Objective: To study the characteristics of mouse embryonic stem (ES) cell line R1 expressing green fluorescent protein (GFP) efficiently and stably. Methods: Four kinds of biological characteristics: growth curve, express level of alkaline phosphatase, karyotype and pluripotentiality of the subclones of R1 ES cell expressing GFP were observed and analysed. Results: No obvious differences between R1 ES cells and the 4 ES cell clones expressing GFP［ESG（+）］ on the proliferation speed, differentiation state and the ratio of normal karyotype were observed. Teratocarcinoma concluded 3 germinal layers could form after inoculating ESG(+) cells to nude mice. Conclusion: The expression of GFP may not make any detectable effect on the proliferation speed, differentiation state and the pluripotentiality of R1 ES cells. This research work ensures efficient utilization of GFP as a reporter gene tracing ES cell in vivo.

12 trace elements from the hairs of 20 cases of aplastic anemia were examined and compared with that from hairs of normal people as control. Results showed that in Yin-deficient type patients, Li, Ca, Si, Cr markedly decreased; in Yangdeficient type patients, Zn, Mg, Ba, Si, Ca, Li markedly decreased, while all 12 trace elements decreased in patients of deficiencies of both Yin and Yang. New approaches were also offered for treatment of aplastic anemia on the basis of differential diagnosis.

BACKGROUND: The environmental change of regeneration of peripheral nerve fiber can accelerate the regeneration of nerve fiber and the recovery of nerve function. Physiotherapy can improve the local microcirculation of the injured area, and promote and stimulate the recovery course of trauma. Therefore, whether choosing different physiotherapies and controlling irradiation dose can speed up the regeneration of nerve fiber?OBJECTIVE: To observe the effects of different physiotherapies in promoting the regeneration of injured nerve fiber.DESIGN:Randomized grouping and controlled animal experiment.SETTING: Laboratory of Anthropotomy and Histo-Embryology Department,Peking University Health Science Center.MATERIALS: The experiment was conducted in the Laboratory of Anthropotomy and Histo-Embryology Department, Peking University Health Science Center, from September 2001 to September 2002. Fifteen adult male SD rats, weighting 185-220 g, were selected.INTERVENTIONS: The model of sciatic nerve injury was established and randomly assigned to 3 groups. Bio-spectrum group and infrared group with 5rats in each received the corresponding physical irradiation. Injury control group was given no irradiation. The slices of sciatic nerve at the operation side of the animals in the three groups were collected on day 14 after operation. The degeneration rate of the injured nerve fiber was observed under the optical microscope to make indirect assessment of the protective effect of physiotherapy after the nerve was injured.MAIN OUTCOME MEASURES: The degeneration rate of nerve fibers at the breakpoint and at 4 points near the spinal cord ( 1,2,3,4 mm) and away from the spinal cord( 1,2,3,4 mm).difference in the degeneration rate of nerve fibers at the breakpoint away from the spinal cord in bio-spectrum group and infrared group as compared with the degeneration rate of nerve fibers in bio-spectrum group(68% ) and infrared group(89% ) was obviously reduced and that in control group was the the spinal cord in the 3 groups showed decreasing trend: the closer to the spinal cord, the fewer the degenerative nerve fibers. The number of degenerative nerve fibers was the smallest in bio-spectrum group, second smallest in infrared group, and the largest in control group.CONCLUSION: The assisting physiotherapy can decrease the number of regenerative nerve fibers, thus promoting and accelerating their recovery. Irradiation with bio-spectrum equipment is more effective.

Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.

Objective: To establish C57BL/6 transgenic mice lineages containing pMTR1 plasmids as an efficient animal model for studying gene mutation in vivo . Methods: The linearized pMTR1 DNAs, constructed by molecular cloning, were injected into 572 fertilized eggs of C57BL/6 mice by microinjection. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant mice respectively, from which 35 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Four mice whose genomes integrated with copies of intact pMTR1 vectors were chosen as founders to establish transgenic mice lineages. The recovery of pMTR1 plasmid were conducted on the genomic DNAs of F1 or F2 transgenic offsprings. Results: It was showed that intact and functional pMTR1 plasmid could be efficiently recovered from the genomic DNAs of these mice. Conclusion: (1) The transgenic mouse lineages containing copies of a stably integrated pMTR1 plasmid is established. (2) The transgenic mice are suitable models for studying gene mutation in vivo .

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.

Aim To investigate the myocardial hypertrophy,the expression of transforming growth factor ?1(TGF-?1),Smad3 and Smad7 proteins in spontaneous hypertensive rats(SHR)and the effects of benazepril and candesartan.Methods SHRs of 12 weeks old were given benazepril and candesartan for 12 weeks.The tail arterial pressure was measured every two weeks.At 12 th weekend,cardiac configuration,heart mass index,area of cadiocytes,concertrations of AngⅡin plasma and myocardium,expressions of TGF-?1、Smad3 and Smad7 proteins were measured respectively.Results The arterial pressure,wall thickness,heart mass index,area of cardiocytes and the expressions of TGF-?1,Smad3 proteins increased in SHRs but were attenuated after the treatment of benazepril or candesartan.After the combined treatment,the synergistic effect could be observed.The levels of cardiac tissue and plasma AngⅡwere reduced.The expressions of Smad 7 were up-regulated after the treatment of benazepril or candesartan,while they were stable after the combined use.Conclusion There is a synergistic effect of attenuating myocardial hypertrophy in SHRs by combined use of benazepril and candesartan.It may be related to the regulation of Ang Ⅱ,decreasing the expressions of TGF-?1 and Smad3.

Objective To observe the expression of the small heat shock protein (HSP27) in the optic chiasma (OC), optic tract (OT), dorsal lateral geniculate body(LG) and superior colliculus (SC) of the adult golden hamster after intraorbital transection of the left optic nerve (ON). Methods The experimental animals were left to survive for l, 2, 3, 4, 5, 6, 8 weeks following ON transection. The animals were perfused with formol-saline and brains were excised, sectioned and stained with the immunohistochemistry. The sections were observed under the light microscope, the optical density (A) was measured and the data were analysed statistically. Results Immunohistochemical results indicated that the HSP27-expressions were not different between the right and left side of the OC, OT, LG and SC in normal or sham-operation controls. However, following transection of the left ON, HSP27 immunohistochemical stainings in the right site of OC, OT,LG and SC were increased, comparing with the left side. The maximum difference of HSP27 immunostaining between the right and left side appeared in the lst week following left ON axotomy. The sharply decrease of the A difference occurred at the 2nd week after axotomy with insignificant changes in the subsequent several weeks. And the significant A difference was observed in most time except 6th week. Most of HSP27-positive cells had morphological appearances similar to astrocytes with smaller cell body and numerous processes. Conclusion After the transection of monolateral ON, HSP27 expressions in the contralateral optic pathway of brain increased and persisted up to 8 weeks. This result suggested that the increase of HSP27 expression had something to do with the injury of the optic pathway, but the mechanism and biological significance of the increase in HSP27 expression level required to be studied further.;

Aim To investigate the protective effect of compound total flavonoids on atherosclerosis in ApoE -/- knockout mice.Methods Seven-week old C57BL/6 mice considered of the normal group (n =1 5 );seven-week old ApoE -/- mice were fed with high-fat diet and were assigned randomly into 5 groups:model group,simvastatin group,the low com-pound flavonoids group,the middle compound fla-vonoids group and the high compound flavonoids group.After 1 6 weeks,mice serum and aortas were harvested.The formation of atherosclerotic plaque was analyzed by HE staining,The serum level of lipids pro-files and superoxide dismutase (SOD )were deter-rnined.The levels of IL-1 βand NF-κB in serum were detected by ELISA assay.Results Area of atheroscle-rotic lesion was significantly less in the compound fla-vones group than in model.The level of TC,TG,LDL-C,IL-1 β,NF-κB in serum of the compound flavonoids group were decreased significantly,while SOD and HDL-C increased significantly compared with the mod-el group,and the difference was significant (P <0.05).Conclusion The compound flavonoids have a good protective effect on early atherosclerosis in mice, which may be due to its alleviating effects on hyperlipi-demia and inflammation and oxidation.

The phospholipase A2 ( PLA2 ) is one of the main constituents of the bee venom. Its hypotensive effect and mechanism are studied in this report.In the anaesthetized rats and cats, the PLA2 of the bee venom given intravenously caused a quick and profound fall in the arterial blood pressure.The results of this study indicate that its hypotensive effect is mainly concerned with releasing the endogenous histamine. If the antagonists of H1 and H2 receptor are administered together, its hypotensive action will be countered.