FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

胃癌患者血清可溶性E选择素的表达及临床意义

目的:研究可溶性E选择素(soluble E-selectin,sE-selectin)在胃癌患者循环血液中的表达及其意义.方法:采用酶联免疫吸附方法,对86例胃癌患者术前、术后1周以及30例健康人的血清sE-selectin水平进行检测,并分析sE-selectin表达与胃癌临床病理特征之间的关系.随访其中12例胃癌患者,比较手术前后血清sE-selectin水平的变化.结果:胃癌患者术前血清sE-selectin水平(51.50±4.39)ng/mL与术后1周水平(54.14±4.32)ng/mL无显著差异(P＞0.05),均显著高于正常人群血清sE-selectin水平[(17.94±5.53)ng/mL(P＜0.001)];胃癌患者术后1个月sE-selectin水平(28.70±5.82)ng/mL与术后2个月水平(22.27±5.43)ng/mL无显著差异(P＞0.05),均显著低于术前(P＜0.005),而与正常人群水平无明显差异(P＞0.05).胃癌患者血清sE-selectin浓度与性别、年龄、幽门螺杆菌感染状态、肿瘤部位、组织学类型及分化程度无关(P＞0.05),而与肿瘤侵犯胃壁深度、区域淋巴结状态、远处转移情况以及TNM分期密切相关(p＜0.05).结论:sE-selectin可能参与胃癌的演进侵袭过程,是反映胃癌病理进展及转移情况的重要指标.     

Stathmin蛋白在结直肠癌组织中的表达及临床意义

目的探讨Stathmin蛋白在结直肠癌组织中的表达及与临床病理因素的关系。方法采用免疫组织化学SP法对58例结直肠癌组织、30例患者癌旁5cm的正常黏膜组织中Stathmin蛋白的表达进行了检测,两组采用卡方检验连续性校正,以P〈0．05有统计学意义。结果Stathmin蛋白在结直肠癌组织中阳性率为69．0％（40／58）,在正常黏膜中的阳性率为33．3％（10／30）,Stathmin在癌组织表达明显高于正常黏膜（r=6．87,P〈0．05）;Stathmin蛋白表达与患者年龄、性别以及肿瘤组织的分化程度、浸润深度、是否有淋巴结转移关系不明显（χ^2=0．26,P〉0．05）,与肿瘤的远处转移有关（χ^2=9．83,P〈0．05）。结论Stathmin蛋白在结直肠癌组织中呈阳性的表达,除了与远处转移有一定关系,尚不能作为判断其余结直肠癌临床病理特性的依据。     

PLAC1在结直肠癌及其肝转移组织中的表达及临床意义

目的探讨胎盘特异性基因PLAC1的表达与结直肠癌及肝转移生物学行为及预后。方法应用免疫组织化学的方法分析PLAC1在62例结直肠癌、13例肝转移癌、28例癌旁10cm正常组织标本的表达情况。结果 PLAC1表达在癌旁10cm正常黏膜、结直肠癌组织、肝转移癌组织中的表达率分别为:0(0/28)、56.5%(35/62)和61.5%(8/13),结直肠癌和肝转移癌组织中的表达显著高于癌旁10cm正常黏膜,其差异有统计学意义(P<0.01);结直肠癌组织和肝转移组织比较,其差异无统计学意义;PLAC1在女性表达高于男性;PLAC1表达增高与结直肠癌浸润深度、淋巴结转移、远处转移等因素相关。结论 PLAC1异常表达可能在结直肠癌及其肝转移的发生发展中起着重要作用。     

p16基因启动子甲基化与结直肠癌发生发展的关系

目的　探讨p16基因启动子甲基化在结直肠癌发生、发展过程中的作用及其临床意义。方法　采用RT PCR、免疫组化方法检测p16基因表达 ,用甲基化特异性PCR(MSP)方法检测p16基因启动子甲基化。结果　 5 8例结直肠癌中 ,癌旁肠粘膜、原发灶、肝转移灶中p16表达阳性率分别为97% (5 6 /5 8)、31% (18/5 8)、7% (2 /2 8) ,原发灶、肝转移灶中p16表达明显降低 (P <0 0 1)。癌旁肠粘膜组织中未发现p16基因启动子甲基化 ,而癌原发灶、肝转移灶中p16甲基化阳性率分别为 5 0 %(2 9/5 8)、75 % (2 1/2 8) ,3种组织p16基因启动子甲基化率的差别有显著性意义 (P <0 0 1)。在 2 8例出现肝转移者外周静脉血、胆汁中可检测到p16甲基化启动子序列者分别为 2 3例 (82 % )、2 0例 (71% )。结论　p16基因启动子甲基化导致p16抑癌基因失活与结直肠癌的发生、转移有密切关系。     

结直肠癌原发灶和正常肠黏膜组织相关差异表达蛋白研究

目的探讨结直肠癌原发灶和正常肠黏膜组织中蛋白质组的表达差异。方法采用双向荧光差异凝胶电泳法(2-DDIGE),对比分析结直肠癌原发灶、癌旁正常肠黏膜组织中蛋白质组的表达差异,经质谱分析鉴定差异蛋白点;采用细胞转染方法,将差异蛋白基因导入结直肠癌细胞,观察细胞生物学行为的改变。结果结直肠癌原发灶与正常肠黏膜组织中蛋白质组分有明显差别。结直肠癌原发灶组织与正常肠黏膜组织比较,差异倍数为1.5倍,差异点数目为60个。共分析了8个差异蛋白点,其中2个蛋白点在原发灶组织中表达下调,6个蛋白点在原发灶组织中表达上调。经质谱鉴定,碳酸酐酶Ⅱ(CAⅡ)、蛋白质二硫键异构酶(PDI)在原发性组织中表达下调,醛缩酶A等在原发灶中表达上调。将CAII cDNA克隆转染结直肠癌细胞后,癌细胞侵袭、趋化运动能力及耐药性均明显降低。结论结直肠癌原发灶和正常肠黏膜蛋白质组表达有显著差异,CAⅡ、PDI表达降低和醛缩酶A表达增强可能与结直肠癌的发生相关。     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

目的探讨直肠癌侵袭转移过程中FasL基因表达对细胞增殖凋亡的影响。方法采用北京军区总医院普通外科实验室构建的4组不同侵袭力的人直肠癌HR-8348细胞亚型HR-8348B、HR-8348L、HR-8348F和HR-8348As,并用化学缺氧法构建上述4组细胞的缺氧12h模型,以Western印迹法验证各组细胞内FasL蛋白的表达水平和缺氧状态下细胞内FasL蛋白的表达,流式细胞仪测定细胞周期分布并计算细胞增殖指数,四甲基偶氮唑盐（MTT）法测定细胞增殖能力改变并计算细胞抑制率,末端标记法（TUNEL）测定细胞凋亡状况。结果Western印迹显示FasL蛋白于40000处显色。常氧条件下HR-8348F细胞内FasL的表达水平显著高于HR-8348B、HR-8348L和HR-8348As（F=361.149。P〈0.01）;缺氧12h时各组细胞均生长良好,形态较常氧状态皱缩;HR-834F,细胞内FasL的表达水平仍显著高于其他3组（F=278.766,P〈0.01）,但其自身常氧与缺氧状态下FasL的水平差异无统计学意义（t=1.762,P〉0.05）。缺氧12h后各组细胞增殖均受到抑制,HR-8348F细胞的增殖指数（60.43±3.72）显著高于HR-8348B（40.01±3.30）、HR-8348L（41.30±4.06）和HR-8348As（35.87±4.39）（F=39.477,P〈0.01）,增殖抑制率（17.30±1.98）和凋亡指数（13.10±1.04）显著低于HR-8348B（33.70±4.33和21.60±1.31）、HR-8348L（34.20±3.92和20.10±1.15）、HR-8348As（38.00±4.55和23.90±1.23）细胞（F分别为28.811和76.462,P〈0.01）。结论缺氧环境中,直肠癌细胞FasL表达增强可导致细胞增殖加速、凋亡减少和侵袭能力增强。促进细胞对微环境缺氧的耐受。     

TTV在急性黄疸性肝炎中的感染状态研究

就ＴＴＶ在各型急性黄疸性肝炎患者中的感染状况进行初步探讨。门诊及住院患者 80例 ,男 4 9例 ,女 31例 ,年龄 9～ 54岁。血清标本包括首次就诊和发病后 3个月 ,标本 -70°Ｃ冰箱保存备检。ＴＴＶ检测采用套式ＰＣＲ法 ;抗 ＨＡＶＩｇＭ、ＨＢｓＡｇ、抗 ＨＢｃＩｇＭ、抗 ＨＣＶ、抗 ＨＤＶＩｇＭ、抗 ＨＥＶＩｇＧ和抗 ＨＧＶＩｇＭ检测采用ＥＬＩＳＡ法。结果表明 ,ＴＴＶ在各型急性黄疸性肝炎患者中的总检出率为 2 8.75% ( 2 3/ 80 ) ,在非甲～庚型肝炎患者中ＴＴＶ阳性率高达 50 % ( 6/ 12 ) ,以下依次为庚型肝炎 33.3% ( 3/ 9)、…     

FasL基因表达对缺氧状态下直肠癌细胞增殖凋亡的影响

Objective To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells. Methods The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348B, HR-8348L, HR-8348F and HR-8348As) with different invasive power were verified by Western blot. Hypoxia models for HR-8348B, HR-8348L, HR-8348F and HR-8348As were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptesis was assessed with TUNEL. Results FasL protein was pigmentized at the position of 40 000 by Western blot, and the expression level of FasL was significantly higher in HR-8348F cells than those in HR-8348B, HR-8348L and HR-8348As cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348F cells than those in other groups (F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia (t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348F (60.43±3.72) than those in HR-8348B (40.01±3.30), HR-8348L (41.30±4.06) and HR-8348As cells (35.87±4.39), respectively (F=39.477, P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348F (17.30±1.98 and 13.10±1.04) than those in HR-834B (33.70±4.33 and 21.60±1.31), HR-8348L (34.20±3.92 and 20.10±1.15), and HR-8348As (38.00±4.55 and 23.90±1.23), respectively (F=28.811 and 76.462, respectively, P<0.01). Conclusion The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apeptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.     

人乳腺癌相关成纤维原代细胞培养的改良方法

目的 通过对常见的人乳腺癌相关成纤维细胞（BCAFs）原代培养方法进行改良,建立一种快速高效的BCAFs原代培养方法。方法 取确诊为浸润性乳腺癌患者的癌组织标本,每个标本随机分成大小、质量相同的改良组和传统组,改良组用改良酶消化法（Ⅱ型胶原酶+透明质酸酶消化10 min）消化,传统组用传统酶消化法（Ⅰ型胶原酶消化8 h）消化。通过细胞计数、MTT和免疫荧光法比较两组消化方法获得的细胞总数、培养48 h后贴壁细胞的活性及BCAFs的纯度并在倒置显微镜下观察BCAFs原代细胞特征。结果 改良酶消化法获得的细胞量、48 h贴壁细胞的活性和细胞纯度均高于传统酶消化法（P<0.001）。获得的BCAFs原代细胞呈纺锤体形或长梭形,胞质内颗粒较多,细胞排列不规则。结论 本研究成功建立了一种高效快速的BCAFs原代培养的方法,为后期探究其在乳腺癌中的功能研究建立基础。