【作者】 刘业强；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 第一部分HIF-1α及其下游靶基因VEGF和GLUT-1在黑素瘤的表达目的研究缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)和葡萄糖转运体-1(GLUT-1)在恶性黑素瘤组织和恶性黑素瘤细胞系A375,A875,KZ28中的表达,探讨HIF-1α作为黑素瘤基因治疗靶点的可能性。方法①应用免疫组织化学方法检测77例恶性黑素瘤组织HIF-1α、VEGF、GLUT-1的表达,分析其表达的相关性以及与临床病理关系,并且以20例色素痣组织作为对照研究。②应用免疫组织化学方法、Western blot方法、ELISA方法对缺氧条件下培养的A375,A875,KZ28中HIF-1α、VEGF、GLUT-1进行检测,并且以常氧条件下培养的上述细胞系作对照。结果①在77例恶性黑素瘤组织中,65例表达HIF-1α,55例表达VEGF,53例表达GLUT-1,而在20例色素痣组织2例表达HIF-1α、4例表达VEGF、3例表达GLUT-1;②在恶性黑素瘤组织中,HIF-1α与VEGF、GLUT-1的表达呈正相关;③在恶性黑素瘤组织中HIF-1α与VEGF、GLUT-1的表达与肿瘤的恶性程度相关。④HIF-1α、VEGF、GLUT-1在缺氧培养条件下的A375,A875,kZ28中表达明显高于上述三株细胞系在常氧培养条件下的表达。⑤分泌型的VEGF和膜型表达的GLUT-1随缺氧时间延长而增加,与HIF-1α表达正相关。结论①HIF-1α、VEGF、GLUT-1可作为区别良、恶性黑素细胞肿瘤的重要指标;②在黑素瘤中HIF-1α的表达影响其下游基因VEGF、GLUT-1的表达③HIF-1α是黑素瘤基因治疗的理想靶点。第二部分HIF-1α-shRNAs表达载体的构建及鉴定目的构建针对HIF-1α的发卡样shRNA真核表达载体HIF-1α-shRNA,并转染恶性黑素瘤细胞,证实其在体外培养恶性黑素瘤细胞中对HIF-1α的干扰作用及引起的效应。方法①人工合成三对互补并编码相应短发夹状HIF-1α-shRNAs的寡核苷酸链,将其插入到Pgenesil-1载体中,经酶切和测序鉴定所构建的重组体是否正确;②采用实时定量逆转录聚合酶链反应(real time RT-PCR)、Western blot分别检测转染细胞HIF-1αmRNA和蛋白的表达变化。对其下游基因分泌型VEGF和膜型GLUT-1蛋白表达的变化分别采用ELISA和流式细胞仪检测技术;③在人恶性黑素瘤细胞系A3758中,用G418筛选沉默效果最佳的HIF-1α-shRNAs的细胞,用FCM检测GFP的表达鉴定稳定转染的单克隆细胞系;④上述稳转细胞系在缺氧情况下培养,观察其凋亡和增殖变化。结果①经酶切和测序证明HIF-1α-shRNAs序列正确;②三株MM细胞系中转染HIF-1α-shRNAs载体后,shRNA1沉默效果最佳,HIF-1αmRNA和蛋白表达均较对照组明显下降(P<0.01),并在三株MM细胞系中具有平行作用,同时也可以下调VEGF和GLUT-1的表达。③荧光显微镜下观察到了G418筛选稳定表达shRNA1的A375细胞,FCM检测GFP表达均在98%以上;④稳定表达shRNA1的A375细胞在缺氧条件培养可以引起细胞凋亡和抑制细胞增殖。结论测序结果表明发卡样的HIF-1α-shRNA真核表达载体构建成功,转染A375、A875、KZ28细胞后获得稳定表达,并可特异性封闭HIF-1α的表达,下调VEGF和GLUT-1的表达,筛选和鉴定出稳定转染单克隆细胞系A375在缺氧条件下可以引起细胞凋亡和抑制细胞增殖,为进一步研究HIF-1α-shRNA载体在恶性黑素瘤治疗中的作用提供了实验基础。第三部分靶向分子TfR在恶性黒素瘤表达目的检测基因治疗靶向分子TfR在恶性黑素瘤组织和细胞系A375、A875、KZ28的表达,以明确TfR作为基因治疗的靶向分子的可能性。方法①应用免疫组织化学方法检测77例恶性黑素瘤组织TfR的表达,并且以20例色素痣组织作为对照研究。②应用免疫组织化学方法、western blot方法、流式细胞仪检测方法对常氧和缺氧条件下培养的A375,A875,KZ28中TfR表达进行检测,并且以已知低表达TfR的卵巢癌细胞系A2780进行对照。③分析在体外常氧和缺氧条件下培养的MM细胞株中TfR表达不同的可能机制。结果①在77例恶性黑素瘤组织中,70例表达TfR,而在色素痣组织只有5例表达;②在恶性黑素瘤组织中,TfR与肿瘤的病理分期相关;③在常氧培养的A375,A875,KZ28中TfR的表达分别为97%,89%,79%,A2780的表达为2%;④免疫组织化学方法、western blot方法检测恶性黑素瘤组织中TfR的表达与流式结果趋势相同。⑤缺氧在A375细胞株中对TfR的表达呈双相作用,早期表达减少,晚期表达增加。⑥缺氧对A375细胞株中对TfR的表达呈双相作用可能与缺氧早期促进凋亡,晚期引起细胞增殖有关。结论①TfR在恶性黑素瘤组织和细胞系A375,A875,KZ28明显高表达,是理想的黑素瘤基因治疗的靶向分子。②缺氧对黑素瘤细胞系TfR的表达呈双相作用,对我们个体化的利用TfR作为靶向分子提供了更加合理的理论依据。第四部分Tf-PEI-HIF-1α-shRNA complex对恶性黑素瘤治疗靶向性实验研究目的研究Tf-PEI-HIF-1α-shRNA complex在TfR高表达的黑素瘤细胞系A375以及在A375荷瘤鼠的体内模型的靶向分布,以期为下一步系统使用Tf-PEI-HIF-1α-shRNA complex治疗A375荷瘤鼠提供理论依据和实验数据。方法①应用FCM方法检测转染不同实验组的GFP的表达,以TfR低表达的A2780细胞系做对照。②应用Real time RT-PCR、western blot方法检测Tf-PEI-HIF-1α- shRNA complex系统治疗的A375荷瘤鼠和A2780荷瘤鼠中肿瘤组织以及重要脏器GFP的表达。结果①在体外实验中,A375组中,Tf-PEI-HIF-1α-shRNA complex转染后GFP表达到达65%,与脂质体转染相当,在A2780组中,Tf-PEI-HIF-1α-shRNA complex转染后均在5%以下。②在体内实验中,Tf-PEI-HIF-1α-shRNA complex系统治疗的A375荷瘤鼠的肿瘤组织较重要脏器肝、脾、心、肾GFP的mRNA及蛋白质具有明显差异(P<0.01),在对照组A2780荷瘤鼠中肿瘤组织与重要脏器肝、脾、心、肾GFP的mRNA及蛋白质没有明显表达差异(P>0.05)。结论①Tf-PEI-HIF-1α-shRNA complex靶向治疗在TfR高表达的黑素瘤细胞系A375有特异性靶向分布,并且转染效率高。②在TfR高表达的黑素瘤细胞系A375荷瘤鼠中,Tf-PEI-HIF-1α-shRNA complex系统靶向治疗具有肿瘤靶向特异性,为黑素瘤基因治疗研究提供了实验依据。第五部分Tf-PEI-HIF-1α-shRNA complex对恶性黑素瘤荷瘤鼠生物学行为影响的实验研究目的观察Tf-PEI-HIF-1α-shRNA complex系统治疗对A375荷瘤鼠的生长抑制及可能的机制进行探讨。方法①分别对接受Tf-PEI-HIF-1α-shRNA complex系统治疗的A375、A2780荷瘤鼠肿瘤大小进行测量,按公式(L×W2)/2计算肿瘤体积大小。②治疗结束后处死荷瘤鼠,采用免疫组化和western blot方法检测HIF-1α、VEGF、GLUT1的表达情况。③治疗结束后处死荷瘤鼠,采用免疫组化方法检测MVD;酶显色法检测肿瘤组织糖酵解产物乳酸含量。末端脱氧核苷酸转移酶标记法(TUNEL)定量检测两组裸鼠模型的肿瘤组织凋亡;结果①体内实验证明,实验组肿瘤生长速度也明显减慢(P<0.01)②实验组HIF-1α、VEGF、GLUT1表达明显低于对照组(P<0.01)③实验组和对照组MVD无显著性差异(P>0.05)。④实验组肿瘤组织匀浆的乳酸含量明显低于对照组(P<0.01)。⑤实验组可见大量凋亡细胞,对照组仅见少许凋亡细胞,实验组凋亡指数与对照组相比有显著性差异(P<0.01)。结论①通过Tf-PEI-HIF-1α-shRNA complex靶向RNA干扰技术不但能阻断HIF-1α的表达,还能沉默其下游靶基因VEGF、GLUT1的表达,从而在体内可显著性抑制恶性黑素瘤生长,为恶性黑素瘤基因治疗研究提供了实验依据。②Tf-PEI-HIF-1α-shRNA complex抑制恶性黑素瘤生长,可能与抑制糖酵解,促进肿瘤组织凋亡有关,未发现与抑制肿瘤血管生成有关。更多还原

【Abstract】 PartⅠExpressions of HIF-1αand its downstream genes VEGF and GLUT-1 in Malignant MelanomaObjectives To study expressions of HIF-1α,VEGF and GLUT-1 and their correlation to tumor clinical-pathology features in human malignant melanoma (MM) and MM cell lines. To analyze the possiblity of HIF-1αbeing an attractive therapeutic molecular target for MMMethods①Tissue sections from 77 MM patients were examined using immunohistochemical technique and morphological quantitative analysis for the protein expressions of HIF-1α,VEGF and GLUT-1 and their corelations to tumor clinical-pathology features in MM were analyzed. And tissue sections from 20 pigmented nevi patients were as controls.②MM cell lines under hypoxia condition were detected using immunocytochemistry ,western blot and ELISA for expressions of HIF-1α,VEGF and GLUT-1.And these cell lines under normoxia were as controls.Results①Positive HIF-1α,VEGF and GLUT-1 immunostaining were observed in 84.4% (65 cases),71.42% (55 cases) and 68.83%(53 cases) of 77 MM lesions respectively,whereas only one case of pigmented nevi expressed HIF-1αand two cases expressed VEGF and GLUT-1 ;②A positive correlation among HIF-1α,VEGF and GLUT-1 was observed in MM;③Expressions of HIF-1α,VEGF and GLUT-1 were positively correlated with malignant degree in MM;④Expressions of HIF-1α,VEGF and GLUT-1 in MM cell lines A375,A875 and KZ28 under hypoxia condition were higher than under normoxia⑤Expressions of secreted VEGF and membrane GLUT-1 in MM cell lines were steadily increased as hypoxia time was enlonged and were corelated to HIF-1αexpression. Conclusions①On the basis of the current data showing that malignant melanocytic tumors displayed strong HIF-1α,VEGF and GLUT-1 expressions, whereas benign melanocytic tumors showed almost no immunoreactivity for HIF-1α,VEGF and GLUT-1,they might be used as discriminating indicators to distinguish malignant melanocytic tumors from benign melanocytic tumors;②The strong upregulation of HIF-1αin MM found in this study suggested that HIF-1αmight be an important factor to MM growth and could be an attractive therapeutic molecular target for MM.PartⅡConstruction and Identification of HIF-1α-shRNAsObjectives Short interfering RNA (siRNA) eukaryotic expression vector for HIF-1αwas constructed and transfected into malignant melanoma cells. To explore its effect of interference for HIF-1αin malignant melanoma cells in vitro and observe its effect to cell apoptosis and proliferation.Methods①Three HIF-1α-shRNAs targeting human HIF-1αmRNA common sequence was synthesized and it was inserted into Bam HI-Hind III linearized Pgenesile-1 vector. The sequence of HIF-1α-shRNA plasmid was analyzed by DNA sequencer and restrict endonuclease cutting .②To screen the best silencing effect HIF-1αshRNA,the alteration of HIF-1αmRNA and protein was checked by real time Rt-PCR and western blot after HIF-1α-shRNAs were transfected in MM cell lines.And the level of secrected VEGF was checked by ELISA and membrance GLUT-1 was analyzed by FCM.③The monoclone A375 cells with stable expression of best effecting shRNA1 were obtained by G418 selection and were identified with checking GFP expression by FCM and observing under Fluorescence microscope④To observe cell apoptosis and proliferation ,shRNA1-A375 stable clone was cultured under hypoxia condition.Results①It was verified that the sequence of constructed recombinant plasmids were correct by DNA sequencing and restrict endonuclease cutting.②HIF-1αshRNAs showed variable efficacy in decreasing HIF-1αmRNA in the three cell lines, relative to the control scrambled shRNA and the mock group. ShRNA1 decreased HIF-1αmRNA by 74%, 69% and 70% in the three cell lines at time point of 24h after transfection, respectively. ShRNA3 decreased HIF-1αmRNA in the three cell lines by 32%, 24% and 22% at the same time point, respectively. Unexpectedly, shRNA2 resulted in a slightly decrease but no significant alteration of HIF-1αmRNA expression in the three cell lines. Among the HIF-1αshRNAs we tested , shRNA1 possessed the strongest inhibitory effect against HIF-1αand had paralleled effect on silencing HIF-1αin the three cell lines.And Silencing HIF-1αby HIF-1αshRNAs could down-regulated secreted VEGF and membrance GLUT-1 in MM cell lines.③We screened and obtained A375 cells with stable expressions of HIF-1α-siRNA as A375 stable clone could observed under Fluorescence microscope and 98% A375 stable clone expressed GFP detected by FCM;④The down-regulation of HIF-1αcould significantly prevent cell proliferation and enhance apoptosis in A375 exposed to hypoxic conditions.Conclusions It indicated that hairpin siRNA eukaryotic expression vector for HIF-1αwould be successfully established,and it played a specific inhibitory role in three kinds of tumor cell lines and had parellel silencing effect and also down-regulated VEGF and GLUT-1. At the same time we obtained cells with the stable expression of HIF-1α-shRNA which could cause cell apoptosis and inhibit cell proliferation under hypoxia condition in A375 stable clone. This study laid experimental foundation for further research of the therapy of HIF-1α-shRNA vector in MM.Part III Expression of TfR in MMObjectives To study expressions of TfR in human malignant melanoma (MM) and MM cell lines and to analyze the possiblity of TfR being an attractive target receptor for MM gene therapy.Methods①Tissue sections from 77 MM patients were examined using immunohistochemical technique and morphological quantitative analysis for the protein expressions of TfR and its corelations to tumor clinical-pathology features in MM were analyzed. And tissue sections from 20 pigmented nevi patients were as controls.②MM cell lines under normoxia and hypoxia condition were detected using immunocytochemistry ,western blot and FCM for expressions of TfR.And A2780 cell lines which TfR expression was low as controls.③To analyze the mechanism of the dual phase change of expression of TfR under hypoxia in different time course by checking cell apoptosis and proliferation under hypoxia.Results①Positive TfR immunostaining were observed in 90.9% (70 cases) of 77 MM lesions,whereas only five case of pigmented nevi expressed TfR ;②Expressions of TfR were positively correlated with tumor graduation in MM;③Expressions of TfR in MM cell lines A375,A875 , KZ28 and A2780 under normoxia condition were 97%,89%,79% and 2%,respectively.④hypoxia has a dual effect on the expression of TfR in human Melanoma A375 cell line during different hypoxic time course which hypoxia down-regulated TfR expression under hypoxia at time points 12 and 24h and up-regulated TfR expression under hypoxia at time point (36,48 and 60h).⑤hypoxia has multiple opposing effects on tumor growth, with a variable net outcome at various time periods and hypoxia might down-regulate TfR expression by inducing apoptosis which decreases the iron requirements in A375 cells in early hypoxic time course, and up-regulates TfR expression by promoting proliferation in A375 cells in late hypoxic time course.Conclusions①On the basis of the current data showing that malignant melanocytic tumors displayed strong TfR expressions,whereas benign melanocytic tumors showed almost no immunoreactivity for TfR,they might be used as target receptor for gene therapy in MM;②Hypoxia has dual effect on the expression of TfR in human Melanoma A375 cell line. These findings may have important implications for more rational, individualized gene-based therapy using TfR as target receptor in melanoma. Part IV To study Tumor-targeted distribution after administration of Tf-PEI-shRNA complex in vitro and in vivoObjectives By detecting the ability of Tf-PEI to deliver shRNA specifically to TfR-high-expressed cells in vitro and TfR-high-expressed cells bearing tumor tissue in vivo, we expect to provide the experimental data and basis of theory for the clinical treatment of malignant melanoma with Tf-PEI-shRNA complex.Methods①t ransfection efficiency for each group was estimated as the mean value of GFP per 10,000 cells by FCM in vitro.②tumors and major organs were excised for GFP mRNA and protein expression by real time RT-PCR and western blot analysis.Results①t ransfected shRNA by Lipofectamine2000 was comparably taken up by ~ 70% of both A2780 and A375. Neither A2780 and A375 appreciably took up shRNA by themselves nor when mixed either with Tf lacking PEI or with unmodified PEI.When mixed with Tf-PEI, did A2780 still not take up shRNA. In contrast, when mixed with Tf-PEI, shRNA was comparably taken up by ~ 52% in A375. Although lower than Lipofectamine2000 ~ 70% transfection in A375, Tf-PEI transfection specific delivery of shRNA into TfR-high-expressed MM cell was verified.②the highest amounts of GFP mRNA and protein were detected in tumor tissues and in major organs such as liver, lungs, heart and kidneys only small amounts of GFP mRNA and protein were detected in the A375 bearing tumor mice at time point of 24h after the single injection. At the same time, the result revealed that the GFP mRNA and protein expression distribution in the tumor tissues and major organs of A2780 bearing tumor mice had no significant differences. The results demostrated that Tf-PEI could deliver shRNA with tumor-targeted specific distribution in the nude mice bearing A375 sc.xenograft tumors.Conclusions these results showed the tumor-targeted relatively specific distribution after administration of Tf-PEI-shRNA complex in vitro and in vivo. Part V Observation of the growth rate of MM xenograft tumor after injection with Tf-PEI- HIF-1αshRNA1 in vivo and exploration of potential mechanismsObjectives To observe growth inhibition of MM xenograft after receiving systemic admnistration of Tf-PEI- HIF-1αshRNA1 and to explore the potential mechanismsMethods①To evaluate therapeutic potential of Tf-PEI-shRNA1 complex in A375 tumor xenograft, the nude mice bearing A375 s.c.xenograft tumors and the nude mice bearing A2780 s.c.xenograft tumors were randomized and divided in three experimental groups, respectively: one group (n=12) was received direct injection of a total of 20μg of“therapeutic”plasmid Tf-PEI- shRNA1 complex conjugate in100μl volume through tail vein, whereas the second group (n=12) was injected with a total of 20μg of negative control Tf-PEI- scrambled shRNA conjugate in 100μl volume through the tail vein and the third group (n=12) was not received any treatment . Tumor diameter was measured at regular intervals with calipers, and tumor length (L) and width (W) were measured at the end of the experiment, and tumor volume was estimated by the formula (L×W2)/2.②At 25 days after injection, mice were sacrificed and tumors were dissected for further analysis.Expression of HIF-1α,VEGF and GLUT-1 and MVD in tumor tissues were detected by immunohistochemistry and western blot.③tumor glucolysis product Lactic Acid (LA) was detected by LA kit.④tumor cell apotosis was detected by TUNEL method.Results①Tumor growth delay was statistically significant (P=0.0014) from day 5 after the beginning of therapy until the day the mice were sacrificed. During day 10-15 after treatment we observed the strongest effect of Tf-PEI-shRNA1 complex; A2780 group tumors in the three groups had a volume of 710±145mm3 , 755±90mm3 and 695±120mm3, which were 7.5-fold larger than the starting volume. Tumor growth delay was not found in the“therapeutic”group.②the immunohistochemistry results shown that in the nude mice bearing A375 s.c.xenograft tumors, HIF-1α,VEGF and GLUT-1 expressions in Tf-PEI-shRNA1 complex group were significantly lower than in Tf-PEI-scrambled shRNA and A375 groups, and no significant difference in HIF-1αexpression was found between Tf-PEI-scrambled shRNA and A375 groups.③But expression of MVD in the three groups has no significant diference (P>0.05)④tumor glucolysis product Lactic Acid (LA) in Tf-PEI-shRNA1 complex group were significantly lower than in Tf-PEI-scrambled shRNA and A375 groups, and no significant difference in HIF-1αexpression was found between Tf-PEI-scrambled shRNA and A375 groups.⑤the number of apotosis tumor cell was larger in Tf-PEI-shRNA1 complex group that other two groups.Conclusions①Tf-PEI was able to deliver shRNAs specifically to TfR-expressed MM cells to suppress MM growth even when administered systemically.②systemic Tf-PEI-HIF-1α-shRNA complex administration in the nude mice bearing A375 sc.xenograft tumors could knock down HIF-1αwhich slowed the growth rate of MM xenograft tumor.③Inhibiting tumor glucolysis ,not tumor angiogenesis maybe involve to enhance tumor cell apotosis and to slow the growth rate of MM xenograft tumor.更多还原