长链非编码RNA反义RNA 1靶向微小RNA-501-3p对食管癌细胞恶性生物学行为影响的实验研究

目的探讨长链非编码RNA(lncRNA)胸腺生成素反义RNA 1(TMPO-AS1)对食管癌细胞恶性生物学行为的影响及其机制。方法2016年6月至2019年12月,实时荧光定量聚合酶链反应(RT-qPCR)检测20例食管癌组织和癌旁组织中lncRNA TMPO-AS1和微小RNA(miR)-501-3p的水平,食管癌Eca109细胞分为lncRNA TMPO-AS1干扰组(转染si-NC和si-TMPO-AS1);miR-501-3p过表达组(转染miR-NC和miR-501-3p);lncRNA TMPO-AS1过表达组(转染pcDNA和pcDNA-TMPO-AS1);lncRNA TMPO-AS1和miR-501-3p共抑制组(转染si-TMPO-AS1+anti-miR-NC和si-TMPO-AS1+anti-miR-501-3p)。噻唑蓝(MTT)法、流式细胞术和Transwell实验分别检测食管癌Eca109细胞增殖、凋亡率、细胞迁移和侵袭能力,蛋白质印迹法(Western blot)检测相关蛋白水平,双荧光素酶报告系统验证lncRNA TMPO-AS1与miR-501-3p的调控关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差进行分析。结果食管癌组织组中的lncRNA TMPO-AS1水平(2.64±0.26)高于癌旁组织(1.00±0.09,t=26.657,P<0.05),miR-501-3p水平(0.44±0.04)低于癌旁组织(1.00±0.07,t=31.063,P<0.05);干扰lncRNA TMPO-AS1的Eca109细胞24 h增殖(0.36±0.03)低于si-NC组(0.39±0.03,t=2.121,P<0.05),48 h增殖(0.45±0.04)低于si-NC组(0.75±0.07,t=11.163,P<0.05),72 h增殖(0.57±0.05)低于si-NC组(1.16±0.09,t=17.191,P<0.05),迁移细胞数[(54.14±5.65)个]低于si-NC组[(113.02±9.87)个,t=15.531,P<0.05],侵袭细胞数[(47.69±5.01)个]低于si-NC组[(96.32±9.88)个,t=13.169,P<0.05],细胞凋亡率[(22.15±2.22)%]高于si-NC组[(6.98±0.69)%,t=19.576,P<0.05];过表达miR-501-3p的Eca109细胞48 h增殖(0.51±0.05)低于miR-NC组(0.77±0.07,t=9.067,P<0.05),72 h增殖(0.65±0.06)低于miR-NC组(1.15±0.09,t=13.867,P<0.05),迁移细胞数[(61.23±6.33)个]低于miR-NC组[(115.25±9.25)个,t=14.458,P<0.05],侵袭细胞数[(53.14±5.33)]低于miR-NC组[(98.32±8.47)个,t=13.543,P<0.05],细胞凋亡[(19.58±1.74)%]高于miR-NC组[(7.23±0.77)%,t=19.471,P<0.05],差异均有统计学意义。结论lncRNA TMPO-AS1通过靶向miR-501-3p促进食管癌细胞的恶性生物学行为,可被视为食管癌患者的潜在治疗靶标。     

胰腺癌SW1990细胞热疗温度及机制初探

目的观察多种温热疗法对人胰腺癌细胞SW1990生长的影响,确定对其进行热疗的最佳温度和时间,并初步探讨其机制。方法MTT检测不同温度、不同作用时间对SW1990细胞生长的抑制作用,光镜、电镜观察不同温度热疗后细胞形态的改变,RT-PCR检测各组凋亡相关基因bcl-2、bax的表达情况。结果MTT示44C对细胞增殖的抑制作用最明显,且44 C的IC50值在1 h左右;光镜示44C 热疗1 h较其他温度作用1 h出现的细胞凋亡小体数多;电镜示44 C热疗1 h细胞的超微结构破坏最明显,呈现明显凋亡。RT-PCR显示随温度的增高,凋亡相关基因bax的表达呈上升趋势。结论44C热疗1 h为SW1990细胞最佳的热疗温度和时间,热疗可能通过上调促凋亡基因bax的表达而诱导SW1990 胰腺癌细胞凋亡。     

肥大细胞在大鼠胰腺组织纤维化形成中的作用及其机制

目的探讨肥大细胞（MC）在大鼠胰腺组织纤维化形成中的作用和机制。方法建立经逆行胆胰管注射2％三硝基苯磺酸（TNBS）诱导大鼠慢性胰腺炎模型，将大鼠分为三组，每组40只，分别用肥大细胞膜稳定剂色甘酸钠和MC激动剂48／80化合物及生理盐水进行干预，并于第3、7、14、21和28天处死动物。H—E染色观察胰腺组织病理学改变；Van Gieson染色观察胰腺组织纤维化情况；硫堇蓝染色观察大鼠慢性胰腺炎过程中MC分布、形态和数量的改变；免疫组化染色观察大鼠慢性胰腺炎α-平滑肌肌动蛋白（α-SMA）、转化生长因子（TGF）β1的表达；逆转录-多聚酶链反应（RT—PCR）观察血管紧张素Ⅱ1型（AT1）和2型（AT2）受体蛋白的表达。结果2％TNBS胰管内注射后可于第4周引起典型大鼠胰腺组织纤维化，在胰腺纤维化区域可见大量Ⅰ型胶原沉积。在此过程中MC存在着活化及脱颗粒。胰腺组织α—SMA、TGFβ1、AT1和AT2 mRNA蛋白制模早期表达即为阳性，至第4周时最强。与对照（生理盐水）组比较，色甘酸钠组MC数量及脱颗粒现象明显减少，α-SMA、TGFβ1蛋白表达和AT1、AT2受体mRNA表达明显减少；48／80化合物组MC数量及脱颗粒现象明显增多，上述各指标的表达均有不同程度的增加。结论MC参与TNBS诱导的大鼠慢性胰腺炎的炎症和纤维化的发生及发展，其机制可能与MC促进胰腺星状细胞活化，上调血管紧张素Ⅱ受体表达等介导了胰腺纤维化的形成。     

微创McKeown食管切除术治疗食管下段癌的临床价值

目的探讨微创McKeown食管切除术治疗下段食管癌的临床价值。方法选取2013-01-2017-06间在郑州大学第一附属医院接受食管切除术的下段食管癌患者。将行微创McKeown手术的患者作为微创组,将行开放Sweet手术的患者作为开放组,倾向得分匹配(PSM)用于降低2组一般资料的统计学差异。比较2组患者的疗效。结果全部患者均顺利完成手术。2组患者的病死率、肺部并发症和吻合口漏发生率差异无统计学意义(P>0.05)。微创组淋巴结清扫数、平均住院费用和手术时间均多于开放组,差异均有统计学意义(P<0.05)。结论微创McKeown食管切除术治疗下段食管癌,较传统开放手术淋巴结清扫数多,但手术时间和治疗费用较多。     

纵隔异位甲状腺癌误诊一例

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基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.     

细胞凋亡在急性坏死型胰腺炎早期肠黏膜上皮细胞死亡中的作用

目的观察急性坏死型胰腺炎(ANP)大鼠早期肠黏膜上皮细胞凋亡的发生及其演变规律,探讨其在肠黏膜屏障功能障碍中的作用.方法Spraque-Dawley大鼠48只,采用胆胰管内逆行注入5%牛磺胆酸钠溶液诱导大鼠ANP模型.假手术组(shamoperation,SO)大鼠仅作剖腹术.术后3、6、12和24h分批处死大鼠,取末端回肠组织,分别应用DNA琼脂糖凝胶电泳,异硫氰基荧光素(FITC)结合的Annexin-V和碘化丙啶(PI)标记细胞作流式细胞仪(FCM)检测和免疫组化(TUNEL法)等技术,研究ANP大鼠肠黏膜上皮细胞凋亡.结果DNA琼脂糖凝胶电泳结果显示,SO组仅于12h出现"梯形(ladder)”条带;ANP大鼠,各时点均可见"梯形”条带.FCM检测结果表明,术后3、6、12和24hSO组肠黏膜上皮脱落细胞凋亡比例分别为(53.7±3.7)%、(27.6±6.0)%、(39.0±4.8)%和(29.0±11.3)%;ANP组分别为(50.3±11.3)%、(79.7±9.2)%、(47.8±17.3)%和(49.6±9.5)%,术后6h达峰值,且较SO组明显增高(P＜0.01),凋亡与坏死细胞(A/N)比值为27.7±17.9.TUNEL法显示,凋亡细胞呈细胞核固缩、断片状,术后3、6、12和24hSO组凋亡指数为6.2±2.4、7.5±1.7、10.5±0.9和5.3±0.8;ANP组为17.5±3.5、20.4±2.9、14.8±1.7和14.2±2.1,各时点均较SO组明显增高(P＜0.01),且于术后6h达峰值.结论ANP大鼠早期肠黏膜上皮细胞凋亡为细胞死亡的主要模式;肠黏膜上皮细胞凋亡在ANP大鼠早期肠黏膜屏障功能障碍发生中可能起重要作用.     

高三酰甘油血症对大鼠胰腺腺泡细胞凋亡的影响

目的:研究细胞凋亡在高三酰甘油(TG)血症损伤大鼠胰腺腺泡细胞中的作用.方法:断奶1周雄性SD大鼠(约70 g)随机分为两组,每组10只,分别给予高脂饲料和普通饲料喂养4周.检测血清TG和胆固醇(TC)水平.用胶原酶消化法分离大鼠胰腺腺泡细胞,用台盼兰染色测定细胞活力.给予不同浓度胆囊刺激素(CCK-8)刺激胰腺腺泡细胞,透射电镜下观察细胞超微结构变化,用RT-PCR方法检测细胞凋亡相关基因bax和bcl-2 mRNA的表达.结果:饲喂大鼠4周高脂饮食后血清TG较正常饮食组明显升高(P＜0.05).体外培养6 h后,高脂饮食组胰腺腺泡细胞活力低于对照组(P＜0.05).与CCK-8培养后,高脂组细胞发生明显病理损害,可见细胞凋亡和坏死表现.RT-PCR结果表明高脂组细胞与CCK-8共培养后,bax与bcl-2比值明显增高.结论:高TG血症可以加重大鼠胰腺腺泡细胞损伤,凋亡相关基因bax与bcl-2比值增高引起细胞凋亡可能参与其发病过程.     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.