The Artificial Synthesis of TAT PTD and Tachyplesin Fusion Gene by Overlap Extension PCR

为探讨HIV-TAT蛋白转导域(PrOTEIn TrAnSduCTIOn dOMAIn,PTd)对中国鲎抗菌肽TACHyPlESIn的协同作用,试验以经毕赤酵母密码子优化后的TACHyPlESIn成熟肽(54 AA)加TAT PTd(11 AA)的CdnA序列为参考模板,设计了6条单链寡核苷酸片段,首尾引物的5'端分别引入ECOr I和XbA I酶切位点,通过重叠延伸PCr方法成功合成了TAT PTd+TACHyPlESIn序列,序列全长219 bP,为其后续功能与协同作用研究奠定了基础。In order to study the coefficient of the TAT PTD(the protein transduction domain in HIV-1 transactivator of transcription protein) to the antibacterial peptide tachyplesin from Tachpleus tridentatus,6 sequence of oligonucleotides with EcoR Iand Xba I in the ends of 5′-end of the first and last primers respectively were designed,based on the TAT PTD and tachyplesin mature peptide cDNA template,which was optimized according to the Pichia pastoris preferred codon.A optimized TAT PTD and tachyplesin fusion gene with 219 base pairs was artificially synthesized by overlap extension PCR,which laid a preliminary foundation for the following functional and coefficient studies.广东省自然科学基金(06025389); 福建省博士后项

Differential regulation of stiffness, topography, and dimension of substrates in rat mesenchymal stem cells

The physiological microenvironment of the stem cell niche, including the three factors of stiffness, topography, and dimension, is crucial to stem cell proliferation and differentiation. Although a growing body of evidence is present to elucidate the importance of these factors individually, the interaction of the biophysical parameters of the factors remains insufficiently characterized, particularly for stem cells. To address this issue fully, we applied a micro-fabricated polyacrylamide hydrogel substrate with two elasticities, two topographies, and three dimensions to systematically test proliferation, morphology and spreading, differentiation, and cytoskeletal re-organization of rat bone marrow mesenchymal stem cells (rBMSCs) on twelve cases. An isolated but not combinatory impact of the factors was found regarding the specific functions. Substrate stiffness or dimension is predominant in regulating cell proliferation by fostering cell growth on stiff, unevenly dimensioned substrate. Topography is a key factor for manipulating cell morphology and spreading via the formation of a large spherical shape in a pillar substrate but not in a grooved substrate. Although stiffness leads to osteogenic or neuronal differentiation of rBMSCs on a stiff or soft substrate, respectively, topography or dimension also plays a lesser role in directing cell differentiation. Neither an isolated effect nor a combinatory effect was found for actin or tubulin expression, whereas a seemingly combinatory effect of topography and dimension was found in manipulating vimentin expression. These results further the understandings of stem cell proliferation, morphology, and differentiation in a physiologically mimicking microenvironment

Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model

Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor alpha or beta_1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment

Effect of transposon-induced motility defect on virulence of Vibrio alginolyticus

采用转座子MInI-Tn10/kM构建了致病性溶藻弧菌的突变库,采用半固体双层平板初筛到73株运动缺陷突变株,初筛的突变株纯化及重复筛选后获得运动性缺陷表型稳定的突变株nd-01MM,SOuTHErn鉴定确定其为单位点插入。野生型菌株nd-01和运动缺陷突变株nd-01MM对致病性溶藻弧菌的天然宿主大黄鱼粘液的趋化、粘附以及在大黄鱼吞噬细胞内存活能力等生理功能的比较研究发现,溶藻弧菌运动缺陷后趋化及粘附能力均极显著下降(P0.05)。采用腹腔注射和浸泡两种方式感染大黄鱼,结果发现运动缺陷对浸泡感染的影响较为明显,但对腹腔注射的感染方式影响不大。Motility is thought to contribute to the virulence of many pathogenic bacteria.Infection studies in many motile pathogens found that in many causes normal motility was required for efficient infection.However,there are few reports on the direct correlation between motility and virulence in Vibrio.In order to understand the role of motility of Vibrio alginolyticus in virulence,the mini-Tn10/Km transposon-induced mutant bank was constructed and 73 motility defective mutants were screened out by double semi-soft agar medium.After purification and repeated screening the mutant ND-01MM with stable motility defect was obtained and identified by Southern blot.The chemotaxis and adhesion of wild-type strain ND-01 and the mutant ND-01MM to mucus of the natural host Pseudosciaena crocea and their persistence in phagocytes of P.crocea were tested.The results showed that the chemotaxis and adhesion of the mutant was highly significantly impaired(P0.05).The results of infection test showed that the virulence of the mutant was decreased by immersion infection,but not different from the wild-type strain by intraperitoneal injection.国家公益性行业专项(200903029);福建省杰出青年基金(2011J06014

创面愈合中力学刺激诱导的表皮细胞非对称迁移

创面修复是机体应答损伤所表现的一个十分复杂的生物学过程,是一个典型的力学-生物学耦合过程,该过程中的生物合成与细胞迁移分化过程均受到生物学和力学因素的协同调控。再上皮化是创面闭合的中心步骤,其主要由表皮细胞迁移完成。表皮细胞是上皮组织中数目最多,分布最广的力学敏感性细胞,在感知和应答力学刺激、调节皮肤创面修复过程中起重要作用。因此,表皮细胞的迁移是组织修复必须的,机械应力和真皮成纤维细胞在创面修复过程中起了重要的作用。因此,表皮细胞迁移动力学是创面修复研究的核心问题