肌腱玻璃化法冷冻保存的体外实验

目的:目前临床上常用低温冷冻法来保存同种异体肌腱,但操作较复杂费时,并且所保存的肌腱活性较低而限制其应用。采用已筛选的玻璃化法冷冻保存鸡屈趾深肌腱,并将复温后的玻璃化肌腱进行体外检测,探索其作为肌腱移植材料的可行性。方法:实验于2003-11/2005-02在解放军总医院骨科研究所完成。①实验材料及分组:来亨鸡16只,雄性,体质量2.5kg左右,随机分为2组,玻璃化组为玻璃化肌腱,新鲜肌腱组为新鲜肌腱,每组8只。②实验过程:切取来亨鸡屈趾深肌腱,置入玻璃化液内快速投入液氮保存2周制备玻璃化肌腱。③实验评估:将2组肌腱在体外进行大体、组织学及超微结构观察,羟脯氨酸含量测定,生物力学性能检测,并对两组肌腱进行细胞培养与鉴定。结果:①玻璃化组肌腱的大体、组织学及超微结构与新鲜肌腱组相似,其细胞及细胞外结构得以良好保存。②应用碱解法测定玻璃化肌腱内的羟脯氨酸含量为69.27mg/g,与新鲜肌腱组间差异无统计学意义。③玻璃化组肌腱破裂强度为165.58MPa,弹性模量1.41GPa,与新鲜肌腱组的力学性能差异无统计学意义。④将玻璃化组肌腱进行细胞培养,细胞第8天自组织块长出,第21天后传代,培养3代后出现明显的退化现象,其生物学特性与新鲜肌腱组相似。⑤将两组肌腱培养的细胞分别进行免疫组织化学染色,经鉴定均为肌腱细胞。结论:玻璃化法保存的肌腱具有良好的细胞活性、细胞外结构及力学性能,其生物学及生物力学特性无明显变异。     

EUROPEAN STROKE PREVENTION STUDY 2: DIPYRIDAMOLE AND ACETYLSALICYLIC ACID IN THE SECONDARY PREVENTION OF STROKE

In 1988, an optimal antiplatelet regimen for secondary stroke prevention remained to be defined. We undertook a randomised, placebo-controlled, double-blind trial to investigate the safety and efficacy of low-dose acetylsalicylic acid (ASA), modified-release dipyridamole, and the two agents in combination. Patients with prior stroke or transient ischaemic attack (TIA) were randomised to treatment with ASA alone (50 mg daily), modified-release dipyridamole alone (400 mg daily), the two agents in a combined formulation, or placebo. Primary endpoints were stroke, death, and stroke or death. TIA and other vascular events were secondary endpoints. Patients were followed on treatment for two years. We concluded that dipyridamole, in a modified-release form, at a dose of 200 mg b.d. and ASA 25 mg b.d., have been shown to be equally effective in the secondary prevention of ischaemic stroke and TIA; that when co-prescribed, the protective effects are additive, the combination being significantly more effective than each agent prescribed singly; and that low-dose ASA does not eliminate the propensity for induced bleeding.     

The 2005 Italian Quality Control Study for the evaluation of CD4 cells in centers involved in the treatment of HIV type 1 patients

We report the results of an external quality control program, including 17 Italian centers involved in the care of patients infected by HIV, to evaluate CD4 T cell count proficiency and reproducibility. The centers received two commercial stabilized blood preparations, one with "normal" and one with "low" CD4 T cell content. The centers were asked to process the samples two times, 1 week apart, with the same procedure used for samples from HIV patients. Most centers showed a good performance of CD4 frequency and absolute count determinations. In particular, the "low" sample was correctly analyzed by all centers; only two underestimated the "normal" sample CD4 frequency, and only one underestimated the CD4 absolute count by >100 CD4 cells/microl. Overall, our data suggest that most Italian laboratories provide reliable and reproducible results in evaluating CD4 T cells in HIV(+) samples.     

Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.