Inhibition of CXCR4 expression by recombinant adenoviruses containing anti-sense RNA resists HIV-1 infection on MT4 cell lines

CXCR4-tropic (X4) variants are associated with faster disease progression than CCR5-tropic variants in HIV infection. We previously reported inhibition of CCR5 expression on U937 cells could protect the cells from HIV-1 infection. Here, we established recombinant adenoviruses containing anti-sense CXCR4 cDNA, to investigate its role in the protection of HIV entering into target cells. A fragment of 636 bp cDNA from CXCR4 mRNA was recombined into adenoviral vector and the recombinant adenovirus was obtained from AD-293 cells. The rates of CXCR4 expression on the MT4 cells infected with recombinant adenovirus were measured by FACS. The MT4 cells infected by recombinant adenovirus were challenged by T-tropic HIV-1 strains and then P24 antigen was assayed. The rate of expression of CXCR4 on MT4 cell infected with recombinant adenovirus was decreased from 42% to 1.12% at 24 h, and to 1.03%, 1.39%, and 1.23% at 48 h, 72 h and 10 days respectively. Compared with Ad-control cells, recombinant adenovirus infected MT4 cells produced much less P24 antigen after being challenged with HIV-1. Furthermore, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the MT4 cells. In conclusion, recombinant adenoviruses containing anti-sense can inhibit CXCR4 expression and resist HIV-1 infection on MT4 cell lines.     

2012年度国家自然科学基金微生物学学科项目资助情况分析

本文介绍了2012年度国家自然科学基金委员会生命科学部微生物学学科项目申请、受理和资助的概况,简单分析了各分支学科和各项目类别申请的特点和存在的问题,说明了学科相关的鼓励政策,希望为今后科研人员的项目申请提供有益的参考.     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cytoskeletal remodelling proteins identified in fetal-maternal interface in pregnant women and rhesus monkeys

The uterus undergoes dramatic remodelling in preparation for embryo implantation and pregnancy establishment. A receptive uterus is pivotal for embryo attachment, implantation and the eventual formation of a hemochorial placenta. We have previously identified by proteomics that tropomyosin alpha-4 chain (TPM4), protein disulfide isomerase A1 (PDIA1) and src substrate cortactin 8 (SRC8) were up regulated in the decidualized stromal cells during the late secretory phase of the menstrual cycle in women. These three proteins are associated with cytoskeletal remodelling. This study determined the localization of these three cytoskeletal proteins in the fetal-maternal interface including the decidual cells in the 1st trimester of pregnancy in women and rhesus monkeys. Immunohistochemical analysis revealed that TPM4, PDIA1 and SRC8 were all expressed by the decidual cells and the wall of the spiral arterioles in pregnant women. Similar expression pattern were also found in the rhesus monkey. In addition, TPM4, PDIA and SRC8 were also localized to the trophoblast cells, further highlighting the importance of these cytoskeletal remodelling proteins in early pregnancy.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

新疆野扁桃天然居群形态变异的研究

为了从数量上分析新疆野扁桃(Amygdalus ledebouriana)天然居群表型性状在居群间和居群内的变异,我们于2007年对分布存新疆的5个野扁桃天然居群的8个表型性状进行了测量和比较分析.结果表明:新疆野扁桃表型性状在居群间和居群内均存在着较丰富的差异,居群内的变异大于居群间的变异,居群间的分化相对较小;利用居群问欧氏距离进行UPGMA聚类分析表明,5个天然居群可以划分为3类,表型性状的欧式遗传距离与地理距离相关不显著.主成分综合分析结果显示:新梢长宽比、叶片长宽比、果核千粒重、果核长宽比及花冠直径等5个表型性状指标是反映新疆野扁桃表型差异的主要因素.     

百合细胞中内生菌的发现

在健康的百合鳞茎、地上茎、叶和幼胚等组织的细胞中发现了“内生菌”。通过光学显微镜、电镜和组培等观察,“内生菌”不影响百合细胞的正常分裂和分化,再生植株健壮。这些细菌还能随着宿主细胞的分裂转移到子细胞。“内生菌”椭圆形或近圆球形。电镜切片菌壁厚,单层,均质,为典型革兰氏阳性菌壁结构。电镜观察结果与光学显微镜观察内生菌的革兰氏染色反应是一致的。     

苜蓿中华根瘤菌代谢组学样品制备方法的研究

代谢组样品制备是代谢组学研究的基础。本文以维生素B12生产菌株苜蓿中华根瘤菌Sinorhizobium meliloti 320为研究对象,通过检测细胞损伤、ATP泄漏、代谢物回收效率以及细胞代谢淬灭效率综合评价细胞淬灭方法,同时对5种提取试剂的提取效率进行比较优化胞内代谢物的提取方法。最终获得苜蓿中华根瘤菌S.meliloti 320的胞内代谢组学样品制备较佳条件:即-20℃40%甲醇淬灭细胞,过滤收集淬灭细胞,甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%甲醇相结合提取胞内代谢物。实验结果显示-20℃的40%甲醇(通过过滤收集细胞)对细胞膜的损伤较小,且细胞代谢淬灭效率和回收效率较高;甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%的甲醇对胞内代谢物的提取效率较高且有互补作用。     

盐生植物海马齿离体再生

建立盐生植物海马齿(Sesuvium portulacastrum)的离体再生体系,为其生物技术改良奠定基础。以海马齿叶片、茎和腋芽为外植体, 在不同激素配比的培养基上进行愈伤组织诱导、继代培养以及不定芽的分化和生根培养。结果表明: 最适愈伤组织诱导的外植体为叶片, 其次为幼嫩的茎段和腋芽。以叶片为外植体, 愈伤组织诱导率最高的培养基为MS+2.0mg·L^–12, 4-D + 0.5 mg·L^–16-BA + 3%sucrose; 芽分化最适培养基为MS + 1.0 mg·L^–1 2, 4-D + 0.2 mg·L^–1 6-BA + 3% sucrose;生根最适培养基为MS + 3%sucrose + 0.1%AC。炼苗移栽后, 成活率可达80%。     

Monitoring and kinetic parameter estimation for the binding process of berberine hydrochloride to bovine serum albumin with piezoelectric quartz crystal impedance analysis

A new method for monitoring, in real time, the drug-binding process to protein with piezoelectric quartz crystal impedance (PQCI) is proposed. The method was used to monitor the binding process of berberine hydrochloride to bovine serum albumin (BSA). BSA was immobilized on the silver electrode surface of a piezoelectric quartz crystal and the optimized experimental conditions were established. The BSA-coated piezoelectric sensor was in contact with berberine solution. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the protein-drug binding were simultaneously obtained. On the basis of the analysis of the multidimensional information provided by PQCI, it was concluded that the observed frequency decrease was mainly ascribed to the mass increase of the sensor surface resulting from the binding. According to the frequency decrease with time, the kinetics of the binding process were quantitatively studied. A piezoelectric response model for the binding was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k(1) and k(-1)) and the binding equilibrium constant (K(a)), were determined. The k(1), k(-1), and K(a) values obtained at 25 degrees C were 67.5 (+/-0.1) (mol liter(-1))(-1) s(-1), 1.7 (+/- 0.1) x 10(-3) s(-1), and 3.97 (+/- 0.06) x10(4) (mol liter(-1))(-1), respectively.