L-OHP及HIF-1α阻断对宫颈癌放疗增敏作用机制研究
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摘要
研究背景与目的
宫颈癌是严重威胁女性健康的一种疾病,全世界每年约有50万的新发宫颈癌患者,每年约有20多万女性死于宫颈癌。我国每年新发现的病例为13.15万,发病率占我国妇女生殖系恶性肿瘤的首位。治疗原则主要是以手术治疗为主,辅以化疗和放疗。化疗耐药是导致治疗效果不佳的一大原因,研究发现在宫颈癌的化疗中,至少80%的患者最终出现耐药,甚至是多药耐药(MDR multidrug resistance),导致复发,晚期病人的5年生存率仅为20%左右。放射治疗是宫颈癌的主要治疗手段之一,但临床上存在一部分患者接受放射治疗后效果不佳,主要原因与肿瘤内存在乏氧细胞有关。肿瘤乏氧在实体肿瘤中是一个常见的现象。实体肿瘤中乏氧细胞约占总细胞数的10%-50%,对放射治疗有明显的抵抗,这些乏氧的肿瘤细胞成为化、放疗后复发及转移的潜在根源。因此探讨耐药机制、增强放疗效果是当今肿瘤治疗研究中的热点问题。乏氧是人类和动物肿瘤的共同特征,实体肿瘤的氧分压(PaO2)低于肿瘤起源的正常组织,是随着瘤体增大和血供不充足所形成的重要生存微环境。为了适应乏氧,增加氧的利用和减少氧的消耗,处于快速增殖的肿瘤细胞很多基因的转录活性发生改变,研究已经发现乏氧在肿瘤的发展、恶性侵袭、远处转移、乃至肿瘤对放疗、光动力学治疗和化疗抵抗等方面都起着极其重要的作用。现在发现乏氧激活的核转录因子：乏氧诱导因子-lα(hypoxia inducible factor la, HIF-la)是维持氧稳态,调节机体或细胞适应整体或局部乏氧、介导细胞生存的关键调控因子,几乎调控了乏氧所诱导的所有基因的转录。有报道宫颈癌组织较腺瘤和正常组织乏氧诱导因子-lαmRNA和蛋白水平的表达明显增高,我们认为乏氧诱导因子-1α、和乏氧可能是导致宫颈癌化、放疗敏感性下降和耐药的重要因素,但有关乏氧、乏氧诱导因子-1α和宫颈癌化、放疗之间的系统研究尚未见报道。本研究以人宫颈癌细胞株HeLa为研究对象,检测常氧和乏氧条件下,HeLa细胞化、放疗敏感性的变化和乏氧诱导因子-1αmRNA、蛋白的表达水平变化,及L-OHP的放疗增敏作用；构建SiHIF-1α.质粒表达载体pSIHIF-1αREN以阻断乏氧诱导因子-1α的表达和功能,从乏氧和L-OHP对细胞周期调控、化、放疗诱导细胞凋亡的改变,探讨乏氧诱导因子-1α、L-OHP的作用,对阐明宫颈癌细胞化、放疗耐受可能机制、筛选放疗增敏剂和锚定以乏氧诱导因子-1α作为肿瘤治疗的新靶点提供实验依据。
第一部分乏氧对HeLa细胞放化疗敏感性的影响
1.目的
乏氧作为实体肿瘤生存的重要微环境因素,有报道乏氧能保护肿瘤细胞,降低对化疗药物的敏感性,本文拟探讨乏氧能否降低体外培养的HeLa细胞对L-OHP、放疗敏感性。
2.方法
胰酶消化收集HeLa细胞接种96孔培养板,每孔1x105个细胞,分成常氧组(含21%02,5%C02和74%N2的细胞培养箱)和乏氧组(1%02,5%C02,94%N2的乏氧细胞培养箱),分别加入L-OHP的浓度为0、2、4、8、16、32、64、128μmol/L,作用24h后行放疗,放疗条件：6MV射线,源皮距(SDD)为97.5cm,放射面积10cm×10cm,放射剂量DT(2Gy),每一浓度设6个复孔,完全培养液和磷酸盐缓冲液(PBS)分别作空白对照和阴性对照。药物放疗作用后24h行MTT检测。并筛选出C1o时L-OHP的浓度(实验浓度)。
3.结果
3.1 MTT结果显示,不同浓度的L-OHP作用于乏氧和正常状态下的Hela细胞,均表现出增殖抑制作用,并随着L-OHP浓度呈梯度增加；常氧培养下的细胞增殖率低于乏氧状态下的Hela细胞,两者相比差异有统计学意义(P<0.05)；在同一浓度L-OHP作用下,常氧培养的细胞抑制率明显高于乏氧培养细胞的抑制率(P<0.05)。
3.2 MTT结果显示,细胞生长抑制率与放射剂量呈正相关,L-OHP可增加正常和乏氧状态下的Hela细胞对放射线的敏感性,且两者相比差异无统计学意义(P>0.05)。C10时L-OHP的浓度(实验浓度)为4μmol/L。
4.结论
乏氧培养的HeLa细胞较常氧培养的细胞对放疗、L-OHP的药物敏感性明显下降,显示了乏氧对HeLa细胞的保护性,推测乏氧可能是导致HeLa细胞对化、放疗敏感性下降的重要原因。
第二部分HIF-1α介导乏氧状态下HeLa细胞放化疗敏感性中的作用
1.目的
检测常氧和乏氧培养时HeLa细胞中乏氧诱导因子-1α的表达,包括mRNA水平、蛋白水平检测,并构建SiHIF-1αRNA的表达载体即pSiHIF-1αREN阻断HIF-la的表达和功能并进行相应的检测。
2.方法
HeLa细胞分为常氧组,乏氧组；常氧+L-OHP:乏氧+L-OHP:常氧+L-OHP +SiHIF.1α组；乏氧+L-OHP+SiHIF.1α组及上述加放疗组。采用RT-PCR检测各组细胞乏氧诱导因子-1amRNA表达水平；免疫细胞化学检测各组细胞乏氧诱导因子-1a的定位表达；Westen bl0t对乏氧诱导因子-1α蛋白进行半定量检测；构建SiHIF-1αRNA的表达载体pSIHIF-1αREN以阻断乏氧诱导因子-1α的表达和功能。
3.结果
3.1乏氧诱导因子-1amRNA表达的检测
采用RT-PCR方法检测各组细胞乏氧诱导因子-1amRNA的表达水平。乏氧较常氧能明显增加HeLa细胞乏氧诱导因子-lamRNA的表达水平(P<0.05)；转染pSIHIF-laREN能明显降低乏氧诱导因子-1amRNA表达水平(P<0.05)；L-OHP对乏氧诱导因子-1αmRNA表达没有明显影响(P>0.05),放疗可调高乏氧诱导因子-1amRNA表达(P<0.05)。
3.2免疫细胞化学检测乏氧诱导因子-1α表达
收集常氧和乏氧培养24h后HeLa细胞和pSIHIF-laREN细胞爬片以免疫细胞化学染色观察乏氧诱导因子-1α的定位表达。发现乏氧培养后乏氧诱导因子-1α主要表达位于细胞核,而常氧培养时乏氧诱导因子-1α仅在细胞浆有微弱表达；pSIHIF-laREN阻断乏氧诱导因子-1α后其表达明显减弱。
3.3采用Western blot方法检测各组细胞乏氧诱导因子-1α蛋白表达水平
HeLa细胞在乏氧较常氧乏氧诱导因子-1α蛋白的表达水平明显增加(P<0.05)；转染pSIHIF-laREN能明显降低乏氧诱导因子-1a蛋白表达水平(P<0.05); L-OHP对乏氧诱导因子-1a蛋白表达没有明显影响(P>0.05),放疗可调高乏氧诱导因子-1α蛋白表达(P>0.05)。
4.结论
4.1乏氧能明显诱导HeLa细胞乏氧诱导因子-1amRNA、蛋白的表达。
4.2构建的SiHIF-laRNA表达质粒pSIHIF-laREN-DNR-DsRedExpress能有效地抑制乏氧诱导因子-1amRNA、蛋白表达水平。4.3放疗可以调高HeLa细胞乏氧诱导因子-1amRNA、蛋白的表达。
第三部分HIF-1α及奥沙利铂对增强放化疗敏感性作用机制研究
1.目的
探讨乏氧诱导因子-1α在乏氧引起HeLa细胞化、放疗敏感性降低和L-OHP放疗增敏中的可能机制：乏氧对HeLa细胞细胞周期的调控；乏氧对L-OHP诱导细胞凋亡的作用；L-OHP放疗增敏作用。
2.方法
2.1 HeLa细胞分为常氧组(含21%02,5%C02和74%N2)和乏氧培养组(1%02,5%C02,94%N2),转染pSIHIF-laREN以阻断乏氧诱导因子-1α的功能,采用流式细胞术分析各组细胞DNA含量和细胞周期的分布。
2.2 HeLa细胞分组：常氧培养；乏氧培养；常氧培养＋放疗；乏氧培养＋放疗；乏氧培养+pSIHIF-1αREN;乏氧培养+pSIHIF-1αREN+L-OHP及相对应加放疗组。采用流式细胞术检测各组细胞凋亡。
3.结果
3.1.乏氧诱导因子-1α在乏氧诱导细胞周期阻滞中的作用
乏氧培养能明显诱导人HeLa细胞阻滞在Go／G1期,常氧培养时Go／G1期细胞含量相比,差异具有显著性意义(P<0.05)；转染pSIHIF-1αREN的乏氧细胞Go／G1期分布与未转染的乏氧细胞之间差异有显著性意义(P<0.05)。
3.2乏氧诱导因子-1α在乏氧影响化疗药物诱导细胞凋亡中的作用
在L-OHP作用下,常氧组HeLa细胞细胞凋亡率明显高于乏氧细胞组(P<0.05)；乏氧细胞转染pSIHIF-1αREN阻断乏氧诱导因子-1α后细胞凋亡率明显增加(P<0.05),而常氧组细胞转染pSIHIF-1αREN细胞凋亡率无明显改变(P>0.05)。
3.3 L-OHP及乏氧诱导因子-1α在乏氧影响放疗中的作用
L-OHP培养能明显诱导HeLa细胞阻滞在G2／M期；经L-OHP作用后接受放疗的HeLa细胞细胞凋亡率明显高于其对照组；在接受放疗的HeLa细胞中乏氧细胞转染pSIHIF-laREN阻断乏氧诱导因子-1α后细胞凋亡率明显增加(P<0.05)。
4.结论
4.1乏氧能诱导HeLa细胞阻滞在G0／G1期,其中乏氧诱导因子-1α可能发挥重要的调控作用。
4.2乏氧促使HeLa细胞抵抗L-OHP和放疗诱导的细胞凋亡,乏氧诱导因子-1α可能发挥了重要的抗凋亡作用。
4.3HeLa细胞在L-OHP的作用下细胞周期阻滞在G2／M期,增强放疗效果。
Background
Cervical cancer(CC) is a serious threat to women's health. There are about 50 million new CC patients worldwide a year. About 20 million women die from CC a year. New cases detected each year are 13.15 million in our country. CC is one of the most common malignant tumors in the women's reproductive system. The main principle of treatment for CC is surgery, supplemented by chemotherapy and radiotherapy. Resistance to chemotherapy leads to a major cause of poor treatment. Recent studies indicate that for chemotherapy in cervical cancer, at least 80%of patients eventually become resistant or even multi-drug resistant (MDR multidrug resistance), Which lead to relapse. The 5-year survival rate of CC patients is only about 20%. Radiation therapy is the primary treatment of CC, but there is a part of patients whose clinical radiotherapy results are very poor, mainly due to lack of anoxic cell in the tumor. Tumor hypoxia in solid tumors is a common phenomenon. Anoxic cells in solid tumors account for about 10%-50%of the total cell numbers, which are apparently resistant to radiation therapy. Anoxic cells are the potential source of relapse and metastasis after chemotherapy and radiotherapy in CC. Therefore, mechanism of drug resistance, enhanced radiation effect in tumor therapy is currently a hot research topic. Hypoxia is one of the characteristics both in human and animal tumors in solid tumor partial pressure of oxygen (PaO2) less than in the normal tissues of tumor origin. Solid tumor of the partial pressure of oxygen (PaO2) is less than the normal tissues of tumor origin, along with the formation of important microenvironment of the increased tumor and blood supply not sufficient to survive. In order to adapt to hypoxia, increased oxygen utilization and reduced oxygen consumption, the transcriptional activity of many genes of tumor cells is in a rapid proliferation changes. Studies have found that hypoxia may play an important role in tumor development, malignant invasion, distant metastasis, and even cancer radiotherapy, photodynamic therapy and chemotherapy resistance, etc. Activation of nuclear transcription factor hypoxia-hypoxia inducible factor-la (hypoxia iriducible factor la, HIF-1α) is to essential in maintaining oxygen homeostasis, regulating the body or cells adapt to hypoxia in whole or in part, mediating survival regulatory factors, controlling almost all of hypoxia-induced gene transcription. It has been reported that comparing with adenoma and normal cervical tissue, HIF-1αmRNA and protein expression is significantly increased. We believe that HIF-1αand hypoxia may be the important factors leading to cervical cellularization and decreasing the sensitivity of radiotherapy and drug resistance, but the relationships of hypoxia, HIF-la and CC, chemotherapy and radiotherapy has not been reported systematically. In this study, human cervical cancer cell lines HeLa as the research object, the changes of HeLa cellularization sensitivity to chemotherapy and radiotherapy, HIF-la mRNA, protein expression and L-OHP radiosensitization effect are investiated in the condition of different gradients of oxygen and hypoxic conditions. SiHIF-la plasmid expression vector pSIHIF-la REN is built to block HIF-1αexpression and function. From hypoxia and L-OHP on cell cycle regulation, chemotherapy and radiotherapy induced apoptosis changes, to investigate the rose of HIF-1α, L-OHP, in order to provide experimental evidence of cervical cellularization, the possible mechanism of radiation tolerance, screening and radiation sensitizers anchored to HIF-1αas a new target for therapy.
Part one The effect of hypoxia to the sensitivity of Hela cell to chemotherapy and radiotherapy
1.Object
Hypoxia is an important micro-environmental factor of the survival of solid tumor. Hypoxia has been reported to protect tumor cells and to reduce the sensitivity to chemotherapy. The current study was performed to investigate whether the hypoxic could decrease the sensitivity of HeLa cells cultured in vitro to radiosensitivity and L-OHP of chemotherapy.
2.Methods
HeLa cells were collected and planted into 96 well plate, 1×105 cells/well, Cells were divided into normoxia group (cell culture box contains 21%O2,5%CO2 and 74%N2) and hypoxia groups (cell culture box contains 1%O2,5%CO2，94%N2), add L-OHP respectively(0、2、4、8、16、32、64、128μmol/L), after 24 hours, underwent radiotherapy(6MV ray, source skin distance 97.5cm, radiation area 10cm×10cm, radiation dose(2Gy), concentration of each compound hole located 6, complete omplete medium and phosphate buffered saline (PBS), respectively, as control and negative control. After 24 hours of radiotherapy MTT test is performed. To select the concentration of L-OHP at the point of C10 (experimental concentration).
3.Result
3.1 MTT results showed that different concentrations of L-OHP role in hypoxia and HeLa cells under normal conditions, have shown proliferation, and with the gradient of L-OHP concentrations increased. Cell proliferation rate of normoxia cultured cells is lower than the rate of HeLa cells under hypoxic conditions, the difference was statistically significant (P<0.05). At the same concentration under the action of L-OHP, inhibition rate of normoxic cultured cells was significantly higher than the inhibition rate of hypoxia (P<0.05).
3.2 MTT results showed that cell growth inhibition rate was positively correlated with radiation dose. L-OHP increase the sensitivity of HeLa cells to radiosensitivity under normal and hypoxic state, and there is no significant difference between the two groups (P<0.05). At the point of C1o the concentrate of L-OHP is 4μmol/L mg/L.
4.Conclusions
Hypoxia decreased the sensitivity of HeLa cells to radiation and L-OHP. Hypoxia on HeLa cells showed the protective effect. Speculated that hypoxia may be the important reasons to lead to the decrease of the sensitivity of HeLa cells to chemotherapy and radiotherapy.
Part two Effect of HIF-la on the sensitivity of HeLa cells to chemotherapy and radiotherapy under the condition of hypoxic
1.Object
To detect the expression of HIF-1αin cultured HeLa cells under the condition of normoxia and hypoxia. To construct SiHIF-1αa RNA expression vector (pSiHIF-1αaREN) and block HIF-1αa expression and function and perform the corresponding test.
2.Methods
HeLa cells were divided into normoxic group, hypoxia group, normoxic+L-OHP group, hypoxia+L-OHP group, normoxic+L-OHP+SiHIF-1αa group, hypoxia +L-OHP+SiHIF-1αgroup, and radiotherapy group. To test the expression of HIF-1αmRNA with RT-PCR, and specific expression of HIF-la with immunocytochemistry, and expression of HIF-la with Western blot.
3.Result
3.1 Test of the expression of HIF-la mRNA
Hypoxia increased the expression of of HIF-la mRNA more than normoxic(P<0.05). Transfection of pSIHIF-laREN significantly lower levels of HIF-1αmRNA expression(P<0.05). L-OHP did not significantly affected on the HIF-1αmRNA expression(P>0.05). Radiotherapy can increase the expression of HIF-la mRNA(P<0.05).
3.2 Test of the specific expression of HIF-la with immunocytochemistry
After hypoxia the expression of HIF-la mainly located in the nucleus, and cultured in normoxia HIF-1αa is only weakly expressed in the cytoplasm. The expression of HIF-1αwas significantly reduced after pSIHIF-laREN blocking.
3.3 Test of the expression of HIF-la with Western blot
HeLa cells in hypoxia compared with normoxic expression of HIF-1αprotein levels were significantly increased (P<0.05). PSIHIF-laREN transfection significantly decreased the protein levels of HIF-la (P<0.05). L-OHP did not significantly affected on the HIF-1αprotein expression (P>0.05). Radiotherapy can increase the expression of HIF-la protein (P>0.05).
4. Conclusions
4.1 Hypoxia significantly induced HIF-la mRNA and protein expression of HeLa cells.
4.2. pSIHIF-laREN-DNR-DsRedExpress can inhibit HIF-la mRNA, protein expression levels.
4.3 Radiation can increase the HeLa cells HIF-la mRNA, protein expression
Part Three Mechanism of Impact of HIF-la in hypoxic chemosensitivity in human cervical cancer
1.Object
To investigate the role of HIF-la in the decreased sensitization of HeLa cells to chemotherapy and radiotherapy caused by hypoxic, and L-OHP radiosensitizer.
2.Methods
2.1 HeLa cells were divided into normoxic group(21%O2,5%CO2,74%N2), hypoxia group(1%O2,5%CO2,94%N2). Transfected pSIHIF-1αREN to block the function of HIF-la. Each group was analyzed cell DNA content and cell cycle distribution by flow cytometry.
2.2 HeLa cells were divided into normoxic group, hypoxia group, normoxic+ radiotherapy group, hypoxia+radiotherapy group, hypoxia+pSIHIF-laREN group+L-OHP, hypoxia+pSIHIF-laREN+L-OHP group and radiotherapy group. To detect apoptosis of each group by flow cytometry.
3.Result
3.1 HIF-la in the role of cell cycle arrest induced by hypoxia.
Hypoxia significantly induced HeLa cells were arrested in Go/G1 phase. Cultured by normal oxygen, content of Go/G1 phase cells is significantly different(P<0.05). Distribution of Go/G1 cells of hypoxia cells transfected by pSIHIF-laREN is significantly different with the no transfected cells(P<0.05).
3.2 HIF-la in the role of cell apoptosis induced by hypoxia effecting chemotherapy.
Under the action of the L-OHP, the rate of normoxic HeLa cell apoptosis was significantly higher than that of hypoxic cell group(P<0.05). Hypoxic cells transfected pSIHIF-laREN blocking HIF-la significantly increased the apoptosis rate (P<0.05). Normoxic cells transfected pSIHIF-laREN do not significantly change in apoptosis rate(P>0.05).
3.3 The role of L-OHP and HIF-la in the hypoxia impact on radiotherapy.
L-OHP significantly induced cultured HeLa cells were arrested in G2/M phase. The apoptosis rate of HeLa cell effected by L-OHP and get radiotherapy was significantly higher than the control group(P<0.05).
4.Conclusions
4.1 Hypoxia can induce HeLa cells were arrested in Go/G1 phase, and HIF-1αmay play an important role.
4.2 Hypoxia results HeLa cell in resistance to apoptosis led by L-OHP and radiation. HIF-1αmay play an important role in anti-apoptosis
4.3 Cell cycles of HeLa cells under the effect of L-OHP arrest in G2/M phase, and the radiation effect is enhanced.
宫颈癌是严重威胁女性健康的一种疾病,全世界每年约有50万的新发宫颈癌患者,每年约有20多万女性死于宫颈癌。我国每年新发现的病例为13.15万,发病率占我国妇女生殖系恶性肿瘤的首位。治疗原则主要是以手术治疗为主,辅以化疗和放疗。化疗耐药是导致治疗效果不佳的一大原因,研究发现在宫颈癌的化疗中,至少80%的患者最终出现耐药,甚至是多药耐药(MDR multidrug resistance),导致复发,晚期病人的5年生存率仅为20%左右。放射治疗是宫颈癌的主要治疗手段之一,但临床上存在一部分患者接受放射治疗后效果不佳,主要原因与肿瘤内存在乏氧细胞有关。肿瘤乏氧在实体肿瘤中是一个常见的现象。实体肿瘤中乏氧细胞约占总细胞数的10%-50%,对放射治疗有明显的抵抗,这些乏氧的肿瘤细胞成为化、放疗后复发及转移的潜在根源。因此探讨耐药机制、增强放疗效果是当今肿瘤治疗研究中的热点问题。乏氧是人类和动物肿瘤的共同特征,实体肿瘤的氧分压(PaO2)低于肿瘤起源的正常组织,是随着瘤体增大和血供不充足所形成的重要生存微环境。为了适应乏氧,增加氧的利用和减少氧的消耗,处于快速增殖的肿瘤细胞很多基因的转录活性发生改变,研究已经发现乏氧在肿瘤的发展、恶性侵袭、远处转移、乃至肿瘤对放疗、光动力学治疗和化疗抵抗等方面都起着极其重要的作用。现在发现乏氧激活的核转录因子：乏氧诱导因子-lα(hypoxia inducible factor la, HIF-la)是维持氧稳态,调节机体或细胞适应整体或局部乏氧、介导细胞生存的关键调控因子,几乎调控了乏氧所诱导的所有基因的转录。有报道宫颈癌组织较腺瘤和正常组织乏氧诱导因子-lαmRNA和蛋白水平的表达明显增高,我们认为乏氧诱导因子-1α、和乏氧可能是导致宫颈癌化、放疗敏感性下降和耐药的重要因素,但有关乏氧、乏氧诱导因子-1α和宫颈癌化、放疗之间的系统研究尚未见报道。本研究以人宫颈癌细胞株HeLa为研究对象,检测常氧和乏氧条件下,HeLa细胞化、放疗敏感性的变化和乏氧诱导因子-1αmRNA、蛋白的表达水平变化,及L-OHP的放疗增敏作用；构建SiHIF-1α.质粒表达载体pSIHIF-1αREN以阻断乏氧诱导因子-1α的表达和功能,从乏氧和L-OHP对细胞周期调控、化、放疗诱导细胞凋亡的改变,探讨乏氧诱导因子-1α、L-OHP的作用,对阐明宫颈癌细胞化、放疗耐受可能机制、筛选放疗增敏剂和锚定以乏氧诱导因子-1α作为肿瘤治疗的新靶点提供实验依据。
第一部分乏氧对HeLa细胞放化疗敏感性的影响
1.目的
乏氧作为实体肿瘤生存的重要微环境因素,有报道乏氧能保护肿瘤细胞,降低对化疗药物的敏感性,本文拟探讨乏氧能否降低体外培养的HeLa细胞对L-OHP、放疗敏感性。
2.方法
胰酶消化收集HeLa细胞接种96孔培养板,每孔1x105个细胞,分成常氧组(含21%02,5%C02和74%N2的细胞培养箱)和乏氧组(1%02,5%C02,94%N2的乏氧细胞培养箱),分别加入L-OHP的浓度为0、2、4、8、16、32、64、128μmol/L,作用24h后行放疗,放疗条件：6MV射线,源皮距(SDD)为97.5cm,放射面积10cm×10cm,放射剂量DT(2Gy),每一浓度设6个复孔,完全培养液和磷酸盐缓冲液(PBS)分别作空白对照和阴性对照。药物放疗作用后24h行MTT检测。并筛选出C1o时L-OHP的浓度(实验浓度)。
3.结果
3.1 MTT结果显示,不同浓度的L-OHP作用于乏氧和正常状态下的Hela细胞,均表现出增殖抑制作用,并随着L-OHP浓度呈梯度增加；常氧培养下的细胞增殖率低于乏氧状态下的Hela细胞,两者相比差异有统计学意义(P<0.05)；在同一浓度L-OHP作用下,常氧培养的细胞抑制率明显高于乏氧培养细胞的抑制率(P<0.05)。
3.2 MTT结果显示,细胞生长抑制率与放射剂量呈正相关,L-OHP可增加正常和乏氧状态下的Hela细胞对放射线的敏感性,且两者相比差异无统计学意义(P>0.05)。C10时L-OHP的浓度(实验浓度)为4μmol/L。
4.结论
乏氧培养的HeLa细胞较常氧培养的细胞对放疗、L-OHP的药物敏感性明显下降,显示了乏氧对HeLa细胞的保护性,推测乏氧可能是导致HeLa细胞对化、放疗敏感性下降的重要原因。
第二部分HIF-1α介导乏氧状态下HeLa细胞放化疗敏感性中的作用
1.目的
检测常氧和乏氧培养时HeLa细胞中乏氧诱导因子-1α的表达,包括mRNA水平、蛋白水平检测,并构建SiHIF-1αRNA的表达载体即pSiHIF-1αREN阻断HIF-la的表达和功能并进行相应的检测。
2.方法
HeLa细胞分为常氧组,乏氧组；常氧+L-OHP:乏氧+L-OHP:常氧+L-OHP +SiHIF.1α组；乏氧+L-OHP+SiHIF.1α组及上述加放疗组。采用RT-PCR检测各组细胞乏氧诱导因子-1amRNA表达水平；免疫细胞化学检测各组细胞乏氧诱导因子-1a的定位表达；Westen bl0t对乏氧诱导因子-1α蛋白进行半定量检测；构建SiHIF-1αRNA的表达载体pSIHIF-1αREN以阻断乏氧诱导因子-1α的表达和功能。
3.结果
3.1乏氧诱导因子-1amRNA表达的检测
采用RT-PCR方法检测各组细胞乏氧诱导因子-1amRNA的表达水平。乏氧较常氧能明显增加HeLa细胞乏氧诱导因子-lamRNA的表达水平(P<0.05)；转染pSIHIF-laREN能明显降低乏氧诱导因子-1amRNA表达水平(P<0.05)；L-OHP对乏氧诱导因子-1αmRNA表达没有明显影响(P>0.05),放疗可调高乏氧诱导因子-1amRNA表达(P<0.05)。
3.2免疫细胞化学检测乏氧诱导因子-1α表达
收集常氧和乏氧培养24h后HeLa细胞和pSIHIF-laREN细胞爬片以免疫细胞化学染色观察乏氧诱导因子-1α的定位表达。发现乏氧培养后乏氧诱导因子-1α主要表达位于细胞核,而常氧培养时乏氧诱导因子-1α仅在细胞浆有微弱表达；pSIHIF-laREN阻断乏氧诱导因子-1α后其表达明显减弱。
3.3采用Western blot方法检测各组细胞乏氧诱导因子-1α蛋白表达水平
HeLa细胞在乏氧较常氧乏氧诱导因子-1α蛋白的表达水平明显增加(P<0.05)；转染pSIHIF-laREN能明显降低乏氧诱导因子-1a蛋白表达水平(P<0.05); L-OHP对乏氧诱导因子-1a蛋白表达没有明显影响(P>0.05),放疗可调高乏氧诱导因子-1α蛋白表达(P>0.05)。
4.结论
4.1乏氧能明显诱导HeLa细胞乏氧诱导因子-1amRNA、蛋白的表达。
4.2构建的SiHIF-laRNA表达质粒pSIHIF-laREN-DNR-DsRedExpress能有效地抑制乏氧诱导因子-1amRNA、蛋白表达水平。4.3放疗可以调高HeLa细胞乏氧诱导因子-1amRNA、蛋白的表达。
第三部分HIF-1α及奥沙利铂对增强放化疗敏感性作用机制研究
1.目的
探讨乏氧诱导因子-1α在乏氧引起HeLa细胞化、放疗敏感性降低和L-OHP放疗增敏中的可能机制：乏氧对HeLa细胞细胞周期的调控；乏氧对L-OHP诱导细胞凋亡的作用；L-OHP放疗增敏作用。
2.方法
2.1 HeLa细胞分为常氧组(含21%02,5%C02和74%N2)和乏氧培养组(1%02,5%C02,94%N2),转染pSIHIF-laREN以阻断乏氧诱导因子-1α的功能,采用流式细胞术分析各组细胞DNA含量和细胞周期的分布。
2.2 HeLa细胞分组：常氧培养；乏氧培养；常氧培养＋放疗；乏氧培养＋放疗；乏氧培养+pSIHIF-1αREN;乏氧培养+pSIHIF-1αREN+L-OHP及相对应加放疗组。采用流式细胞术检测各组细胞凋亡。
3.结果
3.1.乏氧诱导因子-1α在乏氧诱导细胞周期阻滞中的作用
乏氧培养能明显诱导人HeLa细胞阻滞在Go／G1期,常氧培养时Go／G1期细胞含量相比,差异具有显著性意义(P<0.05)；转染pSIHIF-1αREN的乏氧细胞Go／G1期分布与未转染的乏氧细胞之间差异有显著性意义(P<0.05)。
3.2乏氧诱导因子-1α在乏氧影响化疗药物诱导细胞凋亡中的作用
在L-OHP作用下,常氧组HeLa细胞细胞凋亡率明显高于乏氧细胞组(P<0.05)；乏氧细胞转染pSIHIF-1αREN阻断乏氧诱导因子-1α后细胞凋亡率明显增加(P<0.05),而常氧组细胞转染pSIHIF-1αREN细胞凋亡率无明显改变(P>0.05)。
3.3 L-OHP及乏氧诱导因子-1α在乏氧影响放疗中的作用
L-OHP培养能明显诱导HeLa细胞阻滞在G2／M期；经L-OHP作用后接受放疗的HeLa细胞细胞凋亡率明显高于其对照组；在接受放疗的HeLa细胞中乏氧细胞转染pSIHIF-laREN阻断乏氧诱导因子-1α后细胞凋亡率明显增加(P<0.05)。
4.结论
4.1乏氧能诱导HeLa细胞阻滞在G0／G1期,其中乏氧诱导因子-1α可能发挥重要的调控作用。
4.2乏氧促使HeLa细胞抵抗L-OHP和放疗诱导的细胞凋亡,乏氧诱导因子-1α可能发挥了重要的抗凋亡作用。
4.3HeLa细胞在L-OHP的作用下细胞周期阻滞在G2／M期,增强放疗效果。
Background
Cervical cancer(CC) is a serious threat to women's health. There are about 50 million new CC patients worldwide a year. About 20 million women die from CC a year. New cases detected each year are 13.15 million in our country. CC is one of the most common malignant tumors in the women's reproductive system. The main principle of treatment for CC is surgery, supplemented by chemotherapy and radiotherapy. Resistance to chemotherapy leads to a major cause of poor treatment. Recent studies indicate that for chemotherapy in cervical cancer, at least 80%of patients eventually become resistant or even multi-drug resistant (MDR multidrug resistance), Which lead to relapse. The 5-year survival rate of CC patients is only about 20%. Radiation therapy is the primary treatment of CC, but there is a part of patients whose clinical radiotherapy results are very poor, mainly due to lack of anoxic cell in the tumor. Tumor hypoxia in solid tumors is a common phenomenon. Anoxic cells in solid tumors account for about 10%-50%of the total cell numbers, which are apparently resistant to radiation therapy. Anoxic cells are the potential source of relapse and metastasis after chemotherapy and radiotherapy in CC. Therefore, mechanism of drug resistance, enhanced radiation effect in tumor therapy is currently a hot research topic. Hypoxia is one of the characteristics both in human and animal tumors in solid tumor partial pressure of oxygen (PaO2) less than in the normal tissues of tumor origin. Solid tumor of the partial pressure of oxygen (PaO2) is less than the normal tissues of tumor origin, along with the formation of important microenvironment of the increased tumor and blood supply not sufficient to survive. In order to adapt to hypoxia, increased oxygen utilization and reduced oxygen consumption, the transcriptional activity of many genes of tumor cells is in a rapid proliferation changes. Studies have found that hypoxia may play an important role in tumor development, malignant invasion, distant metastasis, and even cancer radiotherapy, photodynamic therapy and chemotherapy resistance, etc. Activation of nuclear transcription factor hypoxia-hypoxia inducible factor-la (hypoxia iriducible factor la, HIF-1α) is to essential in maintaining oxygen homeostasis, regulating the body or cells adapt to hypoxia in whole or in part, mediating survival regulatory factors, controlling almost all of hypoxia-induced gene transcription. It has been reported that comparing with adenoma and normal cervical tissue, HIF-1αmRNA and protein expression is significantly increased. We believe that HIF-1αand hypoxia may be the important factors leading to cervical cellularization and decreasing the sensitivity of radiotherapy and drug resistance, but the relationships of hypoxia, HIF-la and CC, chemotherapy and radiotherapy has not been reported systematically. In this study, human cervical cancer cell lines HeLa as the research object, the changes of HeLa cellularization sensitivity to chemotherapy and radiotherapy, HIF-la mRNA, protein expression and L-OHP radiosensitization effect are investiated in the condition of different gradients of oxygen and hypoxic conditions. SiHIF-la plasmid expression vector pSIHIF-la REN is built to block HIF-1αexpression and function. From hypoxia and L-OHP on cell cycle regulation, chemotherapy and radiotherapy induced apoptosis changes, to investigate the rose of HIF-1α, L-OHP, in order to provide experimental evidence of cervical cellularization, the possible mechanism of radiation tolerance, screening and radiation sensitizers anchored to HIF-1αas a new target for therapy.
Part one The effect of hypoxia to the sensitivity of Hela cell to chemotherapy and radiotherapy
1.Object
Hypoxia is an important micro-environmental factor of the survival of solid tumor. Hypoxia has been reported to protect tumor cells and to reduce the sensitivity to chemotherapy. The current study was performed to investigate whether the hypoxic could decrease the sensitivity of HeLa cells cultured in vitro to radiosensitivity and L-OHP of chemotherapy.
2.Methods
HeLa cells were collected and planted into 96 well plate, 1×105 cells/well, Cells were divided into normoxia group (cell culture box contains 21%O2,5%CO2 and 74%N2) and hypoxia groups (cell culture box contains 1%O2,5%CO2，94%N2), add L-OHP respectively(0、2、4、8、16、32、64、128μmol/L), after 24 hours, underwent radiotherapy(6MV ray, source skin distance 97.5cm, radiation area 10cm×10cm, radiation dose(2Gy), concentration of each compound hole located 6, complete omplete medium and phosphate buffered saline (PBS), respectively, as control and negative control. After 24 hours of radiotherapy MTT test is performed. To select the concentration of L-OHP at the point of C10 (experimental concentration).
3.Result
3.1 MTT results showed that different concentrations of L-OHP role in hypoxia and HeLa cells under normal conditions, have shown proliferation, and with the gradient of L-OHP concentrations increased. Cell proliferation rate of normoxia cultured cells is lower than the rate of HeLa cells under hypoxic conditions, the difference was statistically significant (P<0.05). At the same concentration under the action of L-OHP, inhibition rate of normoxic cultured cells was significantly higher than the inhibition rate of hypoxia (P<0.05).
3.2 MTT results showed that cell growth inhibition rate was positively correlated with radiation dose. L-OHP increase the sensitivity of HeLa cells to radiosensitivity under normal and hypoxic state, and there is no significant difference between the two groups (P<0.05). At the point of C1o the concentrate of L-OHP is 4μmol/L mg/L.
4.Conclusions
Hypoxia decreased the sensitivity of HeLa cells to radiation and L-OHP. Hypoxia on HeLa cells showed the protective effect. Speculated that hypoxia may be the important reasons to lead to the decrease of the sensitivity of HeLa cells to chemotherapy and radiotherapy.
Part two Effect of HIF-la on the sensitivity of HeLa cells to chemotherapy and radiotherapy under the condition of hypoxic
1.Object
To detect the expression of HIF-1αin cultured HeLa cells under the condition of normoxia and hypoxia. To construct SiHIF-1αa RNA expression vector (pSiHIF-1αaREN) and block HIF-1αa expression and function and perform the corresponding test.
2.Methods
HeLa cells were divided into normoxic group, hypoxia group, normoxic+L-OHP group, hypoxia+L-OHP group, normoxic+L-OHP+SiHIF-1αa group, hypoxia +L-OHP+SiHIF-1αgroup, and radiotherapy group. To test the expression of HIF-1αmRNA with RT-PCR, and specific expression of HIF-la with immunocytochemistry, and expression of HIF-la with Western blot.
3.Result
3.1 Test of the expression of HIF-la mRNA
Hypoxia increased the expression of of HIF-la mRNA more than normoxic(P<0.05). Transfection of pSIHIF-laREN significantly lower levels of HIF-1αmRNA expression(P<0.05). L-OHP did not significantly affected on the HIF-1αmRNA expression(P>0.05). Radiotherapy can increase the expression of HIF-la mRNA(P<0.05).
3.2 Test of the specific expression of HIF-la with immunocytochemistry
After hypoxia the expression of HIF-la mainly located in the nucleus, and cultured in normoxia HIF-1αa is only weakly expressed in the cytoplasm. The expression of HIF-1αwas significantly reduced after pSIHIF-laREN blocking.
3.3 Test of the expression of HIF-la with Western blot
HeLa cells in hypoxia compared with normoxic expression of HIF-1αprotein levels were significantly increased (P<0.05). PSIHIF-laREN transfection significantly decreased the protein levels of HIF-la (P<0.05). L-OHP did not significantly affected on the HIF-1αprotein expression (P>0.05). Radiotherapy can increase the expression of HIF-la protein (P>0.05).
4. Conclusions
4.1 Hypoxia significantly induced HIF-la mRNA and protein expression of HeLa cells.
4.2. pSIHIF-laREN-DNR-DsRedExpress can inhibit HIF-la mRNA, protein expression levels.
4.3 Radiation can increase the HeLa cells HIF-la mRNA, protein expression
Part Three Mechanism of Impact of HIF-la in hypoxic chemosensitivity in human cervical cancer
1.Object
To investigate the role of HIF-la in the decreased sensitization of HeLa cells to chemotherapy and radiotherapy caused by hypoxic, and L-OHP radiosensitizer.
2.Methods
2.1 HeLa cells were divided into normoxic group(21%O2,5%CO2,74%N2), hypoxia group(1%O2,5%CO2,94%N2). Transfected pSIHIF-1αREN to block the function of HIF-la. Each group was analyzed cell DNA content and cell cycle distribution by flow cytometry.
2.2 HeLa cells were divided into normoxic group, hypoxia group, normoxic+ radiotherapy group, hypoxia+radiotherapy group, hypoxia+pSIHIF-laREN group+L-OHP, hypoxia+pSIHIF-laREN+L-OHP group and radiotherapy group. To detect apoptosis of each group by flow cytometry.
3.Result
3.1 HIF-la in the role of cell cycle arrest induced by hypoxia.
Hypoxia significantly induced HeLa cells were arrested in Go/G1 phase. Cultured by normal oxygen, content of Go/G1 phase cells is significantly different(P<0.05). Distribution of Go/G1 cells of hypoxia cells transfected by pSIHIF-laREN is significantly different with the no transfected cells(P<0.05).
3.2 HIF-la in the role of cell apoptosis induced by hypoxia effecting chemotherapy.
Under the action of the L-OHP, the rate of normoxic HeLa cell apoptosis was significantly higher than that of hypoxic cell group(P<0.05). Hypoxic cells transfected pSIHIF-laREN blocking HIF-la significantly increased the apoptosis rate (P<0.05). Normoxic cells transfected pSIHIF-laREN do not significantly change in apoptosis rate(P>0.05).
3.3 The role of L-OHP and HIF-la in the hypoxia impact on radiotherapy.
L-OHP significantly induced cultured HeLa cells were arrested in G2/M phase. The apoptosis rate of HeLa cell effected by L-OHP and get radiotherapy was significantly higher than the control group(P<0.05).
4.Conclusions
4.1 Hypoxia can induce HeLa cells were arrested in Go/G1 phase, and HIF-1αmay play an important role.
4.2 Hypoxia results HeLa cell in resistance to apoptosis led by L-OHP and radiation. HIF-1αmay play an important role in anti-apoptosis
4.3 Cell cycles of HeLa cells under the effect of L-OHP arrest in G2/M phase, and the radiation effect is enhanced.
引文
[1]Denko NC. Hypoxia, HIF-1 and glucose metabolism in the solid tumour [J]. Nat Rev Cancer,2008,8(9):705-713.
[2]姜颖,张海青.缺氧诱导因子-1在肿瘤临床方面的研究进展[J].国外医学(肿瘤学分册),2004,(11),789-791.
[3]Brown, JM. Tumor hypoxia in cancer therapy [J]. Methods Enzymol,2007,435:297-321.
[4]Parkin DM, Bray F, Ferlay J,et al. Estimating the World Cancer Burden:Global Cancer 2000 [J]. Cancer,2001,94(2):153.
[5]乐杰.妇产科学[M].6版.北京：人民卫生出版社,2001：314.
[6]Qiao YL, Zhang WH, Li L, et al. A Cross-section Study of Screening Techniques for Cervical Cancer in Shanxi [J].Acta Acad Med Scin,2002,24(1):50-53.
[7]Jian-Jun Zhang. Expression and significance of TLR4 and HIF-la pancreatic ductal adenocarcinoma [J]. World Gastroenterol,2010,16(23):881-888.
[8]Semenza GL. HIF-la inhibitors for cancer therapy:from gene expression to drug discovery[J]. Curr Pharm Des,2009,15(33):3839-3843.
[9]Adams JM, Difazio LT, Rolandelli RH, et al. HIF-la key mediator in hypoxia [J]. Acta Physiol Hung,2009,96(1):19-28.
[10]Hirota K, Semenza GL. Regulation of angiogenesis by hypoxia-inducible factor [J]. Crit Rev Oncol Hematol,2006,59(1):15-26.
[11].宋樱,孙善珍,曲迅,等.乏氧对人舌鳞癌细胞系Tca8113体外黏附和侵袭能力的影响[J].实用口腔医学杂志,2009,25(6)：828-832.
[12]朱世凯.缺氧微环境中HIF-la诱导MT2-MMP表达及对胰腺癌侵袭和转移作用的机制研究[D].武汉：华中科技大学,2010.
[13]Alcazar J L, Casfillo G, Martinez M R. Trans-vaginal color Doppler sonography for predicting response to concurrent chemoradiotherapy for locally advanced cervical cancinoma [J]. Clin Ultrasoun 2007.32(6):267-272.
[14]Toma Dsu I, Dasu A, Karlsson M.The relationship between temporal variation of hypoxia, polarographic measurements and predictions ofturnout response to radiation[J]. Phys Med Biol,2004,49 (19):463-75.
[15]Kanz M, Ibrahim SM. Molecular response to hypoxia in tumor cells [J]. Clin Ultrasoun, 2003,2:23-36.
[16]PASETTO LM, D'ANDRE MR, ROSSI E, et al.Oxaliplatin-related neurotoxicity:how and why? [J]. Crit Rev Oncol Hematol,2006,59(2):159-168.
[17]Bidoli P, Zilembo N, Cortinov D, et al. Randomized phase II three-arm trial with three platinum based doublets in metastatic non-small-cell lung cancer An Italian Trials in Medical Oncology study [J]. Ann Oncol,2007,18(3):461-367.
[18]CASSIDY J, MISSET J L. Oxaliplatin-related side effect:Characteristics and management [J]. Semin Oncol,2002,29(5):11-20.
[19]王琳,秦叔逵,钱军,等.奥沙利铂联合氟尿嘧啶类药物二线治疗晚期胃癌的临床研究[J].现代肿瘤医学,2005,13(3)：361-362.
[20]MCKEAGE M J. New-generation platinum drugs in the treatment of cisplatin-resistant cancers [J]. Expert Opin Investig Drugs,2005,14 (8):1033-1046.
[21]Lordick F, Lorrenzens S, Stollfuss J, et al. Phase II study of weekly oxaliplatin plus infusional fluorouracil and folinic acid(FUFOX regimen)as firstline treatment in metastatic gastric cancer [J]. BrJ Cancer,2005,93 (2):190-194.
[22]周彩存,张捷,徐瑛,等.奥沙利铂联合长春瑞滨一线治疗不可手术的非小细胞肺癌[J].临床肿瘤学杂志,2005,10(5)：481-485.
[23]BELANI C P. Recent updates in the clinical use of platinum compounds for the treatment of lung, breast, and genitourinary tumors and myeloma [J]. SeminOncol,2004,31(14):25-33.
[24]Loncaster J A, Harris A L, Davidson S E.Carbonic anhydrase (CAIX)expression, a potential new intrinsic marker of hypoxia:Correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix [J].Cancer Res,2005, 61(17):6394-6399.
[25]Mizukami Y, Li J, Zhang X, et al. Hypoxia-inducible factor-1-independent regulation of vascular endothelial growth factor by hypoxia in colon cancer [J].Cancer Res.2004,64(5): 1765-72.
[26]Menrad H, Werno C, Schmid T, et al. Roles of hypoxia-inducible factor-lalpha (HIF-lalpha)versus HIF-2alpha in the survival of hepatocellular tumor spheroids [J]. Hepatology, 2010,51(6):2183-2192
[27]No JH, Jo H, Kim SH, et al. Expression of vascular endothelial growth factor and hypoxia inducible factor-lalpha in cervical neoplasia [J]. Ann N Y Acad Sci,2009,16(7),1171: 105-110.
[28]Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1[J]. Biol Chem,1995,270(3):1230-1237.
[29]Semenza GL. Surviving ischemia:adaptive responses mediated by hypoxia-inducible factor 1 [J]. Clin Invest,2000,106(7):809-812.
[30]Mircea I, Keiichi K, Haifeng Yang, et al. HIF-1 Targeted for VHL-Mediated Destruction by Proline Hydroxylation:Implications for O2 [J]. Sensing. Science,2001,292(5516):464-468.
[31]常青,秦仁义,高军,等.反义缺氧诱导因子-1α对胰腺癌细胞BxPC-3化疗敏感性的影响[J].中国普通外科杂志,2006,15(6)：423-427.
[32]Hubert A, Paris S, Piret JP, et al. Casein kinase 2 inhibition decreases hypoxia-inducible fa ctor-1 activity under hypoxia through elevated p53 protein level [J]. J Cell Sci,2006,119 (16):3351-3362.
[33]Takahashi Y, Nishikawa M, Takakura Y. Inhibition of tumor cell growth in the liver by RN A interferencemediated suppression of HIF-lalpha expression in tumor cells and hepatocyte s [J]. GeneTher,2008,15(8):572-582.
[34]Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and therirmetastases [J]. Cancer Res,1999,59:5830-5835.
[35]Fujita M,Yasuda M,Kitatani K,et al.Anup-to-date anticancer treatment strategy focusing on HIF-lalpha su-ppr ession:its application for refractory ovarian cancer[J].Acta Histoche m Cytochem,2007,40(5):139-142.
[36]Hur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilization or DNA binding ability of hypoxia-inducible factorlalpha[J]. Mol Pharmacol,2009,59 (5):1216-1222.
[37]Osada M, Imaoka S, Sugimoto T, et al. NADPH-cytochrome P-450 reductase in the plasma membrane modulates the activation of hypoxia-inducible factor 1[J]. J Biol Chem, 2008,277(26):23367-23373.
[38]Kunz M, Ibrahim SM. Molecular responses to hypoxia in tumor cells [J]. Mol Cancer,2009, 2(1):23-30.
[39]Jach R, Dulinska-Litewka J, Laidler P, et al. Expression of VEGF, VEGF-C and VEGFR-2 in situ and invasive SCC of cervix [J]. Front Biosei (Elite Ed),2010,2:411-423.
[40]Dales JP, BeautilsN, SilvyM, et al. Hypoxia inducible factor lalpha gene(HIF-lalpha)splice variants:potential prognostic biomarkers in breast cancer [J].BMC Med,2010,8(2):44.
[41]沈维干,朱军,于智勇,等.低氧对人肺腺癌A549细胞迁移和黏附的影响[J].肿瘤,2008,28(3)：216-259.
[42]Chi JT, Wang Z, Nuyten DS, et al. Gene expression program s in response to hypoxia:cell type specificity an d prognostic significance in human cancers [J]. Plo S Med,2006, 3(3):395-409.
[43]Ozer A, Wu LC, Bruick RK. The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF) [J]. Proc Natl Acad Sci USA,2005,102(21):7481-7486.
[44]Cayre A, Rossignol F, Clottes E, et al. HIF but not HIF-la transcript is a poor prognostic marker in human breast cancer [J]. Breast Cancer Res,2003,5(6):223-230.
[45]Ritchie W, Flamant S, Rasko JE. mimicroRNA:a microRNA expression profiler and classification resource designed to identify functional correlations between microRNAs and their targets [J]. Bioinformaties,2010,26(2):223-227.
[46]Hanze J, Eul BG, Savai R, et al. RNA interference for HIF-lalpha inhibits its downstream signalling and affects cellular proliferation [J]. Biochem Biophys Res Commun,2003, 312(3):571-577.
[47]Brizel DM, Sibley GS, Prosnitz LR, et al. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck [J]. Int J Radiat Oncol Biol Phys,1997,8(2):285-289.
[48]Hockel M, Vorndran B, Schlenger K, et al. Tumor oxygenation:a new predictive parameter in locally advanced cancer of the uterine cervix [J]. Gynecol Oncol,1993,51(2):141-149.
[49]Goda N, Ryan HE, Khadivi Bk, et al. Hypoxia-inducible factor lalpha is essential for cell cycle arrest during hypoxia [J]. Mol Cell Biol.2003,23 (1):359-369.
[50]Lawrence B, Gardner, Qing Li, et al. Hypoxia inhibits Gl/S transition through regulation of p27 expression [J]. J Biol Chem.,2001,276(11):7919-7926.
[51]Box AH, Demetrick DJ. Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines [J].Carcinogenesis,2004,25(12):2325-2335.
[52]Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia inducible factor-1 dependent regulation of the multidrug resistance (MDR1) gene [J]. Cancer Res,2002,62(12):3387-3394.
[53]Kunz M, Ibrahim SM. Molecular responses to hypoxia in tumor cells [J]. Mol Cancer,2003, 2(1):23-30.
[54]Acs G, Zhang PJ, McGrath CM, et al. Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression [J].Am J Pathol,2003,162 (6):1789-1806
[55]J Bruick RK. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia [J].Proc Natl Aced Sci USA,2000,97(16):9082-9087.
[56]Baek JH, Jang JE, Kang CM, et al. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis [J]. Oncogene,2000,19(40): 4621-4631.
[57]Unruh A, Ressel A, Mohamed HG, et al. The hypoxia-inducible factor-1 alpha is a negative factor for tumor therapy [J]. Oncogene,2003,22 (21):3213-3220.
[58]Achison M, Hupp TR. Hypoxia attenuates the p53 response to cellular damage [J]. Oncogene,2003,29 (22):3431-3440.
[59]Ghafar MA, Anastasiadis AG, Chen MW, et al. Acute hypoxia increases the aggressive characteristics and survival properties of prostate cancer [J]. Cells Prostate,2003,54(1) 58-67.
[60]Mckeown SR, Cowen RL, Williams KJ. Bioreductive drugs:from concept to clinic [J].Clin Oncol(R Coll Radio 1),2007,9(4):427-442.
[61]Hay MP, Hicks KO, Pruijn FB. Pharmacokinetic/pharmacodynamic model-guided identification of hypoxia-selective 1,2,4-benzotriazine 1,4-dioxides with antitumor activity:the role of extravascular transport [J]. Med Chem,2007,50(8):6392-6404
[62]Brown JM. Tumor hypoxia in cancer therapy [J].Methods Enzymol,2007,435(11): 297-321.
[63]Overgaard J. Hypoxic radiosensitization:adored and ignored [J].J Clin Oncol,2007,25(4): 4066-4074.
[64]Anderson P, Aguilera D, Pearson M. Outpatient chemotherapy plus radiotherapy in sarcomas improving cancer control with radiosensitizing agents [J]. Cancer Control,2008, 15(6):38-46.
[65]Mckeown SR, Cowen RL, Williams KJ. Bioreductive drugs:from concept to clinic [J].Clin Oncol(R Coll Radiol),2007,19(7):427-442.
[66]Takahashi, Nishikawa M, Takakura Y. Inhibition of tumor cell growth in the liver by RNA interference-mediated suppression of HIF-lalpha expression in tumor cells and hepatocytes [J]. GeneTher,2008,15(8):572-582.
[67]Bidoli P, Zilembo N, Cortinov is D,et al.Raidolized phase II three-arln trial with three platinum based doublets in metastatic non-small-cell lung cancer [J]. An Italian Trials in Medical Oncology study, Ann Oncol,2007,18(3):461-367.
[68]Furue H. oxaliPlatin [J].GanToKaga-Ryoho,2005,32(8):1195-1202.
[69]Mekeage M J. New-generation platinum drugs in the treatment of cisplatin-resistant caneers [J]. ExPert Opin Investig Drugs,2005,14(8):1033-1046.
[70]Lordiek F, Lorenzen S, Stollfuss J, etal. Phase II study of weekly oxaliplatin Plus Infusional fluorouracil and folinic acid (FIJFOX regimen)as first-line treatment inmetastatic gastric cancer [J].Br J Cancer,2005,93(2):190-194.
[71]CividalliA, CeciarelliF, Livid E, etal. Radiosensitization by oxaliplatin in a mouse adenocarcinoma:influence of treatment schedule [J]. Int-J-Radiat-Oncol-Biol-Phys, 2002,52(4):1092-1098.
[72]吕惠兰,宫良平,邵震宇,等.低剂量奥沙利铂并氟脲嘧啶对晚期复发直肠癌放疗增敏作用的临床观察[J].中国现代普通外科进展,2005：178-182.
[73]江滨,陈书长.抗肿瘤药物临床应用指南[M].北京：中国协和医科大学出版社,2005：228-233.
[1]Kreimer AR, Clifford GM, Boyle P, et al. Human papilomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review [J]. Cancer Epidemiol Biomarkers Prev,2005,14(6):467-475.
[2]Wgngoner S, Chemiky CL. Molceluar biology of cervical and vulvar carcinoma [M].2004, 65-78.
[3]Davey DD. Cervical cytology classification and the Bethesda system [J].Cancer,2003,9 (4):327-334.
[4]Doorbar J. The papilomavirus life cycle [J]. J Clin Virol,2005,32(1):7-15.
[5]Bulk S, Berkhof J, Bulkmans NW, et al. Preferential risk of HPV16 for squarrious cell carcinoma and of HPV 18 for adenocarcinoma of the cervix compared to women with normal cytology in the Netherlands [J]. Br J Cancer,2006,94(1):171-175.
[6]Woodman CB, Collin S, Winter H, et al. Natural history of cervical human papillomavirus infection in young women:a longitudinal cohort study [J]. Lancet,2001,357(9271): 1831-1836.
[7]Clifford GM, Gallus S, Herrero R, et al. Worldwide distribution of human papillomvirus types in cytological normal women in the International Agency for Research on Cancer HPV prevalence surveys:a pooled analysia [J]. Lancet,2005,366(9490):991-998.
[8]Plummer M, Schiffman M, Castle PE, et al. A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significanceor low-grade squamous intraepithelial lesion[J]. J Infect Dis,2007,195(11):1582-1589.
[9]Moore MA, Tajima K. Cervical cancer in the Asian Pacific:epidemiology, screening and treatment [J]. Asian Pac Cancer Prev,2004,5(4):349-361.
[10]Baseman JG, Koutsky LA. The epidemiology of human papilomavirus infection [J]. J Clin Virol 2005,32(l):16-24.
[11]Trottier H, Franco EL.The epidemiology of genital human papilomavirus infection[J]. Vaccine,2006,24(1):1-15.
[12]Nishiwaki M, YamamotoT, one S, et al. Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications [J]. J Clin Microbiol, 2008,46(4):1161-1168.
[13]Weyn C, Boulenouar S, Mathys V, etal. Detection of human papillomavirtts types 45 and 51 by type specific polymerase chain reaction [J]. J Virol Methods,146(2):405-408.
[14]刘翠华.人乳头瘤病毒的诊断研究进展[J].国外医学-妇产科分册,2002,29(4)：213-215.
[15]王勋松,邹学森,袁水斌,HPV与宫颈癌的关系及检测方法的研究进展[J].实验与检验医学,2008,26(2)：166-168.
[16]田永强,朱中元.以PCR为基础的HPV检测及其在宫颈癌筛查和治疗中的价值[J].中国热带医学,2008,8(3)：419-422.
[17]Hesselink AT, VandenBrule AJ, Brink AA, et al. Comparison of hybrid capture 2. with in situ hybridization for the detection of high risk human papillomavirus in liquid based samples [J]. Cance,2004,102(1):11-18.
[18]An HJ, Cho NH, Lee SY, et al. Correlation of cervical cancinoma and precancerous lestions with human papillomavirus (HPV) genotypes detected with HPV chip microarray method [J]. Cancer,2004,102(1):11-18.
[19]陶萍萍,卞美潞,欧华,等.导流基因杂交芯片技术在人乳头瘤状病毒检测中应用的研究[J].中华妇产科杂志,2006,41(1)：43-47.
[20]Perez-Cardenas E, Contreras·Paredes A, Cantu D, et al. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study [J]. Virol J,2007,26(4):18-19.
[21]张建华,王义平,张彩,等.丙戊酸钠联合全反式维甲酸对HeLa细胞的杀伤机制研究[J].肿瘤,2010,30(10)：33-37.
[22]Chen D, Tang Q. An experimental study on cervix cancer with combination of HSV-TK/GCV suicide gene therapy system and 60Co radiotherapy [J]. BMC Cancer,2010, 6(1):609.
[23]Nie CL, Gao GL, Han J, et al. Human papillomavirus 16 E6, E7 siRNAs inhibit proliferation and induce apoptosis of SiHa cervical cancer cells [J]. Chinese Journal of Cancer Research,2008,20(4):301-306.
[24]Jonson AL, Rogers LM, Ramakrishnan S, et al. Gene silencing with siRNA targeting E6/E7 as peutic intervention in a mouse model of cervical cancer [J]. Gynecologic Oncology,2008, 111(2):356-364.
[25]Ritchie W, Flamant S, Rasko JE. mimicroRNA:a microRNA expression profiler and classification resource designed to identify functional correlations between microRNAs and their targets [J]. Bioinformaties,2010,26(2):223-227.
[26]Akao Y, Nakagawa Y, Hirata Ⅰ, et al. Role of anti-on eomirs miR-143 and 145 in human colorectal tumors [J]. Cancer Gene Ther,2010,17(6):398-408.
[27]Xie Z. Piecing the puzzle together:genetic requirements for microRNA biogenesis in Arabidopsis thaliana [J]. Methods Mol Biol,2010(592):1-17.
[28]TianRQ, Wang XH, Hou LJ, et al. MicroRNA-372 is downregulated and targets a) cyclin-dependent kinase 2-CDK21 and cyelin Al in human cervical cancer,which may contribute to tumorigenesis [J]. Biol Chem,2011 (6):123-129.
[29]Lee JW, Choi CH, Choi JJ, et al. Altered micorRNA expression in cervical carcinomas [J]. Clin Cancer Res,2008,14(9):2535-2542.
[30]Wang X, Tang S, Le SY, et al. Aberrant expression of oncogennic and tumor-suppressive microRNAs in cervical cancer required for cancer cell growth [J]. Plos One,2008, (7): 2557.
[31]Yao Q, Xu H, Zhang QQ, et al. MicroRNA-21 promotes cell proliferation and own-regulates the expression of programmed cell death 4(PDCD4)in HeLa cervical carcinoma cells [J]. Biochem Biophys Res Commun,2009,388(3):539-542.
[32]齐广涛,王德华.宫颈癌放疗细胞遗传学损伤的物标志物及剂量-反应关系[J].天津医科大学学报,2009,15(3)：426-429.
[33]魏玮,程玉峰,姜玉华,等Bax、Bel-2ASODN联合转染增强宫颈癌HeLa细胞放射敏感性的实验研究[J].现代妇产科进展,2006,15(11)：841-843.
[34]Huang w, Li MD, Wu QL, et al. Expression and clinical significance of P53 and cerbB-2 in genatric women with cervical carcinoma [J]. Aizheng,2002,21(3):297-300.
[35]CheungTH. C-fos overexpression is associated with the pathoneogenials of invase cervical cancer Gynecol Obstet [J]. Invest,1997,43(3):2000-2003.
[36]Chen SL, Tsai TZ. Mutation analysis of Human papillomanvirus type Ⅱ E5a oncoprotein [J]. J Virol,1996,70(6):2502-2508.
[37]Graflund, Sorbe, Karlssom. Immunohistochemical expression of p53、bc1-2 and p21WAFI/CIPI in early cervical carcinoma:Correlation with clinical outcome [J]. Internat J Gynecol Cancer,2002,30(12):290.
[38]吴明,刘少扬,江大琼,等.Bag-1、bcl-2在宫颈癌中的表达及临床意义….数理医药学杂志,2005,18(4)：323-325.
[39]Skomedal H, Kristensen GH, L ie AK, et al. Aberrant expression of the cell cycle associated proteins TP53, MDM7, P21, P27, CDK4, cyclinDl, Rb and EGFR in cervical carcinoma [J]. Gyneonl Oncol,1999,73(2):223-228.
[40]Krivak TC, Mcbroom JW, Seidman J, et al. Abnormal FH1T expression in advanced cervical carcinoma:a poor prognostic factor [J]. Cancer Res,2001,61(9):4382-4385.
[41]Baykal C, Amhan A, A IA. No relationship is indicated between FHIT expression and clinicopathologic prognosis parameters in early cervical carcinoma [J]. Int J Gynecol Cancer,2003,13(1):192-196.
[42]安锦霞,黎卫平,杨永秀.RASSFIA基因在宫颈癌中的表达及意义[J].第四军医大学学报,2005,26(7)：657-660.
[43]王梅,王冰,王晓丽,et al相关凋亡基因survinin在宫颈癌组织中的表达及其与bcl-2、p53基因相关性的初步研究[J].中华妇产科杂志,2001,36(9)：546-548.
[44]Tugizov S, Berline J, Herrera R, et al. Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex [J]. J Virol,2005, 79(2):1099-1112.
[45]Nawa A, Nishimori K, Lin P, et al. Tumor metastasis-associated human MTA1 gene:its deduced protein sequence. Localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligenucleotides [J]. J Cell Biochem,2000, 79(2):202-212.
[46]Rowhani-Rahbar A, Mao C, Hughes JP, et al. Longer term efficacy of a prophylactic monovalent human papillomavirus typel6 vaccine [J].Vaccine,2009,27(41):5612-5619.
[47]Paavonen J, Naud P, Salmeron J, et al. Efficacy of human papilloma-virus(HPV)-16/18 AS04 adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types(PATRICIA):final analysis of a double-blind, randomised study in young women [J]. Lancet,2009,374(9686):301-314.
[48]Romanowaki B, Borba PC, Naud PS, et al. Sustained efficacy and immunogenicity of the human papillomavirus(HPV)-16/18 AS04-adjuvanted vaccine:analysis of randomized placebo-controlled trial up to 6.4 Years[J], Lancet,2009,374(9706):1975——1985.
[49]Schwarz TF, Spaczynski M, Schreider A, et al. Immunogenicity and tolerability of an HPV-16/18 AS04 adjuvanted prophylactic cervical cancer vaccine in wome aged 15-55 years [J].Vaccine,2009,27(4):581-587.
[50]Rowhani-Rahbar A, Carter JJ, Hawes SE,et al. Antibody responses in oral fluid after administration of prophylactic human papillomavirus vaccines [J]. J Infect Dis,2009, 200(9):1452-1455.
[51]LuD, HooryT, MonieA, et al. Treatment with demethylating agent,5-aza-2'-deoxycytidine enhances therapeutic HPV DNA vaccine potency [J]. Vaccine,2009,27(32):4363-4369.
[52]Cid-Arregui A. Therapeutic vaccines against human papillomavirus and cervical cancer [J].Open Virol J,2009,3(l):67-83.
[53]Schwartz TF, Spaczynski M, Schneider A, et al. Immunogenicity and tolerability of all HPV—16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15-55 years[J]. Vaccine,2009,27(8):581-587.
[54]Munoz N, Manalastas Jr R, Pitisuttitthum P, et al. Safety, immunogenicity and efficacy of quadrivalent human papillomavirus (types 6,11,16,18)recombinant vaccine in women aged 24-45 years:A randomized, double-blind trial [J]. Lancet,2009,373(21):1949-1957.
[1]Chang WC, Li CH, Huang SC, et al. Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma. Cancer.2010 15;116(24):5777-88. doi:10.1002/cncr.25371.
[2]Jemal A, Bray F, Center MM, et al. Global cancer statistics[J]. CA Cancer J Clin,2011, 61(2):69-90.
[3]Zullo MA, Manci N, Angioli R, et al. Vesical dysfunctions after radical hysterectomy for cervical cancer:a critical review[J]. Crit Rev Oncol Hematol,2003,48(3):287-293.
[4]Rob L, Halaska M, Robova H. Nerve-sparing and individually tailored surgery for cervical cancer [J]. Lancet Oncol,2010,11(3):292-301.
[5]Beiner ME, Covens A. Surgery insight:radical vaginal trachelectomy as a method of fertility preservation for cervical cancer[J]. Nat C in Pract Oncol,2007,4(6):353-361.
[6]Dursun P, Ayhan A, Yanik FB, et al. Ovarian transposition for the preservation of ovarian function in young patients with cervical carcinoma[J]. Eur J Gynaecol Oncol,2009,30(1): 13-15.
[7]Liang Z, Xu H, Chen Y, et al. Laparoscopic radical trachelectomy or parametrectomy and pelvic and para-aortic lymhadenectomy for cervical or vaginal stump carcinoma:report of sixcases[J]. Int J Gynecol Cancer,2006,16(4):1713-1716
[8]Ereoli A, Iannone V, Legge F, et al. Advances in surgical management of cervical cancer[J]. Minerva Ginecol,2009,61(3):227-237.
[9]陈春林,郭玉.系统保留盆腔自主神经的广泛性子宫切除术中神经确认与术后膀胱功能评估[J].中国实用妇科与产科杂志,2011,27(3)：163-166.
[10]Ereoli A, Iannone V, Legge F, et al. Advances in surgical managent of cervical cancer[J]. Minerva Ginecol,2009,61(3):227-237.
[11]Zakashansky K, ChuangL, Gretz H, et al. A case controlled study of total laparoscopic radical hysterectomy with pelvic lymphadenectomy versus radical abdominal hysterectomy in a fellowship training program[J]. Int J Gynecol-Cancer,2007,17(5):1075-1082.
[12]Chen Y, Xu H, Li Y, et al. The outcome of laparoscopic radical hysterectomy and lymphadenectomy for cervical cancer:a prospective analysis of 295 patients[J]. Ann Surg Oncol,2008,15(10):2847-2855.
[13]Oleszczuk A, Kohler C, Paulick J, et al. Vaginal robot-assisted radical hysterectomy (VRARH) after laparoscopic staging:feasibility and operative results[J]. Int J Med Robot, 2009,5(1):38-44.
[14]Narducci F, Lambaudie E, Houvenaeghel G, et al. Early experience of robotic-assisted laparoscopy for extraperitoneal para-aortic lymph adenectomy up to the left renal vein[J]. Gynecol Oncol,2009,115(1):172-174.
[15]Chuang LT, Lerner DL, Liu CS, et al. Fertility-sparing robotic-assisted radical trachelectomy and bilateral pelvic lymphadenectlmy in early-stage cervical cancer[J]. J Minim Invasive Gynecol,2008,15(6):767-770.
[16]Magrina JF, Kho RM, Weaver AL, et al. Robotic radical hysterectomy:comparison with laparoscopy and laparotomy[J]. Gynecol Oncol,2008,109(1):86-91.
[17]AltgassenC, Hertel H, Brandstadt A, et al. Multicenter validation study of the sentinel lymph node concept in cervical Cancer:AGO Study Group[J]. J Clin Oncol,2008,26(18): 2943-2951.
[18]Portelance L, Chao KS, Grigsby PW, et al. Intensity-modulated radiation therapy(IMRT) reduces small bowel, recttlm, and bladder doses in patients with cervical cancer receiving pelvic and para-aortic irradjation[J]. Int J Radiat Oncol Biol Phys,2001,51(1):261.
[19]Roeske JC, Lujan A, RotmenschJ, et al. Intensity2modulated whole pelvic radiation therapy in patients with gynecologic malignancies[J]. Int J Radiat Oncol Biol Phys.2000, 48(5):1613-1621.
[20]Teh B. Bloch C. Galli-Guevara M. et al. The treatment of primary and metastatic renal cell carcinoma(RCC)with image-guided stereotactic body radiation therapy(SBRT) [J]. Biomed Imaging IntervJ,2007,3(1):1-9.
[21]郝光军,魏丽春,石梅,等.局部晚期宫颈癌CT图像引导下适形调强后程推量的临床研究[J].现代肿瘤医学,2010,18(12)：2462-2465.
[22]黄曼妮,李明辉,吴令英.宫颈癌调强适形放射治疗与三维适形放射治疗的剂量学比较[J].癌症进展杂志,2009,7(4)：440-442.
[23]Small W, Mell LK, Anderson P, et al. Consensus for delieation of clinical target volume for intenaity modulated pelvic radiotherapy in postoperative treatment of endometrial and cervical cancer[J]1. Int J Radiat Oncol Biol Phys,2008,71(2):428-434.
[24]孔为民,王娟.宫颈癌的放射治疗[J].中国实用妇科与产科杂志,2010,26(3)：174-175.
[25]Rogers L, Siu SS, Luesley D, et al. Adjuvant radiotherapy and chemoradiation after surgery for cervical cancer[J]. Cochrane Database Syst Rev,2009(4):CD007583.
[26]Rosa DD, Medeiros LR, Edelweiss MI, et al. Adjuvant platinumbased chemotherapy for early stage cervical cancer [J]. Cochrane Database Syst Rev,2009(3):CD005342.
[27]Legge F, FuocoG, Lorusso D, et al. Pharmacotherapy of cervical cancer[J]. Expert Opin Pharmacother,2010,11(12):2059-2075.
[28]Lee JW, Choi CH, Choi JJ, et al. Altered micorRNA expression in cervical carcinomas[J]. Clin Cancer Res,2008,14(9):2535-2542.
[29]Wang X, Tang S, Le SY, et al. Aberrant expression of oncogennic and tumor-suppressive microRNAs in cervical cancer required for cancer cell growth[J]. Plos One,2008,3(7): e2557.
[30]Yao Q, Xu H, Zhang QQ, et al. MicroRNA-21 promotes cell proliferation and own-regulates the expression of programmed cell death 4(PDCD4)in HeLa cervical carcinoma cells[J]. Biochem Biophys Res Commun,2009,388(3):539-542.
[31]Nie CL, Gao GL, Han J, et al. Human papillomavirus 16 E6, E7 siRNAs inhibit proliferation and induce apoptosis of SiHa cervical cancer cells[J]. Chinese Journal of Cancer Research,2008,20(4):301-306.
[32]JonsonAL, Rogers LM, Ramakrishnan S, etal. Gene silencing with siRNA targeting E6 /E7 as peutic intervention in a mouse model of cervical cancer[J]. Gynecologic Oncology, 2008,111(2):356-364.
[33]Perez—Cardenas E, Contreras-Paredes A, Cantu D, et al. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study[J]. Virol J,2007,26(4):18-19.
[34]张建华,王义平,张彩,等.丙戊酸钠联合全反式维甲酸对HeLa细胞的杀伤机制研究[J].肿瘤,2010,30(10)：33-37.
[35]Porika M, Tippani R, Mohammad A, et al. Evaluation of sernm human telomerase reverse transcriptase as a novel marker for cervical cancer[J]. Int J Biol Markers,2011,26(1): 22-26.
[36]Humphreys RC, Halpern W. Trail receptors:targets for cancer therapy [J]. Adv Exp Med Biol,2008,615(1):127-158.
[37]Sakurai H, Lirdprapamongkol K, Suzuki S, et al. Vanillin enhances TRAIL-induced apoptosis in cancer cells through inhibition of NF-kappaB activation[J]. In Vivo,2010, 24(4):501-506.
[38]Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4(VEGFR-3) and KDR(VEGFR-2)receptor tyrosine kinases[J]. EMBOJ,1996,15(2):290-298.
[39]Terhi Karpanen, Kari Alitalo, Lymphatic vesseles as targets of tumor therapy [J]. J Exp Med,2001,194(6):F37-42.
[40]Chen D, Tang Q. An experimental study on cervix cancer with combination of HSV-TK /GCV suicide gene therapy system and 60Co radiotherapy[J]. BMC Cancer,2010,6(10): 609.
[41]FranckenaM, Stalpers LJ, Koper PC, et al Long-term improvement in treatment outcome after radiotherapy and hyperthermia in locoregionally advanced cervix cancer:an update of the Dutch Deep HyperthermiaTrial. Int J RadiatOncol Biol Phys,2008,70:1176-1182.
[42]FranckenaM, LutgensLC, Koper PC, et al Radiotherapy and hyperthermia for treatment of primary locally advanced cervix cancer results in 378 patients. Int J RadiatOncol Biol Phys, 2009,73:242-250
[43]FranckenaM, DeWit R, Ansink AC, et al Weekly systemic cisplatin plus locoregional hyperthermia:an effective treatment for patients with recurrent cervical carcinoma in a previously irradiated area. Int J Hyperthermia,2007,23:443-450
[2]姜颖,张海青.缺氧诱导因子-1在肿瘤临床方面的研究进展[J].国外医学(肿瘤学分册),2004,(11),789-791.
[3]Brown, JM. Tumor hypoxia in cancer therapy [J]. Methods Enzymol,2007,435:297-321.
[4]Parkin DM, Bray F, Ferlay J,et al. Estimating the World Cancer Burden:Global Cancer 2000 [J]. Cancer,2001,94(2):153.
[5]乐杰.妇产科学[M].6版.北京：人民卫生出版社,2001：314.
[6]Qiao YL, Zhang WH, Li L, et al. A Cross-section Study of Screening Techniques for Cervical Cancer in Shanxi [J].Acta Acad Med Scin,2002,24(1):50-53.
[7]Jian-Jun Zhang. Expression and significance of TLR4 and HIF-la pancreatic ductal adenocarcinoma [J]. World Gastroenterol,2010,16(23):881-888.
[8]Semenza GL. HIF-la inhibitors for cancer therapy:from gene expression to drug discovery[J]. Curr Pharm Des,2009,15(33):3839-3843.
[9]Adams JM, Difazio LT, Rolandelli RH, et al. HIF-la key mediator in hypoxia [J]. Acta Physiol Hung,2009,96(1):19-28.
[10]Hirota K, Semenza GL. Regulation of angiogenesis by hypoxia-inducible factor [J]. Crit Rev Oncol Hematol,2006,59(1):15-26.
[11].宋樱,孙善珍,曲迅,等.乏氧对人舌鳞癌细胞系Tca8113体外黏附和侵袭能力的影响[J].实用口腔医学杂志,2009,25(6)：828-832.
[12]朱世凯.缺氧微环境中HIF-la诱导MT2-MMP表达及对胰腺癌侵袭和转移作用的机制研究[D].武汉：华中科技大学,2010.
[13]Alcazar J L, Casfillo G, Martinez M R. Trans-vaginal color Doppler sonography for predicting response to concurrent chemoradiotherapy for locally advanced cervical cancinoma [J]. Clin Ultrasoun 2007.32(6):267-272.
[14]Toma Dsu I, Dasu A, Karlsson M.The relationship between temporal variation of hypoxia, polarographic measurements and predictions ofturnout response to radiation[J]. Phys Med Biol,2004,49 (19):463-75.
[15]Kanz M, Ibrahim SM. Molecular response to hypoxia in tumor cells [J]. Clin Ultrasoun, 2003,2:23-36.
[16]PASETTO LM, D'ANDRE MR, ROSSI E, et al.Oxaliplatin-related neurotoxicity:how and why? [J]. Crit Rev Oncol Hematol,2006,59(2):159-168.
[17]Bidoli P, Zilembo N, Cortinov D, et al. Randomized phase II three-arm trial with three platinum based doublets in metastatic non-small-cell lung cancer An Italian Trials in Medical Oncology study [J]. Ann Oncol,2007,18(3):461-367.
[18]CASSIDY J, MISSET J L. Oxaliplatin-related side effect:Characteristics and management [J]. Semin Oncol,2002,29(5):11-20.
[19]王琳,秦叔逵,钱军,等.奥沙利铂联合氟尿嘧啶类药物二线治疗晚期胃癌的临床研究[J].现代肿瘤医学,2005,13(3)：361-362.
[20]MCKEAGE M J. New-generation platinum drugs in the treatment of cisplatin-resistant cancers [J]. Expert Opin Investig Drugs,2005,14 (8):1033-1046.
[21]Lordick F, Lorrenzens S, Stollfuss J, et al. Phase II study of weekly oxaliplatin plus infusional fluorouracil and folinic acid(FUFOX regimen)as firstline treatment in metastatic gastric cancer [J]. BrJ Cancer,2005,93 (2):190-194.
[22]周彩存,张捷,徐瑛,等.奥沙利铂联合长春瑞滨一线治疗不可手术的非小细胞肺癌[J].临床肿瘤学杂志,2005,10(5)：481-485.
[23]BELANI C P. Recent updates in the clinical use of platinum compounds for the treatment of lung, breast, and genitourinary tumors and myeloma [J]. SeminOncol,2004,31(14):25-33.
[24]Loncaster J A, Harris A L, Davidson S E.Carbonic anhydrase (CAIX)expression, a potential new intrinsic marker of hypoxia:Correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix [J].Cancer Res,2005, 61(17):6394-6399.
[25]Mizukami Y, Li J, Zhang X, et al. Hypoxia-inducible factor-1-independent regulation of vascular endothelial growth factor by hypoxia in colon cancer [J].Cancer Res.2004,64(5): 1765-72.
[26]Menrad H, Werno C, Schmid T, et al. Roles of hypoxia-inducible factor-lalpha (HIF-lalpha)versus HIF-2alpha in the survival of hepatocellular tumor spheroids [J]. Hepatology, 2010,51(6):2183-2192
[27]No JH, Jo H, Kim SH, et al. Expression of vascular endothelial growth factor and hypoxia inducible factor-lalpha in cervical neoplasia [J]. Ann N Y Acad Sci,2009,16(7),1171: 105-110.
[28]Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1[J]. Biol Chem,1995,270(3):1230-1237.
[29]Semenza GL. Surviving ischemia:adaptive responses mediated by hypoxia-inducible factor 1 [J]. Clin Invest,2000,106(7):809-812.
[30]Mircea I, Keiichi K, Haifeng Yang, et al. HIF-1 Targeted for VHL-Mediated Destruction by Proline Hydroxylation:Implications for O2 [J]. Sensing. Science,2001,292(5516):464-468.
[31]常青,秦仁义,高军,等.反义缺氧诱导因子-1α对胰腺癌细胞BxPC-3化疗敏感性的影响[J].中国普通外科杂志,2006,15(6)：423-427.
[32]Hubert A, Paris S, Piret JP, et al. Casein kinase 2 inhibition decreases hypoxia-inducible fa ctor-1 activity under hypoxia through elevated p53 protein level [J]. J Cell Sci,2006,119 (16):3351-3362.
[33]Takahashi Y, Nishikawa M, Takakura Y. Inhibition of tumor cell growth in the liver by RN A interferencemediated suppression of HIF-lalpha expression in tumor cells and hepatocyte s [J]. GeneTher,2008,15(8):572-582.
[34]Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and therirmetastases [J]. Cancer Res,1999,59:5830-5835.
[35]Fujita M,Yasuda M,Kitatani K,et al.Anup-to-date anticancer treatment strategy focusing on HIF-lalpha su-ppr ession:its application for refractory ovarian cancer[J].Acta Histoche m Cytochem,2007,40(5):139-142.
[36]Hur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilization or DNA binding ability of hypoxia-inducible factorlalpha[J]. Mol Pharmacol,2009,59 (5):1216-1222.
[37]Osada M, Imaoka S, Sugimoto T, et al. NADPH-cytochrome P-450 reductase in the plasma membrane modulates the activation of hypoxia-inducible factor 1[J]. J Biol Chem, 2008,277(26):23367-23373.
[38]Kunz M, Ibrahim SM. Molecular responses to hypoxia in tumor cells [J]. Mol Cancer,2009, 2(1):23-30.
[39]Jach R, Dulinska-Litewka J, Laidler P, et al. Expression of VEGF, VEGF-C and VEGFR-2 in situ and invasive SCC of cervix [J]. Front Biosei (Elite Ed),2010,2:411-423.
[40]Dales JP, BeautilsN, SilvyM, et al. Hypoxia inducible factor lalpha gene(HIF-lalpha)splice variants:potential prognostic biomarkers in breast cancer [J].BMC Med,2010,8(2):44.
[41]沈维干,朱军,于智勇,等.低氧对人肺腺癌A549细胞迁移和黏附的影响[J].肿瘤,2008,28(3)：216-259.
[42]Chi JT, Wang Z, Nuyten DS, et al. Gene expression program s in response to hypoxia:cell type specificity an d prognostic significance in human cancers [J]. Plo S Med,2006, 3(3):395-409.
[43]Ozer A, Wu LC, Bruick RK. The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF) [J]. Proc Natl Acad Sci USA,2005,102(21):7481-7486.
[44]Cayre A, Rossignol F, Clottes E, et al. HIF but not HIF-la transcript is a poor prognostic marker in human breast cancer [J]. Breast Cancer Res,2003,5(6):223-230.
[45]Ritchie W, Flamant S, Rasko JE. mimicroRNA:a microRNA expression profiler and classification resource designed to identify functional correlations between microRNAs and their targets [J]. Bioinformaties,2010,26(2):223-227.
[46]Hanze J, Eul BG, Savai R, et al. RNA interference for HIF-lalpha inhibits its downstream signalling and affects cellular proliferation [J]. Biochem Biophys Res Commun,2003, 312(3):571-577.
[47]Brizel DM, Sibley GS, Prosnitz LR, et al. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck [J]. Int J Radiat Oncol Biol Phys,1997,8(2):285-289.
[48]Hockel M, Vorndran B, Schlenger K, et al. Tumor oxygenation:a new predictive parameter in locally advanced cancer of the uterine cervix [J]. Gynecol Oncol,1993,51(2):141-149.
[49]Goda N, Ryan HE, Khadivi Bk, et al. Hypoxia-inducible factor lalpha is essential for cell cycle arrest during hypoxia [J]. Mol Cell Biol.2003,23 (1):359-369.
[50]Lawrence B, Gardner, Qing Li, et al. Hypoxia inhibits Gl/S transition through regulation of p27 expression [J]. J Biol Chem.,2001,276(11):7919-7926.
[51]Box AH, Demetrick DJ. Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines [J].Carcinogenesis,2004,25(12):2325-2335.
[52]Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia inducible factor-1 dependent regulation of the multidrug resistance (MDR1) gene [J]. Cancer Res,2002,62(12):3387-3394.
[53]Kunz M, Ibrahim SM. Molecular responses to hypoxia in tumor cells [J]. Mol Cancer,2003, 2(1):23-30.
[54]Acs G, Zhang PJ, McGrath CM, et al. Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression [J].Am J Pathol,2003,162 (6):1789-1806
[55]J Bruick RK. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia [J].Proc Natl Aced Sci USA,2000,97(16):9082-9087.
[56]Baek JH, Jang JE, Kang CM, et al. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis [J]. Oncogene,2000,19(40): 4621-4631.
[57]Unruh A, Ressel A, Mohamed HG, et al. The hypoxia-inducible factor-1 alpha is a negative factor for tumor therapy [J]. Oncogene,2003,22 (21):3213-3220.
[58]Achison M, Hupp TR. Hypoxia attenuates the p53 response to cellular damage [J]. Oncogene,2003,29 (22):3431-3440.
[59]Ghafar MA, Anastasiadis AG, Chen MW, et al. Acute hypoxia increases the aggressive characteristics and survival properties of prostate cancer [J]. Cells Prostate,2003,54(1) 58-67.
[60]Mckeown SR, Cowen RL, Williams KJ. Bioreductive drugs:from concept to clinic [J].Clin Oncol(R Coll Radio 1),2007,9(4):427-442.
[61]Hay MP, Hicks KO, Pruijn FB. Pharmacokinetic/pharmacodynamic model-guided identification of hypoxia-selective 1,2,4-benzotriazine 1,4-dioxides with antitumor activity:the role of extravascular transport [J]. Med Chem,2007,50(8):6392-6404
[62]Brown JM. Tumor hypoxia in cancer therapy [J].Methods Enzymol,2007,435(11): 297-321.
[63]Overgaard J. Hypoxic radiosensitization:adored and ignored [J].J Clin Oncol,2007,25(4): 4066-4074.
[64]Anderson P, Aguilera D, Pearson M. Outpatient chemotherapy plus radiotherapy in sarcomas improving cancer control with radiosensitizing agents [J]. Cancer Control,2008, 15(6):38-46.
[65]Mckeown SR, Cowen RL, Williams KJ. Bioreductive drugs:from concept to clinic [J].Clin Oncol(R Coll Radiol),2007,19(7):427-442.
[66]Takahashi, Nishikawa M, Takakura Y. Inhibition of tumor cell growth in the liver by RNA interference-mediated suppression of HIF-lalpha expression in tumor cells and hepatocytes [J]. GeneTher,2008,15(8):572-582.
[67]Bidoli P, Zilembo N, Cortinov is D,et al.Raidolized phase II three-arln trial with three platinum based doublets in metastatic non-small-cell lung cancer [J]. An Italian Trials in Medical Oncology study, Ann Oncol,2007,18(3):461-367.
[68]Furue H. oxaliPlatin [J].GanToKaga-Ryoho,2005,32(8):1195-1202.
[69]Mekeage M J. New-generation platinum drugs in the treatment of cisplatin-resistant caneers [J]. ExPert Opin Investig Drugs,2005,14(8):1033-1046.
[70]Lordiek F, Lorenzen S, Stollfuss J, etal. Phase II study of weekly oxaliplatin Plus Infusional fluorouracil and folinic acid (FIJFOX regimen)as first-line treatment inmetastatic gastric cancer [J].Br J Cancer,2005,93(2):190-194.
[71]CividalliA, CeciarelliF, Livid E, etal. Radiosensitization by oxaliplatin in a mouse adenocarcinoma:influence of treatment schedule [J]. Int-J-Radiat-Oncol-Biol-Phys, 2002,52(4):1092-1098.
[72]吕惠兰,宫良平,邵震宇,等.低剂量奥沙利铂并氟脲嘧啶对晚期复发直肠癌放疗增敏作用的临床观察[J].中国现代普通外科进展,2005：178-182.
[73]江滨,陈书长.抗肿瘤药物临床应用指南[M].北京：中国协和医科大学出版社,2005：228-233.
[1]Kreimer AR, Clifford GM, Boyle P, et al. Human papilomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review [J]. Cancer Epidemiol Biomarkers Prev,2005,14(6):467-475.
[2]Wgngoner S, Chemiky CL. Molceluar biology of cervical and vulvar carcinoma [M].2004, 65-78.
[3]Davey DD. Cervical cytology classification and the Bethesda system [J].Cancer,2003,9 (4):327-334.
[4]Doorbar J. The papilomavirus life cycle [J]. J Clin Virol,2005,32(1):7-15.
[5]Bulk S, Berkhof J, Bulkmans NW, et al. Preferential risk of HPV16 for squarrious cell carcinoma and of HPV 18 for adenocarcinoma of the cervix compared to women with normal cytology in the Netherlands [J]. Br J Cancer,2006,94(1):171-175.
[6]Woodman CB, Collin S, Winter H, et al. Natural history of cervical human papillomavirus infection in young women:a longitudinal cohort study [J]. Lancet,2001,357(9271): 1831-1836.
[7]Clifford GM, Gallus S, Herrero R, et al. Worldwide distribution of human papillomvirus types in cytological normal women in the International Agency for Research on Cancer HPV prevalence surveys:a pooled analysia [J]. Lancet,2005,366(9490):991-998.
[8]Plummer M, Schiffman M, Castle PE, et al. A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significanceor low-grade squamous intraepithelial lesion[J]. J Infect Dis,2007,195(11):1582-1589.
[9]Moore MA, Tajima K. Cervical cancer in the Asian Pacific:epidemiology, screening and treatment [J]. Asian Pac Cancer Prev,2004,5(4):349-361.
[10]Baseman JG, Koutsky LA. The epidemiology of human papilomavirus infection [J]. J Clin Virol 2005,32(l):16-24.
[11]Trottier H, Franco EL.The epidemiology of genital human papilomavirus infection[J]. Vaccine,2006,24(1):1-15.
[12]Nishiwaki M, YamamotoT, one S, et al. Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications [J]. J Clin Microbiol, 2008,46(4):1161-1168.
[13]Weyn C, Boulenouar S, Mathys V, etal. Detection of human papillomavirtts types 45 and 51 by type specific polymerase chain reaction [J]. J Virol Methods,146(2):405-408.
[14]刘翠华.人乳头瘤病毒的诊断研究进展[J].国外医学-妇产科分册,2002,29(4)：213-215.
[15]王勋松,邹学森,袁水斌,HPV与宫颈癌的关系及检测方法的研究进展[J].实验与检验医学,2008,26(2)：166-168.
[16]田永强,朱中元.以PCR为基础的HPV检测及其在宫颈癌筛查和治疗中的价值[J].中国热带医学,2008,8(3)：419-422.
[17]Hesselink AT, VandenBrule AJ, Brink AA, et al. Comparison of hybrid capture 2. with in situ hybridization for the detection of high risk human papillomavirus in liquid based samples [J]. Cance,2004,102(1):11-18.
[18]An HJ, Cho NH, Lee SY, et al. Correlation of cervical cancinoma and precancerous lestions with human papillomavirus (HPV) genotypes detected with HPV chip microarray method [J]. Cancer,2004,102(1):11-18.
[19]陶萍萍,卞美潞,欧华,等.导流基因杂交芯片技术在人乳头瘤状病毒检测中应用的研究[J].中华妇产科杂志,2006,41(1)：43-47.
[20]Perez-Cardenas E, Contreras·Paredes A, Cantu D, et al. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study [J]. Virol J,2007,26(4):18-19.
[21]张建华,王义平,张彩,等.丙戊酸钠联合全反式维甲酸对HeLa细胞的杀伤机制研究[J].肿瘤,2010,30(10)：33-37.
[22]Chen D, Tang Q. An experimental study on cervix cancer with combination of HSV-TK/GCV suicide gene therapy system and 60Co radiotherapy [J]. BMC Cancer,2010, 6(1):609.
[23]Nie CL, Gao GL, Han J, et al. Human papillomavirus 16 E6, E7 siRNAs inhibit proliferation and induce apoptosis of SiHa cervical cancer cells [J]. Chinese Journal of Cancer Research,2008,20(4):301-306.
[24]Jonson AL, Rogers LM, Ramakrishnan S, et al. Gene silencing with siRNA targeting E6/E7 as peutic intervention in a mouse model of cervical cancer [J]. Gynecologic Oncology,2008, 111(2):356-364.
[25]Ritchie W, Flamant S, Rasko JE. mimicroRNA:a microRNA expression profiler and classification resource designed to identify functional correlations between microRNAs and their targets [J]. Bioinformaties,2010,26(2):223-227.
[26]Akao Y, Nakagawa Y, Hirata Ⅰ, et al. Role of anti-on eomirs miR-143 and 145 in human colorectal tumors [J]. Cancer Gene Ther,2010,17(6):398-408.
[27]Xie Z. Piecing the puzzle together:genetic requirements for microRNA biogenesis in Arabidopsis thaliana [J]. Methods Mol Biol,2010(592):1-17.
[28]TianRQ, Wang XH, Hou LJ, et al. MicroRNA-372 is downregulated and targets a) cyclin-dependent kinase 2-CDK21 and cyelin Al in human cervical cancer,which may contribute to tumorigenesis [J]. Biol Chem,2011 (6):123-129.
[29]Lee JW, Choi CH, Choi JJ, et al. Altered micorRNA expression in cervical carcinomas [J]. Clin Cancer Res,2008,14(9):2535-2542.
[30]Wang X, Tang S, Le SY, et al. Aberrant expression of oncogennic and tumor-suppressive microRNAs in cervical cancer required for cancer cell growth [J]. Plos One,2008, (7): 2557.
[31]Yao Q, Xu H, Zhang QQ, et al. MicroRNA-21 promotes cell proliferation and own-regulates the expression of programmed cell death 4(PDCD4)in HeLa cervical carcinoma cells [J]. Biochem Biophys Res Commun,2009,388(3):539-542.
[32]齐广涛,王德华.宫颈癌放疗细胞遗传学损伤的物标志物及剂量-反应关系[J].天津医科大学学报,2009,15(3)：426-429.
[33]魏玮,程玉峰,姜玉华,等Bax、Bel-2ASODN联合转染增强宫颈癌HeLa细胞放射敏感性的实验研究[J].现代妇产科进展,2006,15(11)：841-843.
[34]Huang w, Li MD, Wu QL, et al. Expression and clinical significance of P53 and cerbB-2 in genatric women with cervical carcinoma [J]. Aizheng,2002,21(3):297-300.
[35]CheungTH. C-fos overexpression is associated with the pathoneogenials of invase cervical cancer Gynecol Obstet [J]. Invest,1997,43(3):2000-2003.
[36]Chen SL, Tsai TZ. Mutation analysis of Human papillomanvirus type Ⅱ E5a oncoprotein [J]. J Virol,1996,70(6):2502-2508.
[37]Graflund, Sorbe, Karlssom. Immunohistochemical expression of p53、bc1-2 and p21WAFI/CIPI in early cervical carcinoma:Correlation with clinical outcome [J]. Internat J Gynecol Cancer,2002,30(12):290.
[38]吴明,刘少扬,江大琼,等.Bag-1、bcl-2在宫颈癌中的表达及临床意义….数理医药学杂志,2005,18(4)：323-325.
[39]Skomedal H, Kristensen GH, L ie AK, et al. Aberrant expression of the cell cycle associated proteins TP53, MDM7, P21, P27, CDK4, cyclinDl, Rb and EGFR in cervical carcinoma [J]. Gyneonl Oncol,1999,73(2):223-228.
[40]Krivak TC, Mcbroom JW, Seidman J, et al. Abnormal FH1T expression in advanced cervical carcinoma:a poor prognostic factor [J]. Cancer Res,2001,61(9):4382-4385.
[41]Baykal C, Amhan A, A IA. No relationship is indicated between FHIT expression and clinicopathologic prognosis parameters in early cervical carcinoma [J]. Int J Gynecol Cancer,2003,13(1):192-196.
[42]安锦霞,黎卫平,杨永秀.RASSFIA基因在宫颈癌中的表达及意义[J].第四军医大学学报,2005,26(7)：657-660.
[43]王梅,王冰,王晓丽,et al相关凋亡基因survinin在宫颈癌组织中的表达及其与bcl-2、p53基因相关性的初步研究[J].中华妇产科杂志,2001,36(9)：546-548.
[44]Tugizov S, Berline J, Herrera R, et al. Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex [J]. J Virol,2005, 79(2):1099-1112.
[45]Nawa A, Nishimori K, Lin P, et al. Tumor metastasis-associated human MTA1 gene:its deduced protein sequence. Localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligenucleotides [J]. J Cell Biochem,2000, 79(2):202-212.
[46]Rowhani-Rahbar A, Mao C, Hughes JP, et al. Longer term efficacy of a prophylactic monovalent human papillomavirus typel6 vaccine [J].Vaccine,2009,27(41):5612-5619.
[47]Paavonen J, Naud P, Salmeron J, et al. Efficacy of human papilloma-virus(HPV)-16/18 AS04 adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types(PATRICIA):final analysis of a double-blind, randomised study in young women [J]. Lancet,2009,374(9686):301-314.
[48]Romanowaki B, Borba PC, Naud PS, et al. Sustained efficacy and immunogenicity of the human papillomavirus(HPV)-16/18 AS04-adjuvanted vaccine:analysis of randomized placebo-controlled trial up to 6.4 Years[J], Lancet,2009,374(9706):1975——1985.
[49]Schwarz TF, Spaczynski M, Schreider A, et al. Immunogenicity and tolerability of an HPV-16/18 AS04 adjuvanted prophylactic cervical cancer vaccine in wome aged 15-55 years [J].Vaccine,2009,27(4):581-587.
[50]Rowhani-Rahbar A, Carter JJ, Hawes SE,et al. Antibody responses in oral fluid after administration of prophylactic human papillomavirus vaccines [J]. J Infect Dis,2009, 200(9):1452-1455.
[51]LuD, HooryT, MonieA, et al. Treatment with demethylating agent,5-aza-2'-deoxycytidine enhances therapeutic HPV DNA vaccine potency [J]. Vaccine,2009,27(32):4363-4369.
[52]Cid-Arregui A. Therapeutic vaccines against human papillomavirus and cervical cancer [J].Open Virol J,2009,3(l):67-83.
[53]Schwartz TF, Spaczynski M, Schneider A, et al. Immunogenicity and tolerability of all HPV—16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15-55 years[J]. Vaccine,2009,27(8):581-587.
[54]Munoz N, Manalastas Jr R, Pitisuttitthum P, et al. Safety, immunogenicity and efficacy of quadrivalent human papillomavirus (types 6,11,16,18)recombinant vaccine in women aged 24-45 years:A randomized, double-blind trial [J]. Lancet,2009,373(21):1949-1957.
[1]Chang WC, Li CH, Huang SC, et al. Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma. Cancer.2010 15;116(24):5777-88. doi:10.1002/cncr.25371.
[2]Jemal A, Bray F, Center MM, et al. Global cancer statistics[J]. CA Cancer J Clin,2011, 61(2):69-90.
[3]Zullo MA, Manci N, Angioli R, et al. Vesical dysfunctions after radical hysterectomy for cervical cancer:a critical review[J]. Crit Rev Oncol Hematol,2003,48(3):287-293.
[4]Rob L, Halaska M, Robova H. Nerve-sparing and individually tailored surgery for cervical cancer [J]. Lancet Oncol,2010,11(3):292-301.
[5]Beiner ME, Covens A. Surgery insight:radical vaginal trachelectomy as a method of fertility preservation for cervical cancer[J]. Nat C in Pract Oncol,2007,4(6):353-361.
[6]Dursun P, Ayhan A, Yanik FB, et al. Ovarian transposition for the preservation of ovarian function in young patients with cervical carcinoma[J]. Eur J Gynaecol Oncol,2009,30(1): 13-15.
[7]Liang Z, Xu H, Chen Y, et al. Laparoscopic radical trachelectomy or parametrectomy and pelvic and para-aortic lymhadenectomy for cervical or vaginal stump carcinoma:report of sixcases[J]. Int J Gynecol Cancer,2006,16(4):1713-1716
[8]Ereoli A, Iannone V, Legge F, et al. Advances in surgical management of cervical cancer[J]. Minerva Ginecol,2009,61(3):227-237.
[9]陈春林,郭玉.系统保留盆腔自主神经的广泛性子宫切除术中神经确认与术后膀胱功能评估[J].中国实用妇科与产科杂志,2011,27(3)：163-166.
[10]Ereoli A, Iannone V, Legge F, et al. Advances in surgical managent of cervical cancer[J]. Minerva Ginecol,2009,61(3):227-237.
[11]Zakashansky K, ChuangL, Gretz H, et al. A case controlled study of total laparoscopic radical hysterectomy with pelvic lymphadenectomy versus radical abdominal hysterectomy in a fellowship training program[J]. Int J Gynecol-Cancer,2007,17(5):1075-1082.
[12]Chen Y, Xu H, Li Y, et al. The outcome of laparoscopic radical hysterectomy and lymphadenectomy for cervical cancer:a prospective analysis of 295 patients[J]. Ann Surg Oncol,2008,15(10):2847-2855.
[13]Oleszczuk A, Kohler C, Paulick J, et al. Vaginal robot-assisted radical hysterectomy (VRARH) after laparoscopic staging:feasibility and operative results[J]. Int J Med Robot, 2009,5(1):38-44.
[14]Narducci F, Lambaudie E, Houvenaeghel G, et al. Early experience of robotic-assisted laparoscopy for extraperitoneal para-aortic lymph adenectomy up to the left renal vein[J]. Gynecol Oncol,2009,115(1):172-174.
[15]Chuang LT, Lerner DL, Liu CS, et al. Fertility-sparing robotic-assisted radical trachelectomy and bilateral pelvic lymphadenectlmy in early-stage cervical cancer[J]. J Minim Invasive Gynecol,2008,15(6):767-770.
[16]Magrina JF, Kho RM, Weaver AL, et al. Robotic radical hysterectomy:comparison with laparoscopy and laparotomy[J]. Gynecol Oncol,2008,109(1):86-91.
[17]AltgassenC, Hertel H, Brandstadt A, et al. Multicenter validation study of the sentinel lymph node concept in cervical Cancer:AGO Study Group[J]. J Clin Oncol,2008,26(18): 2943-2951.
[18]Portelance L, Chao KS, Grigsby PW, et al. Intensity-modulated radiation therapy(IMRT) reduces small bowel, recttlm, and bladder doses in patients with cervical cancer receiving pelvic and para-aortic irradjation[J]. Int J Radiat Oncol Biol Phys,2001,51(1):261.
[19]Roeske JC, Lujan A, RotmenschJ, et al. Intensity2modulated whole pelvic radiation therapy in patients with gynecologic malignancies[J]. Int J Radiat Oncol Biol Phys.2000, 48(5):1613-1621.
[20]Teh B. Bloch C. Galli-Guevara M. et al. The treatment of primary and metastatic renal cell carcinoma(RCC)with image-guided stereotactic body radiation therapy(SBRT) [J]. Biomed Imaging IntervJ,2007,3(1):1-9.
[21]郝光军,魏丽春,石梅,等.局部晚期宫颈癌CT图像引导下适形调强后程推量的临床研究[J].现代肿瘤医学,2010,18(12)：2462-2465.
[22]黄曼妮,李明辉,吴令英.宫颈癌调强适形放射治疗与三维适形放射治疗的剂量学比较[J].癌症进展杂志,2009,7(4)：440-442.
[23]Small W, Mell LK, Anderson P, et al. Consensus for delieation of clinical target volume for intenaity modulated pelvic radiotherapy in postoperative treatment of endometrial and cervical cancer[J]1. Int J Radiat Oncol Biol Phys,2008,71(2):428-434.
[24]孔为民,王娟.宫颈癌的放射治疗[J].中国实用妇科与产科杂志,2010,26(3)：174-175.
[25]Rogers L, Siu SS, Luesley D, et al. Adjuvant radiotherapy and chemoradiation after surgery for cervical cancer[J]. Cochrane Database Syst Rev,2009(4):CD007583.
[26]Rosa DD, Medeiros LR, Edelweiss MI, et al. Adjuvant platinumbased chemotherapy for early stage cervical cancer [J]. Cochrane Database Syst Rev,2009(3):CD005342.
[27]Legge F, FuocoG, Lorusso D, et al. Pharmacotherapy of cervical cancer[J]. Expert Opin Pharmacother,2010,11(12):2059-2075.
[28]Lee JW, Choi CH, Choi JJ, et al. Altered micorRNA expression in cervical carcinomas[J]. Clin Cancer Res,2008,14(9):2535-2542.
[29]Wang X, Tang S, Le SY, et al. Aberrant expression of oncogennic and tumor-suppressive microRNAs in cervical cancer required for cancer cell growth[J]. Plos One,2008,3(7): e2557.
[30]Yao Q, Xu H, Zhang QQ, et al. MicroRNA-21 promotes cell proliferation and own-regulates the expression of programmed cell death 4(PDCD4)in HeLa cervical carcinoma cells[J]. Biochem Biophys Res Commun,2009,388(3):539-542.
[31]Nie CL, Gao GL, Han J, et al. Human papillomavirus 16 E6, E7 siRNAs inhibit proliferation and induce apoptosis of SiHa cervical cancer cells[J]. Chinese Journal of Cancer Research,2008,20(4):301-306.
[32]JonsonAL, Rogers LM, Ramakrishnan S, etal. Gene silencing with siRNA targeting E6 /E7 as peutic intervention in a mouse model of cervical cancer[J]. Gynecologic Oncology, 2008,111(2):356-364.
[33]Perez—Cardenas E, Contreras-Paredes A, Cantu D, et al. The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study[J]. Virol J,2007,26(4):18-19.
[34]张建华,王义平,张彩,等.丙戊酸钠联合全反式维甲酸对HeLa细胞的杀伤机制研究[J].肿瘤,2010,30(10)：33-37.
[35]Porika M, Tippani R, Mohammad A, et al. Evaluation of sernm human telomerase reverse transcriptase as a novel marker for cervical cancer[J]. Int J Biol Markers,2011,26(1): 22-26.
[36]Humphreys RC, Halpern W. Trail receptors:targets for cancer therapy [J]. Adv Exp Med Biol,2008,615(1):127-158.
[37]Sakurai H, Lirdprapamongkol K, Suzuki S, et al. Vanillin enhances TRAIL-induced apoptosis in cancer cells through inhibition of NF-kappaB activation[J]. In Vivo,2010, 24(4):501-506.
[38]Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4(VEGFR-3) and KDR(VEGFR-2)receptor tyrosine kinases[J]. EMBOJ,1996,15(2):290-298.
[39]Terhi Karpanen, Kari Alitalo, Lymphatic vesseles as targets of tumor therapy [J]. J Exp Med,2001,194(6):F37-42.
[40]Chen D, Tang Q. An experimental study on cervix cancer with combination of HSV-TK /GCV suicide gene therapy system and 60Co radiotherapy[J]. BMC Cancer,2010,6(10): 609.
[41]FranckenaM, Stalpers LJ, Koper PC, et al Long-term improvement in treatment outcome after radiotherapy and hyperthermia in locoregionally advanced cervix cancer:an update of the Dutch Deep HyperthermiaTrial. Int J RadiatOncol Biol Phys,2008,70:1176-1182.
[42]FranckenaM, LutgensLC, Koper PC, et al Radiotherapy and hyperthermia for treatment of primary locally advanced cervix cancer results in 378 patients. Int J RadiatOncol Biol Phys, 2009,73:242-250
[43]FranckenaM, DeWit R, Ansink AC, et al Weekly systemic cisplatin plus locoregional hyperthermia:an effective treatment for patients with recurrent cervical carcinoma in a previously irradiated area. Int J Hyperthermia,2007,23:443-450