OBJECTIVE: Embryonic Stem Cell (ES) are characterized by totipotency and normal karyotype and provide the theoretical base in the following fields: embryonic developmeut of mammalian, cell differentiation, expres sion of exogenous genes and utilization of Ess to develop animal model for human inherited diseases. The identification of ES therefore is very important to research and utilize ES.DATA SOURCES: The relevant articles to embryonic stem cell between January 1980 and December 2003 were computer searched for in Medline with the key words "embryonic stem cell, embryo, Alkaline phosphatase,Oct-4"in English. Similarly, the relevant articles to embryonic stem cell between January 1980 and December 2003 were computer searched for in China Journal Full-text Database (CJFD) with the key word "embryonic stem cell" in Chinese.STUDY SELECTION: The articles were browsed firstly. The inclusion criteria were for those articles about the identification of embryonic stem cells. The exclusion criteria were for those about repetitive studies, reviews and other similar articles.DATA EXTRACTION: 16 articles about the identification of embryonic stem cells were collected. Then, the full-texts of the articles were looked through.DATA SYNTHESIS: The selected data were summarized in the following order: ①Preliminary identification of Ess based on morphology and growth;②Immunological valuation; ③Chromosome related identification; ④Identi fication of totipotency and pluripotency.CONCLUSION: The identification of embryonic stem cells is not the result of only one identifying method, but a process of identification. During this process of comprehensive identification, it is recommend to conduct AKP test firstly, karyotype analysis secondly, then examination of surface markers and finally identification of Ess totipotency when Ess are sufficient, takingcare to repeat every identification.

Objective To explore the optimal dose and ways of anesthesia for creating a rat model of spastic paralysis with intermittent bilateral common carotid artery ligation. Methods 60 Wistar rats were randomly divided into groups A, B, C, D, E and F. Group A was anaesthetized with 10% chloral hydrate 5 ml/kg injected subcutaneously, while group B with 4 ml/kg subcutaneously, group C with 4 ml/kg intraperitoneally, group D with 3 ml/kg subcutaneously, group E with 3 ml/kg intraperitoneally, group F with 2 ml/kg subcutaneously. The onset and duration of anesthesia, and intraoperative and postoperative mortality were compared. Results All the rats in the group A died during anesthesia, while the group F did not achieve the depth of anesthesia, and abandoned. The onset time was (6.5±0.7) min, maintained (121.4± 3.9) min, mortality was 0 in the group B, and it was (5.5±1.1) min, (122.0±3.6) min, 30% in the group C; (9.6±0.8) min, (106.7±3.7) min, 0 in the group D, (7.4±1.2) min, (105.3±3.5) min, 20% in the group E. The overall mortality rate was 0 in the groups accepted subcutaneous injected and 25% of intraperitoneal injection. Conclusion Anesthesia with 10% chloral hydrate 4 ml/kg subcutaneous injection is optimal of lower mortality, faster onset and longer maintaining in rats for spastic paralysis model with intermittent bilateral common carotid artery ligation.

ObjectiveTo explore the clinical significance of fms-like tyrosine kinase3(FLT3) expression in evaluation prognosis of acute myeloid leukemia(AML) prognosis.Methods50 patients with AML were selected.AML patients with normal karyotype were 20 cases,the abnormal karyotype were 30 cases.3ml bone marrow before themotherapy was aspirated respectively,and the FLT3 gene expression in leukemia cells was detected with polyenzyme chain react(PCR).ResultsThe FLT3 expression rate in AML patients with normal karyotype was 5.0％,and was 26.7％ in AML patients with abnormal karyotype,33.3％ in AML patients with refractory-relapse,and 4.5％ in AML patients with continue remission.The FLT3 expression rate was related with high leukemia cells percentage in bone marrow and high blood cells count in peripheral blood,and was not related with Franch America British(FAB) classification.The free-disease survival(FDS) and overall survived(OS) was shorter in FLT3 expression AML patients than that in no FLT3 expression AML patients.There was a statistical significance between the former and the latter( x2 =4.17,P ＜0.05 ).Conclusion FLT3 was a kind of worse factor in AML patients prognosis,and could guide clinical individual treatment in AML.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

Gene engineering is the main course of biological engineering. It should be adapted to the demand of innovation spirit, practice ability and comprehensive quality of students. Educational reform of gene engineering conducted by constructing system of theory and practice, optimizing course teaching content, strengthening practice teaching content, using modern teaching technology, strengthening web course construction and improving teaching methods. We pay attention to impart specialty knowledge and learning methods to students. Its aim was to increase teaching effects and meet the demands of bioengineering specialty and qualified personal training in 21 century.

Objective To identify the activity of HCV IRES translation differences and identify the relationship between HCV IRES translation activity and ROS in different concentrations of ferric ammonium citrate ( FAC) in-duction.Methods 1 ) Expression plasmid pCI-Rluc-HCV IRES-Fluc was confirmed by endonuclease digestion as well as luciferase transient expression in Huh-7 cell;2) Controlled by dual-luciferase reporter assay, the differ-ent translation activity of HCV internal ribosomal entry site ( IRES ) was examined in a concentration of 50 μmol/L and 300μmol/L of FAC induction;ROS fluorescent staining method was used to detect the activity of ROS in Huh-7 cells, Western blot method was used to detect the protein expression changes of Nrf2 in Huh-7 cells;3) On the basis of the above experiments, 100 μmol/L DPI was added in 300 μmol/L FAC experimental group, to analyse the changes of HCV replication and ROS production after joining DPI.Results The generation of ROS and the activity of luciferase in the model group were significantly higher than that in the control group ( P<0.05 ) .FAC can enhance the expression of HCV IRES and increase the production of ROS , then causing Nrf2 expression in Huh-7 cell.However,after adding ROS inhibitor DPI, the above functions in Huh-7 cell were weakened.Conclusions The increase of HCV IRES expression induced by FAC is related to excessive ROS pro-duction induced by FAC in Huh-7 cells.

Objective To compare the clinical efficacy and safety of two different conditioning regimens in nonmyeloablative autologous peripheral blood stem cell transplantation (NAST) for the treatment of systemic lupus erythematosus (SLE).Methods Different conditioning regimens were used in two groups:cytarabin combined cyclophosphamide in group 1 and ATG combined cyclophosphamide in group 2.Different recovery time of leucocytes,neutrophils and platelets in the two groups were compared.Statistical analysis were carried out by paired t-test.Results The mean time for peripheral leucocytes reaching 1.0×109/L,neutrophils getting up to 0.5×109/L,platelet raising to 100×l09/L and hemoglobin rising to 120 g/L in group 1 were [(7.2±1.3),(8.0±1.5),(10.5±1.4),(22.1±2.3)days] and [(10.4±2.1),(12.0±1.9),(19.3±2.1),(28.1± 2.4)] days in group 2.The difference was statistically significant (P＜0.01).CD4+ cell count and the ratio of CD4+/CD8+ of pre- and pro-NAST was changed.No significant differences were observed in the two groups.Conclusion For the sake of safety and hematopoietic reconstitution,we recommend cytarabin combined cyclophosphamide as the preferred conditioning regimen.

Objective To investigate the long-term efficacy of nonmyeloablative autologous peripheral blood stem cell transplantation(NAST) to cure refractory autoimmune disease(AD).MethodLong-term follow up of four cases of AD patients with NAST were summarized.The pretreatment regimen was intravenous injection of cytarabin (200 mg· kg-1· d-1 ) and cyclophosphamide (40 mg· kg-1· d-1).The therapeutic effect was evaluated by the change of symptoms and signs and long term complications.Changes of immune function were detected by flow-cytometry.ResultsFive cases of patients had been successfully engrafted.The average time for peripheral leucocytes count to reach 4.0×109/L was 12 days.It needed 10 days for platelets to return to 100×109/L and 22 days for hemoglobin to 120 g/L.Apparent remission of symptoms and signs was observed after transplantation.Lymphocyte subtypes analysis pre- and post- NAST showed that count of CD4+ and the ratio of CD4 +/CD8 + was returned to normal.One patient gave birth to a healthy baby four years after transplantation.Three female patients returned tonormal life. Conclusions Compared with classical myeloablative stem cell transplantation,NAST has a rapid hematopoietic recovery and good long-term therapeutic effect in AD.The quality of life in AD patients treated with NAST is higher than those treated with myeloablative hematopoietic stem cell transplantation.

Objective To evaluate the significance of platelet activation and the expression of inter-leukin (IL)-1β in patients with RA. Methods The activation of platelets and the expression of IL-1β in pla-telets in 50 RA patients(22 high-active, 28 mediate/low active ) and 30 normal controls were determined us-ing flow cytometry. Meanwhile, inflammatory indicators such as erythrocyte sedimentation(ESR), C-reactive protein (CRP) and DAS28 were also recorded. T test and correlation analysis were performed. Results The platelet activation in RA group(19.2±4.8) was higher than the control group(9.0±2.9)(t=10.5, P=0.001). The expression of IL-1β in platelets in RA group(41±11) was higher than control group(21±8)(t=9.01, P =0.000) .The platelet activation in high-active RA group(22 ±4) was higher than mediate/low active RA group(17 ±4)(t =3.96,P =0.001). The expression of IL-1β in platelets in high-active RA group(45 ±10) was higher than mediate/low active RA group (38 ±10)(t =2.329,P =0.024). The expression of IL-1β in platelets in RA group was positively correlated with the level of ESR、CRP and DAS28 (r value and P value were 0.576, 0.578, 0.618 and 0.000, 0.000, 0.000 respectively). Conclusion The platelets of patients with RA are activated and may suggest that IL-1β, which may associate with disease activity. Our research suggest that platelet may play a role in the inflammatory process of RA by secreting IL-1β.

BACKGROUND:It is important to establish an ideal mouse model of severe aplastic anemia for investigating the mechanism and finding new therapies for aplastic anemia. OBJECTIVE:To establish a severe aplastic anemia mouse model by using recombinant human interferon-γand busulfan. METHODS:Sixty healthy Kunming female mice were randomly divided into two groups:model group (n=50) and control group (n=10). The model group was given recombinant human interferon-γat a dose of 1×104 U/d by intraperitoneal injection and busulfan at a dose of 18 mg/(kg·d) through stomach feeding for 7 days. The same volume of physiological saline was given to control group. Multi-parameters, including general condition, body weight, blood cellcount, morphology and biopsy of bone marrow were analyzed in two groups. RESULTS AND CONCLUSION:At day 7 after treatment, the weight, white blood cellcount, hemoglobin, blood platelet, reticulocyte count in model group were significantly lower than control group (P<0.05). Bone marrow smears and biopsy of model group showed marked reduction of bone marrow proliferation and increases of percentages of non-hematopoietic cellclusters and adipose tissue. The oil drop and fat vacuole were apparently seen in the model group. Severe aplastic anemia mouse model can be established by using recombinant human interferon-γand busulfan successful y, which is economic, stable and easy to operate.