腺病毒介导的双自杀基因对胆管癌细胞的体外杀伤作用

目的：研究CD、HSV-tk融合基因对胆管癌细胞QBC939生长的抑制作用。 方法： 构建了含CD、HSV-tk融合基因的重组腺病毒载体，体外转染胆管癌细胞株QBC939，并与单基因转染对照，观察其对胆管癌细胞的杀瘤活性。 结果： CD、HSV-tk融合基因经体外转染可在人胆管癌细胞QBC939中高效表达。与单一自杀基因转染组对照，双基因组对前体药物的敏感性增强，表现出更强的抗肿瘤作用，具有更明显的旁观者效应（BSE）。 结论： CD/HSV-tk双自杀基因对胆管癌细胞具有更强的抗肿瘤作用，是一种较单自杀基因治疗更合理的研究方向。     

胞嘧啶脱氨酶基因/5-氟胞嘧啶联合在肿瘤基因治疗中的研究

临床上许多恶性肿瘤在外科手术治疗后还需进一步作化学治疗,但目前化疗的应用受到很大的限制,其中一个重要原因就是化疗药物在体内杀伤肿瘤细胞的同时往往对机体正常细胞也有明显的损害,使患者难以耐受大剂量的化疗,无法达到彻底杀灭肿瘤细胞的目的,这也是长期以来恶性肿瘤复发率居高不下的原因之一。 因此,如何减少化疗的副作用成为人们普遍关注的问题。近年来随着分子生物学的发展,肿瘤的基因治疗已开始应用于临床。而利用基因治疗与化疗联合应用方案提高化疗的敏感性和选择性,减轻副作用,改善肿瘤疗效,是当前基因治疗中的一个…     

酵母菌胞嘧啶脱氨酶/5-氟胞嘧啶自杀基因系统对人原代T细胞的作用

目的:酵母菌胞嘧啶脱氨酶基因(YCD)是新型自杀基因,利用重组逆转录病毒载体(RV),将YCD高效导入人原代T细胞,并在体内外探讨转基因T细胞的功能及"自杀"效应。方法:构建RV载体MSCV-YCD-puroR,瞬时质粒共转染法制备RV,流动感染法或Retronectin 离心法感染人原代T细胞,嘌呤霉素筛选获得T-YCD-puroR,检测转基因T细胞对肿瘤细胞的杀伤及细胞因子分泌功能,在体外和SCID小鼠体内,探讨前体药物5氟胞嘧啶(5-FC)对T-YCD-puroR细胞的杀伤效应等。结果:病毒滴度为(5.0±3.7)×106TU/mL。流动转染法对人原代T细胞的基因导入率46.0%±9.6%,2轮离心 Retronectin法的基因导入率为60.3%±7.6%(n=3)。经嘌呤霉素筛选48h获得T-YCD-puroR细胞,其基因阳性率96.7%±1.5%,并可大量扩增。与野生T细胞比较,T-YCD-puroR细胞杀伤K562肿瘤细胞的能力及细胞因子分泌功能无统计学意义。体外实验显示,与野生T细胞比较,T-YCD-puroR细胞对puromycin的IC50上升了304.9倍(0.071mg/Lvs22.3mg/L),对5-FC的IC50下降了133.3倍(656.0μmoL/Lvs4.9μmoL/L)。T-YCD-puroR细胞静脉接种于SCID小鼠,7d后每只小鼠腹腔注射5μmoL的5-FC,连续7d,首次给药24h后即可观察到SCID小鼠外周血中CD45 细胞显著下降(P<0.01)。结论:YCD自杀基因导入对人原代T细胞功能无显著影响,T-YCD-puroR细胞可以在体内外被5-FC有效杀伤。本研究为进一步研究打下了良好的基础。     

活化诱导胞嘧啶核苷脱氨酶基因多态性与儿童哮喘的关系

目的　活化诱导胞嘧啶核苷脱氨酶 (activation inducedcytidinedeaminase ,AICDA)是一种RNA编辑脱氨酶 ,在免疫球蛋白类别转换中起重要作用。本文探讨AICDA基因 840 8C T基因多态性与儿童哮喘及血浆总IgE的关系。方法　采用聚合酶链反应 限制性片段长度多态性分析技术(PCR RFLP)检测AICDA基因多态性。结果　 10 2例哮喘患儿AICDA 840 8位T T基因型频率显著高于72例对照组 ( χ2 =10 .31,P <0 .0 1) ,哮喘患者AICDAT T基因型的相对危险度 (RR)为 4.6 ,T等位基因频率也显著高于对照组 ( χ2 =7.736 ,P <0 .0 1) ,5岁以上T T基因型儿童哮喘患者血浆总IgE显著高于C C和C T基因型患者 (P <0 .0 5 )。结论　AICDA 840 8位T T基因型和T等位基因与儿童哮喘发病相关 ;5岁以上儿童哮喘患者T T基因型与高血浆总IgE相关。     

活化诱导的胞嘧啶核苷脱氨酶与免疫球蛋白基因多样性及免疫性疾病的关系

类别转换重组(CSR)和体细胞高频突变(SHM)是活化的B淋巴细胞在免疫球蛋白基因组中启动第2次多样化进程的2个重要过程,然而活化诱导的胞嘧啶核苷脱氨酶(AID)是CSR和SHM过程中必需的酶.研究发现,AID在免疫球蛋白基因多样性中发挥重要作用,并且与免疫性疾病有关.现就AID与免疫球蛋白基因多样性及免疫性疾病之间的关系进行综述.     

构建携带胞嘧啶脱氨酶基因骨髓间充质干细胞及其对胶质瘤细胞的体外抑制作用

背景：转染胞嘧啶脱氨酶基因的骨髓间充质干细胞能有效地将化疗前药5-氟尿嘧啶转化成具有细胞毒性的化疗药物5-氟尿嘧啶,并在体外对胶质瘤细胞有显著的生长抑制作用。 目的：探讨以骨髓间充质干细胞为基因治疗载体表达外源基因胞嘧啶脱氨酶基因对胶质瘤C6细胞增殖的影响。 方法：分离、培养小鼠间充质干细胞,构建胞嘧啶脱氨酶基因与GFP联合的慢病毒载体,通过慢病毒包装法将胞嘧啶脱氨酶基因及GFP转染至小鼠骨髓间充质干细胞,获得稳定表达胞嘧啶脱氨酶基因及GFP的骨髓间充质干细胞,使其与胶质瘤C6细胞共培养,在培养液中加入5-氟胞嘧啶后应用流式细胞仪检测胞嘧啶脱氨酶基因对胶质瘤细胞的增殖影响。 结果与结论：慢病毒介导的胞嘧啶脱氨酶基因及GFP基因成功转染小鼠骨髓间充质干细胞形成C57BL/6 mMSC-codA/eGFP细胞,C57BL/6 mMSC-codA/eGFP在5-氟胞嘧啶的作用下可引起胶质瘤C6细胞的明显凋亡,在5-氟胞嘧啶浓度为1×10⁶ μg/ L条件下C6胶质瘤细胞凋亡率为60%(P < 0.05)。提示,C57BL/6 mMSC-codA/eGFP可将5-氟胞嘧啶转化成5-氟尿嘧啶并对C6胶质瘤细胞生长有显著的限制作用甚至是致死效应。     

活化诱导胞嘧啶核苷脱氨酶基因多态性与成人哮喘及IgE的相关性研究

目的:探讨活化诱导胞嘧啶核苷脱氨酶(Activation-induced ey-tidine deaminase.AICDA)8408C/T基因的多态性与成人哮喘及血浆总IgE的关系。方法:采用聚合酶链反应-限制性片段长度多态性分析技术(PCR-RFLP)检测AICDA基因多态性。结果:成人哮喘患者AICDA 8408位T/T基因型频率与对照组相比较差异有显著性(P<0.05),T等位基因频率与对照组相比较差异无显著性(P>0.05)。成人哮喘组T/T基因型血浆总IgE高于C/C、C/T基因型(P<0.05)。结论:成人哮喘患者AICDA 8408位T/T基因型与成人哮喘的发病及高血浆总IgE均相关。     

5-氟胞嘧啶对胞嘧啶脱氨酶基因修饰的胰腺癌细胞凋亡的诱导作用

目的:探讨5-氟胞嘧啶(5-FC)对基因修饰的胰腺癌细胞凋亡的影响及特征。方法: 以腺病毒介导的胞嘧啶脱氨酶(CD)基因转染胰腺癌SW1990细胞,以Western blot检测目标基因蛋白水平表达,通过细胞形态学、脱氧核糖核酸(DNA)凝胶电泳和流式细胞术观察5-FC对表达CD基因的SW1990细胞凋亡的影响作用。结果: 以含CD基因的重组腺病毒转染的SW1990细胞,给予5-FC100 μmol·L^-1, 培养48 h,细胞出现典型的凋亡形态、DNA梯形改变及凋亡峰, 细胞在G₁,S 和G₂/M各期分别为64%、11%和7%,凋亡率达34.6%。结论:5-FC的上述诱导凋亡作用可能是胰腺癌CD基因疗法的重要机制。     

辐射敏感性启动子调控CD基因表达联合125I照射对膀肤癌EJ细胞的杀伤作用

以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为...     

辐射敏感性启动子调控CD基因表达联合125I照射对膀肤癌EJ细胞的杀伤作用

以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为启动子、CD基因为目的基因的新质粒,并采用DNA测序法测定E8和CD基因的序列;脂质体Lipofectamine2000介导pE8-CD转染膀胧癌EJ细胞,用[3]I照射(吸收剂量为2舜)后,蛋白质免疫印迹分析(Western blot)测定CD蛋白表达;在转染EJ细胞中分别加人不同剂量125 I勺和5-FC,四哇盐比色法(MTT法)测定各组细胞存活率,并以未经125 I勺照射组、未加5-FC组和5-氟尿啼吮(5-FU)组(阳性对照组)进行对照.DNA测序显示构建的pE8-CD质粒含E8启动子及CD基因序列;Westem blot可检测到CD基因表达;125 I加5-FC组细胞存活率明显低于未经125 I照射组及未加5-FC组,与5-FU组相近.这表明放射性核素与基因治疗联合对肿瘤细胞具有协同杀伤作用.     

Circadian gene expression predicts patient response to neoadjuvant chemoradiation therapy for rectal cancer

Preoperative neoadjuvant chemoradiation therapy may be useful in patients with operable rectal cancer, but treatment responses are variable. We examined whether expression levels of circadian clock genes could be used as biomarkers to predict treatment response. We retrospectively analyzed clinical data from 250 patients with rectal cancer, treated with neoadjuvant chemoradiation therapy in a single institute between 2011 and 2013. Gene expression analysis (RT-PCR) was performed in tissue samples from 20 patients showing pathological complete regression (pCR) and 20 showing non-pCR. The genes analyzed included six core clock genes (Clock, Per1, Per2, Cry1, Cry2 and Bmal1) and three downstream target genes (Wee1, Chk2 and c-Myc). Patient responses were analyzed through contrast-enhanced pelvic MRI and endorectal ultrasound, and verified by histological assessment. pCR was defined histologically as an absence of tumor cells. Among the 250 included patients, 70.8% showed regression of tumor size, and 18% showed pCR. Clock, Cry2 and Per2 expressions were significantly higher in the pCR group than in the non-pCR group (P<0.05), whereas Per1, Cry1 and Bmal1 expressions did not differ significantly between groups. Among the downstream genes involved in cell cycle regulation, c-Myc showed significantly higher expression in the pCR group (P<0.05), whereas Wee1 and Chk2 expression did not differ significantly between groups. Circadian genes are potential biomarkers for predicting whether a patient with rectal cancer would benefit from neoadjuvant chemoradiation therapy.     

IL—6转基因表达诱导大肠癌细胞凋亡

通过分子克隆技术将ＩＬ－６基因导入大肠癌细胞ＨＴ－２９，建立了目的基因转导株ＨＴ－２９ＩＬ－６细胞，该转导株接种子裸鼠皮下后，与非转导株相比，长出肿瘤的时间延后，最终瘤径明显较小，而且移植瘤标本镜下可见大量的核固缩，核染色质加深，核断裂等凋亡细胞，用携带ＩＬ－６基因的重组逆转病毒液感染大肠癌细胞Ｌｏｖｏ后，仅以１．２５μｍｏｌ／Ｌ的ＶＵＭＯＮ－２６（简称ＶＭ－２６）即可诱导该转染株发生明显凋亡，而     

Survival in rectal cancer is predicted by T cell infiltration of tumour-associated lymphoid nodules

Lymphoid nodules are a normal component of the mucosa of the rectum, but little is known about their function and whether they contribute to the host immune response in malignancy. In rectal cancer specimens from patients with local (n = 18), regional (n = 12) and distant (n = 10) disease, we quantified T cell (CD3, CD25) and dendritic cell (CD1a, CD83) levels at the tumour margin as well as within tumour‐associated lymphoid nodules. In normal tissue CD3⁺, but not CD25⁺, T cells are concentrated at high levels within lymphoid nodules, with significantly fewer cells found in surrounding normal mucosa (P = 0·001). Mature (CD83), but not immature (CD1a), dendritic cells in normal tissue are also found clustered almost exclusively within lymphoid nodules (P = < 0·0001). In rectal tumours, both CD3⁺ T cells (P = 0·004) and CD83⁺ dendritic cells (P = 0·0001) are also localized preferentially within tumour‐associated lymphoid nodules. However, when comparing tumour specimens to normal rectal tissue, the average density of CD3⁺ T cells (P = 0·0005) and CD83⁺ dendritic cells (P = 0·0006) in tumour‐associated lymphoid nodules was significantly less than that seen in lymphoid nodules in normal mucosa. Interestingly, regardless of where quantified, T cell and dendritic cell levels did not depend upon the stage of disease. Increased CD3⁺ T cell infiltration of tumour‐associated lymphoid nodules predicted improved survival, independent of stage (P = 0·05). Other T cell (CD25) markers and different levels of CD1a⁺ or CD83⁺ dendritic cells did not predict survival. Tumour‐associated lymphoid nodules, enriched in dendritic cells and T cells, may be an important site for antigen presentation and increased T cell infiltration may be a marker for improved survival.     

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non‐small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine‐induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real‐time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL‐expressing cells was evaluated by flow cytometry, and caspase‐8 and caspase‐3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine‐activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti‐Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane‐bound FasL (mFasL) and of mFasL‐positive apoptotic H292 cells, as well as caspase‐8 and caspase‐3 cleavage. Moreover, gemcitabine increased CH11‐induced caspase‐8 and caspase‐3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine‐treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up‐regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas‐dependent apoptosis mediated by caspase‐8 and caspase‐3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine‐induced lung cancer cell killing.     

p53基因与顺铂联合作用对人卵巢癌细胞的杀伤效果

目的 :探讨外源性p5 3基因转染对人卵巢癌细胞系SKOV 3的生物学行为影响及与顺铂联合作用后对人卵巢癌细胞的杀伤效果。方法 :用脂质体介导的转染技术 ,将p5 3基因的真核细胞表达载体 (PcDNA p5 3)导入不表达p5 3的人卵巢癌SKOV 3细胞中 ,经G4 18筛选 ,Northernblot及Westernblot鉴定后 ,观察其对细胞生物学行为的影响及与顺铂联合作用后对细胞集落形成的影响。结果 :转染后获得稳定表达p5 3的转染克隆。外源性p5 3基因的表达明显抑制了卵巢癌细胞的生长及集落形成能力 ,使细胞阻滞于G1 期 ,并增加了卵巢癌细胞对顺铂的敏感性。结论 :外源性野生型p5 3基因能抑制卵巢癌细胞的生长 ,与顺铂联合作用后增强了对人卵巢癌细胞的杀伤效果。     

Cancer stem cell marker CD133+ tumour cells and clinical outcome in rectal cancer

Aims:  The CD133 antigen has been identified as a putative stem cell marker in colorectal cancer tissues. According to the cancer stem cell hypothesis, CD133+ cells determine long-term tumour growth and are therefore suspected of influencing clinical outcome. The aim was to investigate the prognostic value of CD133 expression in rectal cancer patients after preoperative radiation and curative resection.
Methods and results:  The expression of the CD133 stem cell antigen in a series of 73 patients with rectal cancer of various ypTNM stages was analysed by immunohistochemistry on formalin-fixed paraffin-embedded sections. The prognostic value of CD133 expression and other clinicopathological factors was evaluated. On multivariate survival analysis, the proportion of CD133+ cells was a significant ( P  < 0.05) prognostic factor for adverse disease-free survival and overall survival independent of ypTNM stage, tumour differentiation or lymphovascular invasion.
Conclusions:  CD133 stem cell antigen expression correlates with patient survival in rectal cancer, lending support to the current cancer stem cell hypothesis.     

非小细胞肺癌中RET融合基因的研究进展

存在于1%~2%的非小细胞肺癌患者中的RET融合基因具有鲜明的临床病理学特征,且RET抑制剂对其治疗有效,提示RET融合基因已成为该类患者个体化分子靶向治疗的新靶点。本文将对RET融合基因的特点及其在非小细胞肺癌(non-small cell lung cancer,NSCLC)患者中的表达和治疗的研究进展进行阐述。     

Mechanisms of oncogenesis in colon versus rectal cancer

Observations support the theory that development of left- and right-sided colorectal cancers may involve different mechanisms. This study investigated different genes involved in oncogenesis of colon and rectal cancers and analysed their prognostic value. The study group comprised 35 colon and 42 rectal cancers. Rectal cancer patients had been treated with standardized surgery performed by an experienced rectal cancer surgeon. Mutation analysis was performed for p53 in eight colon cancers and for APC and p53 in 22 rectal cancers. MLH1, MSH2, Bcl-2, p53, E-cadherin and beta-catenin were investigated by immunohistochemistry in all colorectal tumours. APC mutation analysis of the MCR showed truncating mutations in 18 of 22 rectal tumours (82%), but the presence of an APC mutation was not related to nuclear beta-catenin expression (p=0.75). Rectal cancers showed significantly more nuclear beta-catenin than colon cancers (65% versus 40%, p=0.04). p53 mutation analysis corresponded well with p53 immunohistochemistry (p<0.001). Rectal cancers showed significantly more immunohistochemical expression of p53 than colon cancers (64% versus 29%, p=0.003). In rectal cancers, a significant correlation was found between positive p53 expression and worse disease-free survival (p=0.008), but not in colon cancers. Cox regression showed that p53-expression (p=0.03) was an independent predictor for disease-free survival in rectal cancers. This study concluded that rectal cancer may involve more nuclear beta-catenin in the APC/beta-catenin pathway than colon cancer and/or nuclear beta-catenin may have another role in rectal cancer independently of APC. The p53-pathway seems to be more important in rectal cancer, in which it also has independent prognostic value. When prognostic markers are investigated in larger series, differences in biological behaviour between colon and rectal cancer should be considered.