^125I放射性粒子对人食管癌裸鼠皮下移植瘤的抑制作用研究

目的：观察^125I粒子植入对人食管癌裸鼠皮下移植瘤的抑制作用。方法：采用雄性BALB／C裸小鼠建立人食管癌Eca-109细胞株的裸鼠皮下移植瘤模型,将荷瘤鼠随机分为5组（每组5只）：对照组（A组）、假手术组（B组）、低剂量组（C组,植入7．4×10^6Bq粒子1枚）、中剂量组（D组,植入14．8×10^6Bq粒子1枚）和高剂量组（E组,植入29．6×10^6Bq粒子1枚）。第30天检测各组移植瘤体积,并观察各组移植瘤组织病理学变化。结果：C、D、E组瘤体积与A组比较显著缩小,差异均有统计学意义（P〈0．05）,D、E组瘤体积与C组瘤体积相比均有统计学意义（均P〈0．05）,但D、E组之间瘤体积相比差异无统计学意义（P〉0．05）。B组和A组瘤体积相比差异无统计学意义（P〉0．05）,C、D、E植入后30d肿瘤体积抑瘤率分别为71．5％、90．8％、92．1％。结论：^125I粒子对人食管癌裸鼠移植瘤具有一定的抑制和杀伤作用。     

Notch1通路活化抑制EC109细胞的增殖及机制探讨

背景与目的：Notch1的活化可以通过下调人类乳头状瘤病毒（human papillomavirus,HPV）早期蛋白E6和E7基因的表达抑制HPV阳性HeLa细胞系的增殖。人食管鳞状细胞癌细胞系EC109细胞为HPV18阳性细胞。本研究将Notch1胞内段（intracellular domain of Notch,ICN）转入EC109细胞,导致EC109细胞中Notch通路的组成性活化,从而探讨Notch通路活化与EC109细胞增殖的关系及机制。方法：用脂质体转染法将ICN转入体外培养的EC109细胞,用MTT法检测细胞增殖率;用流式细胞仪检测细胞周期;用RT—PCR检测HPV18E6／E7基因的表达;用Western印迹法检测周期蛋白激酶抑制因子p53的表达。结果：ICN转染入EC109细胞后,EC109细胞增殖受抑;细胞周期阻滞在G2／M期,转目的基因组G2／M期细胞所占比例[（42．57±1．57）％]与未转染组[（1．88±0．66）％]及转空质粒组[（1．99±1．02）％]相比差异均有显著性（P〈0．01）。E6／E7基因表达降低;p53表达升高,转ICN组（2．15±0．23）与未转染组（0．45±0．07）及转空质粒组（0．46±0．02）相比差异均有显著性（P〈0．01）。结论：Notch1通路活化可以NNHPV18 E6／E7基因的表达,导致p53通路的活化,使HPV18阳性EC109细胞增殖周期阻滞于G2／M期,抑制EC109细胞的生长。     

3-甲基腺嘌呤增强食管鳞癌Eca-109细胞放射敏感性的研究

目的 研究自噬在照射致人食管鳞癌Eca-109细胞死亡过程中的作用。方法 选用食管鳞癌Eca-109细胞,分为对照组、5 mmol/L给药组、10 mmol/L给药组、单纯照射组、照射+5 mmol/L组、照射+10 mmol/L组,共6组。采用Western blot检测不同处理组自噬标志物LC3B表达水平;GFP-LC3转染食管鳞癌Eca-109细胞后监测自噬体数量变化;MTT法检测不同处理组细胞活力;荧光染料Hoechst 33342观察不同处理组细胞凋亡的形态学变化;流式细胞术检测不同处理组细胞周期和细胞凋亡;克隆形成实验检测食管鳞癌Eca-109细胞不同处理组放射敏感性。结果 Western blot显示,照射后自噬水平升高,自噬抑制后LC3BⅡ和LC3BⅡ/LC3BⅠ比值明显下降(F=25.64,P<0.05);GFP-LC3转染食管鳞癌Eca-109细胞后,可见照射后自噬体荧光斑点明显增多,自噬抑制后自噬体明显减少(F=127.36,P<0.05);抑制自噬协同照射后细胞活力明显低于单纯照射组(F=129.54,P<0.05);抑制自噬后也增加了照射诱导的凋亡率和G₂/M期阻滞细胞比;线性二次模型拟合剂量存活曲线显示,抑制自噬能增加食管鳞癌Eca-109细胞的放射敏感性。结论 抑制自噬能提高食管鳞癌Eca-109细胞的放射敏感性,协同杀伤肿瘤细胞,提示抑制自噬或可用于辅助食管鳞癌的放射治疗。     

Acanthamoeba castellanii supports extracellular survival of Helicobacter pylori in co-culture

This study aimed to demonstrate whether Helicobacter pylori is able to survive in co-culture with a protozoan, Acanthamoeba castellanii, in order to further investigate a possible aqueous environmental mode of transmission. Numbers of H. pylori in co-culture with A castellanii were assessed by colony forming unit (CFU) assay and cell morphology was observed by electron microscopy. Viable and intact H. pylori in co-culture were detected and the number of H. pylori in co-culture with A. castellanii was significantly higher than in bacterial single culture. It was also shown that co-culture of H. pylori with A. castellanii physically separated by a filter membrane negated this survival effect, suggesting that adherence of H. pylori to A. castellanii affects its survival. Scanning electron microscopy revealed helical forms of H. pylori in co-culture with A. castellanii, but not in single culture. These results imply that mutual interaction between H. pylori and A. castellanii in the environment is critical for survival of H. pylori. In addition, the H. pylori gene expression profile was found to differ between single and co-cultured cells using RNA-sequence analysis.     

CD109, a novel TGF‐β antagonist,decreases fibrotic responses in a hypoxic wound model

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF‐β co‐receptor and TGF‐β antagonist shown to inhibit TGF‐β‐induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild‐type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post‐wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild‐type mice showed increased levels of HIF‐1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post‐wounding as compared to those in wild‐type mice and to non‐hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.     

RNAI沉默Grp94基因对人食管癌ECA109细胞迁移及侵袭能力的影响

目的 探讨RNAI沉默Grp94基因表达对人食管癌ECA109细胞迁移及侵袭能力的影响和可能机制.方法 设计、合成靶向Grp94基因的siRNA基因片段,应用脂质体包埋转染食管癌ECA109细胞,采用Real-time PCR和Western blot法分别检测转染后的细胞内Grp94、基质金属蛋白酶MMP2及MMP9的表达,采用Transwell实验检测沉默Grp94基因对细胞迁移及侵袭能力的影响.结果 SiGrp94组中Grp94mRNA和蛋白水平显著下调(P＜0.01),MMP2、MMP9表达明显下调(P＜0.05);SiGrp94组细胞迁移及侵袭能力明显降低,差异有统计学意义(P＜0.05).结论 沉默Grp94基因可明显抑制ECA109细胞的迁移及侵袭能力,Grp94可能通过影响MMP2、MMP9参与其中.     

食管鳞癌中Klf17蛋白表达及其对Eca109细胞迁移和侵袭能力的影响

目的探讨KLF17与临床病理特征的关系;探讨KLF17对Eca109细胞迁移和侵袭能力的影响及其作用机制。方法采用免疫组化法,检测KLF17在食管鳞癌及癌旁正常食管上皮中的表达;构建KLF17慢病毒过表达载体感染Eca109细胞,细胞分组为转染组(CON)、空载体转染组(Ad-GFP)和慢病毒转染组(Ad-KLF17)。Real timePCR检测KLF17 mRNA的表达、Western blot检测KLF17、E-cadherin和N-cadherin蛋白的表达;细胞划痕实验及体外Transwell实验观察Eca109细胞迁移和侵袭能力。结果 KLF17蛋白表达与侵袭深度、TNM分期显著相关(P0.05);real time-PCR及Western blot结果显示Ad-KLF17组KLF17及上皮标志物E-cadherin表达明显高于Ad-GFP组及对照组(P0.05),而间质标志物N-cadherin表达明显低于Ad-GFP组及对照组(P0.05);划痕实验与体外Transwell实验结果显示Ad-KLF17组细胞迁移距离及细胞数量明显短于和少于Ad-GFP组和对照组(P0.05)。结论 KLF17能抑制Eca109细胞发生上皮-间质转化,与其迁移、侵袭能力相关。     

Dual effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109

AIM: To investigate the effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109, and the related gene expression. METHODS: The cultured Eca-109 cells were divided into four groups: E1 group (co-cultured with 8-Br-cAMP for 24 h); E2 group (co-cultured with 8-Br-cAMP for 48 h); C1 group (treated without 8-Br-cAMP for 24 h); and C2 group (treated without 8-Br-cAMP for 48 h). The same concentration of cell suspension of each group was dropped separately onto the slides and nitrocellulose membranes (NCM). The biotin-labeled cDNA probes for c-myc, wild-type (wt) p53, bcl-2 and iNOS were prepared for in situ hybridization. The expressions of epidermal growth factor receptor (EGFR), p38 kinase, FAS, FasL and caspase-3 were detected using immunocytochemistry, and the NOS activity and the ratio of differentiated cells/proliferating cells were examined by cytochemistry. Immunocytochemistry, cytochemistry, and in situ hybridization were separately carried out on both slides and NCM specimens for each group. In addition, TUNEL was used to detect the cell apoptosis rate in each group. RESULTS: The apoptotic rate of E2 group was significantly higher compared to E1 group, while there was no difference in the ratio of differentiated cells/ proliferating cells between E1 and E2 groups. The signals of wt p53 and iNOS were markedly stronger, while the signals of c-myc and EGFR were obviously weaker in E1 group than those in C1 group (P<0.05). Moreover, the signals of wt p53, iNOS, p38 kinase, caspase-3 and NOS activity were significantly stronger, whereas, the signals of bcl-2, c-myc and Fas/FasL were markedly weaker in E2 group than those in C2 group (P<0.05). CONCLUSION: The differentiation and apoptosis of human esophageal cancer cell Eca-109 can be induced after 24- and 48-h treatment with 8-Br-cAMP, respectively. Upregulation of wt p53, iNOS and downregulation of c-myc may be associated with differentiation and apoptosis of Eca-109 cells. Furthermore, upregulation of FasL, p38 kinase and caspase-3 as well as downregulation of bcl-2, and Fas may be involved in the apoptosis of Eca-109 cells.     

ZNRD1 might mediate UV irradiation related DNA damage and repair in human esophageal cancer cells by regulation of ERCC1

The downregulation of zinc ribbon domain‐containing 1 (ZNRD1) protein was recently found to partially reverse the resistance of human leukemia cells toward chemical therapeutic drugs. Therefore, the ZNRD1 protein might be involved in the process of DNA damage and repair. To explore the possible protective effects of ZNRD1 on DNA damage induced by ultraviolet (UV)‐C irradiation in human esophageal squamous cancer cell line EC109, we designed and transfected a expression vector into EC109 cells, and established an overexpression cell line. The single‐cell gel electrophoresis (comet assay) was used to investigate the DNA damage and repair in UV‐C‐irradiated control and transfected cells. It was found that the ZNRD1‐expressing cells exhibited a significant enhanced DNA repair capacity. Moreover, the overexpression of ZNRD1 could upregulate the expression of excision repair cross‐complementing 1 (ERCC1) gene. Collectively, these findings suggested that ZNRD1 might play an important role in the process of DNA damage and repair by regulating the expression of ERCC1.