体外Fas介导细胞凋亡的细胞周期特异性的检测方法

Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.     

单臂半环式三维固定胫腓骨骨折支架的生物力学实验及临床应用

为观察自制单臂半环胫腓骨骨折固定器治疗胫腓骨骨折的生物力学效应和临床疗效,选择成人离体新鲜骨1具,新鲜牛胫腓骨4具(其中2具备用),分别制作成斜形、横断形、粉碎性骨折模型,以骨折复位器模拟临床治疗方法将之复位固定后,固定于电子万能试验机上,分别对3种模型模拟骨折固定后的6种受力状态(施加轴向拉伸力、轴向压缩力、前后向弯曲压力、侧向弯曲压力、左扭转力、右扭转力)进行试验,观察骨折端位移,分别记录试验数据、坐标曲线图。同时对50例胫腓骨骨折患者进行临床观察。结果显示,对骨折复位固定后的模型施加较常规外力可增加数倍的力,骨折复位固定器及骨折端本身的位移数均在许可范围。经50例临床应用观察,骨折全部愈合。表明该复位固定器设计力学结构合理,无偏心应力,具有弹性固定功能,性能稳定,能有效维持骨折复位效果的作用。且操作简便、固定牢靠、实用。     

高效液相色谱法测定盐酸达克罗宁乳膏含量

目的:建立盐酸达克罗宁乳膏中盐酸达克罗宁含量测定的高效液相色谱法。方法:采用C18色谱柱,流动相为乙腈-0．02 mol·L-1磷酸二氢钾(用磷酸调pH至3．0)(60:40),检测波长为282 nm,流速为0．9 ml·min-1。结果:盐酸达克罗宁浓度在9．98-49．90 mg·L-1范围内与峰面积呈良好的线性关系(r=0．999 9),平均回收率为98．55％(RSD％为1．19％)。结论:本法简便、快捷,结果准确,适用于该制剂的质量分析检验。     

Interference with the formation of the epididymal microenvironment-a new strategy for male contraception？

The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Recent work in our laboratory has shown that this protein is also expressed by developing germ cells indicating a role of this protein in spermatogenesis. In view of the fact that the CFTR gene has a far reaching and widespread effect on human reproduction, understanding the role of CFTR in the male reproductive tissues and its intervention by pharmacological agents can open a new avenue of research into the development of novel male contraceptives. ( Asian J Androl 2000 ; 2 : 39 - 45 )     

Remembering and knowing in a patient with preserved recognition and impaired recall

ROB is a patient who has a severe deficit in recalling recently presented verbal material following rupture and repair of an anterior communicating artery aneurysm [Hanley JR, Davies ADM, Downes J, Mayes A. Cognitive Neuropsychology 1994;11:543-78; Hanley JR, Davies ADM. In: Parkin A, editor. Case Studies in the Neuropsychology of Memory. Hillsdale, NJ: Lawrence Erlbaum, 1997. p. 111-26]. Despite this, her performance on tests of recognition memory is comfortably within the normal range. In the present series of experiments, we investigated whether or not ROB's performance on tests of recognition memory might be associated with a disproportionately large number of correct decisions made on the basis of familiarity rather than contextual retrieval [e.g. Mandler G. Psychological Review 1980;87:252-71]. Contrary to this hypothesis, the results showed that ROB made a high proportion of remember decisions relative to know decisions in recognition [cf. Gardiner JM. Memory & Cognition 1988;16:309-13] and produced a high recollection score when conscious recollection and familiarity were placed in opposition to one another [cf. Jacoby LL, Woloshyn V, Kelley C. Journal of Experimental Psychology: General 1989;118:115-25.]. ROB's recognition memory performance therefore appears to be qualitatively as well as quantitatively similar to that found in the normal population. As ROB has suffered damage to both the fornix and the anterior thalamus, the results of the present study are consistent with the claim that damage to the extended hippocampal system has a much more severe effect on recall than on recognition [Aggleton JP, Shaw C. Neuropsychologia 1996;34:51-62; Aggleton JP, Saunders RC. Memory 1997;5:49-71]. The present results provide no support, however, for the additional suggestion [Aggleton JP, Brown MW. Behavioral and Brain Sciences 1999;22:425-56.] that the extended hippocampal system is necessary for recognition memory decisions that are based on contextual retrieval.     

Blockade of AMPA receptors and volatile anesthetics: reduced anesthetic requirements in GluR2 null mutant mice for loss of the righting reflex and antinociception but not minimum alveolar concentration

BACKGROUND: The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor mediates fast excitatory neurotransmission in the central nervous system. Many general anesthetics inhibit AMPA receptors in vitro; however, it is not certain if this inhibition contributes to the behavioral properties of these drugs. AMPA receptors lacking the GluR2 subunit are resistant to blockade by barbiturates in vitro. Paradoxically, GluR2 null mutant (-/-) mice are more sensitive to barbiturate-induced loss of the righting reflex (LORR) compared with wild-type (+/+) littermates. To determine if interactions between anesthetics and AMPA receptors account for the increased sensitivity of (-/-) mice, the effects of volatile anesthetics that do not directly inhibit AMPA receptors were examined. METHODS: Isoflurane, halothane, desflurane, or sevoflurane were administered to (-/-) and (+/+) littermate controls. Anesthetic requirements for LORR, movement to tail clamp (minimum alveolar concentration [MAC]), and hind-paw withdrawal latency (HPWL) were determined. Electrophysiologic methods examined the inhibition of AMPA receptors by isoflurane and halothane. RESULTS: Anesthetic requirements for LORR and HPWL were decreased, whereas MAC values were unchanged in (-/-) mice. Isoflurane and halothane caused minimal inhibition of AMPA receptors at clinically relevant concentrations. CONCLUSIONS: Direct blockade of AMPA receptors did not account for the increased sensitivity to volatile anesthetics in GluR2 null mutant mice for HPWL or LORR. Thus, the deficiency of GluR2-containing AMPA receptors increases the sensitivity of neuronal circuitry mediating these end points, but not MAC. GluR2-containing receptors do not contribute appreciably to MAC in this mouse model. These results illustrate the difficulties in attributing behavioral responses to drug-receptor interactions in genetically engineered animals.     

Effects of Mitofusin-2 Gene on Cell Proliferation and Chemotherapy Sensitivity of MCF-7

In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.     

Phosphorothioate oligonucleotide inhibits tissue factor expression in endothelial cells induced by blood flow shear stress in rats

Objective： To determine the effect of antiparallel phosphorothioate triplex-forming oligonucleotide （apsTFO）, which was designed according to shear stress response element （SSRE） in tissue factor （TF） gene promoter region, on the expression of endothelial TF in carotid artery stenosis rats. Methods： Rat model of severe carotid artery stenosis were inflicted by silica gel tube ligation. Half an hour before the model infliction, GT20-apsTFO, GT20-psTFO and GT21-apsTFO labeled with green fluorescence （FITC） were injected into the vena caudalis of rat at a dose of 0.5 mg/kg. Half an hour, 4 or 9 h after the ligation, the distribution of TFO in the common carotid artery, the liver and the kidney was detected with aid of fluorescence microscopy. And the mRNA and protein expressions of TF, Egr-1 and Spl in the above-mentioned organs were determined with in situ hybridization and immunohistochemical assay respectively in 6 h after the model establishment, and the results were analyzed with an image analysis system. Results： Only in 1 h after TFO injection, fluorescent granules appeared in the liver, the kidney and the vascular wall and lumen of carotid artery, and then in 4.5 h, they still deposited in above sites except the vascular lumen. GT20-apsTFO and GT21-apsTFO significant down-regulated the mRNA and protein expressions of TF compared to the rats without treatment （P〈0.05）, and the former apsTFO had a more stronger effect than the later （P〈0.05）. GT20-psTFO had no such effect （P〉0.05）. The 3 TFOs had no inhibition on the mRNA and protein expressions of Egr-1 and Spl. Conclusion： Pretreated apsTFO can partly come into the vascular endothelial cells, and inhibit TF expression induced by shear stress, but had no effect on Egr-1 and Spl gene expressions.     

Plastic Changes of Synapses and Excitatory Neurotransmitter Receptors in Facial Nucleus Following Facial-facial Anastomosis

The remodeling process of synapses and neurotransmitter receptors of facial nucleus were observed. Models were set up by facial-facial anastomosis in rat. At post-surgery day （PSD） 0,7,21and 60, synaptophysin （p38）, NMDA receptor subunit 2A and AMPA receptor subunit 2 （GluR2） were observed by immunohistochemical method and semi-quantitative RT-PCR, respectively. Mean-while, the synaptic structure of the facial motorneurons was observed under a transmission electron microscope （TEM）. The intensity of p38 immunoreactivity was decreased, reaching the lowest value at PSD day 7, and then increased slightly at PSD 21. Ultrastructurally, the number of synapses in nucleus of the operational side decreased, which was consistent with the change in P38 immunoreactivity. NMDAR2A mRNA was down-regulated significantly in facial nucleus after the operation （P〈0.05）, whereas AMPAR2 mRNA levels remained unchanged （P〉0.05）. The synapses innervation and the expression of NMDAR2A and AMPAR2 mRNA in facial nucleus might be modified to suit for the new motor tasks following facial-facial anastomosis, and influenced facial nerve regeneration and recovery.