelerated.PartⅣ:Hepatic Regeneration after Partial Liver Irradiation in Cirrhotic RatsObjective:To investigate if the right half liver were capable of regeneration after sublethal irradiation (IR) and if the IR would trigger regeneration of the left half liver in cirrhotic rat.Material & Methods:This study was designed into two experiments in turn. For experiment one, the cirrhotic rats were divided into control group (without IR), and 5 Gy, 10Gy and 15Gy groups. IR dose of 5 Gy, 10Gy and 15Gy were given as single dose to the right half liver of each group respectively with observations of the endpoints scheduled on 0-day (before IR),30 days,60 days,90 days and 120 days after IR. The results showed a dose-dependent liver proliferation could be stimulated by sublethal partial liver IR of 5Gy to 15Gy, in both irradiated and unirradiated part livers, most notable by 15 Gy. Consequently, the IR of 15 Gy was chosen as a initial stimulus delivered to the right half liver in the experiment two. For experiment two, the cirrhotic rats were divided into sham (OGy) group,2.5 Gy,5 Gy and 7.5 Gy groups according to the IR dose delivered to the left half liver with the right half liver irradiated by 15 Gy. The endpoints same as those in experiment one were observed every 30 days till 150 days after IR. The cirrhotic rats without radiation were chosen to serve as control. The following endpoints were evaluated:(1). Liver injury:Body weight and Serum ALT, AST, ALP and PA. (2). Liver regeneration:liver index; hepatocyte mitotic index (MI); hepatocyte proliferation index (PI); PCNA labeling index (LI); expression of PCNA mRNA; serum HGF, VEGF, TGF-α, IL-6; (3) Serum and in situ TGF-β1.Results:IR of 5 Gy, 10Gy and 15Gy to the right half liver as well as IR of 2.5 Gy, 5 Gy and 7.5 Gy to the left half liver with the right half liver irradiated by 15 Gy could induce hepatic regeneration in both right and left half livers with indicators of liver regeneration including positive staining of PCNA and PCNA mRNA expression, PI and MI, serum HGF, VEGF and IL-6 increasing after IR, and deteriorated liver injury with decrease of ALT, AST and ALP and increase of PA, expression of TGF-β1 in Serum and in situ. The IR-induced hepatic injury and liver regeneration increased accompanying with higher IR dose and whole liver irradiated.Conclusion:Dose-dependent and volume-dependent liver injury and liver proliferation could be stimulated by sublethal partial liver IR of 15Gy in both irradiated and unirradiated portion.更多还原