Global configuration of single titin molecules observed through chain-associated rhodamine dimers

The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding/refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.     

Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations

The bacterial cytoskeletal protein FtsZ is a GTPase that is thought to provide mechanical constriction force via an unidentified mechanism. Purified FtsZ polymerizes into filaments with varying structures in vitro: while GTP-bound FtsZ assembles into straight or gently curved filaments, GDP-bound FtsZ forms highly curved filaments, prompting the hypothesis that a difference in the inherent curvature of FtsZ filaments provides mechanical force. However, no nucleotide-dependent structural transition of FtsZ monomers has been observed to support this force generation model. Here, we present a series of all-atom molecular dynamics simulations probing the effects of nucleotide binding on the structure of an FtsZ dimer. We found that the FtsZ-dimer structure is dependent on nucleotide-binding state. While a GTP-bound FtsZ dimer retained a firm monomer-monomer contact, a GDP-bound FtsZ dimer lost some of the monomer-monomer association, leading to a "hinge-opening" event that resulted in a more bent dimer, while leaving each monomer structure largely unaffected. We constructed models of FtsZ filaments and found that a GDP-FtsZ filament is much more curved than a GTP-FtsZ filament, with the degree of curvature matching prior experimental data. FtsZ dynamics were used to estimate the amount of force an FtsZ filament could exert when hydrolysis occurs (20-30 pN per monomer). This magnitude of force is sufficient to direct inward cell-wall growth during division, and to produce the observed degree of membrane pinching in liposomes. Taken together, our data provide molecular-scale insight on the origin of FtsZ-based constriction force, and the mechanism underlying prokaryotic cell division.     

Effect of recombinant human endostatin onradiotherapy for esophagus cancer

Objective:To investigate the effect of radiotherapy plus recombinant human endostatin(RHendostatin) on esophageal cancer and its mechanism.Methods:A total of SO nudemice were equally randomized into control group,radiotherapy group,and combined therapy group Ⅰ,Ⅱ,and Ⅲ after inoculating with Ecal09 cell suspension(1×10~7 cells/mL).On the day of grouping,control group and radiotherapy group were injected normal saline,while radiotherapy group and 3 combined therapy groups received radiotherapy;besides,combined therapy group Ⅰ,Ⅱ,and Ⅲ was injected RH-endostatin of 2.5,5,10 mg/kg respectively.After 3-week therapy,the tumors of each group were collected and microvessel density and VEGF expression in tumors were determined.In vitro,Eca109 cells were divided into control group,radiotherapy group,and combined therapy group.Forty-eight hours after treatment cell cycle distribution and apoptosis rate were detected,and the activity of VEGF signal paths was semiquantitatively analyzed.Results:Since the 6th day of treatment,the relative tumor proliferation rate of combined therapy group Ⅱ was lower than radiotherapy group(P0.05) and 40%since the 15 th day.Average microvessel density and EGFR expression in combined therapy group Ⅱ were lower than radiotherapy group(P0.05).In vitro,the cell percentage in S and G_2/M phase of combined therapy group cells was lower than that in radiotherapy group cells,while the apoptosis rate and the expression of VEGF,AKT,p-AKT,ERK1/2 and p-ERKl/2 in combined group were higher than that in radiotherapy group(P0.05).Conclusions:RH-endostatin promotes the efficacy of radiotherapy on esophageal cancer,which may be partly realized by inhibiting the activity of VEGF related signal paths.     

人内皮抑素小环载体在真核细胞中的生物活性

目的通过对人内皮抑素小环载体在真核细胞中的表达和生物学效应的研究,探讨小环载体作为生物治疗载体的可行性。方法将mc-hES、pcDNA-hES分别转染人肝癌细胞HepG2后,通过ELISA、RT-PCR和Westernblot观察hES表达。MTT法检测其对人脐静脉内皮细胞（HUVEC）的增殖抑制作用。结果 mc-hES和pcDNA-hES均能表达hES,mc-hES能明显抑制HUVEC的生长,而对HepG2无明显作用（P〈0.05）。在相同条件下,小环较常规质粒在体外能快速介导转基因的表达,且较传统质粒高6～8.3倍。结论 mc-hES较普通质粒pcDNA-hES在肝癌细胞中能高效表达转基因,为小环载体进一步研究提供了很好的实验基础。     

Occurrence of cholesterol crystals in human bile

The occurrence of cholesterol crystals was studied in 20 consecutive gallstone patients with functioning gallbladders. The frequency with which crystals were found rose sharply with the number of stones. Gallbladder bile was found more often to contain cholesterol crystals than hepatic bile of the same individual. Such crystals were absent in T tube drain bile from 10 consecutive choledochostomy patients, studied after the reestablishment of the enterohepatic circulation for at least five days. In gallstone patients in whom the gallbladder was visualized at cholecystography the hepatic bile contained cholesterol crystals more often than in patients with gallbladders not so visualized. In the latter patients the crystals tended to disappear after prolonged fasting. Bile analysis showed hepatic bile of patients with non-functioning gallbladders to be less lithogenic than bile in cases with functioning gallbladders. In the former group bile contained relatively more chenodeoxycholic acid than in the latter. The composition of bile with cholesterol crystals did not differ significantly from that of bile without crystals. In the final analysis it is important to identify possible factors responsible for the precipitation of cholesterol from supersaturated bile.     

重组人血管内皮抑制素对COPD大鼠肺血流动力学指标的影响

目的观察重组人血管内皮抑制素对慢性阻塞性肺疾病（COPD）病程的影响及机制。方法将64只大鼠随机分为8组各8只,其中A组和B组分别于第1、14天气道内注入生理盐水及脂多糖（LPS）溶液;C组同上注入LPS溶液,同时采用香烟暴露法制备COPD模型,共1个月;D组同上注入LPS溶液并行香烟烟雾暴露及18％氧气吸人制备COPD并肺动脉高压（PAH）模型;B1组、C1组、D1组分别按B、C、D组造模,并腹腔注射重组人血管内皮抑制素;Al组处理方式同A组,并腹腔注射生理盐水。采用肺动脉插管法测定各组肺血流动力学指标,采用ELISA法检测肺泡灌洗液（BALF）中血管内皮生长因子（VEGF）蛋白表达情况,采用RT-PCR法、Western-blot法检测肺组织匀浆中VEGF mRNA及蛋白表达,分析VEGF表达与肺血流动力学指标的相关性。结果C组和D组右心室收缩压（RVSP）、平均肺动脉压（mPAP）、右心室肥厚指数（RVHI）均明显高于A组,Cl、D1组此三项指标均较对应C、D组下降（P均〈0．05）;C组和D组BALF中VEGF蛋白表达水平显著高于A组,Cl、D1组VEGF蛋白表达较对应C、D组显著下降（P均〈0．05）;B组、C组和D组肺组织中VEGF mRNA及蛋白表达逐渐增强,B1、C1、D1组VEGF mRNA表达显著低于B组、C组和D组,C1、Dl组VEGF蛋白表达显著低于C组和D组（P均〈0．05）;肺组织中VEGF mRNA及BALF中VEGF蛋白水平均与RVSP、mPAP呈正相关（P均〈0．05）。结论重组人血管内皮抑制素可延缓COPD病情进展,可能机制为下调VEGF表达;此为COPD药物治疗提供了新的思路。     

Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG

Staphylococcus aureus surface protein SasG promotes cell–cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn²⁺ strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell–cell adhesion via specific Zn²⁺-dependent homophilic bonds between β-sheet–rich G5–E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell–cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.The bacterial pathogen Staphylococcus aureus causes a wide range of infections in humans, which are often associated with the ability of the bacteria to form biofilms on indwelling medical devices such as central venous catheters and prosthetic joints (1–4). Biofilm formation involves initial adhesion of the bacteria to surfaces, followed by cell–cell adhesion (aggregation) to form microcolonies and a mature biofilm, and finally dispersal by the detachment of cell aggregates from the biofilm (5). Currently, little is known about the molecular interactions driving biofilm formation by S. aureus due to the paucity of appropriate high-resolution probing techniques. Such knowledge may contribute to the development of novel compounds for therapy.Adhesion and biofilm formation by S. aureus involve a variety of cell wall components. Whereas adhesion to host proteins is mediated by cell-wall–anchored (CWA) proteins (6, 7), intercellular adhesion was until recently thought to be promoted by the expression of the polysaccharide intercellular adhesin (PIA), also known as the poly-N-acetyl-glucosamine (PNAG) (8, 9). This positively charged polymer is able to bind the negatively charged bacterial surfaces. PIA, encoded by genes in the ica operon, represents the most well-understood biofilm-mediating pathway in staphylococci (10, 11). However, many strains do not produce PIA and rely on CWA proteins to promote intercellular adhesion in an ica-independent manner (6, 7).A prototype of biofilm-forming CWA protein is SasG (12–15), which mediates cell–cell adhesion through its “B” multidomain region (5, 7). B repeat sequences contain “G5” domains (∼78 residues) in a tandem array, separated by 50-residue sequences known as the “E” regions (Fig. 1A) (14, 15). SasG forms β-sheet–rich protein fibrils that protrude from the cell surface, which can be visualized by electron microscopy (12). The proposed mechanism for SasG-mediated cell association is based on homophilic protein–protein interactions. SasG is covalently attached to the cell wall and undergoes limited cleavage within the B region to remove the N-terminal “A” region. The cleaved and exposed SasG B domains on neighboring cells interact with each other in a Zn²⁺-dependent manner, leading to cell–cell adhesion (13). The G5–E domains of the related accumulation-associated protein (Aap) of Staphylococcus epidermidis are also responsible for the Zn²⁺-dependent biofilm formation (15). However, recent work also suggests that Aap could bind a ligand protein, the small basic protein (Sbp), which accumulates on the cell surface and within the biofilm matrix (16). Therefore, whereas SasG and Aap are believed to mediate intercellular adhesion via zinc-dependent homophilic bonds between opposing proteins, it is unclear whether this is the only mechanism at play. Also, the mode of action of zinc is controversial. Whereas SasG dimerizes in vitro in a zinc-dependent manner, a direct link between homodimerization and biofilm formation has not yet been established. Rather, it has been suggested that zinc could mediate binding to anionic cell surface components like teichoic acids (14). Direct biophysical analysis of SasG proteins on the surface of living cells would help to clarify these important issues.Open in a separate windowFig. 1.Role of SasG in cell–cell adhesion. (A) Schematic representation of the SasG structure emphasizing the A domain, not engaged in cell–cell adhesion, and the B repeat sequence containing G5 domains (78 residues) in a tandem array, separated by the E regions (50 residues). (B–E) Optical microscopy images of S. aureus cells expressing full-length SasG [SasG₈⁽⁺⁾ cells] after resuspension in TBS buffer (B) or in TBS buffer containing 1 mM of ZnCl₂ (C), after addition of 1 mM EDTA (D), and further addition of 1 mM ZnCl₂ (E). (F and G) Control experiment using S. aureus expressing no SasG [SasG⁽⁻⁾ cells] in TBS buffer (F) or in TBS containing 1 mM of ZnCl₂ (G).Recent advances in atomic force microscopy (AFM) techniques have enabled researchers to gain insight into the biophysical properties and molecular interactions of microbial cells (17, 18), including S. aureus (19–22). A variety of AFM-based force spectroscopy methods have been developed, in which the force acting on the AFM probe is measured with piconewton (10⁻¹² N) sensitivity as the probe is pushed toward the sample, then retracted from it (17). In the past few years, a new force spectroscopy-based imaging mode, multiparametric imaging, has offered the possibility to image the surface structure of living cells, while mapping their mechanical and adhesive properties at unprecedented spatiotemporal resolution (23–28). Unlike in conventional imaging, the method involves recording arrays of force curves across the cell surface, at improved speed, positional accuracy, and force sensitivity (26). As the curves are recorded at high frequency, correlated images of the structure, adhesion, and mechanics of the cells can be obtained at the speed of conventional imaging. This technology has been used to image single filamentous bacteriophages extruding from living bacteria (25) and to map adhesive nanodomains on fungal pathogens (28). Furthermore, recent progress in single-cell force spectroscopy (SCFS) (18, 29, 30) has made it possible to understand the forces driving cell adhesion and biofilm formation. Here, a living cell is attached to the AFM probe, thereby enabling researchers to measure the interaction forces between the cell and a target surface (18). Applying these newly developed modalities to staphylococci is a challenging problem, which would provide novel insights into the molecular bases of biofilm formation and biofilm-associated infections.Here, we combine multiparametric imaging and SCFS to investigate the mechanical strength of SasG on living bacterial cells, thus in its fully functional environment. We use a S. aureus strain carrying a plasmid expressing SasG with eight consecutive G5–E repeats [hereafter S. aureus SasG₈⁽⁺⁾ cells]. We show that intercellular adhesion involves the Zn²⁺-dependent–specific association of G5–E repeats on opposing cells and that the elongated structure and mechanical strength of SasG make it ideally suited for that purpose. In addition, our results show that Zn²⁺ plays a dual role that is more complex than anticipated before: adsorption of Zn²⁺ to cell wall components increases the cohesion of the cell surface, thereby favoring the projection of highly elongated SasG proteins beyond other surface components and making them fully functional for Zn²⁺-dependent homophilic interactions.     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

Retrovirus—mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted. RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.     

持续释放人血管抑素的基因修饰化工程细胞hE/293细胞株的建立

目的:在人胚肾HEK293细胞中转染真核表达载体pSNA2/hEndostatin(hEndostatin,人血管抑素),建立能稳定分泌hES的基因工程细胞株.方法:将含有IL-2分泌肽的人endostatin(ES)全长cDNA插入真核表达载体pSNA2,产生重组质粒pSNA2/hEndostatin;利用阳离子脂质体介导将其转染入HEK293细胞中;用G418筛选出阳性克隆细胞,将其命名为hE/293细胞.用Western blot法检测hE/293细胞培养上清中分泌的hES蛋白.血管内皮细胞(ECV304)增殖抑制试验及鸡胚尿囊膜试验观察其分泌的hES蛋白的抗增殖活性.结果:经过双酶切和DNA测序证实构建出了含hES基因的真核表达载体.通过G418抗性筛选筛选出稳定表达hES的细胞株3株,将其命名为hE/293细胞;Western blot检测该细胞株培养上清中存在分子量为20 kDa的ES蛋白;ECV304增殖抑制试验显示,与HEK293细胞组相比,hE/293细胞组分泌的ES蛋白对bFGF刺激的血管内皮细胞增殖有明显的抑制作用(48h:0.125±0.007 vs 0.159±0.020,P<0.01;72 h:0.088±0.016 vs 0.249±0.070,P<0.01);鸡胚尿囊膜试验证实hE/293细胞分泌的ES蛋白可以抑制鸡胚尿囊膜血管生长.结论:所构建的hE/293细胞株可以稳定的分泌hES蛋白,并能抑制ECV304细胞生长及鸡胚尿囊膜血管生长.     

高糖对人脐静脉内皮细胞内皮抑素表达的影响

目的 研究高糖对人内皮细胞内皮抑素(ES)mRNA及蛋白表达水平的影响及其意义.方法 培养人脐静脉内皮细胞(HUVECs),分为正常糖(5.6mmol/L)对照组,高糖(11.2、22.4mmol/L)组.培养24、48、72、96h后,收集各组不同作用时间点细胞,用RT-PCR法检测ES mRNA表达水平,Western blot法检测ES蛋白的表达.结果 高糖对HUVECs中ES mRNA和蛋白表达水平的影响是双相的:短时间内起促进作用,具有时间依赖性;随作用时间延长转为抑制作用,22.4mmol/L糖培养HUVECs 96h时组ES mRNA和蛋白表达水平较对照组明显降低(P<0.05).结论 糖尿病(DM)慢性病程中,ES表达的降低可能与DM血管病变的发生有关.     

Therapeutic effects of recombinant human endostatin adenovirus in a mouse model of malignant pleural effusion

Purpose  Malignant pleural effusion (MPE) is a common clinical problem in patients with advanced cancer. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the pleural wall are due to high levels of vascular endothelial growth factor (VEGF), which plays an important role in the pathogenesis of MPE. The present study was designed to test whether the recombinant adenovirus-mediated delivery of human endostatin (Ad-hEndo), one of the potent inhibitors of angiogenesis, would inhibit the formation and progression of MPE. Methods  We developed a novel mouse model of MPE by injecting Lewis lung carcinoma (LLC) cells directly into pleural cavity of C57BL/6 mice. To evaluate the therapeutic effects of endostatin in this MPE model, we injected the Ad-hEndo into the pleural cavity of MPE-bearing mice three times with the 3-day interval. Results  We found that this treatment resulted in significant reduction in pleural effusion volume, the number of pleural tumor foci, microvessel density, and vascular permeability, while it significantly prolonged the survival time. In addition, VEGF level of MPE in the group administered with the Ad-hEndo was obviously decreased as compared with that in the two control groups administered with null-adenovirus (Ad-null) or normal saline. Conclusions  Our work provides a rationale for future studies toward evaluating the effectiveness of the adenovirus-based endostatin therapy for MPE. F. Fang, P. Chen and X. Wu have equally contributed to this work.     

重组人血管内皮抑制素联合替莫唑胺治疗复发高级别胶质瘤的临床研究

目的观察重组人血管内皮抑制素(恩度)联合替莫唑胺(TMZ)与单纯应用TMZ治疗复发高级别胶质瘤患者的安全性与疗效。方法将74例复发高级别胶质瘤患者随机均分为两组,一组单纯应用TMZ化疗(37例),另一组应用恩度联合TMZ联合化疗(37例)。TMZ化疗方案的选择基于患者O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)蛋白表达状况和既往TMZ化疗效果,采用个体化的治疗。恩度剂量为15 mg静脉滴注,连续应用14 d,间歇2周重复。结果 37例单纯TMZ化疗患者共接受129周期化疗,中位3次(1~10次)。完全缓解(CR)1例(2.7%),部分缓解(PR)9例(24.3%),微效(MR)3例(8.1%),疾病控制率(CR+PR+MR)为35.1%。中位无进展生存期(PFS)为4个月(95%CI 1.9~6.1个月),6个月的PFS率为27.0%。37例恩度联合TMZ化疗患者共接受200周期化疗,中位6次(2~10次)。CR 3例(8.1%),PR 14例(37.8%),MR 6例(16.2%),疾病控制率(CR+PR+MR)为62.1%。中位PFS为6个月(95%CI 4.9~7.1个月),6个月的PFS率为43.0%。不良反应中,同单纯TMZ组相比,恩度联合TMZ组具有更高的高血压发生率。结论恩度联合TMZ化疗较TMZ单药化疗在复发高级别胶质瘤的治疗有更好的客观疗效,相对延长PFS,且具有较好的安全性。     

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). METHODS: HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2×1010 v.g.) led to a sustained serum endostatin level of approximately (86.71±5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups(P<0.01). CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.     

Optic properties of bile liquid crystals in human body

AIM To further study the properties of bile liquid crystals, and probe into the relationship between bile liquid crystals and gallbladder stone formation, and provide evidence for the prevention and treatment of cholecystolithissis. METNODS The optic properties of bile liquid crystals in human body were determined by the method of crystal optics under polarizing microscope with plane polarized light and perpendicular polarized light. RESULTS Under a polarizing microscope with plane polarized light, bile liquid crystals scattered in bile appeared round, oval or irregularly round. The color of bile liquid crystals was a little lighter than that of the bile around. When the stage was turned round, the color of bile liquid crystals or the darkness and lightness of the color did not change obviously. On the border between bile liquid crystals and the bile around, brighter Becke-Line could be observed. When the microscope tube is lifted, Becke. Line moved inward, and when lowered,Becke-Line moved outward. Under a perpendicular polarized light, bile liquid crystals showd some special interference patterns, called Malta cross. When the stage was tuming round at an angle of 360°, the Malta cross showed four times of extinction. In the vibrating direction of 45° angle of relative to upper and lower polarizing plate, gypsum test-board with optical path difference of 530 nm was inserted, the first and the third quadrants of Malta cross appeared to be blue, and the second and the fourth quadrants appeared orange. When mica test-board with optical path difference of 147 nm was inserted, the first and the third quadrants of Malta cross appeared yellow, and the second and the fourth quadrants appeared dark grey. CONCLUSION The bile liquid crystals were distributed in bile in the form of global grains. Their polychroism and absorption were slight,but the edge and Becke-Line were very clear. Its refractive index was larger than that of the bile.These liquid crystals were uniaxial positive crystals. The interference colors were the first order grey-white. The double refractive index of the liquid crystals was Δn = 0.011-0.015.     

大肠癌组织中uPA及Endostatin的表达及意义

目的研究uPA（尿激酶纤溶酶原激活物）及Endostatin（血管内皮抑素）在大肠癌及正常组织中表达及其与肿瘤浸润生长的关系。方法应用免疫组化SP法检测63例大肠癌组织及23例正常组织中Endostatin、uPA的表达。结果uPA在大肠癌中的表达明显高于正常组织（P〈0．01），且在DukesC＋D期中的表达高于DukesA＋B期，浸及浆膜层组高于未浸及浆膜层组（P〈0．05）。uPA随着组织分化程度降低表达增高，而Endostatin的表达则相反，两者表达呈负相关。结论uPA、Endostatin在大肠癌的侵袭与转移过程中均起着重要作用，uPA促进肿瘤的浸润与转移，而Endostatin则抑制肿瘤的浸润与转移。