【作者】 王悦；

【导师】 乔玉环； 王立东；

【作者基本信息】 郑州大学 ， 妇产科， 2012， 博士

【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。更多还原

【Abstract】 BackgroundCervical 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 radiotherapy1.ObjectHypoxia 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.MethodsHeLa 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.Result3.1 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.ConclusionsHypoxia 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 hypoxic1.ObjectTo 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.MethodsHeLa 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.Result3.1 Test of the expression of HIF-la mRNAHypoxia 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 immunocytochemistryAfter 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 blotHeLa 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. Conclusions4.1 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 expressionPart Three Mechanism of Impact of HIF-la in hypoxic chemosensitivity in human cervical cancer1.ObjectTo 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.Methods2.1 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.Result3.1 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.Conclusions4.1 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-apoptosis4.3 Cell cycles of HeLa cells under the effect of L-OHP arrest in G2/M phase, and the radiation effect is enhanced.更多还原