【作者】 张勇；

【导师】 陈龙华；

【作者基本信息】 南方医科大学 ， 放射肿瘤学， 2011， 博士

【摘要】 研究目的和意义：原发性肝细胞肝癌(hepatocelluar carcinoma, HCC)是世界上最常见的恶性肿瘤之一,全球发病每年约62万例,男女之比为4：1。在我国肝癌发病人数占世界每年新发病患者的55%,居我国癌症发病率的第二位,每年有13万人死于肝癌。在恶性肿瘤中,肝癌是一种生存期较短、死亡率较高、治疗很困难的恶性疾病,提高对肝癌的治疗水平具有十分重要的意义。虽然外科手术是原发性肝癌首选治疗方法,但手术率仅有5%-20%左右,同时手术复发率很高。多数肝癌患者就诊已属中晚期,此类患者能手术切除者仅占30%左右,不能手术切除的原发性肝癌的治疗方法很多,包括肝动脉插管栓塞化疗、病灶局部注射无水酒精、冷冻治疗、瘤体内射频高温治疗和体外超声波聚焦治疗等局部消融治疗都取得了一定的疗效。放射治疗与外科有类似之处,属于局部区域性治疗手段。随着放射治疗技术的进步,适形放疗已逐渐成为非手术治疗肝癌的主要治疗方法之一。然而,放疗的临床治疗效果并不满意。肿瘤组织的放射抵抗是肿瘤放射治疗失败的原因之一,由于肝癌细胞的相对辐射抗拒性,使得肝癌的根治剂量明显高于肝脏的耐受剂量,尤其中晚期肝癌患者多数有肝病基础,又限制了放疗剂量的提高,导致肝癌放疗效果不佳。因此,寻找有效的、专一针对肿瘤组织的放射增敏办法对提高肿瘤组织的放疗敏感性,降低肿瘤组织的放疗抵抗,减低周围正常组织的放疗毒副作用具有重要意义,可为提高肝癌的放射治疗效果提供新的思路和途径。随着分子生物学的研究发展,越来越多的人认识到恶性肿瘤的发生是一种涉及多基因事件的过程。肿瘤的基因治疗逐渐成为肿瘤治疗研究的热点之一,但临床应用效果并不令人满意。同时,分子生物学的发展也为肿瘤放射治疗提供了分子水平的理论依据,指出其杀伤作用的产生有赖于一系列基因功能的正常发挥。为了弥补各自的缺陷和不足,放射治疗与肿瘤基因治疗在自身发展的同时,正在有效地结合起来,以基因治疗靶向性处理肿瘤细胞,提高其对放射线的敏感性,然后联合放疗,有的学者将这种新的治疗模式称为肿瘤的基因-放射治疗(genetic radiotherapy).肿瘤的基因-放射治疗是指将同时具有肿瘤治疗和辐射诱导特性的基因转入体内,在对肿瘤实施局部放疗时诱导肿瘤治疗基因的表达,造成射线和基因对肿瘤的双重杀伤作用。这样一方面可以相对降低等效照射剂量,缓解正常组织损伤；另一方面也可在即使是基因进入体内后均匀分布的情况下,通过射线的局部照射来实现定位表达。因此,将肿瘤的放射治疗与基因治疗相结合,可以解决两者单独应用时各自存在的敏感性和特异性问题。目前,初步的实验研究表明,基因-放射治疗具有提高肿瘤治疗基因的局部表达量、降低有效照射剂量、减轻受照部位正常组织的放射损伤等诸多优点。治疗基因的选择是实现此方案的关键,因此开展新的治疗基因的研究是当前恶性肿瘤基因-放射治疗的研究重点之一。PTEN是迄今发现的第一个具有磷酸酶活性的抑癌基因,被认为是继pRb、p53后的又一重要的肿瘤抑制基因,与多种肿瘤发生、发展密切相关,因此备受关注。PTEN是一种多功能性的蛋白,具有两种磷酸酶的活性：一种是脂质磷酸酶活性,使磷脂酰肌醇-3,4,5-三磷酸去磷酸化,调节第二信使PIP3的水平,阻断了PI3K/AKT途径,参与调节细胞生长、增殖和凋亡,抑制肿瘤生长,诱导肿瘤细胞分化；另一种是蛋白酪氨酸磷酸酶活性,通过靶向于FAK和Shc的蛋白质酪氨酸磷酸酶功能抑制了Ras/Raf/MEK/ERK通路的级联反应和FAK级联反应,影响细胞和细胞间质的相互作用,参与调节细胞粘连、迁移、肿瘤细胞的转移、细胞骨架的组建及MAP kinase的活化和肿瘤血管的形成。在原发性肝细胞癌的研究中,已证实PTEN基因的表达缺失与HCC的发生发展密切相关,且PTEN基因可有效抑制肿瘤细胞的生长。在肝癌细胞系中恢复PTEN的表达,细胞生长受到明显抑制。尽管目前将PTEN基因引入原发性肝癌的放射治疗在国内外尚未见报道,但在对其他多种肿瘤的研究中已发现,PTEN可以增加多种肿瘤细胞的辐射敏感性：在前列腺癌细胞、胶质瘤细胞、结肠癌细胞以及非小细胞肺癌细胞中均发现增加PTEN的表达可以增强放射线对肿瘤细胞的杀伤作用。因此,PTEN强抑癌功能及对肿瘤细胞的放射增敏功能可被引入肝癌的基因治疗,通过转基因技术恢复肝癌细胞中缺失表达的野生型PTEN基因,发挥正常PTEN基因的抑癌功能,同时增强对肝癌细胞的放射杀伤作用。基于肿瘤基因-放射治疗的设想,本研究将具有辐射诱导作用的Egr-1基因启动子辐射诱导增强区域与抑癌基因PTEN相连接,构建辐射诱导表达载体,探讨含PTEN的辐射诱导表达载体稳定转染肝癌细胞系SMMC-7721后,对肝癌细胞的辐射增敏作用及其机制。本研究采用辐射诱导基因调控序列偶联不具备辐射诱导特性的抑癌基因,通过放射线照射调控抑癌基因的表达,这一治疗手段不仅非常有特色,并且可以解决单纯基因治疗存在的特异性不强的问题,从技术手段来讲,对肿瘤放疗研究方面是一次较新的尝试。同时本研究选择抑癌基因PTEN为基因治疗靶点,PTEN基因是目前研究较明确的、具有多途径抑癌作用的强效抑癌基因,目前将PTEN基因引入原发性肝癌的放射治疗在国内外尚未见报道,从治疗基因的选择方面,选择联合PTEN来增强肝癌细胞的放疗敏感性既是对肝癌放疗增敏研究的全新尝试,也是非常可行的研究。另外,研究从肿瘤的放射增敏的分子机制入手,探讨联合PTEN进行放射治疗肝癌细胞的可能机制,丰富了肿瘤基因-放射治疗的理论依据。目前,由于原发性肝癌的治疗效果有限,放射治疗作为非手术治疗患者的首选方法存在相对辐射抵抗性强的问题而限制了其应用,严重影响了肝癌患者的生存质量。本研究的实施将为原发性肝癌的放射治疗提供新的理论和实践视角,在解决肿瘤放射性抵抗问题上起到一定的推动作用,为今后原发性肝癌的基因-放射治疗应用于临床实践提供理论和实践支持。方法：分别构建含野生型PTEN和丧失了脂质磷酸酶活性的突变型PTEN的重组辐射诱导质粒pEgr-PTEN和pEgr-PTEN-G129E,通过荧光素酶报告实验鉴定X线照射对Egr-1启动子活性的影响,寻求最适的照射条件。将构建好的重组质粒分别稳定转染入人肝癌细胞系SMMC-7721细胞,通过Western—blot法检测X线照射对PTEN蛋白表达的影响；MTT实验检测基因稳定转染SMMC-7721细胞后对细胞生长的影响；通过平板克隆实验计数克隆形成数并计算存活分数(SF),利用GraphPad Prism 5软件分别根据线性二次(L-Q)模型和多靶单击模型,得到放射生物学参数并评价转染PTEN基因后对肝癌细胞SMMC-7721细胞的辐射增敏作用；流式细胞仪检测转染PTEN基因后细胞接受X线照射前后细胞周期的变化；TUNEL检测法通过流式细胞仪检测转染PTEN基因并接受X线照射后细胞的凋亡情况；通过免疫荧光法检测细胞在接受8 Gy的X线照射后,于0、7min、1h、2h、4h、6h、12h、24h时细胞的γ-H2AX焦点形成数,进而观察肝癌细胞系SMMC-7721细胞在接受X线照射后的DNA损伤、修复情况。通过Western—blot法检测转染野生型PTEN、突变型PTEN细胞接受照射后总Akt和磷酸化Akt的表达变化,进一步采用PI3K抑制剂LY294002作用于肝癌细胞系SMMC-7721细胞,MTT检测观察应用PI3K抑制剂对受照细胞生长的影响,平板克隆实验计算存活分数,利用GraphPad Prism 5软件分别根据线性二次(L-Q)模型和多靶单击模型,得到放射生物学参数并评价PI3K抑制剂LY294002对SMMC-7721细胞的辐射增敏作用；流式细胞仪检测应用LY294002后细胞接受X线照射前后细胞周期的变化；应用流式细胞仪采用TUNEL法检测应用LY294002并接受X线照射后细胞的凋亡情况；通过免疫荧光法检测LY294002处理细胞在接受8 Gy的X线照射后,不同时间点细胞的γ-H2AX焦点形成数,观察细胞在接受X线照射后的DNA损伤、修复情况,并与转染野生型PTEN细胞组进行比较。结果：1、分别构建出含野生型PTEN和丧失了脂质磷酸酶活性的突变型PTEN的重组辐射诱导质粒pEgr-PTEN和pEgr-PTEN-G129E;2、通过荧光素酶报告实验验证了辐射对所构建的Egr-1启动子区具有一定的诱导表达作用,同时,也寻找到了对SMMC-7721细胞进行照射的最适条件,即细胞转染24小时后,对细胞进行8 Gy的X线照射并于照射后24小时收集细胞进行后续的实验研究；3、Western—blot检测表明转染了pEgr-PTEN和pEgr-PTEN-G129E的SMMC-7721细胞组在未接受照射(0 Gy)时PTEN蛋白的表达量极低,仅见微弱表达,但在接受8 Gy的X线照射后,PTEN蛋白的表达量明显提高,高于转染了pEGFP-PTEN和pEGFP-PTEN-G129E的细胞；4、MTT检测结果表明转染野生型PTEN组细胞在接受了8 Gy的X线照射后,细胞的生长明显较接受了同样剂量的X线照射后的转染空载体pEgr-C1组与转染重组突变质粒pEgr-PTEN-G129E组细胞增殖速度慢；5、转染了野生型PTEN组细胞SF2值(即接受2Gy照射后的细胞存活分数)、D0、Dq值均低于转染突变性PTEN组和未转染PTEN而仅转染了空质粒组细胞,而α值、α/β值均高于转染突变性PTEN组和未转染PTEN而仅转染了空质粒组细胞,表明转染了野生型PTEN组的细胞对X线照射的敏感性提高；6、接受8 Gy的X线照射后,转染了野生型PTEN组细胞G2/M期细胞比例明显提高,出现了明显的G2/M期阻滞；7、细胞接受照射24小时后,就已经有凋亡的出现,且转染野生型PTEN组细胞在接受8 Gy的X线照射后细胞凋亡率较转染空载体pEgr-C1组细胞和转染重组突变质粒pEgr-PTEN-G129E组的细胞凋亡率明显提高,照射后48小时各组细胞在接受X线照射后凋亡率进一步提高,但转染野生型PTEN组细胞在接受X线照射后48小时细胞凋亡率提升最为明显,远较另两组细胞的凋亡率明显提高；8、各组细胞在受到辐射作用后7min均出现DNA双链断裂的发生,随着照射后时间的延长,DNA双链断裂形成的γ-H2AX焦点数逐渐减少,损伤的DNA双链逐渐修复,但在转染了野生型PTEN组、重组突变质粒pEgr-PTEN-G129E组和空载体pEgr-C1组细胞间DNA双链修复却不尽相同：转染野生型PTEN组细胞较另两组细胞受X线照射后,在相同时间点具有更多的未修复的DNA断裂双链,DNA修复能力降低,出现延迟现象；9、Western-blot检测细胞中Total-Akt和磷酸化的Akt (Ser-473)蛋白的表达结果发现,转染野生型PTEN细胞磷酸化的Akt蛋白表达明显减少,细胞中Akt蛋白的磷酸化水平受到抑制,负性调节了PI3K/PTP3/AKT转导通路；10、采用PI3K抑制剂LY294002作用于肝癌细胞系SMMC-7721细胞,发现,与转染野生型PTEN组相似,LY294002处理组细胞磷酸化的Akt蛋白表达明显减少,MTT检测和平板克隆实验结果表明应用了PI3K抑制剂LY294002处理后的细胞在接受X线照射后细胞的生长受到一定的抑制作用、可一定程度地提高SMMC-7721细胞的放射敏感性。其对SMMC-7721细胞的辐射增敏作用也同转染野生型PTEN组细胞相似：PI3K抑制剂LY294002处理后的细胞在接受X线照射后也产生了明显的G2/M期阻滞,细胞的凋亡率明显提高,LY294002处理的细胞在受到辐射作用后也出现了DNA修复延迟现象。进一步表明PTEN可能通过PI3K-Akt信号转导通路增强对肝癌细胞系SMMC-7721细胞的辐射敏感作用。结论：1、PTEN对肝癌细胞具有放疗增敏作用；2、PTEN可以使细胞发生G2/M期阻滞、提高受照细胞的凋亡率、延迟细胞的DNA双链断裂修复；3、PTEN可能通过PI3K-Akt信号转导通路增强对肝癌细胞的辐射敏感作用；4、以PTEN为治疗靶点,联合辐射诱导技术对原发性肝癌进行的实验尝试,可为原发性肝癌的放射治疗提供新的理论和实践视角,为肿瘤基因-放射治疗应用临床提供理论支持。更多还原

【Abstract】 Objective and Significance:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The worldwide estimated incidence of HCC is more than 620,000 per year. The overall male and female sex radio is about 4:1. In China, primary liver cancer is largely a problem in which 55% of the patients reside ranking second among all malignancies in incidence with about 130,000 patients dying of it. Among all malignant tumors, HCC is one of the lesions with low survival rate, high mortality rate and difficulty for therapy. So it is important to improve the therapeutic effects of HCC. Surgical resection is a preferred method for HCC, but feasible only in 5% to 20% of patients. Most patients are firstly diagnosed for HCC with mid- to later-stage and only 30% of them are suitable for operation. Other therapies for inoperable HCC patients such as TACE, PEI, RFA would be the treatment options. Similarly, these techniques are also not suitable for large tumors.Like surgical resection, radiotherapy is also a kind of local therapy. Recent technological and conceptual developments in the field of radiation therapy, such as image-guided radiation therapy (IGRT) have the potential to improve radiation treatment. However the role of radiotherapy for liver tumors had been limited by the tumor cells’relative radioresistance and the risk of radiation-induced liver disease (RILD). Therefore, the amount of the radiation that can be delivered is limited and dose escalation cannot be used to overcome the resistance to radiation. Hence it is important to investigate other strategies for sensitizing HCC to irradiation while decreasing adverse effect and toxic effect of adjacent normal liver.With the development of molecular biology, people realize that the gene changes may involve in the tumorigensis. Many researches for oncology have focused on gene therapy now, but no satisfying results have been achieved. At the same time, the developments of molecular biology also provide a theoretical support for radiotherapy. Combining gene therapy and radiotherapy protocols has the potential to overcome many of the limitations of adverse tumor biology on cancer treatment. Radiation-mediated gene activation, or "genetic radiotherapy", takes advantage of both the killing effect and the precise targeting potential of ionizing radiation to locally regulate transcription of genes encoding toxic or radiosensitizing proteins. X-irradiation activates the transcription of certain genes, including the early growth response gene Egr-1. These findings led us to the concept that promoters from these genes could be used to drive therapeutic transgenes introduced into irradiated tumor cells. In this strategy, designated genetic radiotherapy, radiation is combined with gene therapy, another local/regional modality, to spatially and temporally control transgene expression in the irradiated field. The choice of therapeutic genes is the key for genetic radiotherapy. So it is important to search new effective therapeutic genes.PTEN is the first tumor suppressor gene functioned with a dual-specificity lipid and protein phosphatase. PTEN is a multifunctional phosphatase with a dual-specificity lipid and protein phosphatase. PTEN with its lipid phosphatase activity can dephosphorylate phosphatidylinositol (3,4,5)-triphosphate (PIP-3) to phosphatidylinositol 4,5-bisphosphate (PIP2) which in turn could regulate Akt function and cellular growth. Consistent with its protein phosphatase activity PTEN may function to regulate cell migration/adhesion by decreasing in FAK phosphorylation and also physically interaction with this kinase. Importantly, hepatic deficiency of PTEN leads to the development of liver tumors. Pten +/- heterozygous mice exhibited neoplasms in multiple organs including liver. Frequent genetic alterations and loss of expression of the PTEN gene have been found in a variety of human cancers. In the HCC, PTEN expression is also downregulated mostly because of the promoter methylation and other epigenetic regulation. Although the enhancement of radiosensitivity by the expression of PTEN to HCC cells has not been previously studied, some reports demonstrated that PTEN gene transfer can sensitize cells to irradiation in breast cancer, prostate cancer, non-small cell lung cancer, colorectal cancer and malignant glioma. Considering frequent genetic alterations and loss of expression of the PTEN gene in HCC, we hypothesized that the loss of PTEN expression may effect the radiosensitivity in HCC and transfecting HCC cells with PTEN would sensitize them to the effects of radiation.Based on the theory of genetic radiotherapy, we combined an Egr-1 radio-responsive enhancer with tumor suppressor gene PTEN and constructed radiation-induced expression vectors to test the radiosensitive effect of PTEN in HCC and explore the mechanism on PTEN enhancement of radiosensitivity.At the same time, considering the lack of specificity in gene therapy especially in vivo studies when transfecting to the surrounding normal cells in a nonspecific manner, we also focused on the use of radiation-inducible promoters to activate gene expression. We used the Egr-1 promoter in our system to ensure that PTEN could be regulated by ionizing radiation in temporally, spatially and in a dose-dependent manner. At the same time we chose PTEN gene as target gene for HCC gene therapy. As we known, there have no studies about the enhancement of radiosensitivity by the expression of PTEN to HCC cells. Considering the important inhibition of PTEN on tumor cells, we think it is feasible to use PTEN for radiosensitization HCC. On the other hand, our study may also provide a theoretical support for the clinical application of genetic radiotherapy.Now the role of radiotherapy for liver tumors had been limited by the tumor-cell relatively radioresistance and the risk of radiation-induced liver disease (RILD). And the amount of the radiation that can be delivered is limited and dose escalation can not solve radiation resistance. Our study will give a potential option for HCC radiotherapy and provide a theoretical support for the clinical application of genetic radiotherapy.Methods:Radiation-induced expression vectors of wild-type PTEN (pEgr-PTEN) or mutant PTEN (loss of lipid phosphatase activity) (pEgr-PTEN-G129E) were constructed respectively. Then we evaluated the responses of Egr-1 promoter to radiation treatment by luciferase report assay. After stably transfected, the expressions of PTEN in difference groups of SMMC-7721 cells were detected by Western blot analysis 24 hours after irradiation. To investigate if PTEN could inhibit cells growth, we carried out a MTT assay using SMMC-7721 cells that had been stably transfected with pEgr-PTEN, pEgr-PTEN-G129E or pEgr-C1 (mock) and treated them with 8 Gy irradiation. The radiosensitivity of cells in pEgr-PTEN, pEgr-PTEN-G129E or pEgr-C1 was assessed by clonogenic survival assays and we used GraphPad Prism 5 software to fit cell survival curve in accordance with standard linear-quadratic (LQ) model and one-hit multi-target model and then get the radiation biology parameters. We next determined the effect of PTEN on cell-cycle phases in SMMC-7721 cells by using Fluorescence Activating Cell Sorter (FACS) analysis. To quantitate apoptosis, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP biotin nick end labeling (TUNEL) assay was done. Immunofluorescence was used to detect gamma-histone H2A (γ-H2AX) foci formation and then to track the repair of DSB produced by radiation at several times following irradiation. To explore the mechanism on PTEN enhancement of radiosensitivity we also detected the expression of Akt and its phosphorylated form (pAkt) by Western blot analysis. We also treated cells with LY294002, a specific inhibitor of PI3K/Akt pathway. The cell viability was assessed by MTT assay and the radiosensitivity was assessed by clonogenic survival assays, cell survival curve was fitted in accordance with standard linear-quadratic (LQ) model and one-hit multi-target model using GraphPad Prism 5 software after treating with LY294002. Similarly, the effect of LY294002 on cell-cycle phases in SMMC-7721 cells was determined by using Fluorescence Activating Cell Sorter (FACS) analysis and the apoptosis/rate was quantitated by TUNEL assay. Immunofluorescence was done to detect gamma-histone H2A (y-H2AX) foci formation to detect the repair of DSB at several times following irradiation.Results:1. We constructed the radiation-induced expression vectors of wild-type PTEN (pEgr-PTEN) or mutant PTEN (loss of lipid phosphatase activity) (pEgr-PTEN-G129E) respectively.2. By luciferase activity based promoter assays, we demonstrated that the Egr-1 promoter was induced by irradiation, and the response was dose-dependent. Peak responses were observed in SMMC-7721 cells at 24 h after 8 Gy irradiation.3. Little expression of PTEN was detected with no irradiation, while the response to irradiation was observed where PTEN was expressed higher than that of the non-irradiated cells in the pEgr-PTEN and pEgr-PTEN-G129E groups and that of the pEGFP-PTEN and pEGFP-PTEN-G129E groups after 8 Gy irradiation.4. By MTT assay we found that PTEN expression caused the decreases in the cell viability and proliferation decreased after irradiation.5. The survival fraction of 2 Gy (SF2), DO and Dq in pEgr-PTEN group were lower than that in the pEgr-PTEN-G129E and pEgr-C1 groups, while there was a higher value for both a andα/βin pEgr-PTEN group. Transfection of pEgr-PTEN increased the sensitivity of SMMC-7721 cells to irradiation, whereas that of pEgr-PTEN-G129E and pEgr-C1 induced no changes.6. A significant G2/M arrest at 24 h after irradiation of cells with pEgr-PTEN was found, while cells in the pEgr-PTEN-G129E or pEgr-C1 groups displayed minimal levels of G2/M arrest when compared with the non-irradiated groups.7. We found that the TUNEL positive cells in pEgr-PTEN group were increased at 24 h after irradiation and found a greater number of apoptotic cells at 48h after irradiation.8. During investigations of the DNA repair processes after irradiation in our study, we found that although all the cells incurred an equivalent number of initial DSB, the repair of irradiation-induced DSB was retarded in wild-type PTEN cells, but not in PTEN G129E mutant cells or the mock cells.9. When we detected the expression of phosphorylated Akt, we found that transfecting cells with wild-type PTEN decreased pAkt but not with the PTEN G129E mutant and mock transfections, indicating that PTEN negatively controled the phosphoinositide 3-kinase/PIP3/Akt signaling pathway.10. We treated cells with LY294002, a specific inhibitor of the PI3K/Akt pathway, before irradiation with 8 Gy. By MTT assays, we observed an obvious decrease in absorbance. Correspondingly, the radio sensitivity of cells was enhanced by LY294002 after assessment by clonogenic survival assays. Similarly, treatment of cells with LY294002 and irradiation induced G2/M arrest, and the TUNEL assay showed that the number of apoptotic cells treated with LY294002 only was higher than the other non-irradiated groups which increased after irradiation. The retarding of DSB repair was also seen with LY294002 and irradiation treatments demonstrating that radiosensitivity correlates with an active PTEN-PI3K-Akt pathway.Conlusions:1. Our present study shows that the presence of wild-type PTEN enhances the radiosensitization effects in the HCC line SMMC-7721.2. Restoring PTEN function is correlated with G2/M arrest, increase of apoptosis and the retardation of the repair of radiation-induced DSB.3. PTEN enhanced the radiosensitization effects specifically by its lipid phosphatase activity mediated through the PTEN-PI3K-Akt signaling pathway.4. These findings suggest that strategies designed to restore the expression of PTEN combining with radiation-inducible technique may be promising therapies by sensitizing HCC cells to irradiation and provide a theoretical support for the clinical application of genetic radiotherapy.更多还原