分子生物学实验课程是分子生物学课程的重要组成部分,笔者结合十余年的教学和科研经历,分析分子生物学实验在教学中存在的问题,并进行创新性探索,通过将教学与科学研究有机融合,在前人的教学基础上,加深学生对分子生物学理论知识的吸收掌握,大幅度提升学生的动手操作能力、创新能力和科研思维,从而提高分子生物学实验课程教学质量,为培养兴趣型和实践型高素质生物医药人才奠定基础.

硫酸乙酰肝素蛋白聚糖(Heparan sulfate proteoglycans,HSPG)是由一个核心蛋白和若干条硫酸乙酰肝素(Heparan Sulfate,HS)侧链构成的蛋白聚糖,根据核心蛋白的种类可将HSPG分为4类:多配体蛋白聚糖,磷脂酰肌醇蛋白聚糖,串珠蛋白聚糖和集聚蛋白.硫酸乙酰肝素蛋白聚糖家族是细胞表面和细胞外基质(Extracel-lular Matrix,ECM)中的重要组分,可通过自身HS侧链与细胞因子,生长因子及其受体等多种蛋白结合,从而参与细胞功能,各种信号通路以及细胞基质互作的调节.心肌梗死(Myocardial Infarction,MI)是心脏供血不足,心肌缺氧损伤引起的一种心脏疾病.由于心肌细胞缺乏再生能力,目前临床上还没有一种有效的治疗方法能完全恢复梗死心脏的功能.近年来,对心梗病理的多项研究发现,细胞外基质在心肌梗死早期受损心肌的保护和修复中发挥着积极的作用.而HSPG可通过调节纤维化和激活间质细胞等过程参与ECM的重塑和功能调节,促进心肌修复,预防心室破裂等.文章讨论了不同HSPG在心肌梗死过程中参与ECM重构过程的研究进展,以期为HSPG在心梗中的作用和机制研究提供理论依据,并为心梗的临床治疗提供参考.

糖尿病是当今世界上最常见且普遍的慢性代谢性疾病之一,对于肾脏、心脑血管、神经、眼睛等均有极大的危害性.目前糖尿病主要包括以下四种:1型糖尿病、2型糖尿病、妊娠期糖尿病和特殊类型糖尿病.2017年我国糖尿病患病率持续上升至17.2％,其中2型糖尿病(Type 2 diabetes mellitus,T2DM)比例高达90％以上,因此糖尿病也已成为实施健康中国战略,提升健康水平亟待攻克的难题.肥胖是一种由环境、遗传、饮食、年龄等多种因素相互作用而引起的一种机体代谢紊乱综合征,也是糖尿病发生的重要诱因.T2MD患者和许多肥胖患者都表现出胰岛素刺激的葡萄糖吸收和利用的效率下降,胰岛β细胞出现代偿性增殖并增加总胰岛素的分泌水平从而达到稳定血糖的作用,这一现象被称为胰岛素抵抗(Insulin resistance,IR).胰岛素是通过其信号通路从而在机体中发挥调控作用,如果整个通路中的某一部分受到损害发生损伤,通路因此无法将上游信息正常传递至下游,胰岛素的调控作用将受到削弱甚至可能消失.当IR发生时,若通路受损,胰岛素无法调控全身葡萄糖稳态.随着IR的发展,当胰腺不能提供过量的胰岛素时,就会导致全身葡萄糖稳态的严重失衡,这将会诱发T2MD以及许多代谢性疾病.近年来已有大量以IR病理机制为主题的研究被报道,文章聚焦于以肥胖及胰岛素信号通路受损为主的IR诱导机制,整理并阐述了相关研究进展,以期为未来的进一步研究提供思路与依据.

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.

非编码RNA(non-coding RNA,ncRNA)是一类没有或只有有限编码蛋白能力的RNA,主要包括微小RNA(microRNA,miRNA)、长链非编码RNA(long non-coding RNA,lncRNA)、环状RNA(circular RNA,circRNA)、转运RNA(transfer RNA,tRNA)、PIWI相互作用RNA(PIWI-interacting RNA,piRNA)和核小RNA(small nucleolar RNA,snoRNA).目前,研究已经发现ncRNA在调控胰岛β细胞功能及胰岛素信号通路传导过程中发挥着核心作用,而胰岛素信号传导阻滞是糖尿病发生的重要原因.因此,本文综述了近年来ncRNA与胰岛素信号通路之间的调控关系的研究进展,同时探讨了ncRNA作为糖尿病潜在治疗靶点及临床诊断标志物的可能性,以期为糖尿病的治疗与诊断提供参考.

骨关节炎(OA)是一种好发于中老年人的慢性关节疾病,临床主要表现为关节肿痛、僵硬和活动受限等,是致残的重要原因之一.基质血管组分(SVF)是脂肪组织除去成熟脂肪细胞等成分后的一种异质性细胞群,其中的间充质干细胞赋予SVF干细胞特性,其他多种细胞组分也具有调节机体免疫、促进组织重建等作用.通过对OA的流行病学、发病机制、诊断和治疗,以及SVF治疗OA的临床研究和其治疗机制进行综述,以期为SVF对OA的临床治疗研究提供参考.

脂肪组织易获取、组织相容性好且对供体影响小,可作为获得成体干细胞的重要来源.基质血管组分(SVF)是从脂肪中分离出来的包括脂源性干细胞(ADSC)和基质细胞的异质性细胞群.SVF促进组织的修复和再生已被大量的临床实验所证实,尤其是在美容整形和组织修复中的应用.早期,SVF通过酶消化法获得,随着近年来在临床中扩大应用,为确保患者安全和质量可控,开发出新型自动分离设备.同时,为符合一些国家监管要求,避免酶的使用,采用非酶消化法获取SVF.因此,该文主要针对基于酶消化法和非酶消化法已经发表临床分离方法和上市的相关设备作详细论述.

该研究主要探讨了转化生长因子β(transforming growth factor β,TGFB)信号通路对胰岛β细胞增殖的影响.以Min6为细胞模型,使用TGFB信号通路激活剂TGFβ1、抑制剂SB-431542分别激活和阻断TGFB信号通路,采用CCK-8细胞活性检测法检测Min6细胞活性,并用流式细胞分选术(fluorescence activated cell sorting,FACS)检测Min6细胞中Ki-67阳性(Ki-67+)细胞比例.最后用TGFβ1、SB-431542体外处理C57BL/6J小鼠胰岛48 h,免疫荧光检测小鼠胰岛中胰岛素、Ki-67双阳性(Insulin+&Ki-67+)细胞数量.结果 显示,使用TGFβ1处理Min6细胞、小鼠胰岛,Min6细胞活性下降,Ki-67+ Min6细胞数量减少,小鼠胰岛中Insulin+&Ki-67+细胞数量减少;使用SB-431542处理Min6细胞、小鼠胰岛,Min6细胞活性上升,Ki-67+ Min6细胞数量增加,小鼠胰岛中Insulin+&Ki-67+细胞数量增多.综上所述,抑制TGFB信号通路可促进胰岛β细胞的增殖能力,该研究为胰岛β细胞再生疗法的发展提供了理论支持.

环状RNA(circular RNA)是一类新发现的具有共价闭合环的非编码RNA,在2型糖尿病的发生发展中扮演着重要角色.本文从环状RNA调控胰岛β细胞功能及其调控心脏、肾脏等器官的代谢活动两方面,综述了环状RNA与2型糖尿病的关系,同时指出了环状RNA作为2型糖尿病及其并发症临床诊断标志物的可能性,以期为2型糖尿病的预防、诊断和治疗提供参考和研究方向.

探究miR-802对于胰岛β细胞分泌胰岛素的影响及其作用机制.利用miR-802类似物及miR-802抑制剂分别在胰岛原代细胞及Min6细胞过表达或敲降miR-802;采用ELISA法检测miR-802对胰岛素分泌的影响;通过miRNA靶基因数据库预测、荧光素酶报告法和Western blot法确证miR-802的靶基因;最后开展功能回复实验阐明miR-802调控胰岛β细胞分泌胰岛素的作用机制.结果表明:在胰岛原代细胞和Min6细胞中过表达miR-802能抑制胰岛β细胞分泌胰岛素;qPCR和Western blot实验证明miR-802通过抑制靶基因肝细胞核因子1 B(hepatocyte nuclear factor 1β,Hnf1 B)的转录和翻译,从而抑制胰岛素的分泌.

探究金黄色葡萄球菌核酸酶(SNase)对2,4,6-三硝基苯磺酸(TNBS)诱导小鼠结肠炎的改善作用及机制.用2.5％TNBS溶液灌肠雌性BALB/c小鼠建立结肠炎模型,并连续6 d灌胃给予以重组乳酸菌为递呈载体的SNase.探究SNase介导的中性粒细胞胞外诱捕网(NETs)的降解对小鼠结肠炎的影响.实验分为正常组、TNBS模型组、NZ9000乳酸菌组、表达SNase的重组乳酸菌组.每日观测小鼠体质量、粪便性状和粪便隐血情况,观察期结束取结肠组织进行HE病理分析,检测结肠组织髓过氧化物酶(MPO)酶活和促炎细胞因子的mRNA表达水平,检测血清炎性细胞因子含量,同时免疫组化检测结肠组织中性粒细胞及其NETs标志物的表达水平.结果表明,乳酸菌递呈的SNase能缓解TNBS诱导的结肠炎小鼠体质量下降,降低疾病活动指数(DAI)评分,缓解结肠缩短并减轻病理损伤,降低结肠组织MPO酶活及炎性细胞因子表达,同时改善了血清炎性水平,免疫组化结果表明结肠组织Ly6G和citH3水平下降.初步机制表明,SNase能够下调炎性细胞因子的表达,降低NETs水平从而缓解小鼠结肠炎.

Melanoma is a highly invasive and lethal skin malignant tumor derived from melanocytes. It has the characteristics of high early metastasis and high mortality. In recent years, with the in-depth study of the pathogenesis of melanoma, it has been found that epigenetic modification, especially DNA methylation, is considered to be a universal intrinsic feature of melanoma development and evolution. This article reviews the research progress of abnormal DNA methylation genes in melanoma in detail, and summarizes the biomarker effect of DNA methylation genes, suggesting that the detection of abnormal DNA methylation genes in melanoma patients is hopeful as an early screening index and diagnostic marker for melanoma patients.

探讨长链非编码RNA PLUTO对胰岛β细胞合成及分泌胰岛素作用的影响及机制.利用酶法分离提取db/db、ob/ob及HFD小鼠胰岛细胞,检测PLUTO在不同肥胖模型小鼠胰岛细胞中的表达量;利用糖脂毒性刺激胰岛原代细胞和Min6细胞,检测PLUTO的表达量,阐明肥胖因素诱导PLUTO下调的原因.通过RT-qPCR和ELISA方法分析PLUTO对胰岛素合成及分泌的影响;采用Western blot及功能回复实验阐明PLUTO调控胰岛β细胞的作用机制.结果 表明:与正常小鼠相比,PLUTO在肥胖小鼠模型中表达量显著降低;在胰岛原代细胞和Min6细胞中过表达PLUTO促进胰岛合成和分泌;West-ern blot实验证明PLUTO通过上调相邻基因Pdx1的转录和翻译,促进胰岛素合成及分泌.

Background Bile salt hydrolase plays an important role in bile acid-mediated signaling pathways, which regulate lipid absorption, glucose metabolism, and energy homeostasis. Several reports suggest that changes in the composition of bile acids are found in many diseases caused by dysbacteriosis. Results Here, we present the taxonomic identification of bile salt hydrolase (BSH) in human microbiota and elucidate the abundance and activity differences of various bacterial BSH among 11 different populations from six continents. For the first time, we revealed that bile salt hydrolase protein sequences (BSHs) are distributed in 591 intestinal bacterial strains within 117 genera in human microbiota, and 27.52% of these bacterial strains containing BSH paralogs. Significant variations are observed in BSH distribution patterns among different populations. Based on phylogenetic analysis, we reclassified these BSHs into eight phylotypes and investigated the abundance patterns of these phylotypes among different populations. From the inspection of enzyme activity among different BSH phylotypes, BSH-T3 showed the highest enzyme activity and is only found in Lactobaclillus . The phylotypes of BSH-T5 and BSH-T6 mainly from Bacteroides with high percentage of paralogs exhibit different enzyme activity and deconjugation activity. Furthermore, we found that there were significant differences between healthy individuals and patients with atherosclerosis and diabetes in some phylotypes of BSHs though the correlations were pleiotropic. Conclusion This study revealed the taxonomic and abundance profiling of BSH in human gut microbiome and provided a phylogenetic-based system to assess BSHs activity by classifying the target sequence into specific phylotype. Furthermore, the present work disclosed the variation patterns of BSHs among different populations of geographical regions and health/disease cohorts, which is essential to understand the role of BSH in the development and progression of related diseases.

该研究主要探讨了微小核酸miRNA-193a-5p对人胰腺癌细胞体外迁移和侵袭的促进作用及其机制.采用miR-193a-5p模拟物及miR-193a-5p抑制剂分别上调和下调miR-193a-5p的表达;采用细胞划痕法和Transwell小室法检测miR-193a-5p对细胞迁移和侵袭能力的影响;采用Tar-getScan 7.1数据库预测miR-193a-5p的靶基因;荧光素酶报告法验-miR-193a-5p的靶基因;Westemblot和实时荧光定量PCR验证其表达结果.结果 表明,miR-193a-5p可显著促进细胞的迁移和侵袭能力.TargetScan 7.1软件预测,Prox1可能为miR-193a-5p的靶基因,荧光素酶报告实验显示,miR-193a-5p靶向Prox1基因的3'UTR区.实时荧光定量PCR和Western blot结果显示,miR-193a-5p下调了Prox1的mRNA和蛋白水平的表达.研究结果揭示,在胰腺癌中高表达的miR-193a-5p通过下调Prox1,从而使胰腺癌细胞获得高的迁移和侵袭能力,促进胰腺癌的转移.

研究长链非编码RNA (lncRNA) CTD-3252C9.4对人胰腺癌细胞Pane-1体外迁移和侵袭的抑制作用及其机制.采用培养胰腺癌微球体的三维半固体体系中的表皮生长因子(epidermal growth factor,EGF)刺激培养Panc-1细胞;采用RT-qPCR检测lncRNA CTD-3252C9.4在Pane-1中的表达量和载体转染效率;采用划痕-愈合法和Transwell小室法检测lncRNACTD-3252C9.4及其靶基因骨形态发生蛋白7(bone morphogenetix protein 7,BMP7)对Panc-1细胞侵袭和迁移能力的影响;采用Western blot验证靶基因BMP7和上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白表达量的变化.结果 表明:EGF能显著抑制Panc-1中lncRNA CTD-3252C9.4的表达,且该lncRNA能够通过抑制促癌基因BMP7的转录影响细胞侵袭和迁移能力,并且抑制肿瘤的EMT过程.

考察吉西他滨联合DRibbles疫苗治疗小鼠胰腺癌的作用效果及机制.采用小鼠皮下移植瘤模型,通过观察肿瘤生长情况及小鼠生存期检测吉西他滨联合小鼠胰腺癌细胞panc02来源的DRibble疫苗对小鼠胰腺癌的治疗效果.通过流式细胞术及ELISA检测免疫小鼠体外T细胞增殖能力及IFN-γ的表达水平、抗原特异性B细胞应答水平.通过qRT-PCR观察小鼠肿瘤组织内免疫应答水平,通过天狼猩红染色观察肿瘤组织的胶原分布.实验结果表明,吉西他滨与DRibbles联合用药可显著抑制小鼠panc02皮下移植瘤生长和延长荷瘤小鼠生存期.联合用药主要通过更好地激活效应T细胞增殖能力、肿瘤抗原特异性T细胞和B细胞的应答,及抑制肿瘤内部的胶原结构和增加肿瘤微环境中的免疫活化程度达到更好的治疗效果.因此,吉西他滨联合DRibbles疫苗能够有效抑制小鼠胰腺癌的生长,或许为胰腺癌的治疗提供了一种新的方法.

Objective:To establish a chimeric antigen receptor (CAR) modified T cells specifically targeting CD19 molecule (CD19-CAR-T cells) and to testify their in vitro killing effect on target cells.Methods:CD19-CAR fragments yielded by PCR were constructed into pCDH-GFP lentiviral vectors by molecular cloning technology.The packaged lentiviral particles were transducted into CD3+ T cells of donors.Transduction efficiency was measured by flow cytometry and PCR.The in vitro cytotoxicity of obtained CD19-CAR-T cells against CD19+ Ramos cells was tested by 7-AAD staining.Results:The amplification folds of CD3+ T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days,and about (58.3±5.4)％ cells expressing GFR About (57.4±9.3)％ CD19+Ramos cells were specifically killed by the CD 19-CAR-T cells in vitro at the E∶T ratio of 5∶ 1.Conclusion:This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro,which showed profound efficiency and specific cytotoxity against CD19+ Ramos cells.And this report might provide an experimental evidence for clinical treatment ofCD19+ B cell neoplasmas.

This study aimed at investigating the inhibitory effects and the anti-tumor mechanisms of co-adminis-tration of fusion proteins mGM-CSF/βhCG ( GC ) and hVEGF121/βhCG ( VC ) on RM-1 prostatic cancer and B16 F10 melanoma in the C57 BL/6 J mouse model. Two recombinant stains containing pET-28 a-mGM-CSF-X10-βhCGCTP37 and pET-28 a-VEGF-M2-X10-βhCG-CTP37 were induced by lactose to express fusion proteins. The fusion proteins were separated and purified to prepare the anti-tumor protein vaccines ( VC protein vaccine and GC protein vaccine) , which were then mixed to prepare a combined protein vaccine named VGC protein vac-cine. The prostatic cancer and melanoma tumor-bearing mice C57 BL/6 J were immunized with described vac-cines, then the growth of each tumor was measured;splenocyte proliferation of immunized mice was detected;and the cytotoxic effects of the vaccine on tumor cells were tested. After that, the in vivo concentrations of IFN-γ and anti-hVEGF antibodies were investigated by ELISA. The difference between each experimental group and normal saline group ( NS) was statistically significant in both tumor-bearing mouse models ( P <0. 05) respectively. Besides, VGC group exhibited significantly better anti-tumor effect compared with the GC and VC groups with the anti-tumor rate ( 41. 7 ± 0. 83)% and ( 46. 4 ± 1. 27)% for prostatic cancer and melanoma tumor, respectively. The co-administration of the two proteins, VC and GC, could inhibit the growth of RM-1 prostatic tumor and B16F10 melanoma effectively via anti-tumor immunity and anti-tumor angiogenesis.

Nosiheptide (NOS), a typical member of the thiopeptides, possesses strong activities against multidrug-resistant, gram-positive bacterial pathogens. Similar to other thiopeptides, the biosynthetic pathway of NOS belongs to a ribosomally synthesized and posttranslationally modified peptide system. Bioinformatics analysis of the NOS gene cluster suggests that nosP gene encodes a homologous protein of the Streptomyces antibiotic regulatory protein (SARP) family. In the present study, the actual initiation codon of nosP was identified by comparison of potential initiation codons GUG and AUG. In contrast to previous predictions of starting with GUG, AUG, corresponding to methionine residue as the 53rd residue in the original sequence, is actually the initiation codon of nosP, indicating that a truncated form of NosP (NosP53-323) is a functional protein. For better understanding of the transcriptional regulation for NOS biosynthesis, the binding region was subsequently investigated with NosP53-323, demonstrating that NosP53-323 specifically binds the bidirectional nosL-nosM promoter region. Additionally, NosP53-323 was confirmed to serve as a transcription factor to activate the transcription of all 15 structural genes in the gene cluster. The present study provides new insights into pathway-specific regulation of the biosynthesis of NOS, which would be beneficial to the investigation of the regulatory function of similar SARP proteins in the gene clusters of other thiopeptides.-Wu, X., Jin, L., Zhang, H., Tong, R., Ma, M., Chen, Y. Identification of truncated form of NosP as a transcription factor to regulate the biosynthesis of nosiheptide.