Fas/FasL基因转染联合顺铂对直肠癌细胞杀伤作用的研究

目的探讨Fas/FasL基因转染联合顺铂对直肠癌细胞的杀伤作用。方法构建pcDNA3.1-Fas/FasL真核表达载体,将人Fas/FasL基因通过脂质体导入直肠癌8348细胞中,并利用RT-PCR方法检测直肠癌8348细胞的Fas/FasL基因mRNA表达。用MTT法分析顺铂对转染前后的8348细胞抑制增殖和诱导凋亡的能力。结果Fas/FasL基因转染可明显增强直肠癌8348细胞的Fas/FasL表达。分别加入不同浓度顺铂(1、5、10、20、40μg/ml),Fas转染组8348细胞抑制率分别为47.2%、51.8%、57.2%、65.4%、71.0%;后者细胞抑制率分别为29.6%、33.0%、37.8%、41.4%、47.0%,其差异有显著性意义(t=15.33,P<0.01);FasL转染组8348细胞抑制率分别为11.0%、25.4%、31.2%、37.8%、42.4%;对照组8348细胞抑制率分别为26.1%、34.4%、37.6%、42.9%、53.2%,其差异有显著性意义(t=4.43,P<0.05)。结论转染的Fas/FasL基因可显著上调直肠癌8348细胞的Fas/FasL表达;Fas基因转染联合顺铂对直肠癌细胞有更强的杀伤作用;FasL基因转染可减弱顺铂对8348细胞的细胞毒作用,因此为直肠癌的基因治疗和化疗提供了理论依据。     

FasL cDNA转染对直肠癌细胞顺铂耐药性的影响

目的:探讨FasLcDNA转染和表达对直肠癌细胞耐药性的影响。方法:用RT-PCR方法克隆人FasL全长cDNA,构建pcDNA3.1-FasL真核表达载体,用脂质体法转染HR-8348人直肠癌细胞,采用MTT法检测顺铂对转染和未转染直肠癌细胞的生长抑制率。结果:DNA测序证实克隆FasLcDNA898bp与GeneBank序列完全一致。构建真核表达载体转染HR-8348细胞后,FasLmRNA表达明显增强。在不同浓度顺铂(1、5、10、20、40mg/L)的作用下,FasL转染组直肠癌细胞抑制率分别为11.0%、25.4%、31.2%、37.8%、42.4%:对照组癌细胞抑制率分别为26.1%、34.4%、37.6%、42.9%、53.2%,其差异有显著性意义(t=4.43,P<0.05)。结论:FasL转染HR-8348细胞可增强癌细胞的耐药性,减弱顺铂对HR-8348细胞的杀伤作用。     

结直肠癌中缺氧诱导因子1-α与FasL 表达相关性的研允

目的 探讨结直肠癌侵袭转移过程中缺氧诱导因子1-α(alpha,HIF-1α)与FasL表达的相关性.方法 采用分子克隆方法,将我室已构建的FasL-pcD-NA3.1(+)质粒与pcDNA3.1(一)质粒进行重组,得到新的FasL-pcDNA3.1(一)质粒并加以鉴定;通过脂质体转染法将空质粒、FasL-pcDNA3.1(+)与FasL-pcDNA3.1(一)质粒分别转染人直肠癌HR-8348细胞,构建侵袭力不同的结直肠癌细胞HR-8348L、HR一8348F和HR一8348As,未转染细胞HR一8348B为空白对照,应用Tran-swell,小室检测各组细胞的侵袭能力;采用化学缺氧法构建四组细胞的缺氧模型,Western blot方法定量检测缺氧0h、6h、12h及24h各组细胞内HIF-1α的表达.结果 FasL-pcDNA3.1(一)质粒符合要求,FasL片段大小约900bp,测序结果正确率99.2%;单层细胞体外侵袭实验见HR一8348F细胞穿透Transwell滤膜的细胞数目为(12.930±2.434),显著多于HR-8348B(8.133±1.959)、HR-8348L(7.670±2.093)和HR-8348As(7.870±1.685)细胞(P<0.05);Western blot检测示HIF-1α蛋白于120kD处显色,缺氧0h与6h,各组样品中HIF-α仅表达微量,HIF-1α水平无显著性差异(P>0.05);缺氧12h与24h,HR一8348F细胞内HIF-1α水平较0h和6h时明显增高(P<0.05),而HR-8348B、HR-8348L及HR-8348As细胞内HIF-1α表达与6h时无明显变化(P>0.05),HR-8348F细胞HIF-1α水平显著高于HR-8348B、HR-8348L及HR-8348As细胞(P<0.01).结论 缺氧环境中结直肠癌细胞FasL表达增强是除低氧分压外另一个诱导HIF-1α表达增高的因素,FasL与 HIF-1α水平呈正相关,高侵袭能力的结直肠癌细胞对缺氧的适应能力加强,促进肿瘤的远处转移.     

FasL基因表达对结直肠癌细胞肝转移影响的研究

目的　探讨FasL基因表达对结直肠癌细胞生物学行为的影响及在肝转移中的作用。方法　采用RT PCR方法检测大肠癌原发灶、癌旁肠黏膜、肝转移灶中FasL基因表达。用细胞转染方法 ,将FasLcDNA转染人直肠癌细胞HR 8348,采用四唑蓝法观测FasL表达对癌细胞生长抑制率及对5 FU、卡铂杀伤作用的影响。　结果　结直肠癌原发灶 (5 8例 )、癌旁肠黏膜 (5 8例 )、肝转移灶 (2 8例 )中FasL基因表达阳性率分别为 2 4 % (14 /5 8)、14 % (8/5 8)、10 0 % (2 8/2 8)。肝转移灶中FasL表达阳性率高于癌原发灶 (χ2 =4 3 4 9,P <0 0 1)和癌旁肠黏膜组织 (χ2 =5 7 6 6 ,P <0 0 1)。肝转移组原发灶FasL表达阳性率高于无肝转移组 (χ2 =3 96 ,P <0 0 5 )。转染HR 8348细胞FasL表达为阳性。用 5 FU、卡铂杀伤FasL转染细胞和未转染细胞 ,2组癌细胞生长抑制率有显著性差异 (t=9 0 2、t=11 93,P <0 0 1)。在相同化疗药物浓度下 ,FasL阳性HR 8348细胞存活率高于对照组癌细胞。　结论　FasL阳性癌细胞对化疗药物有较强的耐受性。FasL基因表达能使癌细胞逃避免疫监视和杀伤并对化疗药物产生抗性 ,促进结直肠癌发生肝转移。     

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。     

Fas基因转染联合顺铂对直肠癌细胞杀伤作用的研究

目的探讨Fas基因转染联合顺铂对直肠癌细胞的杀伤作用。方法采用RT PCR技术从健康人的外周血单核细胞中扩增包含全部阅读框架的Fas全长基因 ,与 pGEM TEasy质粒连接 ,经测序验证。构建 pcDNA3 1 Fas真核表达载体 ,将人Fas基因通过脂质体导入直肠癌 8348细胞中 ,并利用RT PCR方法检测直肠癌 8348细胞的Fas基因mRNA表达。用MTT法分析顺铂对转染前后直肠癌细胞抑制增殖和诱导凋亡的作用。结果Fas基因转染可明显增强直肠癌 8348细胞的Fas表达 ,分别用 1、5、10、2 0、4 0mg/L浓度的顺铂作用 ,转染Fas基因的 8348细胞实验组细胞抑制率分别为 4 7 2 %、5 1 8%、5 7 2 %、6 5 4 %、71 0 % ;未转染Fas基因的 8348细胞对照组细胞抑制率分别为 2 9 6 %、33 0 %、37 8%、4 1 4 %、4 7 0 % ,2组相比 ,差异有显著性意义 (t =15 33,P <0 0 1)。结论转染的Fas基因可显著上调直肠癌 8348细胞的Fas表达 ,促进细胞凋亡 ,Fas基因转染联合顺铂对直肠癌细胞有更强的杀伤作用。     

Fas基因转染对直肠癌细胞生物学行为影响的研究

目的　探讨Fas基因转染对直肠癌细胞生物学行为的影响。方法　以正常人外周血单个核细胞 (PBMC)为模板 ,通过RT -PCR方法扩增出Fas全长cDNA ,转染人直肠癌细胞HR - 8348,检测Fas基因表达 ,观察细胞生长状态。采用MTT法观察癌细胞对化疗药物反应及细胞存活率。采用H33342释放法检测PBMC对癌细胞的杀伤活性。结果　未转染的HR - 8348细胞Fas基因表达为阴性 ,转染Fas后HR - 8348细胞Fas表达阳性。两组细胞形态和生长状态无显著性差异。分别加入化疗药物 5 -Fu、卡铂 ,检测细胞存活率。在相同药物浓度下 ,两组细胞存活率有显著性差异 (t =4 .73,3.84 ,P <0 .0 1)。Fas转染组癌细胞存活率低于未转染组 ,Fas表达阳性癌细胞对 5 -Fu、卡铂的敏感性增强。PBMC对Fas转染组HR - 8348细胞的杀伤活性为 5 6 % ,对未转染组的杀伤活性为 2 1% ,两组有显著性差异 (χ2 =2 0 .73,P <0 .0 1) ,PBMC对Fas阳性癌细胞杀伤活性明显高于对照组癌细胞。结论　转染Fas可提高癌细胞对化疗药物的敏感性 ,增强药物对癌细胞的杀伤作用。并促进机体免疫细胞的杀伤活性     

Fas基因转染对直肠癌细胞生物学行为影响的研究

目的：探讨Fas基因转染对直肠癌细胞生物学行为的影响。方法：以正常人外周血单个核细胞（PBMC）为模板，通过RT－PCR方法扩增出Fas全长cDNA，转染人直肠癌细胞HR－8348，检测Fas基因表达，观察细胞生长状态，采用MTT法观察癌细胞对中1芗反应及细胞存活率。采用H3342释放法检测PBMC对癌细胞的杀伤活性，结果，未转染的HR－8348细胞Fas基因表达为阴性，转染Fas后HR－8348细胞Fas表达阳性，两组细胞形态和生长状态无显差异，分别加入化疗药物5－Fu、卡铂，检测细胞存活率，在相同药物浓度下，两组细胞存活率有显性差异（t=4．73，3．84，P＜0．01）。Fas转染组癌细胞存活率低不转染组，Fas表达阳性癌细胞对5－Fu、卡铂的敏感性增强。PBMC对Fas转染组HR－8348细胞的杀伤活性为56％，对未转染组的杀伤活性为21％，两组有显性差异（X^2＝20．73，P＜0．01），PBMC对Fas阳性癌细胞杀活性明显高于对照组癌细胞。结论：转染Fas可提高癌细胞化疗药的敏感性，增强药物对癌细胞的杀伤作用，并促进机体免疫细胞的杀伤活性。     

腺病毒介导胞嘧啶脱氨酶基因对直肠癌细胞的杀伤作用

目的 探讨腺病毒载体介导的胞嘧啶脱氨酶（CD）基因转染和CD/5-FC对直肠癌细胞的杀伤作用。方法 用重组腺病毒介导外源CD基因转移到人直肠癌细胞株HR-8348,通过检测腺病毒的转导效率,CD基因表达,以及集落形成实验,细胞存活率测定,裸鼠移植癌治疗实验,观察分析CD/5-FC对癌细胞的杀伤作用,结果 重组腺病毒介导CD基因转染,在癌细胞中得到高效表达。CD/5-FC系统对转染含CD重组腺病毒HR-8348细胞的集落形成,细胞生长均有明显的抑制作用,而对无CD基因转染癌细胞无影响,在转染和未转染CD基因的HR-8348混合体系中,CD/5-FC除了杀伤转染CD基因的癌细胞外,对周围的无CD转染癌细胞也有明显的杀伤作用。表现出很强的“旁观者效应”。裸鼠皮下移植癌治疗实验结果表明,CD/5-FC对HR-8348实体癌生长抑制率为71.5%。结论 CD/5-FC对腺病毒介导CD基因转染的直肠癌细胞有很强的杀伤作用和显著的“旁观者效应”。     

自身免疫性肝炎和原发性胆汁性肝硬化患者肝组织和外周血单个核细胞Fas及FasL的检测

目的 观察自身免疫性肝炎(AIH)和原发性胆汁性肝硬化(PBC)患者肝组织和外周血单个核细胞(PBMC)Fas、FasL的表达,探讨细胞凋亡在自身免疫性肝病(AILD)发病机制中的作用.方法 采用免疫组织化学技术对10例AIH、20例PBC患者肝组织和PBMC中Fas、FasL进行检测,正常对照为10例健康献血者.结果 AIH 患者PBMC中Fas、FasL检出率分别为100%和90%, Fas、FasL平均阳性细胞率分别为78%和65%;PBC患者PBMC中Fas、FasL检出率均为90%,平均阳性细胞率分别为62%和59%.在肝组织中Fas、FasL表达于肝细胞和淋巴细胞:9例AIH Fas阳性颗粒见于肝细胞浆,其中1例胞膜、胞浆同时阳性;8例FasL阳性颗粒表达于肝细胞浆;Fas、FasL阳性肝细胞多聚集于汇管区周围.同时在汇管区周围浸润的淋巴细胞中也可见Fas或FasL的阳性表达.17例PBC患者Fas阳性颗粒表达于肝细胞浆,其中4例胞膜、胞浆同时阳性;16例PBC FasL阳性,见于肝细胞浆,在肝组织中浸润的淋巴细胞浆中Fas、FasL均有表达.AIH和PBC患者Fas、FasL表达显著高于正常对照.结论 AIH和PBC患者的Fas、FasL表达增加,细胞凋亡可能在AIH和PBC的发病机制中起重要作用.     

Fas-Fas ligand system in the peripheral blood of patients with renal diseases

To clarify the role of the Fas-Fas ligand (FasL) system in the peripheral blood from patients with various renal diseases, the Fas and FasL expression on mononuclear cells (MNCs) and serum levels of soluble Fas (sFas) and soluble FasL were investigated. Patients were selected from those with various types of glomerular diseases showing various degrees of renal function. Fas expression on MNCs was analyzed by a FACScan, sFas and soluble FasL were measured with an ELISA kit, and FasL expression on MNCs was counted using a FACScan after a bioassay. Fas-positive MNCs and sFas increased with statistical significance concomitantly with deterioration in renal function. Moreover, there was a significant correlation between them. sFas- and FasL-positive MNCs were significantly correlated with proteinuria. However, the Fas expression percentage on MNCs and/or serum levels of sFas did not correlate with the number of TUNEL-positive cells in the glomeruli. Also, there was no disease specificity in the activation of Fas. These results indicate that Fas expression on MNCs is activated in accordance with the deterioration in renal function without disease specificity, corresponding to the elevation of serum sFas levels to protect against Fas-mediated apoptosis.     

磷脂爬行酶1在结直肠癌中的表达及其意义

目的 探讨磷脂爬行酶1(PLSCR1)的表达与结直肠癌生物学行为及预后的关系.方法 应用免疫荧光细胞化学方法分析PLSCR1在结直肠癌细胞株Lovo与HR8348中的表达情况,同时应用免疫组织化学方法分析PLSCR1在70例结直肠癌、30例正常黏膜、10例肝转移癌标本中的表达情况.结果 PLSCR1在结直肠癌细胞株Lovo与HR8348中的表达较强,PLSCR1主要定位在结直肠癌细胞株的细胞膜.PLSCR1在结直肠癌和肝转移癌组织中的表达率显著高于正常黏膜(P<0.05).PLSCR1阳性表达与远处转移相关(P<0.05),PLSCR1表达阳性者术后5年生存率相对较低(P<0.05).结论 PLSCR1的异常表达在结直肠癌的发生、进展和转移过程中可能发挥着重要作用.     

Involvement of apoptosis in patients with diabetic nephropathy: A study on plasma soluble Fas levels and pathological findings

Aims: We investigated the relationship between levels of plasma soluble Fas (sFas) and stages of diabetic nephropathy, with special reference to apoptosis and clinical features of diabetic nephropathy in 168 patients with diabetic nephropathy. Results: There was a positive correlation between plasma sFas and creatinine levels, between sFas levels and urinary protein levels, and between sFas levels and urinary albumin. There was a negative correlation between plasma sFas levels and creatinine clearance. Plasma sFas levels in the early stage (stages 1, 2, 3A) and advanced stage (stages 3B and 4) were 2.6 ± 0.1 and 5.4 ± 0.5 ng/mL, respectively. Plasma sFas level of the advanced stage was significantly higher than that of the early stage. The number of proliferating cell nuclear antigen (PCNA) positive cells was significantly lower in the advanced stage than in the early stage. The number of in situ nick‐end labelling (TUNEL) positive cells was also significantly lower in the advanced stage than in the early stage, suggesting the suppression of apoptosis. Conclusion: These data suggest that apoptosis is involved in the advancement of diabetic nephropathy, and that plasma sFas level might be a predicting factor for prognosis.     

Soluble Fas and Fas-ligand in bladder cancer in vitro and in vivo

Circulating soluble Fas (sFas) and expression of Fas-ligand on cancer cells are mechanisms of immune escape. The aim of the present study was to investigate expression and production of Fas and Fas-ligand on bladder cancer cell lines of different grade as a basic mechanism of their secretion in vivo. sFas and sFas-ligand serum levels of patients with different stage of bladder cancer were examined to determine the possible clinical use of these molecules as tumor markers. Bladder cancer cell lines RT4 (G1), RT112 (G1), T24 (G3) and SUP (G4) were analyzed by flowcytometry for Fas and Fas-ligand expression. To determine if the Fas-ligand gene is transcribed in these bladder cancer cell lines, RT-PCR was performed on mRNA extracted from these cell lines. Production of sFas and sFas-ligand was examined in cell culture supernatants of the cancer cells as well as in the serum of 62 patients with bladder cancer by a specific ELISA test. We demonstrate that Fas is expressed in similar levels on all human bladder carcinoma cell lines. In T24 (G3) and SUP (G4) cell lines we were able to detect the Fas-ligand protein, whereas no Fas-ligand protein could be found in RT4 and RT112 (G1) cells. Fas-ligand mRNA was expressed in all bladder cancer cell lines. Furthermore, all bladder cancer cell lines produce sFas but no sFas-ligand in spite of mRNA expression. The range of sFas levels in the serum of all patients with bladder cancer was large and did not show a correlation to the histopathological stage of bladder cancer. Although there is in vitro evidence that sFas and Fas-ligand play a role in bladder cancer, no correlation between the sFas and s Fas-ligand serum levels and the histopathological stage of bladder cancer could be found. Therefore, serum sFas and sFas-ligand have to date limited clinical relevance.     

Transient rise in serum soluble Fas (APO-1/CD95) in patients undergoing cardiac surgery

The Fas molecule, also designated APO-1/CD95, belongs to the tumor necrosis factor (TNF) receptor family. It is a widely expressed membrane-anchored protein that induces apoptosis by Fas/Fas ligand (Fas-L) mediation. It was reported that Fas-mediated apoptosis plays an important role in regulation of the immune system, systemic inflammatory response, and ischemia/reperfusion injury. A soluble form of Fas (sFas) is produced either through the proteolytic cleavage of membrane-bound receptors or by alternative splicing, and sFas is thought to be implicated in apoptosis. In addition, sFas released damaged cells, and elevated serum levels of sFas reflect systemic tissue damage. To examine the specificity of sFas production during cardiac surgery with cardiopulmonary bypass, we serially measured the serum sFas levels in 13 patients during and after surgery. Blood samples were obtained before surgery, at the end of cardiopulmonary bypass, at the end of surgery, and at 12 h after surgery. Levels of serum sFas were determined by sandwich ELISA. Seven patients undergoing other types of surgeries served as controls. Although increased sFas was not observed in the control group, a significantly higher sFas level was detected in cardiac surgical patients at the end of surgery than before surgery (p = 0. 028), and the level decreased at 12 h after surgery. A significant correlation was observed between the maximum sFas values and the length of surgery (r = 0.659, p = 0.012) and cardioplegic arrest (r = 0.559, p = 0.046). Elevated serum sFas levels were observed in patients undergoing cardiac surgery, and these serum sFas levels reflect the severity of a surgery. sFas may play an important role in the pathophysiology of surgical damage caused by cardiac surgery with cardiopulmonary bypass.     

大肠癌浸润淋巴细胞Fas配基表达与癌细胞凋亡的关系

目的　研究结肠直肠癌浸润淋巴细胞Fas配基 (FasL)表达与癌细胞凋亡的关系。　方法　用免疫组化方法检测 86例结肠直肠癌及切缘正常大肠组织的Fas蛋白、FasL的表达 ;用TdT酶介导的DNA3′ OH末端 缺口染色方法检测癌细胞的调亡指数 ,并分析浸润淋巴细胞FasL表达与癌细胞凋亡指数的关系。　结果　 86例结肠直肠癌组织呈Fas蛋白染色阳性 5 3例 (6 1 6 % ) ,正常大肠组织则未见染色 ,癌组织旁浸润淋巴细胞FasL染色阳性 6 9例 (80 2 % )。癌组织Fas蛋白阳性组中 ,浸润淋巴细胞FasL染色程度与癌细胞凋亡指数呈正相关 (P <0 0 1) ,FasL染色越强 ,凋亡癌细胞越多。Fas蛋白阴性组中则不存在这种相关性。　结论　癌旁浸润淋巴细胞通过其表面的FasL与大肠癌细胞表面的Fas蛋白作用 ,是诱导癌细胞凋亡的途经之一。提高癌细胞Fas蛋白的表达及应用特异性Fas抗体 ,将为大肠癌治疗提供新思路。