[Sectional anatomical observation of coronal plane of dacryocystorhinostomy related structures].

Objective:To study coronal plane of dacryocystorhinostomy related structures for clinical practice. Method:Extracted images concluding lacrimal sac and nasolacrimal canal information from head coronal sectional imaging data-set we created.Observed shape and location of the left lacrimal sac,nasolacrimal canal, and surrounding structures.Measured details of the important portion.Result:Images of different layer show the shape and locative relationship of the lacrimal sac, nasolacrimal canal, and surrounding structures precisely.The thinnest thickness of medial wall and lateral wall of the lacrimal sac is 1.2mm and 0.6mm. There is a 2.3mm high longitudinal fold on the lateral wall.The thickness of medial wall and lateral wall of membranous nasolacrimal canal is(1.97±0.47)mm and(1.52±0.17)mm.Transverse diameter of membranous nasolacrimal canal is(1.78±0.12)mm. The thinnest thickness of medial wall and lateral wall of bony nasolacrimal canal is 0.30mm and 0.15mm.Transverse diameter of bony nasolacrimal canal is(5.50±0.12)mm.The proportion of cross-membranous nasolacrimal canal sectional area in bony nasolacrimal canal sectional area is(13.5±2.9)%.Conclusion: Head coronal sectional imaging data-set with high precision can be used for sectional anatomical study of the lacrimal sac, nasolacrimal canal and surrounding structures and measurement of details.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-alpha-induced epithelial-mesenchymal transition of MCF-7 cells.

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-alpha (TNF-alpha). To evaluate the role of TNF-alpha in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-alpha-treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-alpha treatment. These results showed that TNF-alpha can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkappaB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkappaB by the mutant IkappaBalpha also blocked the TNF-alpha-induced upregulation of Snail promoter activity. Thus, the activation of NFkappaB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-alpha-induced EMT. ROS caused by TNF-alpha seemed to play a minor role in the TNF-alpha-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-alpha- and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-alpha (TNF-alpha). To evaluate the role of TNF-alpha in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-alpha-treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-alpha treatment. These results showed that TNF-alpha can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBalpha also blocked the TNF-alpha-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-alpha-induced EMT. ROS caused by TNF-alpha seemed to play a minor role in the TNF-alpha-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-alpha - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7.

BACKGROUND: Adipose-derived adult stem (ADAS) cells are multipotent cells capable of differentiating into osteoblasts, adipocytes and chondrocytes. The aim of this study was to determine whether BMP-7-expressing ADAS cells would elicit bone formation invitro and in vivo. METHODS: ADAS cells were harvested from Lewis rats and transduced with adenovirus carrying the recombinant human bone morphogenetic protein-7 (Ad-BMP-7) gene. Untransduced cells and cells transduced with adenovirus carrying the enhanced green fluorescence protein (Ad-EGFP) gene served as controls. BMP-7 expression was assessed by RT-PCR, immunofluorescence on day 1, and Western blot on days 4, 8 and 12. Alkaline phosphatase (ALP) activity was assayed on days 2, 4, 6, 8, 10 and 12. Osteocalcin production and bone nodule formation were detected by immunohistochemistry and von Kossa stain on day 12. A total of 1 x 10(6) cells mixed with type I collagen were implanted into the subcutaneous pocket in Lewis rat and subjected to histologic analysis 1, 2 and 4 weeks post-implantation. RESULTS: The Ad-BMP-7-transduced ADAS cells expressed BMP-7 at both mRNA and protein levels. ALP activity was detected in Ad-BMP-7-transduced cells from day 2 to day 12, peaking on day 8. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. Histologic examination revealed that implantation of BMP-7-expressing ADAS cells could induce new bone formation in vivo. DISCUSSION: ADAS cells would be a promising source of adult autologous stem cells for BMP gene therapy and tissue engineering.

[Constructing and expression of three zinc-fingers peptide with specific DNA recognition property in Escherichia coli].

For investigating the DNA binding property of classical zinc finger protein Zif268, an in vivo transcription interference experiment was once utilized to develop a genetic selection assay. By screening a library in which the key amino acids of the third zinc finger from Zif268 were randomized, some single fingers with new binding specificity were obtained. In this study, by combining the single fingers, two three-finger peptides cDNA ZF123 and 2ZF123 were constructed by an over-lap PCR technique using the DNA binding domain of Zif268 as the template. After three times PCR, the products were inserted into pUC18 for cloning. The ZF123 and 2ZF123 cDNA were also inserted into pGEX-2T for expression in Escherichia coli after sequencing confirmation. The result showed that the three-finger peptides were expressed at a high level in E. coli JM109. The fusion protein GST-ZF123/2ZF123 have the relative molecular weight of 34.0 kD and consisted about 20% of the total soluble cell protein as detected by SDS-PAGE. After supersonic treatment, the soluble part of the bacterial extract was purified. After two additional thrombin cleavage and Sepharose 4B affinity purification steps, the free three-fingers peptide proteins were also obtained. The construction and obtaining of the three-fingers peptide cDNA and its products will facilitate the in vivo and in vitro DNA binding specificity study and the design of the hybrid transcription factors.

[Inhibition effect in vitro of purified endostatin expressed in Pichia pastoris].

Endostatin is a newly found inhibitor of angiogenesis, which is identified as c-terminal 184 amino acid fragment of collagen XVIII NC1-domain. A 570 bp cDNA fragment of endostatin has been amplified by PCR from a commercial human fetal liver cDNA library. After subcloned into the yeast vector pPIC9 and subsequence to prove its correctness, Pichia pastoris was transformed with the recombinant pPIC9-endostatin. The expressed endostatin in P. pastoris was purified by heparin-sapherose affinity chromatography. It's purity identified by SDS-PAGE thin layer scanning analysis was up to 98.7% and its Mol. Weight measured by MS was 20.34 kD. The expression level was up to 40 mg/L. The first fifteen amino acid sequence of the N-terminal was completely identical with the inner sequence C-terminal fragment of collagen XVIII NC1 domain as has been designed. Bioassay indicated that the recombinant endostatin can inhibit angiogenesis stimulated by bFGF in CAM test and also the proliferation of both HUVEC and ECV304 in an in vitro test.

[Cloning and expressing of an anti-CD5 single chain antibody].

Novel anti-CD5 single-chain fragment of variable domain (ScFv) was cloned and expressed. Anti-CD5 ScFv was constructed with cDNA fragments of heavy and light variable regions (VH and VL) which were reverse-translated from poly (A) mRNA of hybridoma cells producing anti-CD5 McAb. By phage displaying, ScFv in form of ScFv-g3p fusion protein was panned based on its binding capacity to the CD5 antigens on the cell surface of Molt-4 cells. Assayed by the cell-ELISA, 4 clones were found to have high affinity to CD5 antigen. DNA sequencing confirmed that the VH fragment is 339 base pairs, the VL 300 bp, and the ScFv belongs to the mice gene family. In E. coli HB2151 the soluble ScFv-CD5 was expressed mainly in the periplasm.

[Advance in the research of endostatin].

Endostatin was a newly found N-terminal fragment of collagen XVIII NC1 domain, which has distinct anti-angiogenesis character. Its prominent anti-tumor effect in mouse model made its a hot point of research and a promising anti-tumor drug candidate. This article will give some discussion in this field.

[Induction of protective immune response in mice and rhesus monkeys by immunization with fusion protein of cholera toxin B subunit and multiples of Plasmodium falciparum].

Recombinant fusion protein of cholera toxin B subunit (CTB) and poly-valent protective epitopes of plasmodium falciparum was given to i.m. to C57BL/6j mice and rhesus monkeys three times. In rhesus monkeys, high level of antibodies for CTB (1:6400) and malaria epitopes (1:3200) amtobpdoes were elicited as well as the specific CTL activity for P. plasmodium. After the mice were challenged with sporozoites of P. yeolli, about 50% of them were protected from the patent infection. A blood-stage challenge with 10(8) of P. cynomolgi parasite were given to rhesus monkeys, which showed that two animals in control group were patent infection for at least 30 days, in contrast, the two animals immunized were recovered respectively at the day of 11 and 15 after challenges. The results suggested that cholera toxin acts as an effective adjuvent in the development of malaria vaccine.

[A new model of translational control of gene expression in polycistron++ of AB5 entrerotoxin].

The expression level of the B subunit gene of cholera toxin (ctx) and E. coli heat labile toxin (ltx) is five to seven times more than that of A subunit gene. In these studies, a 80 basepair translation regulation element was found located in the structure gene of A gene of both toxin operon which consists of three translation initiation region. Site-directed mutation of the initiation codon of TIR3 resulted in the 9 time decrease of the expression of the downstream cistron which was translational coupled with A gene. The results indicated that translation from the internal of A gene and translation coupling are responsible for the differential expression level of the A and B gene of AB5 enterotoxin.

[Translation initiation function of the regulation element in the operon of cholera toxin A].

To demonstrate that there existed translation coupling between cholera toxin A subunit gene and B subunit gene, and give the answer why the expression level of B gene is five times more than that of A gene, alpha report system for the investigation of translation coupling was constructed by using lacZ gene as reporter. Frame-shift mutation was introduced near the C terminal of ctxA gene, and the ribosome would read through its normal stop codon. The report plasmid was constructed and it was found that the expression level of lacZ gene decreased five times after the frame-shift mutation. The translation of cholera toxin B subunit gene was translational coupled with A subunit gene, and was responsible for the differential expression level of the two genes.

[Overproduction of cholera toxin B subunit by recombinant Escherichia coli MM2 in lactate-containing medium].

The expression of ctb gene in recombinant E. coli MM2 strain is affected by temperature, pH and carbon source. High level production of cholera toxin B subunit (CTB) was investigated in lactate-containing medium which designed by experiments of orthogonal test. Upshifting temperature from 30 degrees C to 37 degrees C could increase the production of CTB by 4 fold, upshifting pH value from 7.2 to 8.4 at the later culture stage could increase the specific expression level of CTB by 2.14 fold, and adding sodium acetate increased the production of CTB by 65%. In 5 L fermentor the cell density was reached at OD600 30, and the 186.7 mg/L of CTB was obtained. The CTB in the culture supernatants was in the form of polymer like the native CTB from Sigma, and also possessed the same antigenity.

[Assessment of malaria DNA vaccines in mice and monkeys].

Cholera toxin B subunit is a good carrier protein and an effective adjuvant which can boost both cellular and humoral immunity. DNA fragments encoding B cell, Th cell and CTL epitopes of P. falciparum CS, MSA-1, MSA-2 and RESA antigens were cloned down-2 stream of cholera toxin B subunit gene in the same reading frame. High titer of anti-malaria epitopes antibodies and strong cellular immunogenicity were elicited after Balb/c mice were immunized three times with 100 micrograms recombinant plasmid DNA dissolved in 100 microliters PBS. A total of 120 vaccinees were challenged with mouse Plasmodium yoelli to investigate if cross protection existed. The protective efficacy was about 60%-80%. Four rhesus monkeys were challenged with 10(8) of P. cynomalgi, better results were obtained in the groups immunized with mixed plasmids including NANP, AWTE.

The effects of calcium phosphate cement particles on osteoblast functions.

Calcium phosphate cements (CPC) are increasingly used in the orthopedic field. This kind of cement has potential applications in bone defect replacements, osteosynthetic screw reinforcements or drug delivery. In vivo studies have demonstrated a good osteointegration of CPC. However, it was also observed that the resorption of CPC could create particles. It is known from orthopedic implant studies that particles can be responsible for the peri-implant osteolysis. Biocompatibility assessment of CPC should then be performed with particles. In this study, we quantified the functions of osteoblasts in the presence of beta-TCP, brushite and cement particles. Two particle sizes were prepared. The first one corresponded to the critical diameter range 1-10 microm and the second one had a diameter larger than 10 microm. We found that CPC particles could adversely affect the osteoblast functions. A decrease in viability, proliferation and production of extracellular matrix was measured. A dose effect was also observed. A ratio of 50 CPC particles per osteoblast could be considered as the maximum number of particles supported by an osteoblast. The smaller particles had stronger negative effects on osteoblast functions than the larger ones. Future CPC development should minimize the generation of particles smaller than 10 microm.

Titanium particles inhibit osteoblast adhesion to fibronectin-coated substrates.

To illuminate the effect of titanium particles on osteoblast function, we compared the adhesion force of neonatal rat calvarial osteoblasts on fibronectin-coated glass after incubation with titanium particles (80% had diameters of less than 5 microm). The cells were incubated with the particles for 1.5-72 hours. Using a micropipette single-cell manipulation system, we showed that the adhesion force of the osteoblasts to fibronectin-coated glass (1.0 microg/ml) was significantly affected by the presence of particulate debris. The adhesion force of the cells incubated with titanium particles for less than 4 hours was not significantly affected by exposure to the particles; after 4 hours, however, it was significantly reduced relative to that of controls. Aspiration of particle-challenged osteoblasts into the micropipette demonstrated that the particles were not stripped from the cell surface and therefore confirmed that the osteoblasts had ingested them. During aspiration, the particles traveled through the cytoplasm rather than on the cell surface. When the osteoblasts were exposed to the particles and cytochalasin D, they exhibited much lower adhesion forces than did the controls or the cells exposed to titanium particles only; this indicates an important role of actin filaments in the osteoblastic response to particles. Staining for F-actin also indicated an influence of internalized titanium particulate on cytoskeletal arrangement and cell spreading. Furthermore, with standard Northern blotting techniques, levels of mRNA for collagen type I and fibronectin were significantly reduced as early as 4 hours after exposure to particles compared with levels in controls, and this effect continued to 72 hours. These data indicate that direct exposure of osteoblasts to titanium particles, which we propose to be ingested by the osteoblasts, can significantly decrease osteoblast adhesion force; this may lead to decreased cellular activity and gene expression of fibronectin and collagen type I in the presence of titanium wear debris.

Time-dependent increases in type-III collagen gene expression in medical collateral ligament fibroblasts under cyclic strains.

Numerous studies have demonstrated the capacity of mechanical strains to modulate cell behavior through several different signaling pathways. Understanding the response of ligament fibroblasts to mechanically induced strains may provide useful knowledge for treating ligament injury and improving rehabilitation regimens. Biomechanical studies that quantify strains in the anterior cruciate and medial collateral ligaments have shown that these ligaments are subjected to 4-5% strains during normal activities and can be strained to 7.7% during external application of loads to the knee joint. The objective of this study was to characterize the expression of types I and III collagen in fibroblast monolayers of anterior cruciate and medial collateral ligaments subjected to equibiaxial strains on flexible growth surfaces (0.05 and 0.075 strains) by quantifying levels of mRNA encoding these two proteins. Both cyclic strain magnitudes were studied under a frequency of 1 Hz. The results indicated marked differences in responses to strain regimens not only between types I and III collagen mRNA expression within each cell type but also in patterns of expression between anterior cruciate and medial collateral ligament cells. Whereas anterior cruciate ligament fibroblasts responded to cyclic strains by expression of higher levels of type-I collagen message with almost no significant increases in type-III collagen, medial collateral ligament fibroblasts exhibited statistically significant increases in type-III collagen mRNA at all time points after initiation of strain with almost no significant increases in type-I collagen. Furthermore, differences in responses by fibroblasts from the two ligaments were detected between the two strain magnitudes. In particular, 0.075 strains induced a time-dependent increase in type-III collagen mRNA levels in medial collateral ligament fibroblasts whereas 0.05 strains did not. The strain-induced changes in gene expression of these two collagens may have implications for the healing processes in ligament tissue. The differences may explain, in part, the healing differential between the anterior cruciate and medial collateral ligaments in vivo.

[Induction of protective immune responses in rhesus monkey by immunization with recombinant plasmids of polyvalent epitopes of Plasmodium falciparum using cholera toxin B as adjuvant].

The immunogenicity and protective efficacy of the DNA vaccine which include cholera toxin B subunit (CTB) and polyvalent protective epitopes of Plasmodium falciparum (awte gene) was assessed using rhesus monkeys as animal models. Recombinant plasmids of pCMV-CTB-AWTE were given to five rhesus monkeys three times with two weeks intervals by intramuscle (i.m.) route, immunization dose was 500 micrograms per plasmid per animal. High levels of anti-CTB and anti-malaria epitopes antibodies and P. falciparum epitope specific CTL activity were elicited. The vaccinated groups was challenged with 1.25 x 10(8) of P. cynomolgi parasites. All monkeys of the control group was patent for at least 34 days, the DNA vaccinated groups wasn't infected during the 60 days we detected. The cocktail DNA vaccine which contains multi-stage and multi-epitope antigen gene shows excellent immunogenicity and protective efficacy, the results also suggests that DNA vaccine plays an important role against malaria infection.

[Cloning of human G-CSF genomic gene and its expression in transgenic mice mammary gland].

Human genomic DNA was used as template of PCR. 1.5 kb G-CSF genomic DNA was obtained using PCR amplification method. Sequence analysis showed that genomic DNA sequence of human G-CSF was correct. The vector of mammary gland expression was constructed and contained whey acid protein (WAP) 5' control region directed human G-CSF genomic DNA. In order to produce transgenic mice, 1200 fertilized eggs were microninjected using WAP-G-CSF fragment. Two male transgenic mice were obtained and identified using PCR method and Southern analysis. Integration rate of human G-CSF gene was 2.37% in mice. Foreign gene could also be identified in F1 and F2 transgenic mice. Expression levels of human G-CSF in transgenic mouse milk were 120-250 ng/ml.

[Studies of foreign gene integeation during embryo early development].

There are still some problems in transgenic animals. Gene transfer, for example, reamins a difficult and costly task for animals, the vectors carrying the gene coding for the proteins of interest are of unpredictable efficiency. Therefore, it is important to identify foreign gene integration in genomose before transferring fertilized eggs to receptors, in order to increase efficiency of producing transgenic animal. In this paper, the construct that mice whey acid protein (WAP) gene promoter directs G-CSF gene was used to microinject fertilized eggs of mice. Fertilized eggs containing foreign gene were measured by using PCR method. The results showed that 100%, 77.7% and 44.4% retentions of foreign gene were achieved in 1, 2 and 8 cell-stage, respectively. Two part homologous recombination fragments were constructed and coinjected in to fertilized eggs of mice. PCR amplification fragment went beyond this homologous recombination area. If foreign gene could not integrate in to genomose, the fragment of PCR amplification could not be produced during embryo development. The results showed that the rationes of foreign gene integrated in to genomoes in 1, 2 and 8 cell-stage were 11.1%, 55.5% and 44.4%, respectively. This method might provide us a way to screen transgenic eggs when we use embryo section technique in farm animal.

Overproduction of nitric oxide inhibits vascular reactivity in portal hypertensive rats.

AIM: To evaluate the relationship between nitric oxide (NO) and hyperdynamic circulatory status in portal hypertension. METHODS: Twenty male Sprague Dawley rats (weighing 200 ± 20 g) were randomized into two groups: portal hypertension group (n = 12) and sham-operated control group (n = 8). The portal hypertensive model was established by means of graded constriction of the portal vein. The concentrations of nitrite (NO2 (-)) in the portal vein and peripheral blood were measured by fluorometric assay to reflect NO levels. The reactivity of isolated abdominal aortic rings from rats with partial portal vein constriction and controls was determined by assessing response to administration of potassium chloride (KCl) (10-80 mmol/L) and phenylephrine (10(-9)-10(-4) mol/L) with or without preincubation with NO synthase inhibitor Nω-nitro-L-arginine (L-NNA). RESULTS: Serum concentrations of NO2 (-) in the portal vein blood (0.766 ± 0.097 µmol/L) and peripheral blood (0.687 ± 0.092 µmol/L) were elevated in portal hypertensive rats, as compared with the concentrations in controls (0.613 ± 0.084 µmol/L and 0.591 ± 0.045 µmol/L respectively, both P < 0.01). In addition, the rates of NO2 (-) in portal vein blood were markedly higher than those in peripheral blood (P < 0.05) in the portal hypertensive rats. Abdominal aortic rings from rats with portal vein constriction exhibited significantly impaired contractility to phenylephrine and KCl, as compared with the control rats. The EC50 values of KCl were markedly higher in the portal hypertensive rings (26.5 ± 0.9 mmol/L) than in the control rings (22.3 ± 1.7 mmol/L, P < 0.01), as were the EC50 values of phenylephrine (37.2 ± 0.4 nmol/L vs control rings: 28.1 ± 0.2 nmol/L, P < 0.01). After preincubation of rings with L-NNA, the difference in EC50 values between portal hypertensive and control rings was no longer statistically significant for either KCl (20.18 ± 0.8 mmol/L vs 19.4 ± 1.2 mmol/L, P > 0.05) or phenylephrine (22.4 ± 1.8 nmol/L vs 21.8 ± 1.4 nmol/L, P > 0.05). However, the maximal concentrations of KCl and phenylephrine for inducing contractions were still significantly lower in the portal hypertensive rings (1.08 ± 0.1 g and 1.43 ± 0.14 g) than in the control rings (1.21 ± 0.11 g and 1.72 ± 0.11 g respectively, both P < 0.05). Thus, addition of the NO synthase inhibitor L-NNA could partially restore contractile responses to KCl and phenylephrine in portal hypertensive rings. CONCLUSION: NO overproduction inhibits the vascular reactivity to vasoconstrictors, and it might be one of the main causes of vasodilatation and hyperdynamic circulatory status in portal hypertension.