等距对称分叶法吻合小血管的实验及临床应用研究

目的：吻合口管壁等距对称分叶后吻合小血管，确保吻合口管壁完全外翻；内膜平整对合，降低血管危象的发生率，提高通畅率。方法：将吻合口管壁纵形剪开分叶，剪开的深度为管壁厚的２倍，小血管分３叶者吻合６针，４叶者８针。用二定点法先缝合叶部，后缝合２叶之间。用此法共吻合大白鼠尾动脉１００个吻合口，免股动脉和肱动脉１２０个吻合口，免耳皮瓣自体交叉移植２０个，用此法进行断手指再植１５例，游离皮瓣移植５例。结果：１００个大白鼠尾动脉吻合后即刻通畅率为１００％，１２０个股、肱动脉吻合后３周其通畅率为９７．５％（３个吻合口因伤口感染栓塞），吻合前、吻合后３周时吻合口内径经ｔ检查无显著差异（Ｐ＞０．０５），兔耳皮瓣全部成活，临床应用２０例全部成功。未发生血管危象。吻合口经扫描电镜观察，愈合过程优于端端吻合法。结论：此法吻合微小血管能确保其吻合口管壁外翻，内膜平整对合，减少缝合针数。有利于吻合口的无创操作，使血管的通畅率提高，血管危象减少     

Evolutionary history of the T cell receptor complex as revealed by small-spotted catshark (Scyliorhinus canicula)

In every jawed vertebrate species studied so far, the T cell receptor (TCR) complex is composed of two different TCR chains (α/β or γ/δ) and a number of CD3 subunits responsible for transmitting signals into the T cell. In this study, we characterised all of the TCR and CD3 genes of small-spotted catshark (Scyliorhinus canicula) and analysed their expression in a broad range of tissues. While the TCR complex is highly conserved across jawed vertebrates, we identified a number of differences in catshark, most notably the presence of two copies of both TCRβ and CD3γδ, and the absence of a functionally-important proline rich region from CD3ε. We also demonstrate that TCRβ has duplicated independently multiple times in jawed vertebrate evolution, bringing additional diversity to the TCR complex. This study reveals new insights about the evolutionary history of the TCR complex and raises new avenues for future exploration.     

肢端皮肤角化过度增生症的手术治疗

目的介绍肢端皮肤角化过度增生症１０例的治疗体会，其中掌跖角化病９例，疣状表皮结构不良１例。方法采用病变皮肤切除皮肤移植术治疗，其中两例有深部组织外露和放射性溃疡选用皮瓣修复，其余行皮片移植。结果１０例均获满意效果，追踪观察８年以上，无复发，能保持接近正常的功能活动。结论手部以皮片效果最佳，足底应用皮瓣修复，耐磨性较皮片为好，但因皮瓣缺乏纤维隔结构，走路时皮瓣与深部组织间产生相对滑动，也容易造成破溃，必须注意妥为保护     

Identifying the Relative Contributions of Rac1 and Rac2 to Osteoclastogenesis

Rac small GTPases may play an important regulatory role in osteoclastogenesis. Our in vitro and in vivo results show that both Rac1 and Rac2 are required for optimal osteoclast differentiation, but Rac1 is more critical. Rac1 is the key Rac isoform responsible for regulating ROS generation and the actin cytoskeleton during the multiple stages of osteoclast differentiation. Introduction: Recent evidence suggests that the Rac small GTPases may play an important regulatory role in osteoclastogenesis. This finding is important because bisphosphonates may regulate their antiresorptive/antiosteoclast effects through the modification of Rho family of small GTPases. Materials and Methods: To elucidate the specific roles of the Rac1 and Rac2 isoforms during osteoclastogenesis, we used mice deficient in Rac1, Rac2, or both Rac1 and Rac2 in monocyte/osteoclast precursors. Macrophage‐colony stimulating factor (M‐CSF)– and RANKL‐mediated osteoclastogenesis in vitro was studied by using bone marrow‐derived mononucleated preosteoclast precursors (MOPs). The expression of osteoclast‐specific markers was examined using quantitative real‐time PCR and Western blot analysis. Free actin barbed ends in bone marrow MOPs after M‐CSF stimulation was determined. The ability of MOPs to migrate toward M‐CSF was assayed using Boyden chambers. Margin spreading on heparin sulfate‐coated glass and RANKL‐induced reactive oxygen species generation were also performed. Functional assays of in vitro‐generated osteoclasts were ascertained using dentine sections from narwal tusks. Osteoclast levels in vivo were counted in TRACP and immunohistochemically stained distal tibial sections. In vivo microarchitexture of lumbar vertebrate was examined using μCT 3D imaging and analysis. Results: We show here that, although both Rac isoforms are required for normal osteoclast differentiation, Rac1 deletion results in a more profound reduction in osteoclast formation in vitro because of its regulatory role in pre‐osteoclast M‐CSF‐mediated chemotaxis and actin assembly and RANKL‐mediated reactive oxygen species generation. This Rac1 cellular defect also manifests at the tissue level with increased trabecular bone volume and trabeculae number compared with wildtype and Rac2‐null mice. This unique mouse model has shown for the first time that Rac1 and Rac2 play different and nonoverlapping roles during osteoclastogenesis and will be useful for identifying the key roles played by these two proteins during the multiple stages of osteoclast differentiation. Conclusions: Rac1 and Rac2 play different and nonoverlapping roles during osteoclastogenesis. This model showed that Rac1 is the key Rac isoform responsible for regulating ROS generation and the actin cytoskeleton during the multiple stages of osteoclast differentiation.     

Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei

In vertebrates, the tumor necrosis factor (TNF)-receptor (TNFR) system participates in diverse physiological and pathological events, such as inflammation and protective immune responses to microbial infections. There are few reports about the role of the invertebrate TNF-TNFR system in immune responses. Here, we isolated and characterized the TNF superfamily (LvTNFSF) gene, TNFR superfamily (LvTNFRSF) gene and lipopolysaccharide-induced TNF-α factor (LvLITAF) gene from Litopenaeus vannamei. LvTNFSF consists of 472 amino acids with a conserved C-terminal TNF domain and has 89.8% identity with the Marsupenaeus japonicus TNF superfamily gene. LvTNFRSF consists of 296 amino acids with a conserved TNFR domain and has 18.0% identity with Chlamys farreri TNFR, 14.6% identity with Drosophila melanogaster Wengen and 14.6% identity with Homo sapiens TNFR1. LvLITAF consists of 124 amino acids with the LITAF domain and shows 62.6% identity with D. melanogaster LITAF and 32.3% identity with H. sapiens LITAF. The promoter region of LvTNFSF was cloned and used to construct a luciferase reporter. In Drosophila S2 cells, the promoter of LvTNFSF can be activated by LvLITAF, L. vannamei NF-κB family proteins (LvRelish and LvDorsal) and LvSTAT. Unlike its mammalian counterparts, LvTNFRSF could not activate the NF-κB pathway in Drosophila S2 cells. Using real-time quantitative PCR, we obtained expression profiles of LvTNFSF, LvTNFRSF and LvLITAF in the gill, intestine and hepatopancreas of L. vannamei after challenge with Gram-negative Vibrio alginolyticus, Gram-positive Staphylococcus aureus, the fungus Candida albicans and white spot syndrome virus (WSSV). Taken together, our results reveal that LvTNFSF, LvTNFRSF and LvLITAF may be involved in shrimp immune responses to pathogenic infections.     

Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spätzle-like Toll ligands (LvSpz1-3) from Litopenaeus vannamei

Toll-like receptor-mediated NF-κB pathways are essential for inducing immune related-gene expression in the defense against bacterial, fungal and viral infections in insects and mammals. Although a Toll receptor (LvToll1) was cloned in Litopenaeus vannamei, relatively little is known about other types of Toll-like receptors and their endogenous cytokine-like ligand, Spätzle. Here, we report two novel Toll-like receptors (LvToll2 and LvToll3) and three Spätzle-like proteins (LvSpz1-3) from L. vannamei. LvToll2 has 1009 residues with an extracellular domain containing 18 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. LvToll3 is 1244 residues long with an extracellular domain containing 23 LRRs and a cytoplasmic TIR domain of 138 residues. The Spätzle-like proteins LvSpz1, LvSpz2 and LvSpz3 are 237, 245 and 275 residues in length, respectively, and all of them have a putative C-terminal cystine-knot domain. In Drosophila Schneider 2 (S2) cells, LvToll1 and LvToll3 were localized to the membrane and cytoplasm, and LvToll2 was confined to the cytoplasm. In Drosophila S2 cells, LvToll2 could significantly activate the promoters of NF-κB-pathway-controlled antimicrobial peptide genes, whereas LvToll1 and LvToll3 had no effect on them. LvSpz1 exerted some degree of inhibition on the promoter activities of Drosophila Attacin A and L. vannamei Penaeidin4. LvSpz3 also inhibited the Drosophila Attacin A promoter, but LvSpz2 could only slightly activate it. LvToll1, LvToll2 and LvToll3 were constitutive expressed in various tissues, while LvSpz1, LvSpz2 and LvSpz3 exhibited tissue-specific expression in the epithelium, eyestalk, intestine, gill and muscle. In the gill, after Vibrio alginolyticus challenge, LvToll1 was upregulated, but LvToll2 and LvToll3 showed no obvious changes. LvSpz1 and LvSpz3 were also strongly induced by V. alginolyticus challenge, but LvSpz2 only showed a slight downregulation. In the gill, after white spot syndrome virus (WSSV) challenge, LvToll1, LvToll2, LvToll3, LvSpz1 and LvSpz3 were upregulated, but LvSpz2 showed no obvious change, except for a slight downregulation at 12 h post-injection of WSSV. These findings might be valuable in understanding the innate immune signal pathways of shrimp and enabling future studies on the host-pathogen interactions in V. alginolyticus and WSSV infections.     

日本沼虾酚氧化酶原基因cDNA的全长克隆及表达分析 

目的 克隆日本沼虾酚氧化酶原（proPO）基因,进行生物信息学及时空表达分析。方法 利用RT-PCR和快速扩增cDNA末端（RACE）技术从血细胞中克隆proPO基因cDNA全长序列,用生物软件对其序列进行生物信息学分析; proPO基因时空表达分析采用RT-PCR和Real-time PCR方法;腹部肌肉注射嗜水气单胞菌（5.0×10SUP>9/SUP>/L）诱导酚氧化酶（PO）,20μl/只,注射后3、6、12、24h取其血淋巴,分别测定血细胞 proPO mRNA水平及血清酚氧化酶（PO）活力。结果 日本沼虾proPO基因cDNA全长2428bp,包含71 bp的 5’UTR、344 bp的 3’UTR 和2013 bp的开放阅读框（ORF）。ORF编码 671 个氨基酸,预测蛋白分子量为76.5kD,理论等电点（pI）约为7.31;含有2个保守的铜离子结合位点和6个组氨酸残基、1个蛋白酶水解位点和硫酯样的基序(GCGWPRHM);含有3个血蓝蛋白结构域;与罗氏沼虾proPO的相似性最高,为93%,与其他甲壳动物proPO相似