Biofunctional hydrogels for skeletal muscle constructs

Hydrogel scaffolds encapsulating C2C12 mouse skeletal muscle cells have been engineered as in vitro constructs towards regenerative medicine therapies for the enhancement and inducement of functional skeletal muscle formation. Previous work has largely involved two‐dimensional (2D) muscle strips, naturally occurring hydrogels and incomplete examination of the effects of the scaffold and/or biological functionalization on myogenic differentiation in a controllable manner. The goal of this study was to identify key properties in functionalized poly(ethylene glycol) (PEG)–maleimide (MAL) synthetic hydrogels that promote cell attachment, proliferation and differentiation for the formation of multinucleated myotubes and functional skeletal muscle tissue constructs. Significant differences in myoblast viability were observed as a function of cell seeding density, polymer weight percentage and bioadhesive ligands. The identified optimized conditions for cell survival, required for myotube development, were carried over for differentiation assays. PEG hydrogels (5% weight/volume), functionalized with 2.0 mm RGD adhesive peptide and crosslinked with protease‐cleavable peptides, incubated for 3 days before supplementation with 2% horse serum, significantly increased expression of differentiated skeletal muscle markers by 50%; 17% more multinucleated cells and a 40% increase in the number of nuclei/differentiated cell compared to other conditions. Functionality of cell‐laden hydrogels was demonstrated by a 20% decrease in the extruded length of the hydrogel when stimulated with a contractile agent, compared to 7% for a saline control. This study provided strategies to engineer a three‐dimensional (3D) microenvironment, using synthetic hydrogels to promote the development of differentiated muscle tissue from skeletal muscle progenitor cells to form contractile units. Copyright © 2014 John Wiley & Sons, Ltd.     

胃癌周围淋巴结组织间充质干细胞的分离和鉴定

目的试分离培养胃癌患者周围淋巴结组织中的间充质干细胞(h GCL-MSCs),并鉴定其生物学特性。方法采用组织块贴壁法分离培养24例胃癌患者周围淋巴结组织,通过测定生长曲线、细胞周期、RT-PCR、染色体分析、免疫组化染色、成骨成脂诱导染色及裸鼠致瘤等实验,评价其生物学特性,并与人骨髓间充质干细胞(h BM-MSCs)进行比较。结果 24例胃癌患者周围淋巴结组织中分离培养出3例h GCL-MSCs;生长曲线试验发现,h GCL-MSCs增殖速率高于h BM-MSCs;染色体分析示其核型正常,裸鼠致瘤实验未见致瘤;RT-PCR检测其表达Oct-4、Nanog、ABCG2、CD44、CD73、α-SMA、Bmi-1等干细胞相关基因;免疫组化染色结果表明,h GCL-MSCs中α-SMA、P53、ABCG2染色呈强阳性,PCNA染色呈弱阳性。结论 h GCL-MSCs与h BMMSCs的生物学特性相似,对研究胃癌微环境及其发生具有重要意义。     

训练免疫在肿瘤综合治疗中的应用研究进展

经典的免疫系统分为固有免疫和适应性免疫。传统观念认为，免疫记忆仅存在于适应性免疫中，然而，在缺乏适应性免疫的生物体中，先天免疫系统的细胞能够在短暂刺激后获得记忆特征，从而在二次抗原激活后增强有效免疫，这种现象被称为训练免疫。训练免疫可以在肿瘤治疗中发挥独特的作用。卡介苗（BCG）诱导的训练免疫已经成为膀胱癌的标准治疗方法；BCG在黑素瘤中的治疗效果也于近年得到肯定；通过不可逆电穿孔（irreversible electroporation, IRE）提高治疗过程中训练免疫效应可以延长胰腺癌患者生存期；全β-葡聚糖颗粒（whole β-glucan particle, WGP）诱导的训练免疫可以有效抑制肿瘤肺转移；通过纳米颗粒（nanoparticles, NPs）引入药物可以提高乳腺癌靶向部位的训练免疫效果。这些研究对改善现有治疗策略、延长患者生存获益具有重要意义。本文重点阐述训练免疫的概念、机制以及在肿瘤治疗领域的现状。     

Relationship between microsatellite status and immune microenvironment of colorectal cancer and its application to diagnosis and treatment

Due to advances in understanding the immune microenvironment of colorectal cancer (CRC), microsatellite classification (dMMR/MSI‐H and pMMR/MSS) has become a key biomarker for the diagnosis and treatment of CRC patients and therefore has important clinical value. Microsatellite status is associated with a variety of clinicopathological features and affects drug resistance and the prognosis of patients. CRC patients with different microsatellite statuses have different compositions and distributions of immune cells and cytokines within their tumor microenvironments (TMEs). Therefore, there is great interest in reversing or reshaping CRC TMEs to transform immune tolerant "cold" tumors into immune sensitive "hot" tumors. This requires a thorough understanding of differences in the immune microenvironments of MSI‐H and MSS type tumors. This review focuses on the relationship between CRC microsatellite status and the immune microenvironment. It focuses on how this relationship has value for clinical application in diagnosis and treatment, as well as exploring the limitations of its current application.     

Reshaping the Immune Microenvironment by Oncolytic Herpes Simplex Virus in Murine Pancreatic Ductal Adenocarcinoma

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Multispectral imaging for quantitative and compartment‐specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients

Semiquantitative assessment of immune markers by immunohistochemistry (IHC) has significant limitations for describing the diversity of the immune response in cancer. Therefore, we evaluated a fluorescence‐based multiplexed immunohistochemical method in combination with a multispectral imaging system to quantify immune infiltrates in situ in the environment of non‐small‐cell lung cancer (NSCLC). A tissue microarray including 57 NSCLC cases was stained with antibodies against CD8, CD20, CD4, FOXP3, CD45RO, and pan‐cytokeratin, and immune cells were quantified in epithelial and stromal compartments. The results were compared with those of conventional IHC, and related to corresponding RNA‐sequencing (RNAseq) expression values. We found a strong correlation between the visual and digital quantification of lymphocytes for CD45RO (correlation coefficient: r = 0.52), FOXP3 (r = 0.87), CD4 (r = 0.79), CD20 (r = 0.81) and CD8 (r = 0.90) cells. The correlation with RNAseq data for digital quantification (0.35–0.65) was comparable to or better than that for visual quantification (0.38–0.58). Combination of the signals of the five immune markers enabled further subpopulations of lymphocytes to be identified and localized. The specific pattern of immune cell infiltration based either on the spatial distribution (distance between regulatory CD8⁺ T and cancer cells) or the relationships of lymphocyte subclasses with each other (e.g. cytotoxic/regulatory cell ratio) were associated with patient prognosis. In conclusion, the fluorescence multiplexed immunohistochemical method, based on only one tissue section, provided reliable quantification and localization of immune cells in cancer tissue. The application of this technique to clinical biopsies can provide a basic characterization of immune infiltrates to guide clinical decisions in the era of immunotherapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

胰腺癌靶向治疗的研究现状

胰腺癌是一种恶性程度高、致死性强的疾病,5年生存率小于5％.虽然外科手术和药物的治疗有了较大的进步,但其预后不佳,这与药物的耐药性有密切关系.随着胰腺癌分子生物学研究的进展,目前可以根据患者的肿瘤组织活检特点进行胰腺癌靶向的个体化治疗.基于此,一些靶向于肿瘤细胞和肿瘤微环境的信号通路的新药正在进行前期临床试验.本文就胰腺癌的靶向治疗作简要综述.     

Importance of tumor/stroma interactions in prognosis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third largest cause of cancer deaths worldwide. It seems to be needed to find new ways to address the mechanisms involved in the progression of HCC, which can provide a prognostic evaluation and new therapeutic targets. Several studies have established that crosstalk between tumor cells and the microenvironment plays a key role in tumor progression and metastasis. In this context, the work of Zhu et al. contributes to assess interactions between tumor and microenvironment associated-macrophages promoting tumor progression and metastasis. Indeed, they concluded that the interplay of osteopontin (OPN) and peritumoral macrophages (PTMs) represents a new insight into tumor progression and therapeutic targets for HCC. Historically, tumor-infiltrating leukocytes have been considered to be manifestations of an intrinsic defensive mechanism against developing tumors, however, now, it is know that that leukocytes infiltration can promote tumor phenotypes, such as angiogenesis, growth, and invasion. Characterization of functional heterogeneity of stromal cell components, and specifically the analysis of stromal fibroblasts can provide a new focus on mechanisms involved in the progression of HCC. All of this opens the possibility to provide prognostic information for HCC based on biological parameters derived from peritumoral status from tumors.     

肿瘤微环境双重响应性的智能型蛋白毒素给药系统的构建和表征

目的 利用蛋白重组技术和PEG定点修饰技术,制备具有肿瘤微环境双重响应性的智能型蛋白毒素给药系统。方法 利用基因重组技术,在天花粉蛋白(trichosanthin,TCS)的C端引入天冬酰胺内肽酶(legumain)的底物天冬酰胺肽段和半胱氨酸残基,将所构建的突变体转化到大肠杆菌中表达目的蛋白并纯化。进一步将重组蛋白末端的半胱氨酸与具有巯基反应性的mPEG-Hz-Mal偶联合成TCS-Asn10-Hz-PEG,采用弱阳离子交换柱纯化TCS-Asn10-Hz-PEG,并在体外考察了TCS-Asn10-Hz-PEG的酸敏感性和酶敏感性。结果 成功制备、分离和纯化得到了TCS-Asn10-Cys,完成了mPEG-Hz-Mal与TCS-Asn10-Cys的定点偶联,得到智能型蛋白毒素给药系统TCS-Asn10-Hz-PEG。TCS-Asn10-Hz-PEG在体外pH 5.6的介质中和天冬酰胺内肽酶的作用下,能够水解或酶解释放出TCS,具有酸敏感特性和酶敏感特性。结论 本实验设计的蛋白毒素给药系统,具有酸敏感和酶敏感双重响应特性。