过敏性鼻炎患者外周血CD4+2型辅助T(Th2)细胞中IL-18的表达升高

目的 探讨过敏性鼻炎(AR)患者外周血2型辅助T(Th2)细胞白细胞介素18(IL-18)、 IL-18结合蛋白亚型a(IL-18BPa)和IL-18受体α(IL-18Rα)的表达水平及过敏原对其表达的影响。方法 采用大籽蒿花粉过敏原粗提液(ASWE)和梧桐花粉过敏原粗提液(PPAE)、室尘螨过敏原粗提液(HDME)激发外周血单个核细胞和免疫磁珠分选后的CD4⁺ T细胞,流式细胞术检测上述标本CD4⁺ Th2细胞中IL-18、 IL-18BPa和IL-18Rα的表达,BioPlex检测血浆IL-4的水平,并分析其与IL-18⁺ Th2细胞比例的相关性。结果 与正常对照相比,AR患者外周血CD4⁺ Th2细胞中IL-18⁺细胞的比例增加,IL-18的平均荧光强度(MFI)增强;IL-18Rα MFI降低。过敏原可直接诱导分选后的HC CD4⁺ Th2细胞表达IL-18和IL-18Rα, AR患者CD4⁺ Th2细胞表达IL-18Rα。A...     

重组rAAV2/eGFP、rAAV2/NGF病毒转染神经干细胞的研究

目的　观察重组rAAV2 /eGFP(含报告基因 )和rAAV2 /NGF(含目的基因 )病毒转染神经干细胞的转染效率 ;探讨神经干细胞治疗神经系统疾病的新途径。方法　分离、培养和鉴定神经干细胞后 ,用重组rAAV2 /eGFP和rAAV2 /NGF病毒转染神经干细胞 ,用荧光显微镜和免疫细胞化学检测GFP和NGF在体外的表达。结果　GFP和NGF于 2 4h后在体外开始表达 ;5d后表达效率高达 89% ,并可持续稳定表达 35d以上 ;MOI(v g/cell)不同 ,表达效率不同。 结论　重组rAAV2 /eGFP和rAAV2 /NGF病毒可转染神经干细胞 ,神经干细胞可稳定表达GFP和NGF ,可用于基因治疗神经系统疾病。     

大鼠创伤性深静脉血栓形成中Ca2+通道相关基因表达研究

分析Ca2 通道相关关键基因在大鼠创伤性肢体深静脉血栓形成中的表达变化,初步探讨该信号通路在此病理过程中的生物学意义。采用定量击打双侧大腿 双后肢石膏固定的方式,对150只SD大鼠造模,建立创伤性深静脉血栓形成动物模型。应用Genechip Rat Genome 430 2.0芯片,检测创伤性深静脉血栓形成过程中8种不同生物学状态下(正常对照、创伤即刻、血栓形成初始期、高峰期血栓形成、高峰期血栓不形成、血栓消退、血栓不消退、血栓不形成)股静脉RNA表达情况,筛查出差异表达基因,并进行pathway分析。重点关注高峰期血栓形成与不形成两种生物学状态下C,a2 通道相关关键基因表达变化。结果显示:高峰期血栓形成组与不形成组比较,Ca2 通道中CaMK、IP3 3K、CaN等多个关键基因表达均呈现下调,可能使包括内皮细胞在内的多种血管细胞,增殖、分化、代谢等功能受到抑制,并影响下游信号通路,引起组织细胞功能失调,最终导致血栓发生。因此,推测Ca2 通道功能受抑制可能与TDVT发生有关。     

IL-2-preS融合蛋白表达质粒的基因枪DNA免疫研究

目的 观察基因枪肌肉人注射ＩＬ－２－ｐｅｒＳ融合蛋白表达质粒（ｐＣＷＩＩＰ）的基因免疫效率,为乙型肝炎基因免疫研究提供基础。方法 设基因枪肌内注射组和正常肌肉注射组,分别用ｐＣＷＩＩＰ质粒及对照质粒ｐＣＩＩＰ－２和ｐＣＩ免疫Ｂａｌｂ／ｃ小鼠,免疫后第４天,取肌肉组织通过免疫组化观察ＩＬ－２－ｐｒｅＳ在肌肉中的表达;另设基因枪、正常肌肉、皮下注射组分别免疫ｐＣＷＩＩＰ及对照质粒在２、４、６、８Ｗ眼后     

干姜吴茱萸水煎液对大鼠结肠平滑肌细胞L型Ca2+通道的影响

目的：探讨干姜吴茱萸水煎液对大鼠结肠平滑肌收缩及平滑肌细胞L型Ca²⁺通道的影响机制。方法：采用0、0.01、0.1、1、10 g/L干姜吴茱萸(1∶1)水煎液依次作用于正常结肠平滑肌,10 g/L水煎液及L型Ca²⁺通道激动剂BAY-K-8644依次作用于氯化乙酰胆碱(Ach)诱导后的正常平滑肌,检测平滑肌收缩张力、频率和振幅;全细胞膜片钳记录L型钙电流;Western blot检测各组细胞中L-型电压依赖钙离子通道α1C亚基(α1c)和钙调蛋白(CaM)蛋白表达情况。结果：1、10 g/L水煎液组能显著降低离体结肠平滑肌的收缩张力(P<0.05),对频率及振幅并无显著影响(P>0.05);与0 g/L水煎液组相比,Ach促进平滑肌收缩张力、频率和振幅显著增加(P<0.05);与Ach组相比,10 g/L水煎液显著降低平滑肌的收缩张力、频率和振幅(P<0.05);与10 g/L水煎液组相比,BAY-K-8644显著增加平滑肌的收缩张力、频率和振幅(P<0.05);与0 g/L水煎液组相比,Ach可显著增加ICa...     

RNA阵列技术检测自发性高血压大鼠肌浆网Ca2+-ATP酶和受磷蛋白基因表达

目的:探讨肌浆网Ca²⁺-ATP酶(SERCA)和受磷蛋白(PLB)在原发性高血压发病过程中的变化特点及其相互关系。方法:提取2、4、6、8、10、12不同周龄雄性自发性高血压大鼠(SHR)和正常血压大鼠(WKY)的心室肌、血管平滑肌、肝脏和肾脏组织的总RNA,共294个样品,利用高通量RNA阵列技术(RNAarray)检测SERCA和PLB基因在不同周龄SHR和WKY中mRNA表达谱改变。结果:SHR在6、8、10、12周龄血压出现显著高于同周龄WKY(均P<0.01),10、12周龄心室肌重量／体重比出现显著增加(均P<0.01),心肌和血管平滑肌SERCA的表达在4、6、8、10、12周龄出现显著高于同周龄WKY(P<0.05或P<0.01)。PLB基因表达在两组间无显著差异(P>0.05)。心肌的SERCA与PLB表达量比值在6、8、10、12周龄出现显著大于同周龄WKY(P<0.05或P<0.01),而血管平滑肌的SERCA与PLB表达量比值在4、6、8、10、12周龄出现显著大于同周龄WKY(P<0.05或P<0.01)。结论:肌浆网SERCAmRNA表达改变及SERCA与PLB比例失常是高血压发生和发展过程中重要的分子生物学机制。     

环氧化酶2和白介素18与急性心肌梗死的关系

目的了解急性心肌梗死(AMI)患者血清环氧化酶2(COX-2)活性和白介素18(IL-18)含量的变化,探讨COX-2和IL-18与AMI患者病情的关系。方法应用酶联免疫吸附双抗体夹心法(ELISA)检测44例AMI患者5个时间点的血清COX-2活性和IL-18含量,即发病第1(治疗前)、2、3、7和14天。24例健康人为对照组。结果AMI组第1、2、3天的COX-2和IL-18含量明显高于健康对照组(P均<0.01);第7、14天的含量则明显下降,与对照组比较差异无统计学意义。AMI存活组的COX-2和IL-18含量显著低于死亡组,差异有统计学意义(P均<0.05)。AMI患者血清CK-MB的变化、发病第1天的临床积分、梗死面积与COX-2和IL-18含量均呈正相关(P均<0.01)。结论血清COX-2和IL-18水平与AMI病情的变化一致,提示COX-2和IL-18参与了AMI的病理过程,可以作为AMI患者病情监测和预后的指标。     

人源性抗乳腺癌Her-2/neu噬菌体单链抗体库构建

目的构建乳腺癌患者抗Her-2/neu全人源性噬菌体单链抗体可变区（ScFv）文库,筛选抗Her-2/neu单链抗体。方法收集40例乳腺癌患者前哨淋巴结（SLN）,提取总RNA,反转录为cDNA作模板,在目前常用的引物基础上重新设计可变区的引物,用PCR扩增全套抗体可变区,构建T载体库。并改造pCANTAB-5E构建了pCANTAB-Linker。将T载体库酶切连接入pCANTAB-Linker构建出ScFv文库。最后构建噬菌体单链抗体库并进行初级筛选。结果成功改良ScFv文库的构建方法。初步得到12株对人乳腺癌组织有高亲和力的Her-2/neu单链抗体。结论改良了ScFv文库的构建方法,可以获得较大库容量的单链抗体库,并从中筛选到人源性抗Her-2/neu单链抗体。     

糖尿病大鼠心肌磷酸受纳蛋白基因表达和肌浆网 Ca2+-ATPase活性的变化

目的：观察糖尿病(DM)大鼠心肌磷酸受纳蛋白（PLB）基因表达和肌浆网 Ca2+-ATPase活性的变化及其与心功能的关系。方法:复制糖尿病大鼠模型，分别于4、6、8周后对糖尿病组和对照组进行左心室血流动力学检测，测定心肌PLB mRNA转录水平以及蛋白表达水平变化，检测心肌肌浆网 Ca2+-ATPase活性。结果:糖尿病大鼠心肌PLB mRNA转录和蛋白表达水平4周时与正常大鼠无明显差异，6周时和8周时明显高于正常大鼠；肌浆网 Ca2+-ATPase活性4周时无明显改变，6周时和8周时明显低于正常大鼠；糖尿病大鼠4周时LVSP、LVEDP、±dp/dtmax与正常大鼠无明显差异，6周、8周时LVSP、±dp/dtmax显著降低，LVEDP显著升高。结论：糖尿病大鼠心肌PLB表达水平升高，肌浆网 Ca2+-ATPase活性降低，引起心功能下降。     

Effect of recombinant adenovirus coding for endomorphin-2 on neuropathic pain in rats

Objective: To construct a transgene expressing human endomorphin-2 by linking the signal peptide of mouse nerve growth factor (PN) to a human endomorphin-2 DNA sequence containing a short linker recognized by the protease FURIN and test the analgesic effect of endomorphin-2 on neuropathic pain. Methods: The transgene was inserted into the cosmid pAxCAwt to generate PN-EM-2-pAxCAwt. The recombinant adenovirus Ad-PNEM2 was packaged and propagated in HEK293 cells. After the Ad-PNEM2-infected NIH3T3 cells had been cultured, protein expression was examined by immunofluorescence and ELISA. A CCI rat model was constructed and the Ad-PNEM2 was administered intrathecally. The rats’ pain thresholds (PWL) were measured and the presence of endomorphin-2 in the cerebrospinal fluid was confirmed through ELISA. Results: The Ad-PNEM2 expressed endomorphin-2 smoothly and abundantly in NIH3T3 cells at a significantly higher rate than the viral control (P<0.01) or blank control (P<0.01). The expressed endomorphin-2 was mainly observed in the cytoplasm. The concentration of endomorphin-2 in the cerebrospinal fluid increased 1 day after injection and peaked between 7 and 14 days after injection. After injection, PWL approached normal levels in the operated study group. No significant change was observed in the control groups. There was a significant correlation between PWL and endomorphin-2 level (r = 0.944, P<0.001). Conclusion: The constructed human endomorphin-2 transgene was expressed effectively, and endomorphin-2 expressed by the recombinant adenovirus altered the threshold to thermal stimulus and showed good analgesic effect.     

Targeting the mitochondrial Ca2+ uniporter complex in cardiovascular disease

Cardiovascular diseases (CVDs), the leading cause of death worldwide, share in common mitochondrial dysfunction, in specific a dysregulation of Ca²⁺ uptake dynamics through the mitochondrial Ca²⁺ uniporter (MCU) complex. In particular, Ca²⁺ uptake regulates the mitochondrial ATP production, mitochondrial dynamics, oxidative stress, and cell death. Therefore, modulating the activity of the MCU complex to regulate Ca²⁺ uptake, has been suggested as a potential therapeutic approach for the treatment of CVDs. Here, the role and implications of the MCU complex in CVDs are presented, followed by a review of the evidence for MCU complex modulation, genetically and pharmacologically. While most approaches have aimed within the MCU complex for the modulation of the Ca²⁺ pore channel, the MCU subunit, its intra- and extra- mitochondrial implications, including Ca²⁺ dynamics, oxidative stress, post-translational modifications, and its repercussions in the cardiac function, highlight that targeting the MCU complex has the translational potential for novel CVDs therapeutics.     

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.     

肺癌组织CDKN2/p16基因点突变的分析

目的 研究我国肺癌组织中CDKN2／p16基因点突变的发生情况。方法 采用聚合酶链反应－单链构象多态性和序列分析的方法，对89例肺癌组织中CDKN2／p16基因第2外显子的点突变进行了研究。结果 在未发生第2外显子缺失的69例肺癌组织中，发现CDKN2／p16基因第2外显子变异或可疑变异者16例；对该16例进行序列分析，共发现有9例存在不同类型的基因点突变。结论 点突变是CDKN2／p16基因失活的一种形式，但不是主要形式。由点突变引起的CDKN2／p16基因失活在肺癌的发生发展中起一定的作用。     

IL-18 time-dependently modulates Th1/Th2 cytokine production by ligand-activated NKT cells

While IL-18 synergizes with IL-12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL-18 on the Th1/Th2 cytokine response. The injection of alpha-galactosylceramide (alpha-GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN-gamma and IL-4. When the mice were treated 2 h before alpha-GalCer challenge with IL-18, IFN-gamma production but not IL-4 production was remarkably up-regulated. In contrast, pretreatment with IL-18 6 h before the challenge enhanced IL-4 production. However, this IL-18-enhanced IL-4 production was not elicited in mice injected with anti-CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL-18 either 2 h or 6 h before challenge were stimulated with alpha-GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up-regulated in the liver MNC from mice pretreated 6 h before with IL-18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up-regulated the phosphorylation of STAT3 and suppressed in vitro IL-4 production by IL-18-primed liver MNC stimulated with alpha-GalCer, but it did not affect IFN-gamma production. These results suggest that IL-18 time-dependently modulates Th1/Th2 cytokine production in ligand-activated NKT cells by regulating/inducing SOCS3 expression.     

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

In recent years, gene editing, especially that using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, has made great progress in the field of gene function. Rapid development of gene editing techniques has contributed to their significance in the field of medicine. Because the CRISPR/Cas9 gene editing tool is not only powerful but also has features such as strong specificity and high efficiency, it can accurately and rapidly screen the whole genome, facilitating the administration of gene therapy for specific diseases. In the field of tumor research, CRISPR/Cas9 can be used to edit genomes to explore the mechanisms of tumor occurrence, development, and metastasis. In these years, this system has been increasingly applied in tumor treatment research. CRISPR/Cas9 can be used to treat tumors by repairing mutations or knocking out specific genes. To date, numerous preliminary studies have been conducted on tumor treatment in related fields. CRISPR/Cas9 holds great promise for gene-level tumor treatment. Personalized and targeted therapy based on CRISPR/Cas9 will possibly shape the development of tumor therapy in the future. In this study, we review the findings of CRISPR/Cas9 for tumor treatment research to provide references for related future studies on the pathogenesis and clinical treatment of tumors.     

自身免疫甲状腺病患者血清中IL-12和IL-18水平的分析

目的:提供自身免疫甲状腺病(AITD)患者体内Th1/Th2平衡紊乱的依据。方法:应用酶联免疫吸附法(ELISA)测定27例Graves病(GD)、24例甲状腺功能正常的桥本甲状腺炎(HT)、25例甲状腺功能低下的HT患者及20例正常对照者血清中IL12和IL18的浓度,并检测GD患者的甲状腺刺激性抗体。结果:GD患者与甲状腺功能正常的HT患者血中IL12、IL18水平无明显差异,但均高于正常对照者的相应水平。甲状腺功能低下的HT患者血中IL12和IL18的水平与正常对照者无差异。在GD和甲功正常的HT,IL18与IL12呈明显正相关。在GD,IL12和IL18均与其甲状腺刺激性抗体(TSAb)活性呈正相关。在甲状腺功能正常的HT还存在IL12和IL18二者与甲状腺球蛋白抗体(TgAb)的显著性正相关。结论:提示Th1型细胞在GD和HT两种AITD的发病中均起重要作用。通过抑制Th2型免疫反应,促进向Th1型的转变来治疗GD时,有可能导致病情恶化。     

单纯疱疹病毒Ⅰ型胸苷激酶基因融合表达产物的免疫学 …

目的 研究ＴＫ基因在真核细胞中的表达。方法 应用已构建和表达的单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ－１ＴＫ）融合蛋白免疫兔制备抗ＨＳＶ－１ ＴＫ血清,并以免疫印迹法鉴定其特异性,以免疫荧光法检测ＨＳＶ－１ ＴＫ基因在转染人肝癌细胞系中的表达。结果 免疫印迹显示,自制和国外提供的抗ＴＫ兔血清,均能特异性地识别Ｍｒ约为９７０００的ＴＫ融合蛋白。免疫荧光染色得到的结果满意,而且自制抗血清的效价和染色强度均优