目的:将吉西他滨+奈达铂+地塞米松化疗方案用于复发难治性淋巴瘤治疗中,对其效果进行评价.方法:对我院收治的67例复发难治性淋巴瘤患者以分组形式进行研究,A组行GDP方案治疗,B组行CHOP方案治疗,对两组化疗效果进行研究.结果:A组疾病缓解率为79.41％(27/34),与B组57.58％(19/33)相比,相对更高(P<0.05);A组骨髓抑制、白血胞减少毒副反应率与B组相比,组间差异不显著(P>0.05),A组为出现心脏毒性、肾毒性毒副反应,与B组9.09％、12.12％相比,组间差异明显(P<0.05).结论:吉西他滨+奈达铂+地塞米松联合化疗方案用于复发难治性淋巴瘤治疗中效果理想,可延缓疾病进展,且不会对心脏、肾脏产生毒副反应,安全性较高,值得应用.

目的:构建蛋白酶体亚基α3(proteasome subunit alpha type 3,PSMA3)基因腺病毒重组载体,并观测腺病毒介导PSMA3基因过表达对肝癌SMMC7721细胞增殖、周期、凋亡及其荷瘤生长的影响.方法:RT-PCR法扩增PSMA3基因全长CDs序列后,克隆入pTG19-T载体,再亚克隆入pAdTrack-CMV腺病毒穿梭载体中,构建重组pAdTrack/PSMA3腺病毒载体,随后与骨架质粒pAdEasy-1同源重组形成pAd/PSMA3重组腺病毒,经包装及滴度测定,获高感染力的pAd/PSMA3病毒.用pAd/PSMA3病毒感染人肝癌SMMC7721细胞,荧光显微镜观测被感染的SMMC7721细胞的绿色荧光蛋白(green fluorescent protein,GFP)的变化情况.Cell counting kit-8(CCK-8)法检测pAd/PSMA3病毒对SMMC7721细胞增殖的影响.Real-time PCR和Westernblot分别检测被感染的SMMC7721细胞的PSMA3基因、增殖细胞核抗原基因(proliferating cell nuclear antigen,PCNA)、Caspase-3促凋亡基因及其编码蛋白的表达情况.流式细胞仪(FCM)检测被感染的SMMC7721的周期、凋亡变化.将经pAd/PSMA3病毒感染的SMMC7721细胞移植裸鼠皮下以生成荷瘤,逐日观察荷瘤的生长情况.所有实验以空病毒感染及未感染的细胞做对照.结果:克隆到768 bp的PSMA3基因,并构建了其具高感染力的pAd/PSMA3重组病毒.CCK-8法结果表明,过表达PSMA3基因的SMMC7721细胞,其增殖明显减慢(P＜0.05).Real-time PCR、Western blot检测显示,pAd/PSMA3重组病毒可介导SMMC7721细胞过表达PSMA3基因,上调其Caspase-3基因及其编码蛋白的表达,并下调其PCNA基因及其蛋白的表达(P＜0.05).FCM检测证实,过表达PSMA3基因的细胞可被阻滞于G1期(细胞周期),并可促使其凋亡,其凋亡率差异有统计学意义(P＜0.05).动物实验证实,pAd/PSMA3病毒可明显抑制裸鼠荷瘤的生长(P＜0.01).结论:重组病毒pAd/PSMA3可将SMMC7721细胞阻滞于G1期(细胞周期),抑制其增殖,促使其凋亡,并可抑制其裸鼠荷瘤的生长,这些可能和下调其增殖相关PCNA蛋白,上调其凋亡相关caspase-3蛋白的表达密切相关.

BACKGROUND:Application of dendritic cells (DC) for cancer immunotherapy involves tumor-associated immunogenic antigens for effective therapeutic strategies. The present study investigated whether DC co-cultured with autologous cytokine-induced killer cells (CIK) could induce a more specific immune response against liver cancer stem cells (LCSC) generated from human hepatocellular carcinoma (HCC) cells in vitro and in vivo.METHODS:Human DC and CIK were generated from peripheral blood mononuclear cells (PBMCs) taken from consenting liver cancer patients. Flow cytometry was used to determine the phenotypes of DC and CIK, and cell proliferation. The tumor growth and anti-tumor activity of these cells were further evaluated using a nude mouse tumor model.RESULTS:We demonstrated that DC and CIK significantly enhanced the apoptosis ratio, depending on DC-CIK cell numbers, by increasing caspase-3 protein expression and reducing proliferating cell nuclear antigen (PCNA) protein expression against LCSC. The in vivo data indicated that DC-CIK exhibited significant LCSC cell-induced tumor growth inhibition in nude mice, which was most significant with LCSC antigen loaded DCs.CONCLUSIONS:The results showed, that DC-CIK cells could inhibit HCC and LCSC growths in vitro and in vivo and the most successful DC triggering of cell cytotoxic activity could be achieved by their LCSC antigen loading.

目的 检测肝细胞癌(hepatocellular carcinoma,HCC)的相关抗原,并予以验证.方法 分别提取15例HCC组织及其癌旁组织总蛋白.Western blot分别检测各总蛋白与其HCC患者自体血清的杂交情况.双向电泳(two-dimensional electrophoresis,2-DE)技术将15例混合总蛋白中的各蛋白进行分离与染色.图像分析法比对染色胶上HCC组织及其癌旁组织蛋白的表达差异,基质辅助激光解吸/电离飞行时间二级质谱(matrix-assisted laser desorption/ionization-time-of-flight with two mass analyzers for tandem mass spectrometry,MALDI-TOF MS/MS)技术及生物信息学网络数据库分别对表达差异蛋白予以分析鉴定与检索匹配,免疫组织化学与Western blot分别检测该差异蛋白在人HCC组织及其癌旁组织中的变化情况.结果 2-DE染色胶图像分析比对结果显示,HCC及其癌旁组织蛋白表达差异斑点共90个,MALDI-TOF MS/MS与数据库对其中19个蛋白斑点分析鉴定与检索匹配发现15个蛋白表达上调,4个蛋白表达下调.免疫组织化学与Western blot检测结果显示,与对照比较,上调蛋白中的DnaJ同源B亚家族成员11 (dnaJ homolog subfamily B member 11,DNAJB11)在HCC组织中的表达明显增强,下调蛋白中的蛋白酶体亚基α3 (proteasome subunit alpha type 3,PSMA3)在HCC组织中的表达则相反.结论 人原发性肝癌相关抗原DNAJB11在HCC组织中的表达上调,PSMA3在HCC组织中的表达下调.

Objective:To investigate the effects of DNAJ homolog subfamily B member 11 (DNAJB11)gene-silencing on proliferation,cell cycle and apoptosis of human hepatocellular carcinoma cell line SMMC7721.Methods:The recombinant lentiviral vector pCDH-Puro/DNAJB11-shRNA carrying the specific shRNA targeting DNAJB11 gene was established.The SMMC7721 cells were infected with high infective lentivirus pCDH-Puro/DNAJB11-shRNA.Then the proliferation of SMMC7721 cells was detected by CCK-8 method.The expression levels of DNAJB11,proliferating cell nuclear antigen (PCNA) and caspase-3 mRNAs and proteins in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.The cell cycle distribution and the apoptosis rate of SMMC7721 cells were analyzed by FCM.Results:The pCDH-Puro/DNAJB11-shRNA was constructed successfully.The proliferation of SMMC7721 cells was significantly inhibited after infection with pCDH-Puro/DNAJB11-shRNA (P ＜ 0.05).In SMMC7721 cells infected with pCDH-Puro/DNAJB11-shRNA,the expressions of DNAJB11 mRNA and protein were silenced effectively (both P ＜ 0.05).After DNAJB11 gene-silencing,the expressions of caspase-3 mRNA and protein in SMMC7721 cells were up-regulated (both P ＜ 0.05),while the expressions of PCNA mRNA and protein were down-regulated (both P ＜ 0.05).Furthermore,the cell cycle was arrested in G1 phase (P ＜ 0.01),and the apoptosis rate was significantly increased (P ＜ 0.01).Conclusion:The DNAJB11 gene-silencing can effectively suppress the proliferation of SMMC7721 cells,and promote their apoptosis.These effects may be related to downregulation of PCNA expression and upregulation of caspase-3 expression in SMMC7721 cells.

Due to non-MHC-restricted and potent cytotoxicity against hematological malignancy, one group of heterogeneous effector T cells with diverse TCR specificities, cytokine-induced killer (CIK) cells are considered as a promising therapeutic approach of leukemia. Recently, it has been found that IL-2/IL-15-activated CIK cells were superior to that of generated with IL-2 protocol, however, the underlying mechanism is not fully elucidated. In this study, we found that in IL-2/IL-15-stimulated CIK cells, autophagy was dramatically down-regulated. We produced recombinant lentivirus containing shRNA specific against Beclin 1. Upon knockdown of Beclin 1 in CIK cells, the cytotoxic activity was considerably promoted. Re-expression of Beclin 1 in Beclin 1-knockdown CIK cells compromised their cytotoxic function, confirming that autophagy decreased the cytotoxic function of CIK cells. 3-MA treatment of CIK cells also enhanced their cytotoxic activity, potentially providing possibility to widespread application. Collectively, our study demonstrates autophagy negatively regulates the cytotoxicity of CIK cells on tumor cells, providing a novel approach for promoting the therapeutic efficacy of CIK cells against hematological malignancy.

目的:探讨细胞因子诱导的杀伤细胞(cytokine induced killer,CIK)对肝癌干细胞(liver cancer stem cells,LCSCs)生长、增殖的影响.方法:用含多种细胞因子(LIF、EGF、bFGF及B27)的无血清培养液(serum-free medium,SFM)诱导SMMC7721人肝癌细胞产生LCSCs,并行LCSCs表面标志CD133、CD90的流式仪(flow cytometry,FCM)检测鉴定与裸鼠致瘤能力观测.以IFN-γ、CD3单克隆抗体(human CD3 monoclonal antibody,hCD3mAb)与IL-2等诱导肝癌患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)的悬浮细胞生成CIK.用Transwell培养小室将LCSCs与不同密度的CIK在同一培养体系内以该小室膜分隔培养24、48、72 h,采用CCK-8试剂盒检测CIK对LCSCs生长、增殖的影响.以RT-PCR与Western blot分别检测与CIK共培养的LCSCs的增殖细胞核抗原(proliferating cell unclear antigen,PCNA)基因及其编码蛋白的表达变化.结果:FCM检测显示,LCSCs高表达干细胞表面标志分子CD133、CD90.CCK-8法检测表明,与对照比较,与相同密度的CIK共培养的LCSCs,其生长、增殖减慢,48 h开始变得更加明显(P＜0.01).RT-PCR、Western blot检测显示,CIK能明显下调LCSCs的PCNA基因及其编码蛋白的表达.结论:成功诱导培养并鉴定到LCSCs.肝癌患者的CIK可明显抑制LCSCs的生长、增殖与下调其PCNA基因及其编码蛋白的表达.

OBJECTIVESTo investigate the prognosis of advanced liver cancer patients treated with CIK-DCs and the mechanism of apoptosis of HEPG 2 cells.METHODS67 patients were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were separated, of which adherent PBMCs used granulocyte 2 macrophage colony2 stimulating factor (GM2CSF), tumor necrosis factor 2α (TNF2α), and interleukin 24 (IL24) to induce DCs, which were sensitized with antigen of autologous or exogenous cancer cells to obtain Ag-DCs; suspended PBMCs used interferon 2γ (IFN2γ), IL-2, and CD 3 monoclonal antibody (CD3mAb) respectively, to induce CIK cells. DCs and CIK cells were cultured together. Flow cytometry was used to detect the phenotypes of DCs and CIK cells, and the blood retransfused into patients. Western blot and flow cytometer were used to analyze the growth cycle of HepG 2 cells and the expression of BAX and PCNA.RESULTSNo patients underwent complete remission, 5 obtained partial remission and 29 had stable disease. Of the 31 patients whose lesions could not be evaluated, 17 received effective treatment, showing that the immune response was enhanced. In vitro laboratory experiments revealed that DC-CIK cells markedly affected the growth cycle of HepG 2 cells. Analysis showed that DC-CIK cells enhanced the gene expression of BAX and inhibited the activity of PCNA.CONCLUSIONSCo-cultured DCs and CIK cells inhibit the proliferation and migration of liver cancer cells by down-regulating PCNA and up-regulating BAX. This approach may be an effective method to treat advanced liver cancer.

目的:探讨树突状细胞(dendritic cells,DCs)-细胞因子诱导的杀伤(cytokine induced killer,CIK)细胞免疫治疗联合化疗对晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)疗效及安全性的影响.方法:将272例晚期NSCLC患者随机分为治疗组(化疗联合DCs-CIK细胞免疫治疗组)和对照组(单纯化疗组).比较2组治疗后的近期疗效,及治疗前后患者免疫功能和生活质量的变化,并分析DCs-CIK细胞免疫治疗的影响因素,观察其不良反应.结果:近期疗效分析结果显示,治疗组和对照组有效率分别为49.26％和37.50％ (P＞0.05),疾病控制率为68.38％和54.41％ (P＜0.05);2组患者治疗前后外周血中表面抗原CD3+、CD8+和自然杀伤细胞所占的比值,差异均有统计学意义(P＜0.05);患者年龄、肿瘤分期和Kamofsky体能状况(Karnofsky performance status,KPS)评分是DCs-CIK细胞免疫治疗疗效的影响因素;治疗组患者生活质量优于对照组(P＜0.05);行DCs-CIK细胞免疫治疗的患者中仅有15例患者出现低热,未见其他的不良反应.结论:DCs-CIK细胞免疫治疗联合化疗可以提高晚期NSCLC的疾病控制率,改善患者的免疫功能和生活质量;患者的年龄、KPS评分及肿瘤分期可影响其疗效.

目的 构建含尿激酶型纤溶酶原激活剂(urokinase plasminogen activator,uPA)裂解位点(uPAcs)和KDEL(Lys-Asp-Glu-Leu)驻留信号序列的丝瓜毒素(Luffin-β)基因的原核载体,表达并纯化其融合毒素蛋白Lufin-β-KDEL-uPAcs(LKP),并探讨融合毒素蛋白LKP抗胃癌SGC-7901细胞的活性.方法 RT-PCR两步法克隆Luffin-β基因,引物延伸法构建Luffin-β-KDEL-uPAcs融合基因并亚克隆至原核表达载体pET-32a(+)中,诱导其表达融合蛋白Trx-EK-Luffin-β-KDEL-uPAcs (TELKP)并纯化TELKP,肠激酶(enterokinase,EK)切割TELKP后,纯化与回收目的毒素蛋白LKP,SDS-PAGE对LKP蛋白予以检测鉴定,高效液相色谱法(HPLC)对其进行纯度检测.采用cell counting kit-8 (CCK-8)、RT-PCR、Western blot等方法,体外检测毒素蛋白LKP经uPA酶裂解后释放Luffin-β的抗胃癌SGC-7901细胞的活性.结果 成功诱导重组载体pET-32a(+)/Luffin-β-KDEL-uPAcs表达相对分子质量约48.8×103含载体表达标签(Trx)的融合免疫毒素TELKP,EK酶切该蛋白获含290个氨基酸,相对分子质量约31.8×103的目的蛋白LKP.SDS-PAGE检测鉴定表明,LKP蛋白与预期大小一致,其纯度达98.8％.CCK-8、RT-PCR、Western blot等法检测显示,LKP经uPA酶体外裂解可释放具杀瘤活性的Luffin-β小分子毒素.结论 成功克隆到Luffin-β-KDEL-uPAcs融合基因,并将其构建于原核表达载体pET-32a(+)中,且诱导该载体表达了相对分子质量约31.8×103的融合毒素LKP.LKP毒素经uPA酶体外裂解能释放具杀瘤活性的Luffin-β小分子毒素.

Objective To construct a double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-βKDEL that containing a single-chain variable fragment(scFv) against vascular endothelial growth factor(VEGF) receptor KDR,urokinase plasminogen activator(uPA) cleavage site(uPAcs),tandemly ligated luffinβ and KDEL(Lys-Asp-Glu-Leu),which is a signal for retention of proteins in the endoplasmic reticulum,and to investigate its cytotoxic effect on non-small cell lung carcinoma(NSCLC) cell line.Methods The complete sequence of KDR scFv gene was synthesized.And luffin-β gene was cloned through reverse transcriptase-polymerase chain reaction(RT-PCR).KDR scFv gene was fused together with luffin-β gene by overlaying PCR.The uPAcs sequence was placed the linking position between KDRscFv and luffin-β gene,and the KDEL sequence was fused at the C-terminal of luffin-β gene.In doing so,the fused gene KDRscFv-uPAcs-Luffin-βKDEL was constructed and cloned into pET-32a(+) vector to form recombinant vector pET-32a(+) / KDRscFvuPAcs-Luffin-β-KDEL.Subsequently,the recombinant vector pET-32a(+) / KDRscFv-uPAcs-Luffin-β-KDEL was transfected into E.coli BL21,and the fusion protein Trx-EK-KDRscFv-uPAcs-Luffin-β-KDEL(TEKPLK) was expressed under induction,and then purified and digested through enterokinase(EK) to produce doubletargeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL(KPLK).Cell count kit-8(CCK-8),RT-PCR and Western blotting were employed to test the cytotoxic effect of KPLK cleavaged by uPA for setting free immunotoxin luffin-β on H460 cells.Results After induction,recombinant vector pET-32a(+)/KDRscFv-uPAcsLuffin-β-KDEL expressed the fusion protein TEKPLK,which contained the Trx tag of vector pET-32a(+),and its relative molecular mass was about 7.5 × 104.The immunotoxin protein KPLK,with its relative molecular mass of about 5.8 × 104,was produced by digesting fusion protein TEKPLK with EK.The results of CCK-8 revealed that immunotoxin KPLK exerted cytotoxic effect on H460 cells in a dose-effect manner.Its IC 50 of antitumor was about 35 ng/mL.RT-PCR and Western blotting certified that immunotoxin luffin-β,which could upregulate the expression of caspase-3 gene and its protein in the tumor cells,was released from KPLK protein cleavaged by uPA in vitro.Conclusion Fused gene KDRscFv-uPAcs-luffin-β-KDEL and its prokaryotic express vector are successfully constructed.Recombinant fusion immunotoxin KPLK(relative molecular mass about 5.8 × 104) is successfully prepared.The immunotoxin luffin-β,possessing cytotoxic effect on tumor cells,can be released from KPLK protein cleavaged by uPA in vitro.

目的 探讨经细胞因子从肝癌患者外周血贴壁单个核细胞(PBMCs)诱导的树突状细胞(DC)与杀伤细胞(CIK)对人肝癌HepG2细胞增殖、迁移的影响.方法 用肝癌患者肝癌组织裂解物(肿瘤抗原)致敏经粒细胞巨噬细胞刺激因子(GMCSF)、肿瘤坏死因子-α(TNF-α)及白细胞介素-4(IL-4)等诱导该患者PBMCs产生的DC.用干扰素-γ(IFN-γ)、IL-2和人CD3单克隆抗体(human CD3 monoclonal antibody,hCD3mAb)等诱导该PBMCs的悬浮细胞产生CIK细胞.用Tanswell培养小室将DC-CIK细胞与HepG2细胞在同一培养体系内经该小室膜分隔培养24、48 h.CCK-8法检测HepG2细胞生长变化,并绘制其生长曲线；划痕实验检测其增殖、迁移能力.RT-PCR、Western blot分别检测其增殖细胞核抗原(PCNA)基因及其编码蛋白的表达.结果 CCK-8法检测显示,DC-CIK细胞对人HepG2细胞生长具显著杀伤作用,与对照组细胞比较,差异有统计学意义(P＜0.01).划痕实验、基因与蛋白水平检测表明,DC-CIK细胞可明显抑制该瘤细胞的增殖与迁移,能在基因与蛋白水平下调该瘤细胞PCNA的表达.结论 DC-CIK细胞可显著下调肝癌细胞的PCNA基因表达,明显抑制其增殖与迁移,以发挥其杀瘤作用.

Objective:To evaluate the results of combined chemotherapy with paclitaxel plus Nedaplat in treatment of advanced nonsmall cell lung cancer in elderly.Methods: All 52 patients with advanced non-samll cell lung cancer were treated with paclitaxel plus nedaplat.PTX 135mg/m2,2 by intravenous infusion on day 1,and NDP 60-80mg/m2,2 by intravenous infusion on day 1.The treatment was repeated every 21or 28 day,up to 2 cycles.Results: All 52 patients were evaluable for efficacy.The overall response rate(RR) was 32.7%(CR:1,PR:16)and 36.3% in first treatment.The major toxicities included myelosuppression,and other side effects were mild.Conclusion: Paclitaxel plus nedaplat were effective and well tolerable regimens in the treatment of advanced non-small cell lung cancer in elderly.