血管内皮生长因子反义RNA对EC9706人食管癌细胞抑制的作用

目的 观察血管内皮生长因子(VEGF)反义RNA对EC9706人食管癌细胞的抑制作用.方法 用脂质体法将反义VEGFcDNA质粒转染至EC9706人食管癌细胞,用噻唑蓝还原法(MTT法)检测EC9706细胞增殖,免疫组化SABC和逆转录-聚合酶链反应(RT-PCR)技术检测VEGF蛋白和VEGF mRNA表达水平.流式细胞术检测细胞凋亡和周期分布.并将转基因EC9706细胞接种于BALB/C裸鼠后肢皮下,4周后观察皮下成瘤情况.结果 被反义VEGFcDNA质粒转染的EC9706食管癌细胞有外源性VEGF反义基因的整合及表达,该细胞VEGF mRNA及蛋白的表达水平降低,但细胞生长增殖能力和增殖周期无明显改变,未发生明显凋亡现象;接种裸鼠28 d后,EC9706-wt组、EC9706-A组及EC9706-E组皮下移植瘤的潜伏期分别为(5.8±2.4)、(12.4±3.6)、(5.3±2.2)d,瘤体重量分别为(2.83 ±0.32)、(0.87±0.14)、(2.62 ±0.68)g,EC9706-A组与其他两组比较差异均有统计学意义(P＜0.05).结论 VEGF反义RNA可抑制EC9706食管癌细胞VEGF表达和裸鼠体内肿瘤生长.     

重组血管内皮抑素对人食管癌细胞株抑制及对乏氧诱导因子-1α和血管内皮生长因子表达的影响

目的 观察重组血管内皮抑素(Endostar)对人食管癌细胞株EC9706细胞体外生长及对乏氧诱导因子-1 α(HIF-1α)、血管内皮生长因子(VEGF)表达的影响.方法 采用噻唑蓝(MTT)法检测不同浓度(12.5、25.0、50.0、100.0、200.0μg/L)Endostar对EC9706细胞增殖24、48、72 h的抑制率;免疫细胞化学和反转录-聚合酶链反应(RT-PCR)实验分为不同浓度(25、50、100 μg/L)实验组和对照组,分别作用48 h后,检测Endostar作用后HIF-1α、VEGF蛋白和基因表达量的变化.结果 Endostar抑制EC9706细胞增殖,呈时间和浓度依赖性,200 μg/L Endostar作用72 h,其抑制率为46.78％;免疫细胞化学检测不同浓度(25.0、50.0、100.0 μg/L)实验组HIF-1α的平均吸光度值分别为0.115 ±0.009、0.089士0.011、0.056±0.009,VGFR的平均吸光度值分别为0.137±0.007、0.102±0.008、0.063±0.007,两者实验各组与对照组比较差异均有统计学意义(P＜0.01);RT-PCR检测不同浓度(25.0、50.0、100.0 μg/L)实验组HIF-1αmRNA的相对表达量分别为0.469±0.530、0.233±0.280、0.171±0.220,VEGF mRNA的相对表达量分别为0.304±0.410、0.199±0.520、0.071±0.310,两者与对照组比较差异均有统计学意义(P＜0.05);Spearman相关分析显示HIF-1α和VEGF蛋白表达与其相应mRNA呈正相关(P＜0.05).结论 Endostar能够抑制EC9706增殖,其作用机制可能与进一步下调了HIF-1α和VEGF的表达有关.     

烟酰胺N甲基转移酶在食管癌中的表达及其机制

本研究通过免疫组织化学检测本医院食管癌患者中烟酰胺N甲基转移酶(NNMT)表达,探讨NNMT对EC9706食管癌细胞增殖、侵袭的影响及其机制。一、材料与方法1.材料:采用免疫组织化学法检测84例食管癌及癌旁正常组织中NNMT的表达,将siRNA NC(对照组)、siRNA NNMT(siRNA组)转染到EC9706食管癌,Real-time PCR检测NNMT mRNA表达,细胞计数试剂盒(cell counting kit-8,CCK-8)实验检测细胞活力,Hoechst染色检测细胞凋亡,Transwell实验检测细胞侵袭能力,蛋白质印迹法(Western blot)检测NNMT、Wnt1a、β-连环蛋白(β-catenin)、细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶(matrix metalloproteinase,MMP)-9蛋白表达。     

烟酰胺N甲基转移酶在食管癌中的表达及其机制

本研究通过免疫组织化学检测本医院食管癌患者中烟酰胺N甲基转移酶(NNMT)表达,探讨NNMT对EC9706食管癌细胞增殖、侵袭的影响及其机制。一、材料与方法1.材料:采用免疫组织化学法检测84例食管癌及癌旁正常组织中NNMT的表达,将siRNA NC(对照组)、siRNA NNMT(siRNA组)转染到EC9706食管癌,Real-time PCR检测NNMT mRNA表达,细胞计数试剂盒(cell counting kit-8,CCK-8)实验检测细胞活力,Hoechst染色检测细胞凋亡,Transwell实验检测细胞侵袭能力,蛋白质印迹法(Western blot)检测NNMT、Wnt1a、β-连环蛋白(β-catenin)、细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶(matrix metalloproteinase,MMP)-9蛋白表达。     

反义微小RNA-21寡核苷酸对食管癌EC9706细胞生长的抑制作用

目的 观察微小RNA-21 (miRNA-21)反义寡核苷酸对食管癌EC9706细胞的生长抑制作用.方法 反义寡核苷酸转染食管癌EC9706细胞,噻唑蓝(MTT)法检测细胞生长抑制率,实时荧光定量聚合酶链反应(FQ-PCR)检测细胞miRNA-21水平,流式细胞仪检测细胞凋亡,FQ-PCR检测程序性细胞死亡因子4 (PDCD4) mRNA表达水平,免疫荧光检测PDCD4蛋白表达.结果 反义寡核苷酸抑制细胞活性的最佳浓度为0.5 μmol/L,转染反义寡核苷酸后细胞内miRNA-21的表达水平明显下调为无义组的0.045 ±0.021,细胞凋亡明显增加为12.95％,生存率显著降低为(48.21±9.56)％,差异有统计学意义(P＜0.05).反义寡核苷酸组中PDCD4 mRNA相对表达增高为0.452±0.002,且蛋白质表达水平增高,差异有统计学意义(P＜0.05).结论 miRNA-21反义寡核苷酸可通过上调PDCD4有效抑制食管癌EC9706细胞增殖并促进凋亡.     

纤维连接蛋白连接片段-1肽段减轻大鼠移植肝缺血再灌注损伤

目的 探讨纤维连接蛋白连接片段-1(CS1)肽段对大鼠肝移植缺血再灌注损伤的影响及其机制.方法 用Wistar大鼠作为供、受鼠,制作肝移植模型.CS1组供鼠分别于术前3d、取肝时和移植前注射CS1肽段,供肝获取后于UW液中保存18h,肝移植术后3d每天注射CS1肽段;对照组用随机肽段替代CS1,其他操作同CS1组.术后6、24、72 h检测受鼠血清转氨酶水平和肝组织病理改变.免疫组织化学染色显示肝脏中的炎症细胞和肝窦内皮细胞.多聚酶链反应检测肝组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和血管内皮细胞生长因子(VEGF) mRNA的表达水平.结果 术后24 h,对照组坏死肝组织面积约占总面积的(25.7±5.4)％,而CS1组为(12.6±3.6)％(P＜0.05).对照组血清转氨酶水平也低于对照组(P＜0.05).术后CS1组移植肝中枯否细胞和中性粒细胞数目均少于对照组(P＜0.05).两组冷缺血18h的供肝中,肝窦内皮细胞均失去正常形态,仅少数内皮细胞特异性标志物阳性.术后72 h,CS1组肝窦内皮细胞基本恢复正常形态,SE-1表达恢复,而对照组肝窦内皮细胞恢复欠佳.术后6和24 h时,CS1组肝组织中TNF-α mRNA的水平低于对照组(P＜0.0)5);术后24 h时,对照组VEGF mRNA的表达高于CS1组(P＜0.05);两组IL-1βmRNA水平的差异无统计学意义(P＞0.05).结论 用CS1肽段处理供、受鼠可以减少炎症因子mRNA的表达,保护肝窦内皮细胞,减轻移植肝缺血再灌注损伤.     

CXCR4/CXCL12信号途径在食管鳞癌浸润转移中的作用机制

目的研究趋化因子受体4(CXCR4)/趋化因子12(CXCL12)信号途径在食管鳞癌浸润转移中的作用机制,为探讨CXCR4成为食管癌治疗新的靶点提供理论依据。 方法取对数生长期的食管鳞癌EC9706细胞,添加趋化因子CXCL12,通过侵袭转移实验、黏附实验分别检测食管鳞癌EC9706细胞株的细胞侵袭、移动和黏附能力。采用RT-PCR和Western Blot技术检测表皮生长因子受体(EGFR)mRNA及蛋白的表达水平。 结果添加不同浓度趋化因子CXCL12(终浓度为5、10 μg/ml),食管鳞癌EC9706细胞株的细胞侵袭、移动和黏附能力均较空白对照组高,并且存在剂量依存关系,即CXCL12浓度越高,细胞的侵袭、移动、黏附能力越强,差异均有统计学意义(P<0.01)。添加不同浓度趋化因子CXCL12,食管鳞癌细胞的EGFR mRNA和蛋白表达水平均较对照组高,并且存在剂量依存关系,即CXCL12浓度越高,EGFR mRNA和蛋白表达水平越高,差异均有统计学意义(P<0.01)。 结论CXCR4/CXCL12信号途径与食管鳞癌细胞的侵袭、转移相关,并且存在剂量依存关系,有可能通过调控EGFR的表达参与食管鳞癌的浸润转移。     

TPX2-siRNA对裸鼠体内移植瘤生长及TPX2表达的影响

我们通过建立裸鼠食管鳞癌细胞EC9706的移植瘤模型,观察TPX2干扰前后对移植瘤的影响和肿瘤组织中TPX2的表达水平. 一、材料与方法 1.材料:人食管癌细胞株EC9706(中国医学科学院肿瘤医院惠赠).裸鼠:BALB/c裸小鼠15只,雄性,4周龄,体质量16～20 g[中国科学院动物所,SCXK(京)2009-0004].TPX2兔抗人多克隆抗体(由德国 Ulrike Bauer惠赠).免疫组织化学试剂盒(北京中山生物技术有限公司).TPX2与β-肌动蛋白(β-actin)引物(北京奥科生物技术有限责任公司),逆转录-聚合酶链反应(RT-PCR)试剂盒(大连TaKaRa公司).     

罗格列酮对大鼠急性胰腺炎肺损伤黏附分子表达的作用

目的 探讨过氧化物酶体增殖物激活受体-γ(PPAR-γ)激动剂罗格列酮(ROSI)对大鼠急性胰腺炎肺损伤(APALI)黏附分子的作用及其机制.方法 雄性Wistar大鼠54只,随机分为假手术组(SO组)、急性胰腺炎组(SAP组)和罗格列酮预处理组(ROSI组).胆胰管逆行注射5%牛磺胆酸钠制备急性胰腺炎模型.ROSI组造模前30 min经股静脉注射10%二甲基亚砜(DMSO)溶解的罗格列酮(6 mg/kg);SO组、SAP组则注射等量10%DMSO.术后3 h、6 h、12 h分批剖杀大鼠,每个时间点6只.检测血清淀粉酶(AMY)、肺组织髓过氧化物酶(MPO)、肺湿干比(W/D),取肺组织行病理学检查;逆转录聚合酶链反应(RT-PCR)检测肺组织细胞间黏附分子-1(ICAM-1)、P-选择素和E_选择素mRNA表达水平.结果 SAP组各时间点AMY、MPO、W/D和肺组织病理评分均较S0组升高(P<0.05);ROSI组上述指标较SAP组下降,AMY、MPO、W/D和病理评分在6 h、12 h差异有统计学意义(P<0.05).SAP组ICAM-1、P-选择素和E-选择素mRNA表达在12 h达高峰,均较SO组12 h升高(P<0.05),ROSI组上述指标mRNA表达在12 h点均较SAP组下降(P<0.05).结论 罗格列酮通过抑制肺组织IcAM-1、P-选择素和E-选择素的表达,减轻急性胰腺炎肺损伤程度.     

罗格列酮对大鼠急性胰腺炎肺损伤黏附分子表达的作用

目的 探讨过氧化物酶体增殖物激活受体-γ(PPAR-γ)激动剂罗格列酮(ROSI)对大鼠急性胰腺炎肺损伤(APALI)黏附分子的作用及其机制.方法 雄性Wistar大鼠54只,随机分为假手术组(SO组)、急性胰腺炎组(SAP组)和罗格列酮预处理组(ROSI组).胆胰管逆行注射5%牛磺胆酸钠制备急性胰腺炎模型.ROSI组造模前30 min经股静脉注射10%二甲基亚砜(DMSO)溶解的罗格列酮(6 mg/kg);SO组、SAP组则注射等量10%DMSO.术后3 h、6 h、12 h分批剖杀大鼠,每个时间点6只.检测血清淀粉酶(AMY)、肺组织髓过氧化物酶(MPO)、肺湿干比(W/D),取肺组织行病理学检查;逆转录聚合酶链反应(RT-PCR)检测肺组织细胞间黏附分子-1(ICAM-1)、P-选择素和E_选择素mRNA表达水平.结果 SAP组各时间点AMY、MPO、W/D和肺组织病理评分均较S0组升高(P<0.05);ROSI组上述指标较SAP组下降,AMY、MPO、W/D和病理评分在6 h、12 h差异有统计学意义(P<0.05).SAP组ICAM-1、P-选择素和E-选择素mRNA表达在12 h达高峰,均较SO组12 h升高(P<0.05),ROSI组上述指标mRNA表达在12 h点均较SAP组下降(P<0.05).结论 罗格列酮通过抑制肺组织IcAM-1、P-选择素和E-选择素的表达,减轻急性胰腺炎肺损伤程度.     

B Cells Help Alloreactive T Cells Differentiate Into Memory T Cells

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B‐cell‐deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T‐cell proliferation and activated CD8 T‐cell survival. These results indicate that B cells help alloreactive T‐cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.     

细胞在胶原复合薇乔网片的吸附研究

目的骨髓细胞、软骨细胞、韧带细胞和滑膜细胞在胶原合成的薇乔网片上培养,研究四种细胞的吸附率。方法细胞在含血清和无血清培养液中,不同的细胞浓度,培养30min和6h,收集未吸附的细胞,统计吸附在合成后的薇乔网片上的细胞吸附率。结果骨髓细胞、软骨细胞、韧带细胞和滑膜细胞在血清和无血清培养液中都可以吸附在胶原复合的薇乔网片上。结论胶原合成的薇乔网片用于组织工程时,应该根据不同的细胞采用不同的培养条件。     

Tolerant T Cells Inhibit Natural Killer Cells Function in Antigen-Presenting Cells in an Independent Fashion

Understanding the relationships among immune cells in the setting of immunologic tolerance is imperative to maintain organ and tissue transplants. T cells and natural killer (NK) cells are responsible for both immune tolerance and immune rejection; however, there is only limited knowledge about the relevance of T and NK cells in tolerance. To address this issue, we explored the possible actions of tolerant T cells on NK cells by the means of mixed lymphocyte co-cultures and NK cytotoxicity assays. We showed that tolerant T cell-induced blockade of the co-stimulatory pathway significantly inhibited NK cell function in vitro regarding antigen-presenting cells. This action was cell-cell-contact dependent. We argue that tolerant T cells and NK cells impart synergistic cooperation to maintain transplant tolerance.     

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

Introduction

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions.

Materials and method

Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein.

Results

The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes.

Conclusion

Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.     

Induction of Porcine Regulatory Cells by Mycophenolic Acid-Treated Dendritic Cells

Tolerance induction in murine allogeneic transplantation is relatively easy, often by induction of regulatory T cells (Treg). Unfortunately, the implementation of these models in clinical situations has not yielded reliable protocols of tolerance induction in humans. Our project sought to create a preclinical model of tolerance induction in large animals. Our current efforts seek to induce and characterize porcine Treg, obtaining dendritic cells (DC) able to preferentially stimulate them. DCs were differentiated from blood monocytes with porcine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 days. These DCs were then stimulated by human CD40 ligand-transfected L cells with or without mycophenolic acid (MPA) for 48 hours. We analyzed surface marker expression, cytokine synthesis, and ability to stimulate allogeneic peripheral blood mononuclear cells (PBMC). The porcine lymphocytes underwent 4 rounds of 1-week stimulation with allogeneic DC treated or not with MPA. At the end of this coculture we analyzed their capacity to suppress allogeneic PBMC proliferation induced by mature DC. Our results showed that porcine DCs pretreated with MPA display a low expression of B7 costimulatory molecules, produce low levels of IL-12, and induce weak proliferation of allogeneic lymphocytes. Moreover, after 4 rounds of stimulation with MPA-treated DCs, PBMCs were able to inhibit an alloreactive response. These preliminary results suggested induction of a regulatory T-cell population that we are currently seeking to characterize.