【作者】 聂春莲；

【导师】 高国兰；

【作者基本信息】 南昌大学 ， 妇产科学， 2009， 博士

【摘要】 背景与目的由于恶性肿瘤增殖失控,新生的血管排列紊乱和功能不良致血供不足,肿瘤组织内不可避免会出现低氧、缺氧。低氧诱导因子(HIF-1)通过调控其靶基因的转录,在肿瘤细胞对低氧的适应过程中起着十分重要的作用。HIF-1由α、β两个亚单位组成,HIF-1α易受多种因素调节。研究显示卵巢癌实体瘤中,低氧诱导了HIF-1α表达增加,从而促进血管内皮生长因子(VEGF)表达增高,HIF-1α表达增高的卵巢癌患者预后差。本研究拟构建人HIF-1α短发夹(sh)RNA表达质粒,并探讨其对HIF-1α基因表达的影响,通过体外和荷瘤裸鼠体内实验了解本研究构建的质粒对卵巢癌细胞增殖、凋亡、对化疗药物敏感性及移植瘤增殖的影响。方法1.根据shRNA对应的DNA模板设计原则,利用在线设计软件,针对HIF-1α的序列设计DNA片段。设计靶向HIF-1α基因的4对候选RNA干扰序列插入pGPU6/GFP/Neo质粒,构建4个重组质粒(H1、H2、H3和H4),经过测序验证。2.在密闭的氮气-二氧化碳-空气混合器中充入氮气和二氧化碳产生低氧环境以模拟体内的低氧环境。3.脂质体法转染构建的质粒入低氧培养的卵巢癌SKOV3细胞株,RT-PCR和Western-blot检测各质粒对癌细胞HIF-1αmRNA、蛋白表达的影响,以优选质粒,进行后续实验。转染质粒后,通过MTT(四甲基偶氮唑蓝)比色法检测并绘制细胞生长曲线及平板克隆形成实验了解其对癌细胞增殖的影响,TUNEL法检测其对细胞凋亡的影响,MTT法检测癌细胞对化疗药物紫杉醇(TAX)、多柔比星(阿霉素,ADM)、顺铂(DDP)的敏感性的影响。4.用卵巢癌SKOV3细胞建立裸鼠皮下移植瘤模型,予DDP和质粒处理,观测肿瘤体积的变化,免疫组化检测移植瘤组织HIF-1α、VEGF蛋白表达水平的变化。结果:1. DNA测序证实本研究构建的4个质粒含有与本研究设计的针对HIF-1α的DNA序列完全一致的序列,说明我们已把合成的63bp寡核苷酸链插入了pGPU6/GFP/Neo质粒中,成功构建了靶向HIF-1α的H1、H2、H3和H4 RNAi表达质粒。将构建的质粒转染卵巢癌SKOV3细胞,在荧光倒置显微镜下见多数细胞中出现绿色荧光,表明构建的质粒转染成功,并且可以正常表达其功能。2.常氧培养条件下卵巢癌SKOV3细胞可检测到低水平的HIF-1αmRNA和蛋白表达。采用本研究的低氧条件培养卵巢癌SKOV3细胞24小时,与常氧条件下培养比较,RT-PCR结果显示HIF-1αmRNA表达水平无明显差异(p>0.05);Western blot结果显示低氧培养时HIF-1α蛋白表达水平显著升高(p<0.05)。3.脂质体法转染构建的构建的质粒48小时后,RT-PCR和Western blot结果显示H3和H4质粒转染组细胞HIF-1αmRNA和蛋白表达均明显下调(p<0.05),脂质体对照组、阴性对照质粒组、H1组和H2组之间HIF-1αmRNA和蛋白表达水平均无明显差异(p>0.05)。选用H3质粒(pGPU6-H质粒,简称H质粒)进行后续实验。4.细胞生长曲线和平板克隆形成实验结果显示,转染H质粒能明显抑制SKOV3细胞的增殖,而阴性对照质粒(NC质粒)对细胞的增殖无明显影响。TUNEL法检测细胞凋亡的结果显示转染H质粒组细胞48小时、72小时平均凋亡率分别为19.2%、33.09%,比CTRL组、Lipo组和NC对照组细胞高,差异有统计学意义(p<0.05);且转染质粒后72小时细胞凋亡率高于转染后48小时,差异有统计学意义(p<0.05)。5. MTT体外药敏实验结果显示:H质粒转染组SKOV3细胞对紫杉醇/多柔比星/顺铂作用24小时的IC50值明显降低,与Lipo组, NC质粒组相比差异具有统计学意义(p<0.05),而相应的紫杉醇/多柔比星/顺铂组、Lipo组、NC质粒组之间化疗药物作用24小时的IC50值差异具无统计学意义(P>0.05)。Western-blot结果显示,与CTRL组、Lipo组和NC对照组细胞相比,H质粒转染组的P-gp表达水平明显降低,差异有统计学意义(F=7.24,p=0.01),而脂质体组、NC对照组与空白对照组之间的P-gp表达水平相比差异无显著性(p>0.05)。6. SKOV3细胞裸鼠移植瘤体内实验结果显示:随着时间的延长,NS组、脂质体组和NC质粒组后一时相与前一时相相比,3组裸鼠的肿瘤体积逐渐增大(p<0.05),各个时相脂质体组和NC质粒组与NS组肿瘤体积差异无统计学意义(p>0.05)。3周前H质粒组肿瘤体积增长受到明显抑制。而3周后H质粒组肿瘤生长加速;4周后DDP组肿瘤肿瘤生长加速。H质粒+DDP组各个时相的肿瘤体积均显著小于单用H质粒和DDP组(p<0.05)。移植瘤组织免疫组化结果显示:H质粒+DDP组、H质粒组和DDP组裸鼠移植瘤组织HIF-1α、VEGF蛋白阳性率比NS组、Lipo组和NC质粒组移植瘤组织的低,差异有统计学意义(p<0.05);H质粒+DDP组的阳性率比H质粒组和DDP组低,差异有统计学意义(p<0.05)。结论:1.低氧可诱导卵巢癌SKOV3细胞HIF-1α蛋白水平表达显著增高,而低氧对HIF-1αmRNA的表达水平无明显影响;HIF-1αRNAi表达质粒可下调低氧培养的卵巢癌细胞SKOV3 HIF-1α基因mRNA和蛋白的表达,从而抑制癌细胞增殖,促进癌细胞凋亡。表明下调HIF-1α基因的表达可部分逆转卵巢癌细胞的恶性表型。2.通过HIF-1αRNAi表达质粒下调卵巢癌细胞SKOV3 HIF-1α基因的表达后,低氧培养的卵巢癌细胞P-gp表达下降,可显著提高癌细胞对化疗药物紫杉醇、多柔比星和顺铂的敏感性。3.通过HIF-1αRNAi表达质粒下调卵巢癌细胞SKOV3 HIF-1α基因的表达后,卵巢癌细胞皮下移植瘤的生长速率均显著减慢,癌组织VEGF的表达降低。表明下调HIF-1α基因的表达可降低SKOV3细胞的致瘤性;HIF-1αRNAi质粒联合DDP对肿瘤的抑制作用强于单用质粒和DDP,表明采用RNA干扰技术阻滞HIF-1α基因的表达联合化疗可望成为改善卵巢癌疗效的一个有前景的措施。更多还原

【Abstract】 Background and purposeWith the over-proliferation of malignant tumors with disorganized, insufficient blood supply,hypoxic and or anoxic regions will inevitably develop. Hypoxia inducible factor-1(HIF-1) plays a key role in the adaptation of tumor cells to hypoxia by activating the transcription of the targeted genes. HIF-1 is a heterodimer composed of HIF-1αand HIF-1β. The expression of HIF-1αis regulated by various factors. Some studies have revealed a significant association between the expression of HIF-1αand VEGF within the ovarian cancer tissues and the poor prognosis of the patients with ovarian cancer. To investigate the effect of HIF-1αshort hairpin RNA(shRNA) expressing plasmid on ovarian cancer in vitro and in vivo.Methods1. Oligos for hairpin RNA expression which targeted HIF-1αgene were designed and selected based on the well-known principle using online software and synthesized and annealed. Annealed oligos were inserted into the down stream of human U6 promoter of the pGPU6/GFP/Neo plasmid to construct H1,H2,H3 and H4 recombinant plasmids.The recombinant vectors were verified by sequencing.2. Ovarian cancer SKOV3 cells were cultured in mimic hypoxia condition using N2-O2-Air Mixing chamber.3. The plasmids were transfected into SKOV3 cells using lipofectmine. 4. The expression of HIF-1αmRNA and protein was assayed by RT-PCR andWestern-bloting respectively. Cell apoptosis was evaluated by TUNEL.Cell viability and chemotherapy drug sensitivity were determined by methylthiazoly- ltetrazolium(MTT) assay.5. The nude mice with SKOV3 cell subcutaneous xenograft tumor were treated with the recombinant plasmids and DDP. Tumor sizes were measured every week.The HIF-1αand VEGF expression of xenograft tumor tissue were evaluated with immunohistochemistry.Results:1. Recombinant plasmids were confirmed successful by sequencing analysis.2. There are low level of expression of HIF-1αmRNA and protein in normoxic SKOV3 cells. The hypoxic environment significantly induced the expression of protein HIF-1α(P<0.05),while the HIF-1αmRNA expression were not significantly changed(P>0.05) .3. Compared to that of lipofectmine(Lipo) group, negative control(NC) group,H1group and H2 group , HIF-1αmRNA and protein expressions of H3 and H4 group were significantly downregulated at 48 h after transfection with the recombinant plasmids (P<0.05), and that were not significantly defference among Lipo group, NC group,H1group and H2 group (P>0.05). H3 plasmid(named H plasmid)was selected for the latter experiment.4. Compared with that of Lipo group and NC group ,growth and clone formation of SKOV3 cell was markedly inhibited after transfection with H plasmid and viable cells were significantly declined (P<0.05).And the apoptosis rate of SKOV3 cell was19.2%,33.09 % at 48h,72h after transfection with H plasmid respectively,which was significantly increased (p<0.05), apoptosis rate of SKOV3 cell at 72h posttransfection was significantly higher than that of SKOV3 cell at 48h posttransfection (p<0.05).5. Compared with that of Lipo group and NC group, IC50 of H plasmid group SKOV3 cells to paclitaxel,cisplatin and doxorubicin is significantly lower (P<0.05),and there were not significantly difference among the IC50 of Lipo group , NC group and the corresponding paclitaxel,cisplatin and doxorubicin group (P>0.05).6. In vivo tests,the tumor volume growth rates of H plasmid +DDP group ,DDP group and H plasmid group were significantly lower than that of NS group,Lipo group and NC group within 3 weeks after treatment(P<0.05) ;and compared with that of DDP group and H plasmid group,the tumor growth rate in H plasmid +DDP group was significantly lower (P<0.05). The expression of HIF-1αand VEGF protein of H plasmid +DDP group ,DDP group and H plasmid group xenograft tumor tissue detected by immunohistochemistry were all significantly reduced (P<0.05) when compared with those of NS group,Lipo group and NC group.Conclusions:1. These data indicate that the HIF-1αprotein expression of SKOV3 cells is significantly up-regulated by the hypoxic environment.2. RNAi expressing plasmid directed against HIF-1αcan effectively inhibit the growth and enhance the apoptosis of SKOV3 cells through down-regulating the expression of HIF-1αmRNA and protein. After selectively silencing HIF-α, hypoxia-induced expression of its target genes such as P-glycoprotein were markedly attenuated. Moreover, HIF-1αknockdown was found to increase drug sensitivity of SKOV3 cells to paclitaxel,cisplatin and doxorubicin.3. Intratumoral injection of HIF-1αRNAi expression plasmid can effectively activate RNAi-mediated down-regulation of HIF-1αand VEGF in vivo.HIF-1a knockdown can effectively curb the growth of SKOV3 cells xenograft and increase the sensitivity of SKOV3 cells xenograft tumor to DDP, and which suggest a promising combination of both downregulating HIF-1αexpression by RNA interference and traditional chemotherapy to improve cancer treatment.更多还原