Based of the study on the impact of protoplasts under different conditions of enzymolysis solution, culture time, enzymolysis time and enzymolysis temperature in sequence using orthogonal test. The best optimal condition of producing protoplast was :0.5% helicase add 0.5% lysozyme and 1.0% cellulase, enzymolysis time was 3 hours , enzymolysis temperature was 32℃,under these condition the production of protoplast can be 4.25?10~(6 )per mL. Ca~(2+) and PEG can increase the regeneration ratio of protoplast.After induced mutation by complex factor under DES,UV and LiCl,the mutagenesis strain which own increasing enzyme activity and transmissibility steaty was obtained.

Objective: To find the reason of risk on medical equipment. Methods: Based on RCA, to adopt the event classification to evaluate the X-ray equipment, high-frequency electro tome, electro-cardiac monitor, infusion pump, ventilator, defibrillators and infant incubator, discuss the root cause of medical risk. Results:To deduce the medical equipment risk by PM and quality control. Conclusion:With the analysis of quality control, the application of RCA is gradually explained in management of medical equipment.

In our university,the training for geriatric postgraduates of clinical medicine is now in the initial stage.Therefore,it is important to explore the training model to improve the quality of postgraduate.The program focused on cultivating geriatric postgraduates' ability of diagnosis and treatment,helping them have the concept of integral service and enhancing their teaching and thinking ability of clinical sciences to provide high-level comprehensive geriatric doctors for the society.

Fusion PCR, which employs chimeric primers to generate PCR products with complementary ends in its amplifications, is a rapid and flexible method in joining different DNA fragments. This method can assemble DNA fragments without the treatment of restriction endonucleases and T4 DNA ligase. It offered a shortcut for the construction of homologous recombinant fragments. Through assembling three recombinant fragments successfully, fusion PCR procedure was improved and manipulation essentials of fusion PCR were described in detail. The results indicated that the improved procedure can assemble three or four fragments simultaneously, and the length of each fusion product is above 4.5 kb. The result recombinants were proposed to be use in further experiments, which structures had been confirmed by sequencing.

Based on a ?-tubulin gene EST sequence from Trichoderma harzianum mycelium cDNA library,a 1.74kb ?-tubulin gene coding region,together with a 1.5kb 5'-flanking region and a 1.0kb 3'-flanking region was cloned from T.harzianum by IPCR methods.The gene encodes a 446 amino acid polypeptide,which shows a high degree of homology with other fungal ?-tubulins.A three-dimensional model of the T.harzianum ?-tubulin was built based on the crystal structure of bovine tubulin using homology modeling method.Tertiary protein structure of the ?-tubulin should provide a basis for understanding a significant body of research on tubulin's properties and interactions with antimicrotubule agents.

Objective To explore effect of miR‐302a on proliferation and apoptosis in folate deficiency mouse embryonic stem cell (mESC) .Methods The cases were divided into complete culture medium group (control group) ,folate‐deficient culture medi‐um group (folate‐deficient group) ,folate‐deficient culture medium plus miR‐302a mimic group (miR‐302a group) .The expression of miR‐302a was examed by RT‐PCR in control group and folate‐deficient group .To construct miR‐302a mimic and then was trans‐fected into the folate‐deficient culture medium mESC .Effect of miR‐302a mimic on mESC viability was detected by MTT assay ,the effect of miR‐302a mimic on mESC apoptosis was examed by Annexin V‐FITC/PI flow dual‐staining method ,the effect of miR‐302a mimic on mESC cycle was examed by flow cytometry .The activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) and expression of CyclinD1 ,p21 and p27 was assayed by Western blot . Results The expression of miR‐302a was lower in folate‐deficient group(P<0 .01) .Compared with control group ,the viability of mESC was lower ,the apoptosis of mESC was higher ,the cell cycle was arrested in G1 phase ,the level of phosphorylation of AKT and mTOR was lower ,the expression of CyclinD1 was lower ,the expression of p21 and p27 was higher in folate‐deficient group , with statistical significance (P<0 .01) .Compared with folate‐deficient group ,the viability of mESC was higher ,the apoptosis of mESC was lower ,G1 phase was shortened ,the level of phosphorylation of AKT and mTOR was higher ,the expression of CyclinD1 was higher ,the expression of p21 and p27 was lower in miR‐302a group with statistical significance (P<0 .01) .Conclusion These results suggested miR‐302a exerted anti‐apoptosis and promote cell proliferation in folate‐deficient culture medium ,which might be related to PI3K/AKT/mTOR signal pathway .

BACKGROUND:Studies have found that bone marrow mesenchymal stem cel s, under certain conditions, can be induced to differentiate into neurons and glial cel s, which to some extent solves the problem of the source of seed cel s. Induction methods currently used are different, and their efficiencies are not the same.
OBJECTIVE:To observe the effects of different antioxidants on differentiation of rat bone marrow mesenchymal stem cel s into neuron-like cel s in vitro.
METHODS:Bone marrow mesenchymal stem cel s from Wistar rats were divided into four groups:non-intervention group,β-mercaptoethanol group, retinoic acid group,β-mercaptoethanol+retinoic acid group. Changes in cel morphology and positive rate of neuron-specific enolase and microtubule-associated protein 2 were observed and detected at 5 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, and 10 days after induction.
RESULTS AND CONCLUSION:Except non-intervention group, bone marrow mesenchymal stem cel s in the other three groups were gradual y becoming spindle-shaped, and gave birth to many smal protrusions that were interconnected into a network, showing neuron-like cel morphology. Immunocytochemical staining showed that the efficiency of theβ-mercaptoethanol+retinoic acid group was the highest at 10 days after induction, and the positive rates of neuron-specific enolase and microtubule-associated protein 2 were 71.63%and 79.72%, respectively. The results show thatβ-mercaptoethanol can be combined with retinoic acid to accelerate the differentiation of bone marrow mesenchymal stem cel s into neuron-like cel s.

Objective To assess the effects of hydrocortisone (HC) on brain injury in septic shock rats induced by lipopolysaccharide (LPS).Methods Pathogen-free Wistar male rats were chosen to produce septic shock model by injection of LPS (Escherichia coil,O55:B5),25 mg/kg LPS was given by femoral vein.All rats in accordance with the random number table,were randomly divided into 3 groups:normal control group,LPS group,and low dose of HC group (HC 5 mg/kg).Every group had ten rats.Six hours after the septic shock model was established,the blood sample and brain tissue were collected to perform histopathological examination and measure the serum level of neuron specific enolase (NSE).Results The serum level of NSE,brain water content,inflammation score,neural damage score,vascular reaction score in LPS group were significantly higher than those in control group [(10.078 ± 0.526) μg/L vs.(3.719 ± 0.602) μg/L,(82.328 ±0.011) ％ vs.(78.896 ±0.005) ％,(2.750 ±0.463) scores vs.(0.125 ±0.345) scores,(2.875 ± 0.353) scores vs.(1.125 ± 0.354) scores,(2.875 ± 0.463) scores vs.(0.500 ± 0.534) scores,P ＜ 0.01,respectively].After HC treatment,the serum level of NSE,brain water content,and brain tissue pathological score [(6.297 ± 1.393) μg/L,(80.283 ± 0.005) ％,(0.625 ± 0.744) scores,(1.375 ± 0.744) scores,(1.250 ± 0.463) scores] were significantly lower than those in LPS group (P ＜ 0.05,respectively).Conclusion After giving low dose of HC,the serum level of NSE,brain edema,brain pathological changes all got improvement,which indicated that low dose HC could have protect effect on septic encephalopathy induced by LPS.

BACKGROUND:It is confirmed that the onset of osteoarthritis is closely related to inflammatory cytokines. OBJECTIVE: To investigate the levels of interleukin-6 and matrix metaloproteinase-3 in the synovial fluid of patients with osteoarthritis and their correlation with the severity of osteoarthritis. METHODS: Synovial fluid specimens of 78 patients were harvested to detect the levels of interleukin-6 and matrix metaloproteinase-3 using ELISA assay. The severity of osteoarthritis was estimated by the Hospital for Special Surgery (HSS) score. Pearson correlation analysis was adopted to explore the pairwise correlations between interleukin-6, matrix metaloproteinase-3 and HSS score. RESULTS AND CONCLUSION:The levels of interleukin-6 and matrix metaloproteinase-3 in the synovial fluid were (13.1±5.7) and (989.3±429.5) μg/L, respectively; the HSS score was (56.4±12.0) points. Pearson correlation analysis showed that the expression levels of interleukin-6 and matrix metaloproteinase-3 had a significant positive correlation (r=0.70,P< 0.001); the levels of interleukin-6 and matrix metaloproteinase-3 had a negative correlation with the HSS scores (r=-0.70,r=-0.75,P < 0.001). Experimental findings show that there is a positive correlation between interleukin-6 and matrix metaloproteinase-3 in the synovial fluids, and these two cytokines also have a positive correlation with the severity of osteoarthritis, indicating that to measure interleukin-6 and matrix metaloproteinase-3 levels in the synovial fluids of osteoarthritis patients is important for early diagnosis and treatment of osteoarthritis.

Objective To investigate the clinical effect of insulin on gestational diabetes mellitus(GDM).Methods GDM patients were randomly divided into group A and group B,group B were treated with short term intensive insulin pump therapy,and group A were treated with insulin pump in short term combined with biosynthetic human insulin therapy.Results The body mass index and diabetes family history rates pre-pregnancy in group B were significantly higher than group A(P<0.05).Logistic analysis showed that family history of BMI pre-pregnancy and diabetes family history were independent risk factors for GDM drug treatment(P<0.05).Compared with the group B,the improvement of blood glucose levels and the incidence of complications in patients with group A were better than the former(P<0.05).Conclusion Rational application of insulin can improve the efficacy of clinical treatment of GDM, reduce the incidence of complications, with high safety.