Endoproteinase Lys-C/trypsin sequential digestion and trypsin digestion were used in 293 T cell proteomics sample preparation and the results of Lys-C/trypsin sequential digestion and trypsin digestion in proteomics sample preparation was systematically evaluated. It was found that the number of identified peptides and proteins increased significantly, and missed cleavage sites, especially K sites decreased dramatically through Lys-C/trypsin sequential digestion. And the average sequence coverage of identified proteins in Lys-C/trypsin sequential digestion sample was higher than that in trypsin digestion sample. Besides, different amount of enzymes was tested to select the optimal usage of enzymes in Lys-C/trypsin sequential digestion. This study provided the references for proteomics sample preparation.

Objective To investigate the correlation between serum homocysteine (Hcy) and macrophage migration inhibitory factor(MIF) with carotid atherosclerosis and between serum Hcy and MIF ,and to study whether serum Hcy influence the carotid atherosclerosis formation by MIF .Methods 258 inpatients and outpatients were performed the color ultrasound examination to ob-serve the carotid arterial vascular anatomic form ,endomembrane circumstance ,plaque and the plaque echo nature ,and the carotid in-tima-media thickness(IMT) was measured .According to the results of color ultrasound ,the cases were divided into three groups :IMT normal group(control group) ,thickening group and plaque group .According to the plaque echo characteristics ,the plaque group was redivided into two subgroups :stable plaque group and unstable plaque group .Serum Hcy and MIF levels and biochemical parameters were measured simultaneously in all cases .The differences of serum Hcy and MIF levels were compared between groups .The correlation coefficients among serum Hcy levels ,MIF levels and IMT were calculated .Results The serum Hcy and MIF levels in the control group ,thickening group and plaque group were increased in turn ,the difference among groups was statisti-cally significant(P<0 .01);which in the unstable plaque group was significantly higher than that in the stable plaque group ,the difference between them was statistically significant (P<0 .01) .The positive correlation was found among serum Hcy levels ,serum MIF levels and IMT (r=0 .584 ,0 .562 ,0 .607 ,P<0 .01) .Conclusion The serum Hcy and MIF levels are closely related with the carotid atherosclerosis degree and the plaque stability ;the serum Hcy and MIF levels are positively correlated with the carotid arte-rial IMT ;serum Hcy is positively correlated with the MIF level ,Hcy may cause the carotid atherosclerosis formation via MIF .

Objective To investigate the trend of early neural development in premature infants. Methods At the age of 12 months and 24 months, Bayley Scales of Infant Development were used to assess the mental development index (MDI) and the psychomotor development index (PDI) in preterm (corrected age) and full-term infants. Results At 12 months, there was no significant difference in corrected age PDI scores among different gestational age groups (<32 , 32–33+6 and 34–36+6 weeks) (P=0.820). The actual age MDI and PDI scores of full-term infants and premature infants in 34~36+6 weeks group were significantly higher than those of premature infants in <32 and 32-33+6 weeks groups, and the PDI score of full-term infants was significantly higher than that of premature infants in 34-36+6 weeks group (P<0.05). There was no significant difference in actual age PDI scores among different birth weight groups (P=0.166). The actual age MDI and PDI of full-term infants and premature infants in birth weight≥2500 g group were significantly higher than those of premature infants in <1500 g, 1500~1999 g and 2000~2499 g groups (P<0.05). At 24 months, the actual age MDI scores of full-term infants were significantly higher than those of premature infants in different gestational age and birth weight groups (P<0.05). The actual age MDI curve of premature infants in birth weight <1500g group showed a downward trend, while the actual age PDI curve showed a significant upward trend. Conclusion The neurodevelopment of preterm infants at the corrected age of 12 and 24 months reaches the level of full-term infants.

Aim To investigate the effects of berberine combined with bone marrow mesenchymal stem cells ( BM-MSCs) on the energy metabolism of human um-bilical vein endothelial cells ( HUVECs ) under the condition of high glucose. Methods ①The state of cell reproduction and cell proliferating activity were de-termined by MTT assay. ②The cell cycle was detected by flow cytometry ( FCM ) . ③DNA damage of cells was measured by comet tail assay. ④The contents of ATP, ADP and AMP were determined by high perform-ance liquid chromatography ( HPLC ) and the level of energy charge ( EC) was calculated. ⑤The expression of CCR and COX mRNA was detected by reverse tran-scription-polymerase chain reaction ( RT-PCR) . ⑥The expressions of COX and CCR were detected by Western blot. Results ①The proliferating activity of HUVECs declined apparently and the proliferation decreased af-ter high glucose intervention. Meanwhile, the quantity of cells during S +G2 dropped dramatically and there was certain degree of damage to DNA. The berberine and BM-MSCs respectively improved the proliferating activity and the proliferation in different degrees, in-creased the quantity of cells during S+G2 and promo-ted the repair of DNA ( P <0 . 01 ) , and so did the combination of the two, with a better effect than each of them alone. ②After high glucose intervention and the damage caused, the content of both ATP and ADP of HUVECs was reduced, and EC level also declined significantly, while the content of AMP increased. The berberine and BM-MSCs respectively up-regulated the content of ATP and ADP ( P<0. 01 ) , and so did the combination of the two, with a better effect than each of them alone. ③After high glucose intervention and the damage caused, the expression of COX, CCR mR-NA and protein decreased obviously. Yet, all of the three gained a dramatic increase when the berberine, BM-MSCs or the combination of the two were added ( P<0. 01 or P <0. 05 ) , among which the combination worked more effectively. Conclusions The berber-ine, BM-MSCs and the joint use of the two could im-prove the energy metabolism of HUVECs, which had been damaged by high glucose, probably because the berberine and BM-MSCs could up-regulate the expres-sion of COX, CCR mRNA and protein, which leads to the hydrolyzation of glucose oxide and thus the im-provement of blood environment and the enhancement of glucose's supply and intake of HUVECs. Then, here comes the final result: the improvement of the energy metabolism of damaged vascular endothelial cells by high glucose.

AIM:To construct a recombinant eukaryotic expression vector pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/? and detect its expression in NIH 3T3 cells.METHODS:CD28-? cDNA was amplified from the plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/IgGFc/CD80 gene.The recombinant plasmids were transfected into NIH 3T3 cells,and resistant clones were obtained by G418 selection.The gene expression of the fusion protein was determined by RT-PCR and FACS.RESULTS:The recombinant eukaryotic vector was constructed successfully,determined by PCR and enzyme digestion analysis.The target gene was amplified from NIH 3T3 cells transfected with the vectors by RT-PCR.The FACS showed that recombinant protein was expressed in NIH 3T3 cells.CONCLUSION:Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/? expression vector and its expression in NIH 3T3 cells lay the foundation for further research of generation of modified T lymphocytes to CD20 positive lymphoma.

OBJECTIVE: To explore the relationship between chromosome anomaly and spontaneous abortion, and to provide useful information for genetic counseling and prenatal diagnosis in reproductive clinic. METHODS: A total of 1 780 patients who had a history of spontaneous abortion before 24 weeks of gestation were enrolled. The lymphocyte culture and harvest were performed according to standard methods. Karyotypes were analyzed by G-banding in all cases and C- banding in some cases in addition. RESULTS: Altogether 57 abnormal karyotypes were found and the overall incidence of chromosomal abnormalities was 3.20% (women 3.32%; men 2.12%). Among them 23 cases were the balanced translocation; 14 cases were the Robertsonian translocation, 3 cases were the complex chromosomal rearrangement, and the other 17 cases were the other abnormalities. In women with 1, 2, 3 or more spontaneous abortion, the incidence of chromosomal abnormalities was 1.7%, 2.3%, and 5.8%, respectively. CONCLUSION Translocations are the major abnormal karyotpes associated with spontaneous abortions. The chance of finding chromosomal aberration increases with the number of abortions. Chromosomal abnormalities are more common in women with 3 or more spontaneous abortions.
Abortion, Spontaneous ; genetics ; pathology ; Adult ; Chromosome Aberrations ; Cytogenetic Analysis ; Female ; Humans ; Karyotyping ; Pregnancy ; Prenatal Diagnosis ; Translocation, Genetic

Objective To establish an isolated rat pancreas perfusion technique,a method for the precise measurement of insulin secretion in vitro.Methods An isolated rat pancreas perfusion technique was applied in the study of insulin secretion from?-cells in 10 high-fat diet-induced obese Wistar rats.Results For the assessment of the functional integrity of the perfused pancreas,the isolated pancreas of 6 rats met all the criteria: (1)The constancy of perfusion pressure was kept over the whole experiment time[(70?5)mm Hg,1 mm Hg= 0.133 kPa].(2)The duodenal peristaltic activity of isolated pancreas and duodenum block was present after perfusion experiment.(3)Total insulin response to arginine stimulation was significantly increased as compared with glucose stimulation[maximum insulin secretion rate:(987?100)?U/min vs(545?50)?U/min,P

OBJECTIVETo observe the effects of ginsenoside Rb1 (GRb1) on the oxidative stress in the skeletal muscles of rats with postoperative fatigue syndrome (POFS) and to study its anti-fatigue mechanisms.

METHODSThe POFS model was established using resection of 70% of mid-small intestine. Ninety-six Sprague-Dawley (SD) rats were screened using grasping test. The rats were randomly divided into the control group, the model group, and the GRb1 treated group (at 10 mg/kg) by the body weight. The maximum grip strength of rats was detected on the 1st, 3rd, 7th, and 10th day after operation, respectively. The contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) were detected.

RESULTSCompared with the model group, the maximum grip strength was obviously enhanced on the postoperative day 7 and 10 (P < 0. 05), the MDA content obviously decreased on the postoperative day 3 and 7 (P < 0.05), the SOD activity obviously increased in the GRb1 treated group (P < 0.05). There was no obvious change in the activities of CAT and GSH-PX among the three groups at each time point (P > 0.05).

CONCLUSIONGRb1 could reduce the oxidative stress injury in the skeletal muscles, improve the activities of antioxidant enzymes, and enhance the functions of the skeletal muscles in POFS rats, which may be important reasons for fighting against POFS.

Animals ; Catalase ; metabolism ; Fatigue ; etiology ; metabolism ; Ginsenosides ; pharmacology ; Glutathione Peroxidase ; metabolism ; Male ; Malondialdehyde ; analysis ; Muscle, Skeletal ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Postoperative Period ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the factors affecting the management and treatment of immigrant workers with tuberculosis, to determine the most effective measures and to provide an academic basis for tuberculosis control and prevention strategies targeting the immigrant population.

METHODSA self-designed questionnaire was administered to 1364 immigrant workers with tuberculosis (study group) and 436 local residents (control group) with tuberculosis.

RESULTSWhether a patient's tuberculosis status was discovered or not was related to the medical facilities initially visited, job mobility, and the individual worker's economic condition. The percentage of cases discovered was relatively low among those who made their first visit to a private clinic (39.0%), and 58.4% (796) of immigrant workers delayed their first consultation mainly due to neglecting symptoms (55.9%, 445), subjective perception of symptoms being not severe enough (19.3%, 154) to visit a physician and the inconvenience of visiting a hospital because of its being far away from home (15.8%, 47). As comparing immigrant workers with local residents, the differences of the delay reason between the two groups were statistically significant (chi(2) = 21.49, P < 0.01). And 39.9% (544) of immigrant workers and 49.1% (214) of local residents had had late confirmation. The differences of the delay reasons between the two groups were insignificant (chi(2) = 7.31, P = 0.293). And 17.6% (240) patients of immigrant workers and 13.1% (57) patients of local residents did not keep to their drug regimens in a timely fashion. The differences of the reasons between the two groups were insignificant (chi(2) = 6.66, P > 0.05). And 77.5% (1057) of immigrant workers and 31.8% (138) of local residents were considered that taking medicine in supervision spot might have impact on their lives and work. The differences of the reasons between the two groups were significant (chi(2) = 9.71, P < 0.05). All 79.2% (1080) of immigrant workers and 63.3% (276) of local residents did not obtain medicine according to prescriptions. The differences of reasons between the two groups were statistically significant (chi(2) = 24.84, P < 0.01). And 51.2% (699) of immigrant workers and 46.1% (201) of the registered population did not follow up with doctors' directions for lab tests. The differences of the reasons between the two groups were insignificant (chi(2) = 3.26, P > 0.05).

CONCLUSIONThe influential factors in tuberculosis management and treatment were complex. Prevention strategies should focus on health education and promotion activities to improve awareness in seeking medical services. Also, developing and standardizing reference mechanisms for patients, and alleviating the economic burden of the workers will be critical to reduce the tuberculosis incidence.

China ; epidemiology ; Factor Analysis, Statistical ; Humans ; Transients and Migrants ; Treatment Outcome ; Tuberculosis, Pulmonary ; epidemiology ; prevention & control ; therapy ; Urban Population

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.
Antibodies, Neoplasm ; genetics ; immunology ; isolation & purification ; Bacteriophages ; genetics ; Brain Neoplasms ; immunology ; pathology ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Glioma ; immunology ; pathology ; Humans ; Peptide Library