毛细管电泳检测肺癌及癌旁正常组织蛋白质混合物差异

以建立的毛细管电泳(CE)-激光诱导荧光(LIF)检测蛋白质的方法对提取肺癌及癌旁正常组织蛋白质混合物(变 性/活性)差异进行检测. 采用异硫氰酸荧光素(FITC)为衍生剂, 电泳缓冲液为1×TBE (TBE为Tris-硼酸-EDTA, 变性电泳pH 10.0, 活性电泳为pH 8.3且含有2 mg/L考马斯亮蓝), 分离电压15 kV, 柱温15 ℃, 电动进样(10 kV×10 s), 激发波长/发射波长＝488/520 nm检测时, 肺癌及癌旁正常组织蛋白质混合物样品得到较好分离且有明显差异. 与目前常用蛋白分析方法: 变性SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及活性蓝绿温和胶电泳(BN-PAGE)进行比较. BN-PAGE结果显示肺癌组织相比正常组织有较明显蛋白种类差异|SDS-PAGE结果表明一些蛋白质表达量差异也是肺癌及癌旁正常组织的显著差别, 且主要集中在20～116 kDa. CE-LIF检测结果与PAGE结果大致相同, 且CE-LIF检测蛋白质的灵敏度高于PAGE, 能更准确反映肺癌及癌旁正常组织的蛋白质差异. 结论是CE-LIF可用于蛋白质差异检测, 时间短, 效果较好, 对活性蛋白质进行分析体现了其优点: 可提供较强的动力——电压, 及强动力下良好的温度稳定性.     

蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

HCV全基因组培养细胞的比较蛋白组学研究

利用比较蛋白质组技术研究了转染丙型肝炎病毒(Hepatitis C virus, HCV)全基因组的人肝癌细胞系Huh7细胞模型中蛋白质表达谱的变化, 建立了Huh7-HCV的双向凝胶电泳蛋白质表达图谱和数据库. 通过双向凝胶电泳分离和图像分析, 对表达差异2倍以上蛋白质点进行了胶内酶解和MALDI-TOF MS鉴定. 得到包括与细胞骨架蛋白、细胞周期、凋亡和信号转导等相关的14个蛋白质, 并且用Western blot验证了热休克蛋白70的蛋白质组研究结果. 利用HCV全基因组培养系统, 采用蛋白质组学技术, 为研究HCV病毒和宿主细胞相互作用提供了新的实验数据, 为深入研究HCV病毒复制和分子致病机理奠定了基础.     

鼠肝癌淋巴道转移细胞模型的蛋白质组学研究

对2株来源于同一亲本细胞但淋巴道转移力显著不同的小鼠肝癌腹水型细胞株Hca-F(淋巴结转移率75%)和Hca-P(淋巴结转移率25%), 采用荧光差异双向凝胶电泳(2D DIGE)和DeCyder定量分析软件及HPLC-nESI-MS/MS技术, 定量分析和鉴定了小鼠肝癌细胞Hca-F和Hca-P的差异表达蛋白. 结果显示, 有116个蛋白质点表达水平存在明显差异(p<0.05), 在Hca-F中表达上调蛋白质点62个, 下调蛋白质点54个. 对所有116个蛋白质点进行了电喷雾串联质谱鉴定, 共鉴定出109种单一(Unique)蛋白. 其中部分蛋白已被报道与不同类型肿瘤的发生、浸润和转移相关, 多数蛋白质被首次报道与肝癌的淋巴道转移过程直接相关.     

在镉盐胁迫下扇贝鳃组织应激蛋白的研究

采用透射电子显微镜观察了虾夷盘扇贝(Patinopecten yessoensis)鳃组织细胞的超微结构, 发现镉盐能胁迫鳃组织中的腮丝、细胞核和线粒体产生病变. 利用双向凝胶电泳(2D-PAGE)优化分离扇贝鳃组织的全蛋白, 获得约800个蛋白质斑点, 并筛选出37个由于镉盐胁迫而产生的差异蛋白质斑点. 选用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF MS)技术和数据库检索鉴定差异蛋白, 结果发现7个与镉毒性密切相关的蛋白质, 即热休克蛋白70和β-淀粉酶等上调蛋白质及原肌球蛋白、肌动蛋白和钙活化核苷酸酶1等下调蛋白质. 此外, 还发现转录调节子Crp/Fnr家族为低表达蛋白质, 而ABC转运子为高表达蛋白质. 在这些差异蛋白中, 部分蛋白质适合作为连续监测流动海水中镉污染程度及评价其危害性的蛋白指示物.     

凝胶电泳-毛细管液相色谱-质谱联用技术分析大肠癌肿瘤与癌旁组织的差异蛋白质组

通过凝胶电泳将大肠癌患者的手术标本组织(肿瘤与癌旁)中的总蛋白按照分子量分成不同的组分,经胶内水解后,利用毛细管液相色谱-质谱联用技术对各个组分的多肽混合物进行蛋白质组学分析,共鉴定出29种差异表达的蛋白质,其中肿瘤组织中表达上调的蛋白质19种,如Fibrinogen beta chain,Vimentin,Fi-brinogen gamma chain,Histone H2A type 1-B/E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Al-pha-enolase,Protein disulfide-isomerase A3等;在肿瘤组织中表达下调的有10种蛋白质,如Sarcomeric tropomy-osin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C等.通过蛋白质印迹实验对其中的踝蛋白1(Talin-1)和烯醇化酶(Alpha-enolase)的蛋白质组学鉴定结果进行了验证.生物信息学分析表明在大肠癌组织中表达上调的蛋白多具有胞外分泌的属性,提示这些蛋白很有可能会渗透到机体内的循环系统中,从而在患者血液或者腹水中能够被检测到;而在大肠癌组织中表达下调的蛋白则多为细胞骨架、胞外基质纤维等的组成成分,这些蛋白的下调常与肿瘤细胞向周围组织的侵袭和转移有密切关系.本研究为大肠癌的早期诊断、大肠癌初筛等方面的研究等提供了基础数据,对进一步深入研究大肠癌发病机制具有重要意义.     

蛋白质组学技术鉴定与分析三七粉诱导海兔神经连索表达的差异蛋白质

RP-HPLC分离三七粉提取液,并鉴定含有Rb1、Rg1、Re、R1等皂甙成分。以蓝斑背肛海兔(Notarcusleachii cirrosus Stimpson,NLCS)为分析模型,三七粉提取液为诱导剂,选用蛋白质组技术研究NLCS神经连索诱导前后所表达的差异蛋白质。通过优化双向凝胶电泳分离NLCS神经连索全蛋白质组技术,获得496个蛋白质斑点。采用肽指纹图谱技术和数据库检索比对法,初步鉴定了NLCS受三七粉提取液诱导前后,其神经连索表达13个差异蛋白质,其中较高的匹配率蛋白质为肌动蛋白、3-羟酯酰辅酶A脱氢酶、ATP结合转运子和甲基转移酶12。选用LOC tree软件对13个差异蛋白质进行亚细胞定位,认为它们在保护神经系统中发挥重要的调节作用。     

肝移植后人血清中差异表达蛋白质的纳升超高效液相色谱-电喷雾串联质谱鉴定

建立了基于质谱的人肝移植后血清中差异表达蛋白质的分析方法,肝移植前后的人血清蛋白质用双向凝胶电泳分离,AgNO3染色,差异蛋白质斑点用胰蛋白酶进行胶内酶解后,采用纳升超高效液相色谱-电喷雾串联质谱进行鉴定。鉴定了8个显著差异表达的蛋白质,Mascot检索分值101~1543分,肽段匹配数5~34个,序列覆盖率10%~43%。所发现的差异蛋白质多与免疫调节有关,可为深入研究肝移植术后免疫排斥的分子机理提供线索。     

Ⅱ型糖尿病大鼠神经视网膜组织中差异蛋白质分析

以高脂饮食联合小剂量链脲菌素(Streptozotocin, STZ ) 注射方法诱导大鼠II型糖尿病 (T2DM) 模型为研究对象, 采用比较蛋白质组学技术鉴定和分析早期糖尿病神经视网膜差异表达蛋白.通过联用液氮内研磨、冰浴超声、高速离心提取全蛋白技术,提取每只大鼠神经视网膜全蛋白质总量约900 μg.采用双向凝胶电泳(2-DE) 分离技术,获得2500个以上可辩认的蛋白质斑点.通过蛋白质组学比对技术筛选出糖尿病状态下神经视网膜差异表达蛋白,用基质辅助激光解吸电离飞行时间串联质谱 (MALDI-TOF- MS/MS) 和肽质量指纹图谱 (PMF) 鉴定出20个差异蛋白点.按照Gene Ontology (GO)分类体系,对差异蛋白归类分析,揭示其亚细胞定位和分子功能.应用Pathway Studio软件对差异表达蛋白的生物学意义进行分析,认为凋亡是早期糖尿病视网膜病变(Diabetic retinopathy,DR) 重要的细胞事件;AnnexinΙ、CRYAB、mtHsp70蛋白是与病理过程相关的关键蛋白,对研究DR致病机制有重要意义.     

钆对费氏中华根瘤菌抑制效应的蛋白质组分析

运用双向凝胶电泳和MALDI-TOF质谱技术,分析了Gd3+对费氏中华根瘤菌USDA 205的抑制效应。结果表明,经1 mmol.L-1Gd3+处理12 h后,22个蛋白质表达量有显著变化,其中13个蛋白质表达量增加,9个蛋白质表达量下降。这些蛋白质可根据功能分为8类,包括转运蛋白、胁迫相关蛋白、代谢相关蛋白等。     

5-epi-Sinuleptolide from Soft Corals of the Genus Sinularia Exerts Cytotoxic Effects on Pancreatic Cancer Cell Lines via the Inhibition of JAK2/STAT3, AKT,and ERK Activity

Pancreatic ductal adenocarcinoma is one of the most lethal malignancies: more than half of patients are diagnosed with a metastatic disease, which is associated with a five-year survival rate of only 3%. 5-epi-Sinuleptolide, a norditerpene isolated from Sinularia sp., has been demonstrated to possess cytotoxic activity against cancer cells. However, the cytotoxicity against pancreatic cancer cells and the related mechanisms are unknown. The aim of this study was to evaluate the anti-pancreatic cancer potential of 5-epi-sinuleptolide and to elucidate the underlying mechanisms. The inhibitory effects of 5-epi-sinuleptolide treatment on the proliferation of pancreatic cancer cells were determined and the results showed that 5-epi-sinuleptolide treatment inhibited cell proliferation, induced apoptosis and G2/M cell cycle arrest, and suppressed the invasion of pancreatic cancer cells. The results of western blotting further revealed that 5-epi-sinuleptolide could inhibit JAK2/STAT3, AKT, and ERK phosphorylation, which may account for the diverse cytotoxic effects of 5-epi-sinuleptolide. Taken together, our present investigation unveils a new therapeutic and anti-metastatic potential of 5-epi-sinuleptolide for pancreatic cancer treatment.     

Bio-assembled smart nanocapsules for targeted delivery of KRAS shRNA and cancer cell bioimage

The five-year survival rate for pancreatic cancer is less than 5%. However, the current clinical multimodal therapy combined with first-line chemotherapy drugs only increases the patient's median survival from 5.0 months to 7.2 months. Consequently, a new strategy of cancer treatments is urgently needed to overcome this high-fatality disease. Through a series of biometric analyses, we found that KRAS is highly expressed in the tumor of pancreatic cancer patients, and this high expression is closely related to the poor prognosis of patients. It shows that inhibiting the expression of KRAS has great potential in gene therapy for pancreatic cancer. Given those above, we have exploited the possibility of targeted delivery of KRAS shRNA with the intelligent and bio-responsive nanomedicine to detect the special oxidative stress microenvironment of cancer cells and realize efficient cancer theranostics. Our observations demonstrate that by designing the smart self-assembled nanocapsules of melanin with fluorescent nanoclusters we can readily achieve the bio-recognition and bioimaging of cancer cells in biological solution or serum. The self-assembled nanocapsules can make a significant bio-response to the oxidative stress microenvironment of cancer cells and generate fluorescent zinc oxide Nanoclusters in situ for targeted cell bioimaging. Moreover, it can also readily facilitate cancer cell suppression through the targeted delivery of KRAS shRNA and low-temperature hyperthermia. This raises the possibility to provide a promising theranostics platform and self-assembled nanomedicine for targeted cancer diagnostics and treatments through special oxidative stress-responsive effects of cancer cells.     

MS analysis reveals O-methylation of L-lactate dehydrogenase from pancreatic ductal adenocarcinoma cells

L-lactate dehydrogenase (LDH) converts pyruvate to lactate when oxygen is absent or in short supply, and the enzyme plays a crucial role in cancer metabolism. The functions of many mammalian proteins are modulated by posttranslational modifications (PTMs), and it has been reported that LDH was subjected to several PTMs, including phosphorylation, acetylation, and methylation. In this present work, we characterized the PTMs of LDH from pancreatic ductal adenocarcinoma (PDAC) cells by electrophoresis and mass spectrometry, and identified 13 O-methylated residues from the enzyme. In addition, our qualitative analysis revealed differential methylation of LDH from normal duct cells. The preliminary findings from this study provide important biochemical information toward further understanding of the LDH modifications and their functional significance in pathophysiological processes of pancreatic cancer.     

Metabolomic Profiling of Serum from Human Pancreatic Cancer Patients Using <Superscript>1</Superscript>H NMR Spectroscopy and Principal Component Analysis

Pancreatic cancer is a malignant tumor with the worst prognosis among all cancers. At the time of diagnosis, surgical cure is no longer a feasible option for most patients, thus early detection of pancreatic cancer is crucial for its treatment. Metabolomics is a powerful new analytical approach to detect the metabolome of cells, tissue, or biofluids. Here, we report the application of ¹H nuclear magnetic resonance (NMR) combined with principal components analysis to discriminate pancreatic cancer patients from healthy controls based on metabolomic profiling of the serum. The metabolic analysis revealed significant lower of 3-hydroxybutyrate, 3-hydroxyisovalerate, lactate, and trimethylamine-N-oxide as well as significant higher level of isoleucine, triglyceride, leucine, and creatinine in the serum from pancreatic cancer patients compared to that of healthy controls. Our data demonstrate that the subtle differences in metabolite profiles in serum of pancreatic cancer patients and that of healthy subjects as a result of physiological and pathological variations could be identified by NMR-based metabolomics and exploited as metabolic markers for the early detection of pancreatic cancer.     

Gastrointestinal cancer organoids—applications in basic and translational cancer research

Cancer is a major health problem and a leading cause of death worldwide. Early cancer detection and continuous changes in treatment strategies have improved overall patient survival. The recent development of targeted drugs offers new opportunities for personalized cancer treatment. Nevertheless, individualized treatment is accompanied by the need for biomarkers predicting the response of a patient to a certain drug. One of the most promising breakthroughs in recent years that might help to overcome this problem is the organoid technology. Organoid cultures exhibit self-renewal capacity, self-organization, and long-term proliferation, while recapitulating many aspects of their primary tissue. Generated patient-derived organoid (PDO) libraries constitute “living” biobanks, allowing the in-depth analysis of tissue function, development, tumor initiation, and cancer pathobiology. Organoids can be derived from all gastrointestinal tissues, including esophageal, gastric, liver, pancreatic, small intestinal and colorectal tissues, and cancers of these tissues. PDOs are amenable to various techniques, including sequencing analyses, drug screening, targeted therapy testing, tumor microenvironment studies, and genetic engineering capabilities. In this review, we discuss the different applications of gastrointestinal organoids in basic cancer biology and clinical translation.Subject terms: Gastrointestinal cancer, Cancer models     

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.     

Photo-responsive NIR-II biomimetic nanomedicine for efficient cancer-targeted theranostics

In pancreatic cancer, the special barrier system formed by a large number of stromal cells severely hinders drug penetration in deep tumor tissues, resulting in low treatment efficiency. Cell membrane protein-camouflaged liposomal nanomedicines have cancer cell targeting abilities, whereas near-infrared two-zone (NIR-II) fluorescence imaging can achieve deep tissue penetration due to its long light wavelength (1,000–1,700 nm). To combine the cell membrane-based biomimetic technology with NIR-II fluorescence imaging, we constructed a biomimetic nanomedicine (BLIPO-I/D) by camouflaging indocyanine green-doxorubicin (ICG-DOX) liposomes with SW1990 pancreatic cancer cell membrane. The nanomedicine exhibited light-controlled DOX release and high pancreatic cancer treatment efficiency in vitro and in vivo. BLIPO-I/D showed the ability of targeted delivery of a large number of liposomes to pancreatic tumor tissues through homologous targeting of SW1990 cell membranes, which increased the NIR-II fluorescence imaging intensity. Irradiation of the liposomes taken up by pancreatic tumor tissues with near-infrared light (808 nm) triggered the rapid release of DOX from the liposomes, induced the photothermal and photodynamic effects of ICG, which exerted anti-tumor effects. Therefore, the fabricated biomimetic liposomal nanomedicine BLIPO-I/D is expected to achieve precise theranostics of pancreatic cancer.     

毛细管电泳-激光诱导荧光检测法分析胃癌组织及癌旁正常组织蛋白质的差异性

胃癌是临床常见的恶性肿瘤之一。近年来寻找肿瘤相关特异蛋白质是蛋白质组学研究的热点。本文通过考察毛细管动态涂层方法、筛分介质聚环氧乙烷(PEO)的浓度、缓冲液的pH值、分离电压、温度及荧光染料对分离效果的影响,建立了毛细管电泳-激光诱导荧光法分离胃癌组织及癌旁正常组织蛋白质的方法;通过分离检测,获得两者的蛋白质指纹图谱。经分析,两者的指纹图谱相似度达到0.8以上,差异蛋白质分子质量集中在50000~100000 Da之间,提示某些小分子蛋白质可能是和肿瘤发生相关的特异蛋白质,从而缩小了特异性分子标记物的筛选范围。病理组织学分型及蛋白质电泳峰数目的统计结果验证了该方法的可靠性。该方法具有临床应用的潜力。     

Gd3+与RGD共修饰量子点用于胰腺癌细胞的荧光及MR双模态成像

结合磁共振成像(MRI)和荧光成像技术,以钆离子(Gd3+)、量子点及精氨酸(R)-甘氨酸(G)-天冬氨酸(D)(RGD)多肽等为功能单元,采用纳米载体组装技术构建了MRI弛豫率/荧光效率高和靶向性强的Gd3+与RGD共修饰的量子点双模态纳米探针(QDs@Gd3+-RGD),并将其用于胰腺癌细胞的双模态成像.实验结果表明,QDs@Gd3+-RGD双模态纳米探针具有较高的弛豫率,且能对胰腺癌patu8988细胞进行荧光和T1-weighted MR成像.     

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC₅₀) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.