肌动蛋白与真核生物的进化

以肌动蛋白氨基酸取代与真核生物进化年代呈线性关系为依据，收集原生生物界、真菌界、植物界和动物界等四界74种生物的128个肌动蛋白序列，通过对其氨基酸序列同源性进行比较，制作出肌动蛋白的分子进化树，并依此进化树从分子水平对真核生物的进化进行一些探讨。从总体上看，肌动蛋白分子进化树较好地反映了真核生物间的进化关系，为确定某些生物的进化位置及进化关系提供了分子证据．

兔角膜基质飞秒激光扫描后Ki-67、转化生长因子β2及平滑肌肌动蛋白的表达

目的研究角膜基质飞秒激光扫描后Ki-67、转化生长因子β2及平滑肌肌动蛋白（α-SMA）的表达情况，探讨角膜基质内扫描后角膜创伤愈合反应机制。方法20只纯种新西兰大白兔随机编号，35只眼用60kHzIntralase飞秒激光进行角膜基质内扫描（其中5只兔只做右眼），未手术的5只左眼作为正常对照组。激光参数为：点／线间距10μm，扫描直径8．5mm，扫描能量1．3μJ，扫描深度为135μm，不进行角膜边缘切削。术后在30min、1h,2h、1个月和3个月用裂隙灯显微镜观察角膜的透明程度。分别在术后1d．3d、1周、1个月和3个月各取6只术眼的角膜组织，做免疫印迹法蛋白定量检查，各时间点取1只术眼做免疫组织化学检查，分别观察Ki-67、TGF-β2及α-SMA的表达情况。用两组完全随机化设计资料均数的t检验进行统计学处理。结果免疫印迹法蛋白定量及免疫组织化学检查显示：Ki-67在基质扫描后第1天开始表达增加（0．0670±0．0008），扫描后3d达最高峰（0．6923±0．0051），随着时间推移其表达逐渐减少，但直至术后3个月和对照组相比差异均有统计学意义（t=24．12，57．22，43．26，39．78，18．35；P〈0．05）。TGF-β2（t=0．933，0．856，0．934，0．970，1．132）及OL-SMA（t=1．126，1．235，0．993，1．175，1．211）在术后各时间点的表达和对照组相比差异均无统计学意义（P〉0．05）。结论角膜基质内飞秒激光扫描后Ki-67表达增加，角膜基质细胞活化、增殖；TGF—β2被完整的角膜上皮所屏障，活化和增生的角膜基质细胞没有向肌成纤维母细胞转化，损伤修复愈合过程中无haze形成。

Endoscopic therapy for gastric stromal tumors originating from the muscularis propria

AIM:To explore endoscopic therapy methods for gastric stromal tumors originating from the muscularis propria.METHODS:For 69 cases diagnosed as gastric stromal tumors originating from the muscularis propria,three types of endoscopic therapy were selected,based on the size of the tumor.These methods included endoscopic ligation and resection(ELR),endoscopic submucosal excavation(ESE) and endoscopic full-thickness resection(EFR).The wound surface and the perforation of the gastric wall were closed with metal clips.Immunohistostaining for CD34,CD117,Dog-1,S-100 and smooth muscle actin(SMA) was performed on the resected tumors.RESULTS:A total of 38 cases in which the tumor size was less than 1.2 cm were treated with ELR;three cases were complicated by perforation,and the perforations were closed with metal clips.Additionally,18 cases in which the tumor size was more than 1.5 cm were treated with ESE,and no perforation occurred.Finally,13 cases in which the tumor size was more than 2.0 cm were treated with EFR;all of the cases were complicated by artificial perforation,and all of the perforations were closed with metal clips.All of the 69 cases recovered with medical treatment,and none required surgical operation.Immunohistostaining demonstrated that among all of the 69 gastric stromal tumors diagnosed by gastroscopy,12 cases were gastric leiomyomas(SMA-positive),and the other 57 cases were gastric stromal tumors.CONCLUSION:Gastric stromal tumors originating from the muscularis propria can be treated successfully with endoscopic techniques,which could replace certain surgical operations and should be considered for further application.

Comparative studies on the biochemical characteristics of natural actomyosin isolated from PSE and normal pork

Biochemical changes of natural actomyosin from fresh pale, soft, exudative (PSE) and normal pork were studied,and the effects of different storage temperatures and different incubation temperature and times on sample superprecipitation,total sulfhydryl (-SH) content, and ATP (adenosine triphosphate) sensitivity were investigated. The results demonstrated that ATPase activity and thermal stability of PSE actomyosin were lower than those of normal pork; and that PSE actomyosin had higher -SH content than that of normal pork at all incubation temperatures and times tested.

Involvement of Lipid Rafts and Cellular Actin in AcMNPV GP64 Distribution and Virus Budding

GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.

Study progress of cell endocytosis

Endocytosis is a process through which extracellular materials are transported into cell through membrane deformation. This process is not a simple step-by-step process in which a series of proteins function according to the chronological order, but rather a complex process comprising many members which are regulated precisely. The role of endocytosis is broadly divided into two categories, phagocytosis and pinocytosis, the latter is divided into four species in accordance with the size of endocytosis substances： clathrin dependent endocytosis, the diameter of clathrin-coated vesicle is 100-150 nm; caveolin dependent endocytosis, the diameter of caveolin protein-coated vesicle is 50-100 nm; macropinocytosis, the diam- eter of macropinocytosis is generally 0.5-2 μm, sometimes up to 5 μm; clathrin and caveolin independent endocytosis. Many proteins including endophilin A1, A2, A3, and endocytotic proteins B, B1a, and Blb as well as dynamin, actin and Rab protein families are involved in endocytosis and play an important role in different stages. The abnormal endocytosis may be involved in the development of certain diseases.

Gastrointestinal stromal tumor of duodenum:a cause of upper gastrointestinal hemorrhage

Objective:The aim of the study was to report an anemia patient with melena for five years caused by duodenal gastrointestinal stromal tumor (GIST), who required surgical treatment. Methods: A 44-year old man present with anemia appearance was admitted to our center (Department of Hepatobiliary Surgery, Union Hospital, Huazhong University of Science and Technology, China) due to sustaining melena for five years. Endoscopy found no special mucosal abnormalities in the duodenal lumen. Computed Tomography showed a well-demarcated mass, 7.4 cm in diameter, located between the C loop of duodenum and pancreatic head. Pylorus-preserving pancreaticoduodenectomy and right hemicolectomy were performed when the patient's general conditions were improved. He recuperated successfully and was discharged on the 21st postoperative day. No complications happened during the period of hospital stay. Results: Histological and immunohistochemical study revealed a high risk invasive duodenal GIST which was positive for CD117, CD34, α-smooth muscle actin and negative for S-100. Conclusion: Duodenal GIST can be a source of upper gastrointestinal hemorrhage; surgical treatment is still a reasonable choice for the patients with invasive duodenal GIST.

绿色荧光蛋白标记的Raw264.7体外诱导分化为破骨细胞的实验研究

目的 观察增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）标记的Raw264.7体外分化为破骨细胞的能力,拟为EGFP基因作标志物对外源性破骨前体细胞进行体内追踪做准备.方法 利用反转录病毒介导pEGFP-Lifeact基因转染Raw264.7细胞;采用有限稀释技术,荧光显微镜下获得EGFP稳定转染的G3单克隆细胞并观察其形态,将稳定转染成功的细胞定为G3-EGFP转染组,未转染野生细胞作为对照组;核因子κB受体激活子配体（receptor activator of nuclear factor kappaB ligand,RANKL）诱导G3细胞向破骨细胞分化,通过抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色、蛋白质印迹法检测组织蛋白酶K、骨吸收陷窝实验观察转染后对破骨细胞形成和骨吸收功能的影响;激光共聚焦显微镜实时拍摄转染细胞向破骨细胞分化过程中伪足小体结构变化的动态影像.结果 Raw264.7细胞内成功转染EGFP-Lifeact,传至20代以上仍可稳定表达EGFP,建立了G3-EGFP单克隆细胞株;转染细胞无明显形态学变化,能诱导形成TRAP染色阳性的多核巨细胞,转染组细胞融合率为（35±5）％,对照组细胞融合率为（39±5）％,二者差异无统计学意义（P＞0.05）;免疫印迹实验中对照组与转染组中组织蛋白酶K/β-肌动蛋白半定量分析比值分别为0.83±0.07、1.02±0.08,两组细胞组织蛋白酶K表达差异无统计学意义（P＞0.05）;对照组与转染组骨吸收总面积分别为272 252±36 193和262 408±23 243（P＞0.05）,骨吸收陷窝数目分别为320±51和339±55 （P＞0.05）,表明转染不影响破骨细胞形成与骨吸收功能;激光共聚焦显微镜实时拍摄显示转染细胞向破骨细胞分化时不断发生细胞融合,伪足小体作为动态自组装结构不断发生改建,最初聚集形成伪足小体簇,逐渐形成环状结构,最终于成熟破骨细胞周围形成相对稳定的伪足小体带.结论 EGFP可成功标记Raw264.7细胞;转染并不影响Raw264.7细胞向破骨细胞分化的能力.