结直肠癌血清肿瘤标记物液相芯片制备条件的优化及在CEA抗原检测中的初步应用

液相芯片技术由于其高通量,灵敏度高,信噪比高,液相条件下反应,操作简便,耗时短等优点,已被美国FDA批准成为临床的检测手段。本文主要介绍了结直肠癌血清肿瘤标记物液相芯片制备条件的优化及其在CEA抗原检测中的初步应用。本研究首先将CEA抗原的捕获抗体与微球载体进行偶联,制备液相芯片,然后对影响反应的微球与抗原的反应时间,生物素化检测抗体的浓度及avidin-PE荧光染料的反应浓度等因素进行正交设计,确定出最优的反应条件;用该液相芯片反应体系检测55例临床样本,与ELISA试剂盒检测结果相比：在同样的样本浓度范围内,两者的检测结果基本一致,但液相芯片检测的浓度范围更大而且液相芯片可将多种肿瘤标记物在一个反应进行检测,节省检测的时间和人力。     

蛋白激酶C对鼻咽癌细胞c－myc，c－fos表达的影响

用蛋白激酶Ｃ（ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴ,抑制催化亚基）与Ｓｐｈｉｎｇｏｓｉｎｅ（ＳＳ,抑制调节亚基）处理人鼻咽癌细胞系ＣＮＥ－２Ｚ,经点印迹后扫描定量和免疫细胞化学检测ｃ－ｍｙｃ、ｃ－ｆｏｓ表达及其分布。结果发现：（１）ｃ－ｍｙｃ蛋白：主要分布于胞浆,４０．７％的细胞核与胞浆均为阳性;经ＳＳ或ＳＴ处理后,表达明显减弱,且核阳性率分别为１７．３％与２３．３％。扫描定量在ＳＳ组为对照组的６０％±２５．７％（Ｐ＜０．０５）,ＳＴ组为对照组的５５％±２５．９％（Ｐ＜０．０５）。（２）Ｃ－ｆｏｓ蛋白：主要分布于胞浆,２９．４％的细胞核与胞浆均为阳性,经ＳＳ或ＳＴ处理后,表达明显减弱,且核阳性率分别为９％与１０．２％。扫描定量ＳＳ组为对照组的５８．６％±２５％（Ｐ＜０．０５）,ＳＴ组为对照组的５９．７％±２６．２％（Ｐ＜０．０５）。结果表明：使用ＰＫＣ抑制剂后,两种核癌基因产物量均有不同程度的减少,并且存在细胞内分布的改变,特别是其发挥效应的核内表达明显减少。结合我们以前所发现的ＰＫＣ抑制剂明显抑制ＣＮＥ—２Ｚ细胞生长的结果,提示这些癌基因可能参与了ＰＫＣ对ＣＮＥ—２Ｚ细胞生长的调节。     

Extracellular membrane-proximal domain of HAb18G/CD147 binds to metal ion-dependent adhesion site (MIDAS) motif of integrin β1 to modulate malignant properties of hepatoma cells

Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp(179) in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells.     

Gekko-sulfated glycopeptide inhibits tumor angiogenesis by targeting basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a therapeutic target of anti-angiogenesis. Here, we report that a novel sulfated glycopeptide derived from Gekko swinhonis Guenther (GSPP), an anticancer drug in traditional Chinese medicine, inhibits tumor angiogenesis by targeting bFGF. GSPP significantly decreased the production of bFGF in hepatoma cells by suppressing early growth response-1. GSPP inhibited the release of bFGF from extracellular matrix by blocking heparanase enzymatic activity. Moreover, GSPP competitively inhibited bFGF binding to heparin/heparan sulfate via direct binding to bFGF. Importantly, GSPP abrogated the bFGF-stimulated proliferation and migration of endothelial cells, whereas it had no inhibitory effect on endothelial cells in the absence of bFGF. Further study revealed that GSPP prevented bFGF-induced neovascularization and inhibited tumor angiogenesis and tumor growth in a xenograft mouse model. These results demonstrate that GSPP inhibits tumor angiogenesis by blocking bFGF production, release from the extracellular matrix, and binding to its low affinity receptor, heparin/heparan sulfate.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.     

Initial experience of hypofractionated radiation retreatment with true beam and flattening filter free beam in selected case reports of recurrent nasopharyngeal carcinoma

AimTo show our preliminary experience in using TrueBeam with RapidArc technology and FFF beam for stereotactic re-irradiation of nasopharyngeal carcinoma.BackgroundThanks to new advanced techniques, as well as intensity modulated radiation therapy, it is possible to approach head and neck recurrences in selected patients. Volumetric Modulated Arc Therapy (VMAT) in its RapidArc^® format, permits to reduce significantly the time to deliver complex intensity modulated plans, allowing to treat hypofractionated regimes within a few minutes. With TrueBeam it is possible to perform photon beams without usage of the flattening filter. It seems possible to expect a reduction of out-of-field dose when flattening filter free (FFF) beams are used. While research into the physics domain for FFF beams is increasing, there are very few clinical data where FFF beams are applied in clinical practice.Materials and methodsWe present here the cases of 4 patients with local or regional recurrence of nasopharyngeal carcinoma. All patients were treated using TrueBeam with RapidArc technology and FFF beam for stereotactic hypofractionated re-irradiation.ResultsAll patients concluded SBRT and showed good tolerability. During follow-up, complete response at imaging evaluation (PET and/or MRI) for all treated patients was documented.ConclusionsOur preliminary experience using TrueBeam with RapidArc technology and FFF beam for stereotactic hypofractionated re-irradiation of nasopharyngeal carcinoma was safe and effective in all 4 treated patients. Longer follow-up and a larger population of study is needed to confirm these promising results.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

CD4 and CD8 T cell responses to tumour-associated Epstein–Barr virus antigens in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.