金纳米棒标记的人CIK细胞用于胃癌靶向光声成像与免疫治疗

目的:利用金纳米棒标记的人细胞因子诱导杀伤细胞(CIK)主动靶向体内胃癌细胞的特性,实现胃癌的局部光声成像,观察金纳米棒在胃癌组织中的分布,测量血清中细胞因子浓度变化.方法:取健康志愿者的全血提取外周血单个核细胞,在多种细胞因子的诱导下扩增出CIK细胞;采用种子合成法制备金纳米棒并对其表面进行二氧化硅修饰与表征;利用胃癌细胞系MGC803细胞建立裸鼠胃癌皮下瘤模型;制备金纳米棒标记的CIK细胞,尾静脉注射进入荷胃癌的裸鼠模型,注射后不同时间点,进行光声成像观察金纳米棒标记的CIK细胞在肿瘤部位的分布;注射后3d,收集裸鼠血清,测量IL-2、IL-17、γ-IFN的浓度,评判免疫治疗效果.结果:CIK细胞成功制备;制备的金纳米棒长为60 nm,宽为8 nm,二氧化硅厚度为20 nm;制备的金纳米棒被人CIK细胞内吞;金纳米棒标记的人CIK细胞能够主动靶向体内胃癌组织,在胃癌组织中大量聚集,呈现出增强的光声成像信号;细胞因子IL-2、IL-17与γ-IFN的浓度显著升高(P＜0.05).结论:金纳米棒标记的CIK细胞能够主动靶向体内胃癌组织,高效聚集并呈现出增强的光声成像信号,同时升高血液中细胞因子水平,增强免疫治疗功能.     

介孔二氧化硅包覆金纳米棒增强人三阴性乳腺癌细胞放射敏感性研究

目的 探讨介孔二氧化硅包覆金纳米棒(GNRs@mSiO₂)联合6 MV X射线对人三阴性乳腺癌细胞MDA-MB-231的放射增敏作用及其机制。方法 选用透射电子显微镜观察GNRs@mSiO₂的形态和在细胞内的分布。原子吸收分光光度计检测细胞对GNRs@mSiO₂的摄取量。CCK-8实验检测不同浓度GNRs@mSiO₂对细胞的毒性。克隆形成实验检测GNRs@mSiO₂对细胞放射敏感性的影响。流式细胞仪检测细胞周期、凋亡情况及活性氧自由基的产生水平。行成组t检验差异。结果 MDA-MB-231细胞对GNRs@mSiO₂ 的摄取在24 h时达峰值,摄取量是空白对照组的(7.09±0.26)倍,且明显高于其余各组(P=0.002~0.014)。12.5~50.0 μg/mlGNRs@mSiO₂孵育细胞24 h后细胞存活率>90%。GNRs@mSiO₂联合照射组的D₀、D_q、SF₂值均低于单纯照射组,放射增敏比为1.27(SF₂之比)。GNRs@mSiO₂联合照射组细胞凋亡率为(30.4±0.68)%。G₂+M期细胞比例为(31.25±0.75)%,均明显高于其余各组(P=0.003~0.033)。结论 介孔二氧化硅包覆金纳米棒可有效增强人三阴性乳腺癌细胞MDA-MB-231对高能6 MV X射线的放射敏感性。     

靶向HER2的抑制胃癌细胞功能性单抗的制备和鉴定

目的: 制备可特异识别人表皮生长因子受体2(human epidermal growth factor receptor2,HER2)膜抗原的单克隆抗体，筛选出亲和力较高并对胃癌细胞有明显抑制作用的克隆。方法：以高表达HER2膜抗原的人胃癌细胞系NCIN87免疫BABL/c小鼠，免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2     

载双歧杆菌胞外多糖纳米粒子诱导人胃癌裸鼠移植瘤细胞的凋亡

目的: 制备载双歧杆菌胞外多糖纳米粒子（Bifidobacterium exopolysaccharideloaded nanoparticles，B.EPSNPs)，探讨B.EPSNPs对人胃癌细胞裸鼠移植瘤增殖和凋亡的影响。方法：取Fe3O4纳米粒子和B.EPS，采用乳化高温固化法制备B.EPSNPs。建立人源胃癌细胞(BGC823)裸鼠移植瘤模型，接种6 d后随机分为5组，分别以生理盐水、NPs、环磷酰胺（cytoxan,CTX）、游离B.EPS及B.EPSNPs进行灌胃治疗。观察各组瘤体的生长情况，TUNEL法检测细胞凋亡，透射电镜观察细胞超微结构，免疫组化法检测增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)及凋亡调控基因bcl2、bax蛋白表达水平。结果：空载NPs和B.EPSNPs平均粒径分别为（560±21）nm、（960±32）nm，B.EPSNPs载药率为30％。B.EPSNPs对移植瘤的抑瘤率为（46.4±2.94）%，高于游离B.EPS组的(32.0±1.62)％和NPs组的(4.9±0.98)％。B.EPSNPs组移植瘤细胞凋亡指数明显高于B.EPS组\[（66.8±5.58）％ vs （41.3±4.26）％\]（P<0 .01)。透射电镜观察到B.EPSNPs治疗后的移植瘤细胞核染色质凝集成块状，出现了典型的凋亡特征。与B.EPS相比，B.EPSNPs组PCNA及bcl2的表达水平有所降低（P<0.01），bax的表达水平有所提高（P<0 .01）。结论：纳米粒子运载B.EPS增强了后者对胃癌细胞(BGC823)移植瘤的增殖抑制作用和促凋亡作用，提高了B.EPS的抗肿瘤活性。      

人胃癌裸鼠移植瘤中标记滞留细胞的初步鉴定(英文)

Objective:This study has investigated the existence of label-retaining cell and its distribution in gastric cancer,in the hope that this information will assist investigations on gastric cancer stem cells.Methods:The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice,which developed tumors.Label-retaining cells were quantified by immunohistochemical methods.Results:BrdU positive cells constituted about 96%of the cells in xenograft tumors after 10 days.Subsequently,BrdU positive cells gradually decreased,at the 80th day,labelretaining cells steadily occupied about 0.5%.This set of population cell localized in the margin of cancer nests,which had no difference in cellular morpha.Conclusion:The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells,which are most likely the cause for spread,metastasis and recurrence.     

Tumor progression-dependent angiogenesis in gastric cancer and its potential application

Despite improvements in the early diagnosis, prognosis and therapeutic strategies for gastric cancer (GC), human GC remains one of the most frequently diagnosed malignant tumors in the world, and the survival rate of GC patients remains very poor. Thus, a suitable therapeutic strategy for GC is important for prolonging survival. Both tumor cells themselves and the tumor microenvironment play an important role in tumorigenesis, including angiogenesis, inflammation, immunosuppression and metastasis. Importantly, these cells contribute to gastric carcinogenesis by altering the angiogenic phenotype switch. The development, relapse and spreading of tumors depend on new vessels that provide the nutrition, growth factors and oxygen required for continuous tumor growth. Therefore, a state of tumor dormancy could be induced by blocking tumor-associated angiogenesis. Recently, several antiangiogenic agents have been identified, and their potential for the clinical management of GC has been tested. Here, we provide an up-to-date summary of angiogenesis and the angiogenic factors associated with tumor progression in GC. We also review antiangiogenic agents with a focus on the anti-vascular endothelial growth factor receptor (VEGFR)-mediated pathway for endothelial cell growth and their angiogenesis ability in GC. However, most antiangiogenic agents have reported no benefit to overall survival (OS) compared to chemotherapy alone in local or advanced GC. In phase III clinical trials, only ramucirumab (anti-VEGFR blocker) and apatinib (VEGFR-TKI blocker) have reported an improved median overall response rate and prolonged OS and progression-free survival outcomes as a 2nd-line agent combined with chemotherapy treatment in advanced GC. By providing insights into the molecular mechanisms of angiogenesis associated with tumor progression in GC, this review will hopefully aid the optimization of antiangiogenesis strategies for GC therapy in combination with chemotherapy and adjuvant treatment.     

Expression and regulatory function of miRNA-34a in targeting survivin in gastric cancer cells

We aimed to investigate the expression of microRNA-34a (miR-34a) in human gastric cancer cells and to evaluate the effects of miR-34a, acting via its gene survivin, on gastric cancer cell HGC-27 to provide potential new strategies for treating gastric cancer. In vitro cultures of the human gastric cancer cell lines MGC80-3, HGC-27, NCI-N87, and SGC-7901 and the normal human gastric epithelial cell line GES-1 were established. The expression of miR-34a in each gastric cancer cell line and GES-1 normal human gastric epithelial cell line was detected using quantitative real-time polymerase chain reaction (qRT-PCR). After the HGC-27 cells were transfected with a miR-34a mimic for 48 h, the changes in the expression levels of miR-34a were detected using qRT-PCR. The effect of miR-34a on HGC-27 cell viability was measured using a tetrazolium-based colorimetric [?(4,5)-dimethylthiahiazo-(?z-y1)-3,5-di-phenytetrazoliumromide (MTT)] assay. Flow cytometry was used to analyze the effects of miR-34a on HGC-27 cell proliferation. Annexin V/propidium iodide double staining and flow cytometry were used to analyze the effects of miR-34a on HGC-27 cell apoptosis. A Transwell invasion chamber was used to detect the effects of miR-34a on HGC-27 cell invasion. Finally, western blotting was used to analyze the effects of miR-34a on survivin protein expression. The qRT-PCR test determined that miR-34a expression in gastric cancer cells was significantly reduced compared to the normal gastric epithelial cell line GES-1 (p?<?0.01). Compared to the control group, cellular miR-34a expression levels were significantly increased in HGC-27 human gastric carcinoma cells after transfection with a miR-34a mimic for 48 h (p?<?0.01). The MTT assay demonstrated that after overexpressing miR-34a in HGC-27 cells, cellular viability was significantly reduced (p?<?0.05). Flow cytometry analysis determined that upon miR-34a overexpression, the proliferation index decreased significantly (p?<?0.05), and cellular apoptosis was significantly increased (p?<?0.01). The Transwell invasion chamber assay illustrated that after increasing the expression of miR-34a, the number of cells passing through the Transwell chamber was significantly reduced (p?<?0.01). Based on western blotting, compared with the control group, survivin protein expression levels were significantly decreased in the HGC-27 cells transfected with the miR-34a mimic for 48 h (p?<?0.01). In conclusion, the expression level of miR-34a was downregulated in human gastric cancer cell lines. miR-34a can negatively regulate survivin protein expression and inhibit gastric cancer cell proliferation and invasion. Therapeutically enhancing miR-34a expression or silencing the survivin gene may benefit patients with gastric cancer.     

miR-21通过靶向SOX2增强胃癌细胞的迁移能力

目的:探讨miR-21在胃癌细胞系中对SOX2的调控作用及其对胃癌细胞迁移的影响。方法:在胃癌细胞系AZ-521、AGS中通过转染miR-21 mimic或者miR-21 antagomir构建miR-21过表达和低表达模型,RT-qPCR检测转染效果,并通过蛋白免疫印迹法检测SOX2在转染细胞中表达的变化。构建miR-21双荧光素酶报告基因,在工具细胞HEK293T中检验miR-21是否通过直接结合SOX2的3′-UTR区域来对其实现调控。应用Transwell迁移实验检测miR-21及SOX2对胃癌细胞迁移能力的影响。结果:miR-21直接结合于SOX2的3′-UTR区域来抑制SOX2在胃癌细胞系中的表达。迁移实验发现上调miR-21或者下调SOX2都能促进胃癌细胞的迁移。结论:miR-21可促进胃癌细胞的迁移能力,其机制可能通过抑制SOX2的表达而实现。     

二烯丙基二硫对人胃癌细胞裸鼠移植瘤的抗肿瘤作用

背景与目的:既往研究发现二烯丙基二硫(diallyldisulfide,DADS)在体外可抑制多种肿瘤细胞生长,但在体内抗肿瘤作用的研究报道较少。本实验旨在探讨DADS对人胃癌细胞移植瘤在BALB/C裸鼠体内生长的影响。方法:未经药物处理和经30mg/LDADS处理1天的胃癌细胞MGC803接种于裸鼠皮下;观察体外DADS处理MGC803细胞裸鼠移植瘤的成瘤情况和未处理MGC803细胞移植瘤成瘤后腹腔注射DADS对胃癌移植瘤在BALB/c裸鼠体内生长情况的影响。Westernblot检测瘤组织中增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)的表达情况。结果:30mg/LDADS处理的MGC803细胞移植裸鼠体内无一成瘤。荷瘤裸鼠腹腔注射DADS剂量为50、100和200mg/kg时的抑瘤率分别为27.8%、66.1%和73.0%,同时可抑制移植瘤癌细胞PCNA的表达。结论:DADS可明显降低胃癌细胞裸鼠移植瘤的成瘤性,并对移植瘤生长有明显抑制作用。     

Noninvasive imaging of apoptosis and its application in cancer therapeutics.

PURPOSE: Activation of the apoptotic cascade plays an important role in the response of tumors to therapy. Noninvasive imaging of apoptosis facilitates optimization of therapeutic protocols regarding dosing and schedule and enables identification of efficacious combination therapies. EXPERIMENTAL DESIGN: We describe a hybrid polypeptide that reports on caspase-3 activity in living cells and animals in a noninvasive manner. This reporter, ANLucBCLuc, constitutes a fusion of small interacting peptides, peptide A and peptide B, with the NLuc and CLuc fragments of luciferase with a caspase-3 cleavage site (DEVD) between pepANLuc (ANLuc) and pepBCLuc (BCLuc). During apoptosis, caspase-3 cleaves the reporter, enabling separation of ANLuc from BCLuc. A high-affinity interaction between peptide A and peptide B restores luciferase activity by NLuc and CLuc complementation. Using a D54 glioma model, we show the utility of the reporter in imaging of apoptosis in living subjects in response to various chemotherapy and radiotherapy regimens. RESULTS: Treatment of live cells and mice carrying D54 tumor xenografts with chemotherapeutic agents such as temozolomide and perifosine resulted in induction of bioluminescence activity, which correlated with activation of caspase-3. Treatment of mice with combination therapy of temozolomide and radiation resulted in increased bioluminescence activity over individual treatments and increased therapeutic response due to enhanced apoptosis. CONCLUSION: The data provided show the utility of the ANLucBCLuc reporter in dynamic, noninvasive imaging of apoptosis and provides a rationale for use of this technology to optimize dose and schedule of novel therapies or to develop novel combination therapies using existing drugs.     

多西紫杉醇对胃癌细胞作用及其机制的研究

目的:研究多西紫杉醇对人胃癌中分化细胞株SGC7901的作用效果及机制。方法:MTT法检测多西紫杉醇对胃癌细胞株SGC7901的增殖抑制作用。AnnexinV方法检测药物作用后胃癌细胞的凋亡。PI染色法检测凋亡晚期的胃癌细胞。采用流式细胞仪检测多西紫杉醇作用前后细胞凋亡相关分子Fas蛋白、Bcl2蛋白表达水平的变化。结果:0.92、3.7、14.8、59.2μg/mL多西紫杉醇作用于SGC7901细胞72h,抑制率分别为13.3%、21.6%、57.5%、61.0%。多西紫杉醇可诱导胃癌细胞株SGC7901发生细胞凋亡。多西紫杉醇使SGC7901凋亡分子Fas表达增加,作用前后分别为(26.5±7.2)%、(37.9±7.0)%;多西紫杉醇作用SGC7901前后Bcl2表达分别为(38.9±9.1)%、(31.2±5.6)%,差异无显著性。结论:多西紫杉醇对胃癌中分化细胞株SGC7901生长有明显的抑制作用,可诱导胃癌细胞系SGC7901凋亡,凋亡作用的发生可能与多西紫杉醇诱导Fas分子表达有关。     

表没食子儿茶素没食子酸酯诱导人胃癌细胞裸鼠移植瘤凋亡的研究

背景与目的探讨绿茶提取物表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)诱导人胃癌细胞裸鼠移植瘤凋亡及分子机制。方法建立人胃腺癌(SGC7901)细胞裸鼠异种移植瘤模型,用不同剂量的EGCG进行治疗,并设对照,用流式细胞分析术检测肿瘤组织中细胞凋亡情况,免疫组织化学方法检测肿瘤组织的凋亡相关基因Bcl-2、Bax、Caspase-3的表达情况。结果裸鼠异种移植瘤治疗实验结果显示,EGCG对移植瘤生长有明显抑制作用(其中20mg/kg、EGCG抑制率54.64%与对照组比较P<0.05);PI染色FCM分析发现,EGCG20mg/kg能诱导移植瘤细胞凋亡率17.2%与对照组比P<0.05;SP免疫组织化学结果表明EGCG可上调移植瘤细胞中Bax、Caspase-3蛋白的表达,下调Bcl-2蛋白的表达。结论表没食子儿茶素没食子酸酯(EGCG)具有体内诱导人胃腺癌细胞凋亡的作用,其机制可能与Bcl-2/Bax比值降低导致Caspase-3的活化从而启动细胞凋亡有关。