前列腺特异性抗原体外诱导结肠癌特异性CTLs

目的检测前列腺特异性抗原(PSA)在结肠癌中的表达水平,探讨PSA作为结肠癌主动免疫治疗新靶点的可能性。方法用RT-PCR方法检测结肠癌细胞系中PSAmRNA的表达水平;免疫组织化学方法检测结肠癌细胞中PSA蛋白的表达水平。利用PAP表位肽对结肠癌患者的PBMCs进行体外诱导,ELISA法检测PSA特异性IFN-γ分泌水平;51Cr释放法检测PSA多肽特异性CTLs对结肠癌细胞的细胞毒性。结果4种结肠癌细胞(colo201,colo205,SW480和SW620)表达PSA mRNA和PSA蛋白。HLA-A+24结肠癌患者的PBMCs经体外诱导产生的CTLs可特异性杀伤HLA-A24+/PSA+的结肠癌细胞,CTLs的细胞毒活性依赖于CD8+的T淋巴细胞。结论结肠癌患者的外周血中存在PSA特异性CTLs前体细胞,PSA有可能成为结肠癌特异性免疫治疗的靶点。     

产气肠杆菌磷酸酶基因克隆表达及其特性

产气肠杆菌(E.aerogenes IAM1183)的酸性磷酸酶,具有特异转移磷酸基团到核苷5′-位的功能,是极具应用价值的酶。本研究以产气肠杆菌(E.aerogenes IAM1183)的酸性磷酸酶(PhoC)为研究对象,采用基因工程手段,克隆了phoC基因,构建成酸性磷酸酶(PhoC)与麦芽糖结合蛋白(MBP)的融合表达载体pMKL-c2x-phoC,把其转化到不同的宿主,进行催化研究。结果发现构建成的基因工程菌具有高于原始菌催化5′-IMP生成的能力、水解5′-IMP的活性降低、酶促反应的最适pH值近中性等特点。     

酸性离子液体及其在催化酯化反应中的应用研究进展

本文介绍了酸性离子液体的分类与合成方法,综述了近年来Lewis酸性离子液体、Br?nsted酸性离子液体、B-L双酸性离子液体在酯化反应中的研究进展,对酸性离子液体的酸性表征方法进行总结,提出酸性离子液体在酯化反应中的问题与研究方向。     

Caspase-3、Bcl-2和Bak在胃癌中的表达及意义

目的 研究Caspase-3,Be1-2.Bak等凋亡相关基因蛋白在胃癌中的表达与胃癌临床病理特征和预后的相关性.方法 将100例无术前放化疗史的胃癌手术标本和30例对照组胃粘膜制成组织芯片,取样针直径2.0 mm.用免疫组化方法分别检测Caspase-3.Bel-2,Bak的表达;用原位末端标记法进行细胞凋亡指数捡测.对随访14个月至13年的47例作生存分析.结果 获得2个组织芯片蜡块,分别含114和1164个位点.Caspase-3在正常粘膜的表达(90%)显著高于胃癌组织(56%)(P＜0.01),其表述与胃癌组织学分化.血管神经侵犯相关(P＜0.05),与淋巴结转移和国际抗癌联盟(UICC)临床分期及预后无关.Bl-2和Bak在胃癌中的表达分别为42%和47%,表达均与组织学分化相关(P＜0.05),与淋巴结转移、血管神经侵犯、UICC分期及预后无关.结论 Caspase-3.Bcl-2和Bak均参与细胞凋亡调控和胃癌生物学进程,但其表达与胃癌预后无关.UICC分期仍然是胃癌的独立预后指标.     

人重组酸性成纤维细胞生长因子在大肠杆菌中的表达及鉴定

利用逆转录-DNA聚合酶链式反应，从体外传代培养的人肺成纤维细胞中钓出人酸性成纤维细胞生长因子（aFGF）全编码区的cDNA，将其克隆人pKK223-质粒，在大肠杆菌JM105中高效表达出人重组aFGF。此aFGF经Heparin－Sepharose纯化后，表现了很好的生物学活性。     

重组靶向毒素hIL6(T22)-PE38在大肠杆菌中的表达及其细胞毒性

目的 在大肠杆菌中表达、纯化重组靶向毒素hIL6(T22)-PE38,并检测其细胞毒性。方法以PHis-hIL6(T22)-PE38质粒为模板,PCR扩增hIL6(T22)-PE38基因,插入表达载体pET-28a(+)的NcoⅠ和XhoⅠ多克隆位点,构建重组表达质粒,分别转化至大肠杆菌BL21(DE3)、Rosettablue(DE3)和BL21(DE3)pLysS中,IPTG诱导表达,并对表达条件进行优化。表达产物经亲和层析纯化后,检测其细胞毒性。结果重组表达质粒pET-28a-hIL6(T22)-PE38经双酶切和测序证明构建正确。表达的重组毒素相对分子质量约56 000,在大肠杆菌Rosettablue(DE3)中表达量最高。最适诱导表达条件为:1 mmol/L IPTG 28℃诱导4 h。重组毒素能选择性地杀伤人骨髓瘤细胞U266和鼠骨髓瘤细胞SP2/0,体外对U266和SP2/0细胞的IC50分别为0.5～1.0和1.0～1.5μg/ml。结论重组靶向毒素hIL6(T22)-PE38在大肠杆菌中成功获得可溶性表达,纯化的蛋白可明显选择性杀伤U266和SP2/0细胞。     

真核表达质粒pcDNA3.1(+)-Vasostatin的构建及其在293T细胞中的表达

目的构建真核表达质粒pcDNA3.1(+)-Vasostatin并检测其在真核细胞内的表达水平。方法将带有信号肽的Vasostatin基因片段克隆至pcDNA3.1(+)真核表达载体上,经酶切鉴定及测序分析证明构建成功后,以脂质体介导法转染293T细胞,通过Westernblot法,检测其在293T细胞内的表达水平。结果所构建的真核表达质粒pcDNA3.1(+)-Va-sostatin转染293T细胞后,在其裂解的上清液中,检测到目的基因的表达。结论已成功构建了pcDNA3.1(+)-Vasostatin表达载体,并在真核细胞中表达了目的蛋白。     

小鼠H22肝癌移植瘤模型在PD-1单抗治疗肝癌中的应用

目的 探讨小鼠H22肝癌移植瘤模型在PD-1单抗治疗肝癌中的应用.方法 制备小鼠H22肝癌移植瘤模型,用淋巴细胞分离液分离肿瘤浸润淋巴细胞(tumor-infiltrating lymphocytes,TILs),然后采用流式细胞术分析TILs中T细胞、CD8+T细胞、CD4+T细胞和调节性T细胞比例,以及肿瘤组织和脾...     

PAFC的制备及其在酸性红73废水处理中的应用

以钢铁镀件前处理产生的酸洗废液和工业废铝为原料,采用氧化聚合法制备聚合氯化铝铁(PAFC)絮凝剂,并用PAFC处理低浓度的酸性红73,考察各因素对去除效果的影响。实验结果表明:在最佳条件下,PAFC处理10 mg/L的酸性红73的去除率高于85%,处理效果优于市售PAC,但与市售PAFC相当;铝铁比为9∶1的PAFC处理5~20 mg/L的酸性红73,其上清液出水色度均达到了《纺织染整工业水污染物排放标准》(GB 4287—2012)中的特别排放限值要求;达到了以废治废的目的。     

Human Prostatic Acid Phosphatase: Structure,Function and Regulation

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.     

Survivin-siRNA真核表达载体的构建及其对胃癌SGC-7901细胞增殖和凋亡的影响

目的构建Survivin-siRNA真核表达载体,并探讨其对胃癌SGC-7901细胞增殖和凋亡的影响。方法化学合成4条能转录出siRNA的模板DNA,各75个碱基,退火形成2条双链DNA,双酶切后插入pSUPER.basic载体。将阳性重组质粒转染SGC-7901细胞,进行细胞计数,并用MTT法检测细胞的增殖活性;半定量RT-PCR检测细胞Survivin基因mRNA的转录水平;流式细胞术检测细胞周期的变化。结果PCR和酶切鉴定表明Survivin-siRNA真核表达载体构建正确,其能下调SGC-7901细胞Survivin基因mRNA的转录水平,抑制SGC-7901细胞生长和增殖,并促进细胞凋亡,使G0/G1和亚G1期细胞增多,S期细胞减少。结论已成功构建了Survivin-siRNA真核表达载体,其能下调SGC-7901细胞中Survivin基因mRNA的转录水平,使细胞增殖减弱,凋亡增加,为RNA干扰技术应用于胃癌的基因治疗提供了一定的实验依据。     

Establishment of In Vitro and In Vivo Anticolorectal Cancer Efficacy of Lithocholic Acid-Based Imidazolium Salts

Imidazolium salts (IMSs) are the subject of many studies showing their anticancer activities. In this research, a series of novel imidazolium salts substituted with lithocholic acid (LCA) and alkyl chains of various lengths (S1–S10) were evaluated against colon cancer cells. A significant reduction in the viability and metabolic activity was obtained in vitro for DLD-1 and HT-29 cell lines when treated with tested salts. The results showed that the activities of tested agents are directly related to the alkyl chain length, where S6–S8 compounds were the most cytotoxic against the DLD-1 line and S4–S10 against HT-29. The research performed on the xenograft model of mice demonstrated a lower tendency of tumor growth in the group receiving compound S6, compared with the group receiving 5-fluorouracil (5-FU). Obtained results indicate the activity of S6 in the induction of apoptosis and necrosis in induced colorectal cancer. LCA-based imidazolium salts may be candidates for chemotherapeutic agents against colorectal cancer.     

南蛇藤提取物诱导SGC-7901胃癌细胞凋亡及其机制

目的观察南蛇藤乙酸乙酯提取物和正丁醇提取物在体外诱导SGC-7901胃癌细胞凋亡的作用,并初步探讨其分子机制。方法制备南蛇藤乙酸乙酯和正丁醇提取物,采用MTT法检测二者对SGC-7901胃癌细胞增殖的影响;应用FITC-AnnexinⅤ及碘化丙锭(PI)双标法,通过流式细胞仪(FCM)观察SGC-7901细胞的凋亡率及P53蛋白的表达。结果不同浓度的南蛇藤乙酸乙酯和正丁醇提取物(15、30、60、120μg/ml)对SGC-7901细胞的增殖均有一定的抑制作用,且呈剂量依赖性。两种南蛇藤提取物(60、120、240、480μg/ml)可剂量依赖性地诱导肿瘤细胞凋亡,且随着浓度的升高,SGC-7901细胞P53蛋白的表达也增加,浓度为240和480μg/ml的提取物组与对照组相比,差异有统计学意义。结论南蛇藤乙酸乙酯提取物和正丁醇提取物在体外可诱导SGC-7901细胞凋亡,上调细胞中P53基因的表达可能是其抗肿瘤作用的分子机制之一。     

Synthesis,In Vitro,and Computational Studies of PTP1B Phosphatase Inhibitors Based on Oxovanadium(IV) and Dioxovanadium(V) Complexes

One of the main goals of recent bioinorganic chemistry studies has been to design and synthesize novel substances to treat human diseases. The promising compounds are metal-based and metal ion binding components such as vanadium-based compounds. The potential anticancer action of vanadium-based compounds is one of area of investigation in this field. In this study, we present five oxovanadium(IV) and dioxovanadium(V) complexes as potential PTP1B inhibitors with anticancer activity against the MCF-7 breast cancer cell line, the triple negative MDA-MB-231 breast cancer cell line, and the human keratinocyte HaCaT cell line. We observed that all tested compounds were effective inhibitors of PTP1B, which correlates with anticancer activity. [VO(dipic)(dmbipy)]·2 H₂O (Compound 4) and [VOO(dipic)](2-phepyH)·H₂O (Compound 5) possessed the greatest inhibitory effect, with IC₅₀ 185.4 ± 9.8 and 167.2 ± 8.0 nM, respectively. To obtain a better understanding of the relationship between the structure of the examined compounds and their activity, we performed a computer simulation of their binding inside the active site of PTP1B. We observed a stronger binding of complexes containing dipicolinic acid with PTP1B. Based on our simulations, we suggested that the studied complexes exert their activity by stabilizing the WPD-loop in an open position and limiting access to the P-loop.     

MMP-2反义寡核苷酸对人胃癌SGC7901细胞增殖和侵袭能力的影响

目的研究基质金属蛋白酶-2(MMP-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖及侵袭能力的影响。方法将不同浓度的硫代磷酸化修饰的MMP-2ASODN转染入人中低分化胃腺癌SGC7901细胞株,RT-PCR法检测细胞MMP-2基因mRNA的转录水平;Transwell试验检测细胞体外迁移和侵袭能力变化;MTT法检测细胞的增殖活性。结果转染MMP-2ASODN后,SGC7901细胞中MMP-2基因mRNA的转录水平显著降低,细胞的体外迁移和侵袭能力受到抑制,且抑制作用随ASODN浓度的增加而增强;而细胞的增殖活性无明显变化。结论MMP-2ASODN可抑制胃腺癌SGC7901细胞MMP-2基因mRNA的转录水平及细胞的体外迁移和侵袭能力,但与胃癌细胞的增殖活性无明显相关性。     

WT1蛋白CTL表位肽基因载体的构建与鉴定

目的构建WT1(Wilms’tumor gene 1)蛋白CTL表位肽基因载体,并检测其在293T细胞中的转录。方法设计分别含WT1-126肽、WT1-235肽以及这两种肽的基因载体,并加入Th通用表位Pan-DR-Th(PADRE),应用蛋白酶体切割软件PAProC和NetChop优化各表位和间隔序列,DNA疫苗在线预测工具DyNAVacS优化真核密码子后,人工合成核苷酸序列,分别插入pUC57载体,构建pUC57-WT1质粒,测序鉴定后,酶切回收各目的片段,亚克隆至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒,转染293T细胞,RT-PCR检测各目的基因在293T细胞中的转录。采用无内毒素质粒大量提取试剂盒提取各重组质粒,采用紫外分光光度计测定质粒的纯度和浓度。结果各重组真核表达质粒经双酶切及测序鉴定证实构建正确;重组质粒携带的目的基因可在293T细胞中成功转录;各重组质粒DNA的纯度均合格,浓度在864.6～883.9μg/ml之间。结论成功构建了WT1蛋白CTL表位肽基因载体,并能在真核细胞中正常转录,为下一步在小鼠体内探讨特异性不同的CTLs群发挥抗肿瘤作用的机制奠定了基础。     

紫杉醇联合亚叶酸钙和氟尿嘧啶治疗晚期胃癌的临床观察

目的评价紫杉醇(TAX)联合亚叶酸钙(CF)和氟尿嘧啶(5-FU)方案治疗晚期胃癌的近期疗效和毒性反应。方法TAX135mg/m2,静脉滴注d1,CF200mg/m2,静滴2h,5-Fu400mg/m2,静推后继之5-Fu600mg/m2,静滴22h连续3d,每3周重复。结果可评价疗效者38例,获得CR2例(5.26%),PR19例(50.00%),NC10例(26.30%),PD6例(15.80%),总有效率(CR PR)为55.30%,中位TTP6.2个月,MST9.5个月。主要毒性反应为骨髓抑制。结论TAX联合CF、5-Fu治疗晚期胃癌患者疗效较好,毒副反应轻,可以耐受。     

Molecular Radiotherapy with 177Lu-Immunoliposomes Induces Cytotoxicity in Mesothelioma Cancer Stem Cells In Vitro

Malignant mesothelioma (MM) is a lethal tumor originating in the mesothelium with high chemotherapeutic resistance. Cancer stem cells (CSCs) persist in tumors and are critical targets responsible for tumor resistance and recurrence. The identification and characterization of CSCs may help develop effective treatment for MM. The objective of this study was to evaluate the therapeutic effect of molecular targeted radiotherapy by ¹⁷⁷Lu-labeled immunoliposomes (¹⁷⁷Lu-ILs) on CSCs of mesothelioma. MM CSCs were sorted based on CD26/CD24 expression level and their functional significances were established by small interference RNA. CSC potential of MM was evaluated for drug resistance, cell invasion, and cell growth rate in vitro. CSC metabolism was evaluated with the uptake of ¹⁸F-FDG. Therapeutic effects of ¹⁷⁷Lu-labeled immunoliposomes targeting CD26 and CD24 were evaluated in vitro through proliferation and apoptotic assays. CSCs sorted from H28 cells exhibited significant drug resistance and enhanced proliferative activity as well as increased metabolism indicated by higher ¹⁸F-FDG uptake. Treatment with ¹⁷⁷Lu-ILs, compared with ¹⁷⁷Lu-CL and ILs, showed enhanced therapeutic effects on inhibition of proliferation, up-regulation of apoptosis, and suppression of CD26 and CD24 expression. Thus, our results suggest that molecular radiotherapy targeting both CD26 and CD24 could be a promising approach for CSC-targeting therapy for MM.