转基因技术及骨髓间充质干细胞在骨缺损修复中的应用与展望

骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)是一类存在骨髓内的非造血干细胞,具有较强的增殖功能及多向分化潜能。在一些细胞因子的诱导下可以向成骨系细胞、内皮细胞、成纤维系细胞、成软骨系细胞等方向分化,近年来已成为生物学和医学的研究热点。而转基因技术是一项目前较为热门的生物科技技术。本文简要介绍了不同基因如骨形态发生蛋白(bone morphogenetic protein,BMP)、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、软骨源性形态发生蛋白1(cartilage-derived morphogenetic protein 1,CDMP-1)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、成骨生长肽(osteogenic growth peptide,OGP)等通过转染后对BMSCs增殖、分化的影响及其在骨缺损修复中的应用。

单细胞全基因组扩增技术与应用

同一组织中的细胞往往具有类似的结构和功能,然而通过对单个细胞进行测序分析后,发现每个细胞都具有一定异质性.单细胞全基因组扩增技术是进行单细胞测序的前提,该技术可用于揭示单细胞基因组结构差异,同时在肿瘤研究、发育生物学、微生物学等研究中发挥重要作用,并成为生命科学研究技术的热点之一.单细胞全基因组扩增技术的难点在于单细胞的分离和全基因组的扩增.本文介绍了单细胞全基因组扩增技术中常用的单细胞分离技术和单细胞全基因组扩增技术,并对各技术间的优缺点进行比较,同时着重讨论该技术在肿瘤研究、发育生物学和微生物学研究中的应用.

微生物单细胞基因组技术及其在环境微生物研究中的应用

单细胞及单细胞基因组学研究是近年生命科学研究的热点之一,微生物单细胞基因组学研究是继微生物元基因组学(又称宏基因组学,Metagenomics)之后新发展起来的,可有效获取环境中大量无法培养的微生物遗传信息的技术。微生物单细胞基因组技术包括单细胞获取、全基因组扩增、全基因组测序以及数据分析等步骤,目前该技术在环境微生物研究中的应用主要集中于探索未被元基因组技术或其它常规技术探测到的新型功能基因,或是对环境中物种丰度极小的未培养微生物的发现,以及对微生物细胞生命进化过程的研究等。本文对微生物单细胞基因组技术中单细胞获取和全基因组扩增所涉及到的不同方法以及应用此技术对环境微生物取得的主要研究进展进行综述。

两种不同方式介导IL-12真核表达载体抑制肝移植瘤生长的研究

目的 :比较两种不同方式介导IL 12真核表达载体抑制肝移植瘤生长的效果。方法 :利用鼠肝癌细胞系H2 2建立肝移植瘤模型 ,以脂质体介导IL 12质粒腹腔注射或直接瘤内注射给荷瘤鼠 ,每周 1次 ,共 2次。治疗后每周两次测量瘤体大小 ,取血利用ELISA法检测血清中IL 12及IL 2的含量变化 ;MTT法检测NK细胞杀伤活性。结果 :直接瘤内注射组瘤体积明显小于腹腔注射组 (P <0 .0 5 ) ,直接瘤内注射组血清IL 12、IL 2浓度有明显的周期变化 ,但二者NK细胞杀伤率无明显差异。结论 :脂质体介导直接瘤内注射IL 12真核表达载体可有效抑制肝移植瘤的生长。

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Evolutionary dynamics of the Wnt gene family:implications for lophotrochozoans

Genes encoding Wnt ligands, which have important roles in cell communication and organ development, are restricted to multicellular animals. We systematically studied W nt genes from eumetazoan genomes, with emphasis on the poorly studied superphylum Lophotrochozoa(four annelids, seven mollusks, eight platyhelminths, one bdelloid rotifer, and one brachiopod species). Between 3 and 39 W nt loci were identified in each genome, and the protostome-specific loss of Wnt3 genes was confirmed. We identified gastropod-specific loss of Wnt8, refining the previously proposed mollusk-specific loss. Some duplicated Wnt genes belonging to a same subfamily or closely related subfamilies showed tandem distribution in the lophotrochozoan genomes, indicating tandem duplication events during Wnt family evolution. Members of the conserved Wnt10-Wnt6-Wnt1-Wnt9 cluster showed highly correlated expression patterns over time in two assayed lophotrochozoans, the oyster C rassostrea gigas and the brachiopod L ingula anatina, reflecting the possible similar function of the clustered W nt genes.

Advantages and Disadvantages of Transgenic Animal Technology with Genetic Engineering

Transgenic animal technology has been one of the fastest growing biotechnology areas. The exogenous genes have been introduced into the animal genome by genetic engineering, so that these genes can be inherited and expressed by offspring to produce desired traits or evaluate function in elite livestock breeds. There are several methodologies for the production of transgenic animals, i.e., （1） microinjection of genes into pronuclei of fertilized ova; （2） DNA transfer by retroviruses; （3） injection of embryonic germ （EG）/embryonic stem （ES） cells previously treated with foreign DNA; （4） DNA transfer into cells and embryos with using liposomes; （5） exogenous DNA transfer while in vitro fertilization by using sperm; （6） electroporation of DNA into sperm, embryos or ova; （7） biolistics; （8） nuclear transfer （NT） with somatic cells, EG or ES cells; （9） germ line stem cell-mediated; （10） gene targeting; （! 1） gene silencing technology with RNA interference; （12） induced pluripotent stem cell; （13） zinc-finger nuclease gene targeting technology. Gene farming is one of the newest and most promising areas in modern biotechnology. Cattle, goats, sheep, pigs and rabbits are the main farm livestock species and fish is also used in transgenic technology. The question of ＂why make transgenic animals？＂ is very important. Some of the answers to this question are： （1） to obtain new knowledge; （2） to solve the genetic code; （3） to create genetic disease models; （4） to study the genetic control of physiological systems; （5） to improve animal production traits; （6） to produce new animal products. Transgenic technology is one of the main and important tools in the finding solutions to problems of growing population with their applications to different organisms, and takes more attention and interest every day. Transgenic technology creates opportunities and areas to play with organisms to fulfill the demands of people. Because of this, this paper based on mainly transgenic applications to take people＇s attention and exhibit its importance.

Expression of human acidic fibroblast growth factor in Pichia pastoris

Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.

Cloning,expression and function of the extracellular fragment of human TRAIL gene

To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC） by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 （DE3）. Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.

Elevated interleukin-13 in patients with active lupus nephritis

Objective To investigate the significance of interleukin-13 (IL-13) in patients with active lupus nephritis (LN).Methods Ten healthy volunteers and 16 patients with active LN were included in this study. The protein level of IL-13 in plasma was examined by enzyme linked immunosorbent assay (ELISA), and gene expression of IL-13 in peripheral blood mononuclear cells (PBMCs) by reverse transcription polymerase chain reaction (RT-PCR). Expression of IL-13 mRNA in renal tissue was studied by in situ hybridization (ISH) techniques.Results The level of IL-13 in plasma and the expression of IL-13 mRNA in PBMCs were significantly higher in LN patients than those in the controls ( P ＜ 0.001 ). Increased expression of IL-13 mRNA was detected in renal tissue of active LN patients compared to those in the controls ( P ＜ 0.001 ). Analysis of the linear correlation indicated that the level of IL-13 mRNA in the tubulointerstitial area in patients with active LN correlated with the concentration of serum creatinine (Scr), the glomerular activity index (GAl),the activity index of tubulointerstitium, and the level of serum C3 ( P ＜ 0.05 for each).Conclusion The elevation of IL-13 may play an important role inthe molecular pathogenesis of active LN.

Single cell sequencing: technique, application,and future development

The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification techniques bring sequencing technology to the single-cell level. Single cell genome sequencing allows us to study tumor evolu- tion, gamete genesis, somatic mosaicism at genome-wide level; single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel- opment, differentiation and reprogramming; single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies. Lots of attempts are still being made in other dimensions of sequencing. The increasing need for single cell sequencing requires the future techniques with the following features： （1） high accuracy and fidelity; （2） able to perform multiple omics analyses in one cell; （3） high degree of automation and standardized pipeline. These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories. The wide application of single cell sequencing techniques will substantially change biomedi- cal research in future.