【作者】 任鹏；

【导师】 郝希山； 于振涛；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 博士

【摘要】 目的：研究食管鳞状细胞癌及正常食管组织中HIF-1α和VEGF的表达情况,两者表达的相关性及其与临床病理特征和放疗反应之间的关系；检测RNA干扰HIF-1α后HIF-1α和VEGF的变化、及其对细胞增殖、细胞周期的影响；通过体外和体内实验检查KYSE-150细胞在RNA干扰后放射敏感性的变化,初步探讨其影响放射敏感性的机制,为食管癌放射增敏及基因治疗提供新的靶点。方法：选取42例接受手术的食管鳞状细胞癌新鲜标本,通过实时定量PCR、免疫组化检查癌组织和正常组织中的HIF-1α及VEGF的表达情况,分析其相关性及与临床病理特征的联系；另选接受单纯放疗的食管癌标本60例,免疫组化检测HIF-1α及VEGF的表达,分析其与放射反应及预后的关系。针对HIF-1α基因的mRNA序列设计三个siRNA序列构建质粒,转染KYSE-150食管癌细胞系,潮霉素筛选获得稳定表达siRNA的细胞克隆,实时定量PCR、Western-blot方法检测细胞中HIF-1αmRNA及蛋白表达,验证干扰效果并筛选能够成功沉默HIF-1α的序列；实时定量PCR、Western-blot方法检测RNA干扰后VEGF的变化,MTT法、流式细胞仪检测siRNA对细胞增殖、细胞周期的影响；体外使用不同剂量的X线分别照射KYSE-150细胞和稳定表达siRNA的细胞克隆,平板克隆形成实验及MTT法检测放射线照射后的细胞存活情况。采用KYSE-150食管癌细胞及KYSE-150/HIF-1α(-)细胞建立裸鼠食管癌移植瘤模型,分组后进行不同剂量的放射线照射,建立生长曲线,对比研究两组肿瘤照射后的放疗反应,根据生长延缓和照射剂量分析其放射敏感性差异；对两种肿瘤标本进行HE及免疫组化染色,比较其HIF-1α、Ki67及CD34表达差异。结果：HIF-1α和VEGF在食管鳞状细胞癌中的表达率分别为50.0%和76.2%,而在正常食管黏膜组织中表达率为14.3%和33.3%,二者在食管癌中的表达存在强相关性,而在正常食管黏膜组织中弱相关。免疫组化显示食管鳞状细胞癌中HIF-1α蛋白在胞浆与胞核中均有阳性表达,但胞核较胞浆表达强；食管正常粘膜细胞中HIF-1α蛋白表达不稳定且表达较弱。食管鳞状细胞癌中HIF-1α蛋白表达与淋巴结转移及肿瘤组织分化相关,VEGF表达与临床病理特征无关,有淋巴结转移及肿瘤组织分化差者较无淋巴结转移及肿瘤组织分化好HIF-1α蛋白表达率高。HIF-1α和VEGF在接受单纯放疗的食管癌中的表达与放疗敏感性及预后有关,阴性表达较阳性表达放疗敏感性高,预后较好。成功构建了三个HIF-1α的靶向siRNA,并与pGCsi-H1/Hygro/GFP质粒连接,通过电转染食管癌KYSE-150细胞,经筛选后得到稳定的干扰后细胞,实时定量PCR、Western-blot结果证实shRNA3能够使HIF-1α表达沉默,并能抑制VEGF表达,MTT法示转染shRNA3组细胞在缺氧条件下增殖活力减弱,流式细胞仪检测示细胞周期中G0／G1期比例降低,而凋亡增加；平板克隆形成实验及MTT法示shRNA3组细胞照射后存活分数降低,计算结果放射敏感性提高。接种KYSE-150/HIF-1α(-)细胞的裸鼠皮下移植瘤生长明显慢于接种KYSE-150细胞组,两组移植瘤的免疫组化染色显示,接种KYSE-150/HIF-1α(-)细胞的肿瘤中HIF-1α、Ki67和CD34的表达均明显低于KYSE-150组。X线照射可以使两组移植瘤均出现生长缓慢甚至体积减小,KYSE-150/HIF-1α(-)组放射照射后生长延缓更加明显,计算结果SER大于1。结论：HIF-1α和VEGF在食管鳞状细胞癌中均呈高表达,且二者的表达呈强相关性。食管鳞状细胞癌中HIF-1α蛋白在胞浆与胞核中均有阳性表达,胞核较胞浆表达更强,更特异。食管鳞状细胞癌中HIF-1α表达与淋巴结转移及肿瘤组织分化有关,HIF-1α高表达时淋巴结转移率更高,肿瘤组织分化更差。在单纯接受放疗的食管癌中HIF-1α和VEGF的表达与放疗敏感性及预后有关,阴性表达较阳性表达放疗敏感性高,预后较好。在食管癌KYSE-150细胞中通过RNA干扰能够沉默HIF-1α的表达,抑制VEGF的表达,并能使其在缺氧条件下增殖活力减弱、凋亡增加、细胞周期的分布也发生改变。RNA干扰HIF-1α后能够在体内外抑制肿瘤的增殖和血管生成。RNA干扰HIF-1α有放射增敏作用,在体内外均能提高食管癌KYSE-150细胞的放疗敏感性,机制可能与其减少血管生成、延缓增殖有关。更多还原

【Abstract】 Objective:To study the expression of HIF-1αand VEGF in esophageal squamous cell carcinoma (ESCC), the relationship between them, and their relationship with pathological features and radiotheraputic response. To study the changes of HIF-1αand VEGF after RNA interference, its effect on proliferation and cell cycle. To study the change of radiosensitivity of KYSE-150 cell after RNA interference in vivo and in vitro, explore the mechanisms of radiosensitization and provide a new target for enhancing radiosensitivity and gene therapy on esophageal cancer.Methods:To choose 42 fresh specimens from patients performed resection for ESCC, detect the expression of HIF-1αand VEGF by immunohistochemistry and real-time PCR, analysis the corelation between them and their relationship with pathological features. To choose 60 specimens from patients received radiotherapy for ESCC, detect the expression of HIF-1αand VEGF by immunohistochemistry, and analysis their relationship with radiotheraputic response and prognosis. To design three short hairpin sequences targeting HIF-1αand construct recombinant plasmids expressing HIF-1αshRNA, transfect them into KYSE-150 cell, screen stable transfected cells with Hygromycin. To detect the expression of HIF-1αand validate the effect of RNAi by real-time PCR and Western-blot, detect the expression of VEGF by real-time PCR and Western-blot. To analyse the proliferation and cell cycle distribution of KYSE-150 cells after transfection by MTT assay and flow cytometry. Irradiated with different doses of X-rays, the survival fraction of KYSE-150 cells were examined by MTT and clonogenic assays. The subcutaneous xenograft models in nude mice were established with KYSE-150 and KYSE-150/HIF-1α(-) cell lines and irradiated with single doses of 5Gy,10Gy and 20Gy. To observe the tumor inhibitions by radiotherapy between two models, analyse the improvement of radiosensitivity on KYSE-150 cell after HIF-1αsiliencing by tumor growth delay and radiation dose. To detect the expression of HIF-1α, Ki67 and CD34 by immunohistochemistry between two models. Results:The positive expression rates of HIF-la and VEGF were 50.0% and 76.2% in ESCC respectively, while 14.3% and 33.3% in normal tissues. The expression of HIF-1αwas positively correlated with VEGF in ESCC with a very high degree, while a very weak positive relationship was found in normal tissues. HIF-1αcan be seen in the cytoplasm and nucleus of ESCC, which in the nucleus it is stronger than in the cytoplasm. The expression of HIF-1αin normal esophageal tissues is unstable and low. The expression of HIF-1αin ESCC was related with lymphatic metastasis and histological differentiation, the expression rate of HIF-1αin the cancers with lymphatic metastasis and worse differentiation was higher than that in the cancers with no lymphatic metastasis and better differentiation. The expression of HIF-1αand VEGF were related with radiosensitization and prognosis of ESCC after radiotherapy, those with negative expression had good radiosensitization and prognosis. Three HIF-1αshRNA were constructed and linked to pGCsi-Hl/Hygro/GFP plasmids, which were transfected into KYSE-150 cell. The stable transfected cells RNAi were screened. Real-time PCR and Western-blot confirmed that shRNA3 can silence HIF-1αand inhibit the expression of VEGF, MTT assay and flow cytometry showed that the proliferation attenuated, the ratio of G0/G1 stage cell decreased, and apoptosis increased in shRNA3 group. MTT and clonogenic assays showed the survival fraction in shRNA3 group after irradiation was lower than control group, which showed that the radiosensitivity was improved. The tumor xenografts derived from KYSE-150/HIF-1α(-) cell grew slowly than that from KYSE-150 cell. The result of immunohistochemistry showed the staining of HIF-1α, Ki67 and CD34 in KYSE-150/HIF-1α(-) tumors was weaker than in KYSE-150 tumors. Irradiation can inhibit the growth of both tumors, and decrease the volume of the tumors. The tumor growth delay was more obvious in KYSE-150/HIF-1α(-) group than KYSE-150 group, its sensitivity enhancement ratio was larger than 1.Conclusion:The expression rates of HIF-1αand VEGF were both high in ESCC, there was a strong correlation between them. HIF-la can be seen in the cytoplasm and nucleus of ESCC, while its expression is stronger and more characteristic. The expression of HIF-1αin ESCC was related with lymphatic metastasis and histological differentiation, the cancers with higher expression rate of HIF-1αhad more lymphatic metastasis and worse differentiation. The expression of HIF-1αand VEGF were related with radiosensitization and prognosis of ESCC received simple radiotherapy, those with negative expression had good radiosensitization and prognosis. In KYSE-150 cell the RNAi can silence the expression of HIF-1α, inhibit the expression of VEGF, attenuate the proliferation, increase apoptosis and change the distribution of cell cycle in hypoxia. RNAi on HIF-1αcan inhibit the proliferation and angiogenesis of tumor in vitro and in vivo. It can also enhance radiosensitization of KYSE-150 cell in vitro and in vivo, which mechanisms may be related with the inhibition on proliferation and angiogenesis.更多还原