Enzyme immobilization is the core technology of biocatalysis. Over the past few decades, enzyme immobilization research mainly focused on single enzyme immobilization. In recent years, multi-enzyme immobilization attracts more and more attention as it could increase the local concentration of reaction and improve the reaction yield. In this review, a summary of the recent progress, together with our research, is presented. Special emphasis is placed on four methods in multi-enzymes co-immobilization, namely, the nonspecific covalent co-immobilization, the nonspecific non-covalent co-immobilization, the non-covalent encapsulation co-immobilized and the site specificity co-immobilized. Finally, some industrial uses of immobilized multi-enzymes were addressed and the application prospect of multi-enzyme immobilization was highlighted.
Biocatalysis ; Enzymes, Immobilized

Objective:To probe into the expression and the clinic significance of LEA on colorectal carcinoma. Methods:The expression of LEA and CEA in 140 colorectal cancer specimens and 100 colorectal non-cancerous specimens had been detectd by immunohistcchemistry S-P method.Results:The expression of LEA was relative to tumor differentiation degree and exhibits higher selectivity in high differentiation ade-nocarcinoma ( P 0.05) . The positive rate of LEA in adenoma was much higher than surrounding non-cancerous mucosa and normal mucosa. In normal mucosa the positive rate of LEA was obviously lower than that of CEA ( P

Objective　To observe the ER ultrastructures in humn embryonic epithelial cells of colonic mucosa、renal tubule and hepatocytes and also their alterations in embryogenesis.Methods　Transmission electromicroscopy technique.Results　With the embryonic development, the ER increased in amount, became complex in structure and with its characteristic ER structures in different cells. Conclusion　The changes of ER structures are one of the characters during cell differentiation.

There are more prognostic factors affecting the patients with cerebral hemorrhage, including bleeding volume, bleeding site, cause of bleeding, and blood pressure regulation during acute phase. Among them, the regulation of blood pressure during acute phase is a man-controlled important factor. However, the ideal blood pressure level has not yet to he determined at present. Therefore, more clinical trials are needed to determine the exact time window of blood pressure management in patients with intracerebral hemorrhage during acute phase and the range of blood pressure control.

OBJECTIVE:To observe the clinical efficacy and safety of triple therapy of aspirin,clopidogrel and urinary kallidi-nogenase in the treatment of recurrent transient ischemic attack. METHODS:180 patients with recurrent transient ischemic attack were randomly divided into control group and observation group. Control group was orally treated with Aspirin enteric-coated tab-lets 100 mg,once a day + Clopidogrel hydrogen sulfate tablets 75 mg,once a day. Based on the treatment of control group,obser-vation group was additionally treated with Urinary kallidinogenase for injection 0.15 PNA unit adding into Sodium chloride injec-tion 100 ml by intravenous injection,once a day. The treatment course for both groups was 2 weeks. The clinic data was observed, including clinical efficacy,and LDL,HDL TC and TG levels before and after treatment,recurrence rate of cerebral ischemia,inci-dence of cerebral infarction and adverse reactions after 6 months of follow-up. RESULTS:The total effective rate in observation group was significantly higher than control group,and the recurrence rate of cerebral ischemia and incidence of cerebral infarction were significantly lower than control group(P<0.05). After treatment,HDL level in 2 groups were significantly higher than be-fore,and observation group was higher than control group;levels of LDL,TC and TG were significantly lower than before,and observation group was lower than control group(P<0.05). There were no severe adverse reactions in groups during treatment. CON-CLUSIONS:Triple therapy of aspirin,clopidogrel and urinary kallidinogenase has significant efficacy in the treatment of transient ischemic attack,with good safety.

AIM:To develop a new method to determine cucurbitacin B in Pedicellus Melo by capillary zone(electrophoresis(CZE).) METHODS:All the samples were analysed on a fused silica capillary(75 cm?75 ?m I.D.) by using 50 mmol/L sodium borate solution(containing 5% acetonitrile) as the background electrolyte with the running voltage at 12.0 kV and detection wavelength of 265 nm.And clorprenaline hydrochloride was applied as the internal standard. RESULTS:The results showed a good linear correlation between relative peak area of cucurbitacin B(y) and its concentration(x) over the range of 0.15～1.2 mg/mL.Regression equation was y=(0.003 4x)+0.058,r=0.999 7,and the average recovery for baicalin was 100.2% with the RSD at(2.11%). CONCLUSION:The method is rapid,simple,reproducible and produces low pollution.It serves as a novel way to determine cucurbitacin B.

The structure of social network and its information spreading characteristics in 41 users of Sina microb-logs were studied , which showed that the exchange level was quite low in the 41 users of health information in Sina microblogs, usually in small groups.Although the 41 users were found in the core area, their exchange was sporadic even with no exchange in the core nodes or in other nodes, thus effective information sharing could not be achieved and information could not be widely spread .

Objective　To prevent the after cataract induced by lens epithelial cells proliferation from postoperative cataract, monoclonal antibody (McAb) specific for rabbit lens epithelial cell is made, it will be the carrier further to be conjugated with cytotoxin. The conjugations will inhibit lens epithelial cells growth and not damage the other tissues of eye. Thereby McAb is the experimental bases of preventing after cataract.Methods　BALB/c mice were immunized by mixture of rabbit lens epithelial cells and Freund's adjuant. The immunized mouse spleen cells were fused with parental mouse myeloma cells (BALB/c　SP2/0) using polyethylene glycol (PEG-4000). The fused cells were selectively cultured by hypoxanthine, aminopterin and thymidine (HAT) culture medium. The specificities of the supernatant from hybridomas were tested by indirect immunofluorescence and immunohistochemistry SP (Streptavidin peroxdase conjugated method). The positive hybridomas were further cloned three times by methylcellulose culture medium to ensure monoclonality. At last, the consensual reaction of McAb was tested on human eye tissues.Results　Hybridomas were produced by fusion of spleen cells of immunized mice and mouse myoloma cells (SP2/0) with PEG-4000, and grown selectively in medium containing HAT after 16 days. Antibodies of the supernatant from hybridomas were tested on frozen sections of rabbit lens epithelial cell by indirect immunofluorescence. Only a positive clone secreted McAb against antigen of rabbit lens epithelial cell. The specificity of McAb was tested on paraffin sections of whole rabbit eye and whole human eye by immunohistochemistry SP. The results indicated that McAb was only positive to rabbit lens epithelial cell membrane and it was negative to the other tissues of rabbit or whole human eye tissues.Conclusions　McAb specific for rabbit lens epithelial cell was manufactured successfully. The specificity of McAb was strong. There was no consensual reaction on human eye tissues. It might be the experimental bases of further targeting chemotherapy on after cataract.

BACKGROUND: The islet cell transplantation has provided a solid basis for diabetic therapy, but the insufficient donor limits its development.OBJECTIVE: improving the method of isolating and purifying islets to observe the transplantation effect.DESIGN: A laboratory animal research.SETTING: Key Laboratory of Animal and Department of Cell Biology, China Medical University.MATERIALS: The experiment was carried out in the Key Laboratory of Animal and Department of Cell Biology in China Medical University between January and October in 2006. Donors were Wistar rats of either gender, weight 250-300 g;Acceptors were SD male rats, weight 180-220g. The two kinds of rats were all common closed population and from the Experimental Animal Department of China Medical University (The Admission Number of Experimental Animal Institute is SYXK(LIAO)2003-0013).METHODS: ①Isolation and exaltation of islet cells as well as the functional evaluation of pancreas: After etherisation, the Wistar rat without fasting was executed. A little cut was made on the beginning of the biliary pore, then the little cut lumbar anesthesia ductus, which were connected with a 1-mm-diameter syringe and full of cold collagenase solution (1.5 g/L), was inserted directly to dilate pancreas thoroughly. The pancreatic gland was isolated and digested in the water of centrifuge, when doing that, 1 mol/L NaOH was put interruptedly into the centrifuge tube to keep the pH value of the solution at 7.8±1.0. The rat pancreas purified by centrifugation of Ficoll density gradient: The identification of purified islets was evaluated by dithizone staining. The viability of islet was assessed by fluorescence staining of aridine orange and propidium iodide. The motility rate=the total number of live cells/(the total number of live cells + the total number of dead cells)×100%. Pancreatic activity was calculated: insulin release index=the level of insulin at the third hour (high concentration glucose)/the level of insulin at the second hour (low concentration glucose). ②The blood from vena caudalis of SD rat was sampled and measured the blood sugar after the intraperitoneal injection of streptozocin. The rat was diagnosed as DM when blood sugar was more than 16.7 mmol/L twice without fasting. The DM rats were divided into two groups, every group 8 rats. The experimental group rats were injected about 1 000 islet cells into the location below renal capsule, and the control group rats were injected the same volume of 1640 cultu re solution. Eight normal rats, whose glucose concentration ≤ 5.5 mmol/L, were taken randomly as normal controlled group. The blood sugar was measured every day after the surgery. The blood sugar less than 11.1 mmol/L without fasting was taken as the sign of successful islet transplantation. Intravenous sugar tolerance test was applied to the rats of normal control group, DM control group and experimental group 3 days after islet transplantation. Fasting for 12 hours before test, the blood sugar was measured at 0, 15, 30, 60, 90 and 120 minutes.MAIN OUTCOME MEASURES: Purity quotient, survival rate and activity of islet cells.RESULTS: All 24 SD acceptor rats were involved in the result analysis without miss.①The total number of purified islets of one pancreas was (1 150±141) in well morphology. The purity of islets was more than 95%. The viability of islets was more than 98%. ②The insulin secretion response to glucose challenge in vitro showed the mean value of insulin in the low-glucose medium was (70.5±6.9) mlU/L, while that of high-glucose medium was (321.4±11.6) mlU/L, the insulin release index was 4.6±0.52, that meant the beta cell of islet functioned well. ③The blood glucose level and the insulin level in plasma of the transplanted recipients restored to normal 3 days after transplantation. The survival period of transplanted islets was (6±2) days. But there was not any change in the concentration of blood sugar in the control group (16.7 mmol/L). The intravenous glucose tolerance test showed the identical outcome between the islet splantation group and the normal control group.CONCLUSION: There are high yield and high purify of islet cells in rats, which are isolated by in situ perfusion and purified by Ficoll density gradient centrifugation.

Objective: To study effect of small ubiquitin related modifier protein 1 (SUMO1) in inflammatory reactions mediated by tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB in myocardial damage of rats with type 2 diabetes mellitus (T2DM). Methods: A total of 20 Goto-Kakizaki (GK) rats with spontaneous diabetes mellitus (DM) were randomly divided into group DM1 (pure DM group, n=10) and group DM2 (DM+high-fat diet group, n=10), and another 10 normal Wistar rats were regard as healthy control group. Expressions of SUMO1, TNF-α and NF-κB were measured by immunohistochemical method. Results: 1. Levels of blood glucose and TG in group DM1 and group DM2 were significantly higher than those of healthy control group, and those of DM2 group were higher than of DM1 group ,P<0.05 all; 2. Myocardial cells lined up in order and there was no hypertrophy in group DM1; but those in group DM2 showed cells loosely lined up and hypertrophy under light microscope; 3 Immunohistochemical assay indicated that expression of SUMO1 in group DM2 and DM1 group were significantly higher than those of healthy control group [(44.5±1.1) vs. (27.2±2.2) vs. (21.7±3.0)], and of group DM2 was significantly higher than that of DM1 group (P<0.01 all); expression of TNF-α in group DM2 and group DM1 were significantly higher than that of healthy control group [(27.5±1.5) vs. (20.2±2.7) vs. (13.1±1.6)], and of DM2 group was significantly higher than that of group DM1 (P<0.01 all)；expression of NF-κB in group DM2 and group DM1 were significantly higher than that of healthy control group [（30.1±1.7）vs.40.7±1.5）vs.（16.0±2.6）], but of group DM1 was significantly higher than that of group DM2 (P<0.01 all). Conclusion: There are obvious metabolic disorders of glucose and lipid in T2DM rats, and complicated morphological changes of myocardial tissues similar to myocardial lesions in DM humans; the expressions of SUMO1, NF-κB and TNF-α significantly increase, suggest SUMO1 takes part in inflammatory reaction mediated by NF-κB, TNF-α in myocardial lesion of rat with T2DM，and may inhibit NF-κB, possesses effect of protect myocardium.