GnRH and VEGF have been investigated as prostate carcinoma enhancers that support tumor spread and progression. Although both have documented roles in prostate carcinoma and many cancer types, the weak immunogenicity of these peptides has remained a major challenge for use in immunotherapy. Here, we describe a novel strategy to inhibit GnRH and VEGF production and assess the effect on the immune responses against these hormones using the RM-1 prostate cancer model. We designed a novel recombinant fusion protein which combined GnRH and VEGF as a vaccine against this tumor. The newly constructed fusion protein hVEGF121-M2-GnRH3-hinge-MVP contains the human vascular endothelial growth factor (hVEGF121) and three copies of GnRH in sequential linear alignment and T helper epitope MVP as an immunogenic vaccine. The effectiveness of the vaccine in eliciting an immune response and attenuating the prostate tumor growth was evaluated. Results showed that administration of a new vaccine effectively elicited humoral and cellular immune responses. We found that, a novel fusion protein, hVEGF121-M2-GnRH3-hinge-MVP, effectively inhibited growth of RM-1 prostate model and effectively promoted immune response. In conclusion, hVEGF121-M2-GnRH3-hinge-MVP is an effective dual mechanism tumor vaccine that limits RM-1 prostate growth. This vaccine may be a promising strategy for the treatment of hormone refractory prostate malignancies.

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, puri-fication, and refolding of other bacterial toxic proteins.

This study aimed at investigating the inhibitory effects and the anti-tumor mechanisms of co-administration of fusion pro-teins gonadotropin-releasing hormone(GnRH)and hVEGF-GnRH3(VG)on B16F10 melanoma in C57BL/6J mice model.The metastasis of melanoma can be promoted by overexpression of the targeting antigens GnRH and VG.VG vaccine has been shown to elicit an immune inflammatory response against prostate cancer.Hence the authors hypothesized VG also has potential to prevent melanoma angiogenesis,progression and recurrence.Recombinant protein hVEGF-GnRH3 was extracted by E.coli expression system and purified by DEAE anion exchange chromatography to prepare protein vaccine.B16F10 melanoma cells were transplanted into male C57BL/6J mice(1 x 106 cells per mouse)to construct a melanoma model.The tumor-bearing mice were immunized with VG protein vaccine for three cycles.Thereafter,the tumor suppressing effect of the vaccine was evaluated from the aspects of tumor growth,angiogenesis and tumor histology,periphery and splenocytes humoral,cellular,and intracellular immunomodulation also included.The results showed that the volumes and angiogenesis of melanoma were significantly inhibited in VG group(P＜0.01).Compared to the PBS group,VG increased the levels of anti-GnRH,anti-VEGF IgG and circulating interferon-gamma(IFN-γ)in mice serum,while the level of IL-6 was significantly decreased.In the spleen-related experiments,the MTT assay showed that VG vaccine promoted splenocytes prolife-ration and cytotoxicity.At the same time,further analysis of intracellular cytokines revealed a significant increase in CD3+CD4+INF-γ+cells.The results above indicate that VEGF-GnRH3 is a promising vaccine for the treat-ment of melanoma.

目的 通过综述目前新兴的1型糖尿病(T1DM)疗法,为T1DM的临床治疗提供参考.方法 利用GeenMedical及知网等检索平台,查阅相关文献42篇,总结近年来T1DM治疗方法的最新进展.结果 近年来出现的钠-葡萄糖协同转运蛋白抑制剂(sodium-glucose cotransporters inhibitor,SGLTi)、二甲双胍、抗CD3单克隆抗体、闭环胰岛素泵系统、胰岛移植及干细胞治疗等新兴疗法在T1DM的临床前或临床应用中均显示良好的治疗效果.而肠道菌群和胰岛干细胞为人类防治T1DM提供了新的方向.结论 除传统胰岛素治疗外,新型治疗方法对未来T1DM的治疗具有重大意义.

实验探究了重组Monellin菌株的最佳培养条件,并对其进行了甜味阈值确认和血糖值影响实验.实验所用菌种为实验室构建保存的BL21(DE3)-pET28a Monellin菌种,利用不同浓度的乳糖和IPTG诱导该菌种生成目的蛋白,探究其最佳诱导条件,利用镍柱亲和层析纯化目的蛋白后进行相对甜度和甜味阈值实验,随后设置甜度梯度,对小鼠进行灌胃比较蔗糖和Monellin的升血糖作用及不同浓度Monellin对血糖的影响.实验结果表明,0.9 mmol/L IPTG诱导5 h的Monellin表达量最多,其甜味阈值为100μg/mL,相对甜度为蔗糖的800倍.灌胃结果表明,Monellin升血糖作用不显著,并且在一定范围内对血糖的影响不随其浓度的改变而发生变化.该实验为Monellin在糖尿病等代糖食品市场上的应用提供了科学的理论依据.

前列腺六段跨膜上皮抗原(Six-segment transmembrane epithelial antigen of prostate,STEAP)家族属于跨膜蛋白,其在细胞与细胞之间的通讯方面起着重要作用.STEAP具有离子通道功能,在体内可发挥金属氧化还原酶活性,广泛参与调控细胞增殖、凋亡、迁移和侵袭等生命活动.在多数肿瘤细胞中STEAP均呈现过表达的趋势,与正常的组织细胞表达量形成显著差异,这种表达特异性,预示其可作为治疗癌症的潜在靶标.文章主要针对STEAP家族中的成员STEAP1,对其蛋白结构功能、正常细胞与肿瘤细胞之间的表达差异以及靶向策略作一简要综述,用以评估其作为肿瘤治疗靶点的可行性.

凝血因子Ⅶ是一种维生素K依赖型的单链糖蛋白,在凝血过程中发挥着极其重要的作用,在临床上有广泛的应用,可用于伴有抑制物的血友病、先天性FⅦ缺乏症、血小板无力症及外科手术或严重外伤导致的创伤出血等止血用途.基因重组技术提供了能够大规模制备人凝血因子Ⅶ的有效途径,近年来已尝试并建立了多种人凝血因子Ⅶ的重组表达系统.对重组人凝血因子Ⅶ蛋白在酵母细胞、哺乳动物细胞、转基因动物这三类表达系统中的发展和应用概况进行了综述,介绍了不同表达系统的特点并比较了FⅦ在不同哺乳动物细胞系中蛋白质翻译后修饰的差别,为重组人凝血因子Ⅶ在重组表达系统上的进一步开发提供参考.

淋巴细胞活化因子-3(lymphocyte activation gene-3,LAG-3)又名CD223,为一类免疫检查点受体蛋白,主要表达于活化的免疫细胞中.LAG-3分子在生理条件下可维持机体免疫稳态,在肿瘤微环境中可介导肿瘤细胞的免疫逃逸,因此,可将LAG-3作为肿瘤免疫治疗的新靶点进行研究.LAG-3阻断性抗体可有效缓解其他免疫检查点抑制剂导致的患者耐受,且在药物联用方面具有较好的临床疗效.目前,国内外有多款抗体药物处于研发阶段,部分已进入临床后期阶段.该文从结构、表达、功能等多个方面对LAG-3分子进行综述,并阐述其相关抗体药物的临床研究进展以及市场情况.

促红细胞生成素(EPO)通过结合促红细胞生成素受体(EPOR)的同型二聚体(EPOR)2发挥促红作用,最初被用作贫血治疗.近二十年来,大量研究证实EPO可以抗凋亡、减轻炎症反应及通过刺激细胞增殖和分化以支持受损功能的重建,从而对多种组织系统起到保护作用,特别是神经系统.进一步研究表明,这种组织保护作用主要是由EPOR与β共同受体(βCR)组成的异质二聚体EPOR/βCR介导的.为避免EPO治疗组织损伤过程中产生的促红细胞生成的副作用,不同形式的EPO衍生物被开发出来.本文旨在综述EPO及其衍生物在神经系统内的保护作用与机制,希望为EPO在神经疾病中的临床应用提供指导.

近年来,连续型细胞培养由于其高单位体积产量、稳定的产品质量属性以及潜在的成本节约效应正成为生物大分子制药生产的工艺焦点。相比传统的流加培养模式,灌流培养因培养的连续性、操作的复杂性,致使其反应器规模培养需消耗大量培养基,产生更高人力成本,不能满足当今加速化高效化的工艺开发需求。为获得稳健的灌流培养工艺并控制较低成本,高通量灌流培养模型被用于批量化的小规模灌流培养,进行灌流培养前期的克隆筛选、培养基筛选及工艺参数优化等工作,为后期大规模培养提供实用性数据支持,同时也被用于预测大规模培养的细胞表型和产品质量属性。重点介绍了当前高通量系统包括摇瓶/摇管系统、多平行自动化系统以及微流控体系用作灌流培养的特征、具体应用及比较,同时论述当前高通量灌流培养系统在生物工艺领域发展所面临的机遇及挑战,并展望其应用前景。

以大学生创新训练项目为契机,依托国家级生物制药实验教学示范中心平台,围绕“双靶点抗肿瘤疫苗研制”项目指导大学生进行相关理论知识的学习、科研思路的设计,并完成实验操作、数据整理、专利申请和论文写作等训练.初步选择鼠源粒细胞巨噬细胞集落刺激因子(mGM-CSF)和促胃泌素释放肽(GRP)为双靶点,完成了基因工程菌的构建、目标蛋白的制备、细胞培养、动物肿瘤模型的建立、肿瘤疫苗药效初步分析与探索.通过指导学生申请专利、发表文章、参加药苑论坛、挑战杯等各类比赛完成对学生的训练,提升其自主设计实验、自主完成实验、自主管理实验等综合能力.

To establish an automated high-throughput mimic perfusion scale-down model (SDM) in ambr® 15 system. An optimized SDM for mimic perfusion was developed in ambr® 15 system. Cell retention in ambr® 15 was realized by sedimentation and supernatant removal with a retention rate > 95%. Although the SDM couldn’t reach the viable cell density (VCD) at a bench scale bioreactor (BR), it maintained VCD at approximately 30 × 106 cells/mL with a cell bleeding rate estimated theoretically and predicted the cell specific perfusion rate (CSPR). A base-feeding strategy was developed to alleviate the pH drop during sedimentation which would adversely have an impact on cell growth, and showed an apparent cell viability improvement from 79.6% (control) to 90.1% on Day 18. The optimized SDM for mimic perfusion was employed for media screening in two cell lines. A small-scale high-throughput perfusion model in ambr® 15 was developed, optimized to improve cell viability, and as a result, utilized for media screening in two cell lines.

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.

目的 通过对达托霉素产生菌进行诱变选育,并对其发酵条件优化,以提高其达托霉素发酵水平,降低生产成本.方法 以玫瑰孢链霉菌(DT-9#F2)为出发菌株,分别采用紫外诱变、微波诱变、LiCl诱变及复合诱变并结合链霉素抗性筛选进行菌株选育,同时,对其发酵培养基中氮源、碳源及生长因子进行优化,以进一步提高其发酵产量.结果 得到一株达托霉素高产突变株DT-37,其摇瓶发酵产量达到12.2mg/L,较出发菌株提高20.79％;优化后的发酵培养基为:酵母粉(YP300)1.65％、FeSO4 0.043％、葡萄糖1.50％、玉米淀粉7.20％、糖蜜0.72％,VB12 1.50μg/mL、硫辛酸5.00μg/mL.其摇瓶发酵产量达到30.56mg/L,较初始培养基提高了297.92％.经100L发酵罐上放大验证,达托霉素的产量达到了2872mg/L,较优化前提高了14.88％.结论 紫外、微波及LiCl复合诱变后经抗生素抗性筛选,结合发酵培养基优化,可有效提高菌株的达托霉素发酵产量.

Acute infections with enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) usually cause Hand, foot and mouth disease (HFMD) among infants and young children with several large outbreaks worldwide. Unfortunately, the molecular mechanisms underlying enterovirus infections remain largely unknown. In this study, we analyzed the genome-wide DNA methylation patterns of host cells in response to EV71 and CVA16 infections using the Illumina Infinium HumanMethylation450 BeadChip. Of over 480,000 loci studied, significant differential methylation was observed between EV71 infected-cells and control cells at 3957 CpG sites, out of which 2478 were hypermethylated and 1479 were hypomethylated, whereas CVA16 infection resulted in methylation level changes of 5194 CpG sites with 4288 hypermethylated and 906 hypomethylated. These differential methylated loci displayed a wide range of genomic distributions in chromosomes, inside and surrounding areas (shores and shelves) of CpG islands, as well as functional gene regions including promoter, gene body and 3’UTR. Based on methylation alterations, 1189 genes were identified to be potentially co-associated with the replication processes of two enteroviruses. GO function annotation and enrichment analysis of 1189 common differentially methylated genes reflected a broad spectrum of biological regulatory events during viral infection. KEGG pathway analysis indicated the involvement of diverse signaling pathways including viral myocarditis, Notch signaling and antigen processing and presentation. Our present study provides a novel insight into enterovirus-host interaction network at epigenetic profile, thus contributing to improved understanding of HFMD pathogenesis.

黑色素瘤是一种侵袭性强的恶性肿瘤,且是发病率增长最快的恶性肿瘤之一,但目前尚无良好的治疗方法.通过对大量相关黑色素瘤文献的收集阅读,分析了中国及世界的黑色素瘤发病与死亡状况,发现黑色素瘤发病率与死亡率与人种、年龄、性别、居住区域的环境状况密切相关,总结了黑色素瘤的发病机制,包括强烈紫外线照射、基因突变(BRAF、NRAS和NF1基因突变)和分子信号通路(MAPK通路和PI3K通路)等.文章介绍了目前黑色紊瘤的治疗方法的现状,如手术、放疗、化疗、中药治疗、靶向治疗和免疫治疗,并分析了其优劣势.提出了新靶点、多靶点联合治疗、将传统的治疗手段与新兴的治疗方法相结合的新思路,为黑色素瘤的新药研发和临床治疗提供参考.

探究促性腺激素释放激素(GnRH)与胃泌素释放肽(GRP)偶联的融合蛋白(G3G6)和热休克蛋白65(HSP65)与六跨膜前列腺上皮抗原(STEAP1)偶联的融合蛋白(HST1)致敏树突状细胞(DC)的效果和致敏后DC对B16F10黑色素瘤的抑制杀伤作用.利用实验室现存工程菌表达融合蛋白G3G6和HST1,按未致敏DC (US-DC)组,G3G6融合蛋白致敏DC(G3G6-DC)组,HST1融合蛋白致敏DC (HST1-DC)组和G3G6及HST1联合致敏DC(GH-DC)组致敏小鼠骨髓来源的DC分化成熟,获得融合蛋白致敏的相应DC疫苗.将B16F10黑色素瘤细胞以1×106个/只移植于C57BL/6J雄性小鼠构建黑色素瘤模型,DC疫苗免疫治疗,体内外实验探究DC疫苗的抗肿瘤药效.流式细胞术分析证明,融合蛋白有效刺激DC分化成熟;动物实验显示,与US-DC组相比,G3G6-DC黑色素瘤抑制率为35.75％,HST1-DC组为34.03％,GH-DC组为55.74％.研究结果初步证明,G3G6-DC和HST1-DC均可有效抑制黑色素瘤B16F10细胞小鼠移植瘤的生长(P＜0.05),且联合用药优于单独用药(P＜0.01).

The research of tumor vaccine plays a crucial role in tumor immunotherapy. This study has constructed and prepared a fusion protein vaccine of heat shock protein 65 (HSP65) and the octapeptide epitope 186-193 of the six transmembrane epithelial antigen of the prostate 1 (STEAP1 186-193), and investigated the inhibitory effect of the fusion protein on mouse RM-1 prostate cancer and B16F10 melanoma xenografts. The fusion protein His-HSP65-STEAP1 186-193 (HHST1), His-HSP65-2×STEAP1 186-193 (HHST2) and His-HSP65-6×STEAP1 186-193 (HHST6) were obtained by setting different copy number of STEAP1 186-193 and adding His purification tag before HSP65. Firstly the inhibitory effect of fusion protein on mouse RM-1 prostate cancer xenografts has been studied, which could be the basis of the study the inhibitory effect of the best fusion protein on mouse B16F10 melanoma xenografts. All studies compared with the fusion protein His-HSP65 (HHSP65), the fusion proteins HHST1, HHST2 and HHST6 all could significantly inhibit the growth of mouse RM-1 prostate cancer xenografts. In addition, the fusion protein HHST2 was proved to be the best compared with the fusion proteins HHST1 and HHST6 (P<0.05). Apart from this, compared with the fusion protein HHSP65, the fusion protein HHST2 also significantly inhibited the growth of mouse beared B16F10 melanoma. The results above indicate that HSP65 and STEAP1 186-193 can significantly inhibit the growth of mouse RM-1 prostate cancer and B16F10 melanoma xenografts, and the appropriate increase of copy number can effectively improve that the fusion protein has an excellent anti-tumor ability.

Background: Type 1 diabetes is a polygenic disease that is influenced by environmental factors. A cure is not available, and patients depend on lifelong insulin injections. There is no clinical drug that can both promote the proliferation of islet beta cells and eliminate the immune attack of autoreactive T cells on beta cells. This study aimed to design a new bifunctional fusion polypeptide that is expected to have a good effect on type 1 diabetes. Methods: Our previous studies found that VP peptide, an immunomodulatory peptide that can eliminate inflammatory microenvironment, and EGLP-1, a structural analogue of GLP-1 that has the effect of protection of the pancreatic beta cells. So, we combined VP peptide and EGLP-1 to form a novel bifunctional peptide P30, hoping that VP may be an appropriate target as an immuno-modulatory arm, while GLP-1 analog may be suitable as a beta cell protective arm in combination therapy for T1D. The protective effect of each group on islets was determined by H&E and immunofluorescence, and the proportion of Treg/Th17 and Th2/Th1 cells were detected by flow cytometry. Findings: Our data demonstrate that P30 treatment significantly reduced the morbidity and mortality in NOD mice and alleviated pancreatitis symptoms. In addition, P30 has the function of enhancing immunoregulation involving increasing the ratio of Treg/Th17 cells and Th2/Th1 cells. Interpretation: These findings indicate that P30 represents a very promising strategy for prevention type 1 diabetes in mouse models of NOD. Funding: National Natural Science Foundation of China (Grant No. 81570696 and No. 31270985); Qing Lan Project; Program for Jiangsu Province Innovative Research Team; the Fundamental Research Funds for the Central Universities (2016ZPY010, 2632017PY06). Declaration of Interest: The authors declare that there is no duality of interest associated with this manuscript. Ethical Approval: All studies were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (Nanjing, China).