Child, Preschool ; Chyle ; Female ; Humans ; Infant ; Male ; Mesenteric Cyst ; diagnosis ; surgery

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.

Objective: To analyze the correlation between third molar agenesis and craniofacial morphology by studying the location and number of congenital missing third molars and results of craniofacial cephalometric measurement. Methods: A total of 123 patients were included, including 64 patients in the control group without congenital third molar absence and 59 patients in the absence group with at least one third molar absent. Cephalometric measurements included FMA, IMPA, AR-Go, GoGn-Sn, Co-A, Co-Gn, ANS-Me, Go-Me, SN-MP, Ar-Go-Me, SNA, SNB, ANB, Y-axis angle, Y-axis length, Ar-Go, Go-Me, MP-OP, FH-PP, FH-OP, a total of 18 bone tissue indicators, U1-SN, U1-L1, U1-NA, L1-NB, U1-APo and L1-APo, a total of 6 dental indicators, and UL-EP, LL-EP and nasolabial angle, a total of 3 soft tissue indicators. The correlation between congenital agenesis of third molars and craniofacial morphology was analyzed. Results: The most common missing location of the third molar occured in the upper jaw and the most common number of missing teeth was one. In control group, Ar-Go-Me and SN-MP were larger (P<0.05), U1-SN, U1-NA, L1-NB, UL-EP and LL-EP were larger (P<0.05), and U1-L1 was smaller (P<0.01). There were no significant differences in Ar-Go and Go-Me between the two groups(P>0.05). Conclusion Patients with four third molars are more likely to have backward and downward rotation of the mandible and are more likely to develop into a convex facial type than patients with missing third molars, which has a higher correlation with hyperdivergent growth pattern and convex facial type.

Objective To investigate the expressions of PTEN and MDM2 in bladder transitional cell carcinoma (BTCC) and their clinical significance.Methods The expressions of PTEN and MDM2 were detected by tissue immunohistochemistry test (SP method) in BTCC (n =80) and normal bladder tissues (n =20).The relationship between PTEN and MDM2 as well as their correlations with clinical pathological features were analyzed.Results The positive rate of PTEN in different pathological grading (G1,G2,G3)and clinical staging [superficial (Tis ～ T1),infiltration (T2 ～ T4)] was (86.20％,74.07％,37.50％ ;80.00％,46.67％),respectively,with a significant difference (x2 =15.004,P ＜ 0.01 ; x2 =9.497,P ＜0.01).The positive rate of MDM2 in different pathological grading(G1,G2,G3) and clinical staging [superficial (Tis ～ T1),infiltration (T2 ～ T4)] was (82.75％,55.55％,37.50％ ; 70.00％,43.35％),respectively,with a significant differcnce(x2 =11.543,P ＜ 0.01 ; x2 =5.556,P ＜ 0.05).The expression of PTEN was negatively correlated with that of MDM2 in BTCC (r =-0.611,P ＜ 0.05).Conclusions Expressions of PTEN and MDM2 might be involved in the BTCC pathogenesis.The combined detection of PTEN and MDM2 might be of great value in the prediction of tumor behavior and prognosis.

Animals ; Hepatitis B, Chronic ; immunology ; Humans ; Immune System Phenomena ; Toll-Like Receptors

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

AlM:To evaluate the efficacy of retrobulbar injection of triamcinolone acetonide ( TA ) combined with 577nm laser macular grid photocoagulation for the treatment of cystoid macular edema.
METHODS: Fifty-eight cases ( 66 eyes ) with cystoid macular edema caused by different diseases were recruited in this study. The included patients were treated with both retrobulbar injection of triamcinolone acetonide and 577nm laser macular grid photocoagulation. The best corrected visual acuity, macular thickness, fundus and intraocular pressure were observed in the 1wk, 1 and 3mo after the treatment in all of the included cases.
RESULTS: After treatment, all of the 66 eyes showed cystoid macular edema partially or completely subsided according to optical coherence tomography and fluorescence fundus angiography; 54 eyes ( 82%) visual acuity improved, 12 vision remained the same.
CONCLUSlON: Retrobulbar injection of triamcinolone acetonide combined with 577nm laser macular grid photocoagulation has good curative effect, simple operation procedure and rare complications in the management of cystoid macular edema.

OBJECTIVE: To study the expression of S100B and glial tibrillory acidic protein (GFAP) atter primary brainstem injury in rat and discuss the changes with brainstern injury time and their mechanism in the injury. METHODS: The brainstem injury animal model was established using the mechanical impacting method. The HE staining, Gless argentaffin staining and SP immunohistochemical method were applied to observe the changes of S100B and GFAP at different injury time. The immunostaining results were measured statistically with imaging analysis technology. RESULTS: A large number of S100B positive cells could be seen in 30 min. Afterward, expression increased gradually with time and peaked up in 24 h, and reversed back the normal in 72h. The GFAP positive cells showed rise continually in 30 min, and reached the peak in 48 h, then started to decrease, but still higher than that in control. CONCLUSION The expression of S100B and GFAP is correlated with post traumatic intervals after brainstem injury in rat, and may be useful in estimation post traumatic intervals and nerve regeneration.
Animals ; Brain Injuries/metabolism* ; Brain Stem/metabolism* ; Disease Models, Animal ; Glial Fibrillary Acidic Protein/metabolism* ; Immunohistochemistry ; Nerve Growth Factors ; Neuroglia ; Rats ; S100 Calcium Binding Protein beta Subunit/metabolism* ; S100 Proteins

Objective To investigate clinical effect and safety of ostium secundum atrial septal defect(ASD) treatment via one -stop hybrid and classical surgical procedures.Methods 45 patients were diagnosed ostium secun-dum simple ASD by ultrasound cardiogram and clinical manifestation,they were divided into one -stop hybrid proce-dure group (n =20)and classical surgical procedure (n =25).Age,gender,weight,post operation hospital day,on -pump time,blood transfusion amount,drainage flow,incision length and incidence of complication between the two groups were compared.Results Age and weight had no difference between the two groups(t =0.40 and 1.64,P >0.05),but the proportion of female cases in one -stop hybrid procedure group was higher than post operation(χ2 =9.45,P <0.05).Hospital day was shorter(t =11.11,P <0.05),drainage amount was fewer(t =81.68,P <0.05), incision length(t =22.51,P <0.05)was shorter.Incidence of complication had no statistically significant difference between the two groups(χ2 =0.35,P >0.05).And one -stop hybrid procedure group was off -pump without blood transfusion.Conclusion One -stop hybrid procedure was simple,could make a quick recovery post operation and was an ideal method for ostium secundum ASD treatment.

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.