Specific anti-tumor effect induced by attenuated <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1＋ VEGFR2（n1-7） was transformed into competent attenuated Salmonella typhimuriurn SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1＋VEGFR2（n1-7）. To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy （TEM） was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an enhanced accumulation of CD8^＋ cytotoxic T lymphocytes, as well as an increase in CD4^＋ cells in the tumore of animals treated with the oral gene vaccine compared to tumors from control group mice. UI- trestructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the r     

一种中药复方对小鼠脾脏淋巴细胞PD-1/PD-L1表达的影响

为了探讨中药复方对小鼠脾脏淋巴细胞PD-1/PD-L1的表达情况,以及CD3~+CD4~+和CD3~+CD8~+T淋巴细胞比例的影响,采用传统熬制中药的方法得到中药复方提取液,按低、中、高3个浓度对小鼠灌胃给药(1次/d),2周后,采用脑脊髓脱臼法处死小鼠,利用Real-time PCR和流式细胞仪检测脾脏PD-1/PD-L1的表达情况以及淋巴细胞亚型的变化。结果表明,随着中药提取液浓度的增加,脾脏淋巴细胞PD-1的表达显著降低,而PD-L1的表达无显著变化;CD3~+CD4~+型和CD3~+CD8~+型T淋巴细胞的比例均显著增加。所研制的中药复方能抑制淋巴细胞PD-1的表达,激活免疫辅助细胞和效应细胞,有望应用于抗肿瘤的免疫治疗。     

Carboxymethytl Pachymaram Up-Regulates Dendritic Cell＇s Function in Hepatitis B Virus Transgenic Mice

The effect of carboxymethytl pachymaram (CMP) on the function of dendritic cells(DCs) derived from spleens of hepatitis B virus transgenic mice are studied in vitro. The phenotypes of DCs are tested by flow cytometry (FCM), cytokines measured by ELISA. The expression of DCs’ phenotypes in HBV transgenic mice are low (CD80⁺CD11c⁺:59.12±11.53 vs 9.60±4.53, p<0.01; CD80⁺ MHC-II⁺: 44.86±12.31 vs 9.80±5.72, p<0.01, normal mice vs HBV transgenic mice), the ability of DCs stimulating T lymphocytes proliferation decreases (0.37±0.11 vs 0.20±0.11, p<0.05, normal mice vs HBV transgenic mice), levels of IL-12 and IFN-γ decrease whereas the level of IL-10 increases; CMP can enhance DCs’ ability of stimulating T lymphocytes proliferation, facilitate the secretion of IL-12 and IFN-γ, inhibit the secretion of IL-10, thus up regulates DCs function. The results show a good prospective use of CMP on the treatment of chronic hepatitis B. Biography: HOU Anji (1963–), male, Ph.D. candidate, Associate professor, research direction: clinical virology.     

Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

A series of retro viral vectors encoding humanmdr1 gene alone as wetl as in combination with either humanmgmt gene or human mutantSer ³¹-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34⁺ cetls. It has been shown that expression of dual drug resistance genes in transduced cetls confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in anin vivo model in which transduced hematopoietic cetls will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cetls more effectivety.     

CD8<Superscript>+</Superscript> T cell response mediates the therapeutic effects of oncolytic adenovirus in an immunocompetent mouse model

The role of anti-tumor immune responses in oncolytic adenoviral therapy has not been well studied due to lack of efficacious tu- mor model in immunocompetent mice.Here,we evaluated the contributions of immune components to the therapeutic effects of oncolytic adenoviruse in an immunocompetent murine tumor model permissive for infection and replication of adenovirus.We found that CD8+T cells were critical mediator for antitumor efficacy by oncolytic adenovirus.Intratumoral viral therapy induced intensive infiltration of CD8+T cells in tumor,increased tumor-specific IFN-?(interferon-?)production and CTL(cytotoxic T lymphocyte)activity of lymphocytes,and generated a long-term tumor-specific immune memory.Boosting CD8+T cell responses by agonistic anti-4-1BB(cluster differentiation 137,CD137)antibody showed synergistic anticancer effects with oncolytic viro- therapy.Our results provide insight into antitumor mechanisms of oncolytic adenovirus in addition to their direct oncolytic effect.     

基于TCGA数据库分析UHRF1基因在恶性胸膜间皮瘤中的表达及预后意义

利用生物信息学数据库分析泛素样含PHD和环指结构域蛋白1（UHRF1）在恶性胸膜间皮瘤（MPM）中的表达水平及临床意义。基于TCGA数据库和GTEx数据库差异表达分析UHRF1 mRNA在MPM组织和正常肺组织中的表达水平;使用R软件分析UHRF1 mRNA表达量与临床病理参数的相关性;构建Kaplan-Meier模型和单因素多因素COX回归模型分析UHRF1基因在MPM中的预后;利用TIMER2.0数据库分析UHRF1基因与免疫细胞浸润的关系; GSEA分析UHRF1基因发挥功能的主要富集通路。选取8例MPM组织及4例非MPM胸膜组织,通过RT-qPCR的方法验证UHRF1在MPM与非MPM胸膜组织的表达情况。数据库分析结果表明,与正常肺组织相比,UHRF1 mRNA在MPM组织中高表达; UHRF1高表达患者提示MPM患者预后不良;UHRF1基因表达量与CD4⁺辅助型T细胞2、CD4⁺效应记忆性T细胞、巨噬细胞等多种免疫细胞浸润水平具有显著的相关性（P ＜ 0.01）,且显著影响MPM患者的预后。功能富集分析显示,UHRF1主要在DNA复制、蛋白酶体、同源重组等通路中起作用。在收集到的病例样本中,与非MPM胸膜组织相比,UHRF1 mRNA在MPM组织中的表达显著增高（P ＜ 0.001）。UHRF1在MPM组织中高表达,可能通过调节DNA甲基化和免疫细胞浸润来影响MPM患者预后,有望成为MPM治疗和预后评估的潜在靶点。     

STAT1 signaling is required for optimal Th1 cell differentiation in mice

Although IFN-γ alone does not prime type I T helper cell (Th1) differentiation, the loss of IFN-γ signaling leads to impaired Th1 phenotype: IFN-γ receptor-deficient (Ifngr-/-) Th1 cells fail to permanently repress IL-4 expression. They can differentiate into IL-4-producing cells under Th2-inducing conditions. These observations suggest that IFN-γ signaling plays a critical role in si- lencing Il4 gene in Th1 cells and stabilizing Th1 phenotype. IFN-γ signaling has been further shown to inhibit IL-4 express...     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

有效微生物(EM)在中华绒螯蟹养殖中的应用

为探讨有效微生物( EM)对蟹塘水质和螃蟹成活率的改善作用,在南京市高淳区螃蟹文化园开展了EM应用试验研究。试验结果表明：在中华绒螯蟹养殖过程中投加EM,能有效降低水体中污染物的质量浓度,提高螃蟹成活率,但EM投加质量浓度不宜过大。当籽( EM)=1.5 mg/L时,蟹塘水体中NH+4-N、NO-2-N、COD和TP的质量浓度比不投加EM的对照塘降低12.7%~29.2%,螃蟹成活率提高2.4%;籽( EM)=6.0 mg/L时,NH+4-N、COD和TP的质量浓度分别降低1.1%、14.4%、0.03%,而NO-2-N 升高15.6%,螃蟹成活率升高1.0%。由此可见,当 EM 投加质量浓度过高(6.0 mg/L)时,水质指标及成活率改善效果不如EM投加质量浓度较低时(1.5 mg/L),且会引起NO-2-N的累积。     

模拟微重力大鼠T淋巴细胞发育异常的初步研究

对模拟空间微重力条件下大鼠T细胞发育受阻的神经免疫调节机理进行初步探究.采用流式细胞检测技术测定模拟微重力大鼠胸腺中不同发育阶段T淋巴细胞的数量变化,用ELISA法测定胸腺组织中去甲肾上腺素(NE)和内源性糖皮质激素(GC)的含量.结果表明模拟微重力模型大鼠出现明显胸腺萎缩(p<0.05),CD3⁺CD4⁺CD8^-和CD3⁺CD4^-CD8⁺T细胞数量明显减少(p<0.05);模拟微重力大鼠胸腺中NE和GC含量有升高的趋势.模拟微重力大鼠胸腺中发生了从双阳性T细胞向单阳性T细胞的发育阻滞,这种异常可能与神经内分泌免疫调控相关.     

镉盐及镉与锌联合应用对lewis肺癌小鼠的影响

探讨4种镉盐对lewis肺癌的作用,以及锌与镉联合应用时锌的保护机制.建立lewis肺癌小鼠模型,随机分成不同的给药组.16 d后检测瘤重、胸腺指数、脾脏指数以及肝脏和肿瘤中的金属硫蛋白含量.氯化镉组对lewis肺癌抑制作用明显并可以增强荷瘤小鼠的免疫功能.1 mg·kg^-1氯化镉组与联合应用组相比肿瘤抑制率无显著性差异,而且后者小鼠肝脏中MT表达量显著增加.结果表明氯化镉可有效抑制lewis肺癌细胞的增殖,最佳抑制剂量为1 mg·kg^-1;在联合应用组中,氯化锌在不影响镉抗lewis肺癌效果的同时还能够进一步减轻镉对机体正常组织的毒性作用.     

红树蚬表型性状对秋季软体部重量的影响效果分析

在广西北部湾随机抽取红树蚬(Polymesoda erosa)171个,测量壳长(X1)、壳宽(X2)、壳高(X3)、活体重量(X4)和软体部重量(Y)5个性状,采用相关分析、通径分析和回归分析等方法研究各表型性状对软体部重量的影响。结果表明,各测量性状间的相关系数均达到了极显著水平(P<0.01);活体重量对软体部重量的直接影响最大,其次为壳长,壳宽和壳高对软体部重量的直接影响不显著(P>0.05)。采用逐步回归分析方法建立估计软体部重量的多元回归方程为:Y=-5.286+0.133 X1+0.012 X3+0.096 X4(R2=0.758,P<0.01)。     

黑鲷NKCC1分子特征及其对急性盐度胁迫的表达响应

为了解黑鲷(Acanthopagrus schlegelii)在盐度胁迫过程中的适应机制,本研究利用基因扩增、聚类分析、荧光定量PCR等技术对NKCC1基因进行生物信息学分析以及在不同组织中的表达特征研究,并探讨其在急性盐度胁迫下的表达机制.结果表明NKCC1基因的开放阅读框(Open Reading Frame,OR...     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

Effect of MTEC1 on the functional expression of TCRaβ+ 3G11− 6C10− CD4+ CD8− thymocyte subset

A murine CD4⁺ thymocyte subset with phenotype of TCRαβ⁺ 3G11⁻ 6C10⁻ CD4⁺ CD8⁻ CD69^+/- HSA^med/lo contains the cells in relatively functional matured status. The functional property of the cells in this subset is characterized by the unique pattern of cytokine production at transitional stage from Th0 to Th2 type with the latter being the dominant type. After being co-cultured with murine thymic medullary epithelial cell line (MTEC1) cells, a murine thymic medullary type epithelial cell line, the TCRαβ⁺ 3G11⁻ 6C10⁻ CD4⁺ CD8⁻ CD69^+/- HSA^med/lo thymocytes, has exhibited significantly higher levels of proliferation capability and IL-6 production, whereas the production of IL-4 and IL-10 is suppressed after co-culturing with MTECl. By contrast, MTECl could not induce thymocytes to secrete Thl type of cytokines. The results suggest that MTECl can regulate functional status of this thymocyte subset and induce them to develop into a specialized Th2 subset.     

Phenotypic analysis of the medullary-type CD4−CD8+ thymocytes in mice

Phenotypic analysis of the medullary-type CD4 CD8⁺ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CDS⁺T cells is TCRαβ⁺CD3⁺Qa-2⁺HSA 3G11⁻6C10, whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2⁺; 33%, HAS; 30%, 3G11; and 70% are 6C10. The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four cell surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10⁺HSA⁺3G11 Qa-2→ 6C10⁺HSA⁺ 3G11⁺Qa-2 → 6C10 HSA⁺3G11⁺Qa-2 → 6C10⁻HSA⁻3G11⁺Qa-2 → 6C10⁻HSA⁻3G11 Qa-2 → 6C10⁻HA S 3G11 Qa-2⁺, the cells in the last subgroup exit the thymus and home into periphery.     

糖蜜酒精废液脱钾树脂的解吸动力学

【目的】研究糖蜜酒精废液脱钾树脂BK-001中K~+的静态解吸过程。【方法】考察洗脱剂温度、浓度及树脂粒径对K~+解吸过程的影响,用动边界模型描述K~+的解吸过程。【结果】确定糖蜜酒精废液脱钾树脂BK-001中K~+的解吸过程为颗粒扩散控制,该反应的表观活化能为40.9kJ/mol,反应级数为1.19,表观频率因子为5.27×10~4 min~(-1),假二级动力学模型更适合描述K~+的解吸过程(R~20.995)。K+解析过程的总动力学方程式为1-3(1-F)~(2/3)+2(1-F)=5.27×104r20[H2SO4]~(1.19)e_((-4.09×10~4))/RT。【结论】脱钾树脂BK-001的解吸动力学方程为糖蜜酒精废液脱钾树脂的再生及钾盐资源的综合利用提供理论依据。