A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts

The bilayer structure of chitosan film and sponge was designed as a scaffold of skin tissue engineering and a dermis substitute. It was processed successively via the formation of a dense chitosan film by casting method and a porous chitosan sponge by lyophilization. The dry thickness of the film layer was 19.6 microm and that of the sponge layer was controlled at 60-80 microm. Porogens such as sodium chloride, glucose, and sucrose were used to create large pores of the chitosan sponge layer. Human neofetal dermal fibroblasts were seeded in the chitosan sponge layer and cultured for 4 weeks. It was found that the cells could grow and proliferate well in an extended shape on the flat bottom of large pores with 15-100 microm width and in spherical form on the rough pore walls or at the edges of micropores less than 5 microm. Fibroblasts after the culture could bind tightly with the sponge layer via newly formed extracellular matrices to give a living cell-matrix-chitosan composite. The bilayer chitosan material remained stable in shape and size during the cell culture. The results suggested that the bilayer chitosan material would be an alternative of collagen materials which was obviously contracted during cell culture.     

胶原基真皮再生支架的微结构控制

综述了影响胶原基真皮再生支架微结构的各种因素及其控制方法。胶原基真皮支架的生物活性和修复创面的能力受到诸如除胶原外的其它主要材料 (第二组分 )、支架的孔径和孔隙率、支架厚度、生物活性因子以及交联度等多方面因素的综合影响。从材料学、生物学和医学的角度综合地应用物理、化学和生物学的手段探索各种影响因素的控制方法是组织工程皮肤研究的重点。     

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG.

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.     

Inhibitory effect of ketamine on phosphorylation of the extracellular signal-regulated kinase1/2 following brain ischemia and reperfusion in rats with hyperglycemia

To determine if the inhibitory effects of ketamine on the extracellular signal-regulated kinase (ERK) 1/2 are involved in reduction of the hyperglycemia-exaggerated cerebral ischemic lesion, rats with normoglycemia, hyperglycemia, or hyperglycemia supplemented with ketamine were subjected to 15 min of forebrain ischemia, and then, reperfusion for 0.5, 1, and 3h. Phosphorylation of ERK1/2 in the brain tissues was assessed by immunohistochemistry and Western blot analysis. In rats with normoglycemia, we demonstrated a moderate increase of the ERK1/2 phosphorylation in the cingulum cortex and hippocampus CA3 following an ischemic intervention. It quickly dropped to control levels after reperfusion for 0.5h. In rats with hyperglycemia, however, the increase of the ERK1/2 phosphorylation in these areas was significantly higher in all animals reperfused. The neuronal death, detected by the TdT-mediated-dUTP nick end labeling assays, was found in the cingulum cortex (5.23+/-2.34, per high power feild) and hippocampus CA3 areas (6.29+/-3.68, per 1mm(2)) in hyperglycemic group after reperfusion for 3h. With ketamine treatment, the ERK1/2 phosphorylation in cingulum cortex and hippocampus CA1 and CA3 areas was found to be the same as that in normoglycemia rats. Our results suggest that hyperglycemia may increase the ischemic insult through modulation of the signal transduction pathways involving ERK1/2. The inhibitory effects of ketamine on the hyperglycemia-activated ERK1/2 phosphorylation are probably through inhibition of the N-methyl d-aspartate-mediated calcium influx, which subsequently reduce the hyperglycemia-exaggerated cerebral damage.     

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl^– for gluconate^– shifted the reversal potential, while Cl^– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca²⁺ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca²⁺-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.     

A constrained tracking algorithm to optimize plug patterns in multiple isocenter gamma knife radiosurgery planning

We developed a source blocking optimization algorithm for Gamma Knife radiosurgery, which is based on tracking individual source contributions to arbitrarily shaped target and critical structure volumes. A scalar objective function and a direct search algorithm were used to produce near real-time calculation results. The algorithm allows the user to set and vary the total number of plugs for each shot to limit the total beam-on time. We implemented and tested the algorithm for several multiple-isocenter Gamma Knife cases. It was found that the use of limited number of plugs significantly lowered the integral dose to the critical structures such as an optical chiasm in pituitary adenoma cases. The main effect of the source blocking is the faster dose falloff in the junction area between the target and the critical structure. In summary, we demonstrated a useful source-plugging algorithm for improving complex multi-isocenter Gamma Knife treatment planning cases.     

API0134对大鼠血浆和血小板血栓素B2生成与血小板聚集的影响

用放射免疫分析法和比浊法测定ＰＡＩ０１３４对大鼠血浆血栓素Ｂ２浓度，血小板ＴＸＢ２生成血小板聚集的影响。静脉注射ＡＰＩ０１３４７０ｍｇ或１００ｍｇ．ｋｇ显著降低血浆ＴＸＢ２浓度，其降低２率分别为３８．８％和５１．６％，ＡＰＩ０１３４明显抑制ＡＤＰ诱导的大鼠血小板聚集和ＴＸＢ２生成，抑制率分别为２７．８％，３９．５％，和４１．４％，５３．６％，两抑制率间呈显著正相关。     

Ultrastructure of Amelanotic Melanocytes from Human Hair Follicles

Objective: To investigate the ultra structure of amelanotic melanocytes (AMMC). Methods: The hair follicles obtained from normal human scalp by 0.50% collagenase type V treatment were washed with 0.1mol/L phosphate buffer salt (PBS). Hair-follicle cell suspensions were prepared by trypsin treatment and cultured in melanocyte medium. Remaining keratinocytes were removed by differential trypsinization. 100μg/ml geneticin was used to eliminate the contaminating fibroblasts. At third passage, the cells were trypsinized, and then washed in phosphate-buffered saline and processed for transmission electron microscopy. Results: Under transmission electron microscope, the cultured cells showed round or oval shape, with single large nuclear and the karyotheca were double deck. There were obvious euchromosome within the nucleus, and sparse heterochromosome. There were various organelles in the cytoplasm, including plentiful melanosomes with nearly similar size, mitochondria, rough endoplasmic reticule (RER) and ribosome. The electron density granules in most of the melanosomes disposed along concentric circularities. Golgi apparatus in the cells was inconspicuous. Conclusion: The ultra structure of AMMC from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional immature of AMMC.     

Human heat shock protein 60 (409-424) fragment is recognized by serum antibodies of patients with acute coronary syndromes.

Acute coronary syndromes (ACS), including unstable angina (UA) and acute myocardial infarction (MI), are clinical manifestations of a progressive atherosclerotic process. Antibodies (Ab) to heat shock proteins (hsp) have been reported to be associated with atherosclerosis. Blood samples from 35 patients with ACS and 20 healthy volunteers were tested for Ab to human hsp60 by an enzyme-linked immunosorbent assay (ELISA). Levels of specific serum Ab against hsp60 were significantly elevated in patients with ACS when compared to clinically healthy subjects. To determine the antigenic determinants recognized by these Ab, antibody binding to seven peptides, selected from the hydrophilic and acrophilic regions of the human hsp60 molecule, was assessed. Despite the individual variation in the immune response among patients, one immunodominant region was revealed corresponding to the hsp60 (409-424) peptide. The identification of this epitope may be important for understanding the function of this protein in the atherosclerotic process.