OBJECTIVE: To discuss the influences of the concentrations of sodium cholate, cholesterol and drug, preparation temperature, ultrasonic time and power on the particle size and encapsulation efficiency of Flos Magnoliae volatile oil nano-liposome through film-ultrasonic wave technique together with membrane filter method. METHODS: The nano-liposomes were prepared by film-ultrasonic wave technique together with membrane filter method. The drug content in liposomes was determined by gas chromatography and the particle size was determined by laser light scattering granularity equipment. RESULTS: By film-ultrasonic wave technique together with membrane filter method, the liposomes in the range of 30-40 nm were prepared. The prepared nano-liposomes demonstrated good decentralization and homogeneity, and the encapsulation efficiency was (91.7+/-3)%. CONCLUSION: Film-ultrasonic wave together with membrane filter method is a simple method to fabricate nano-magnoliae liposome.

Objective: To probe into the relationship between integrin β1 expression in human cervical carcinoma HCE1 multicellular spheroids (HCE1/MCS) and their invasion into human umbilical vein endothelium cells (HUVEC), so as to assess the inhibitory effect of anti-integrin β1 monoclonal antibody P5D2 on the invasion of HCE1/MCS into HUVEC. Methods: A model of HUVEC monolayer invaded by HCE1/MCS was established (in brief, the HCE1 invading model). Morphology changes of the HCE1 invading model were observed under inverted microscope and analyzed by Motic Med System after P5D2 treatment. The expressions of β1 integrin on HCE1 monolayer cells, HCE1/MCS, HVUEC monolayer cells, and P5D2-treated HCE1 invading models were measured by immunocytochemistry SABC assay. Results: A HCE1 invading model was successfully established. The HCE1/MCS proliferated rapidly after culture, and on the 7th day the invading area of HCE1/MCS was 40.42 folds larger than the original HCE1/MCS. Invading areas of P5D2-treated HCE1/MCS were significantly smaller than that of the control group after 1, 4, 7 day (P<0.05, or P<0.01), with the invading area after 7 days reduced by (84.68±0.08) % compared with the control group. Integrin β1 expression in HCE1/MCS was significantly higher than that in HCE1 monolayer cells (P<0.01), and the expression was negative in HUVEC. Integrin β1 expression in P5D2-treated HCE1/MCS was significantly lower than that the in untreated HCE1/MCS (P<0.01). Conclusion: Upregulated expression of integrin β1 in HCE1/MCS and HCE1 invading model may be associated with their enhanced adhesion and invasion abilities. Anti-integrin β1 monoclonal antibody P5D2 can partially block the invasion of HCE1/MCS into HUVEC.

Objective To evaluate the expression of Integrin α2β1 in serum of osteosarcoma patients and to investigate the effect of Integrin α2β1 on metastasis of osteosarcoma. Methods Sandwich enzymelinked immunosorbent assay was applied to test Integrin α2β1 in serum of 26 healthy controls (male 16, female 10; age 18-32, average age 23.54±3.82) and 43 osteosarcoma patients (male 28, female 15;age 8-47,average age 18.42±9.10)before and after the surgery and the chemotherapy. The patients included osteoprogenitor cells type in 24 cases, fibroblast type in 9 cases, chondroblast type in 7 cases, and other types in 3cases; with Enneking stage ⅡA in 13 cases, stage ⅡB in 26 cases, stage Ⅲ in 4 cases. Among them, 22 patients receive the neo-adjuvant chemotherapy. After that, we compared the data of different groups. At the same time, in accordance with tumor size, position, clinical stage, histology grade and metastasis situation to categorize groups compared among the groups of serum Integrin α2β1 differences in order to explore its relationship with patients' prognosis and metastasis. Results The Integrin α2β1 of osteosarcoma patients in blood before the surgery was expressed highly. It was significantly higher than the control group. The expression before surgery had significant correlation with osteosarcoma Enneking clinical stages, tumor size and metastasis. And it was independent of tumor position and Dahlin histology grade. The expression of Integrin α2β1 in blood of the osteosarcoma patients after limb salvage surgery decreased significantly. And the expression of Integrin α2β1 in blood of patients receiving new adjuvant chemotherapy was significant differences before and after the chemotherapy. Conclusion The expression of Integrin α2β1 has positive correlation with the occurrence, development and metastasis of the osteosarcoma. It is probably a new biological indicator for diagnosis and estimating prognosis of osteosarcoma. Simultaneously surgery and new adjuvant chemotherapy are still effective methods to treat osteosarcoma.

BACKGROUND: Arterial injury surrounding the mandibular angle is very common during mandibular angle ostectomy. The retromandibular vein is susceptible to injury owing to thin vascular wall and deep location. Anatomical studies regarding the retromandibular vein are rarely reported.OBJECTIVE: To observe the construction, branches, backstreaming of the retromandibular vein and its relation to the facial nerve and mandibular angle.METHODS: Twenty formalin-fixed adult cadavers (40 samples) were randomly selected. Bilateral retromandibular veins and their branches were cleaned. The construction, branches, backstreaming of the retromandibular vein and its relation to the facial nerve and mandibular angle were anatomically observed.RESULTS AND CONCLUSION: The retromandibular vein originated constantly and provided an average length of (4.51±2.01) cm, with an average outer diameter of (0.52±0.28) cm at the origination part. A total of 34 samples showed that the retromandibular vein was formed by the union of the superficial temporal and maxillary veins, and six samples showed that the retromandibular vein originated from the superficial temporal vein. The retromandibular vein divided into two types according to the backstreaming and course: one type consisted of the anterior and posterior branches: the anterior branch mostly drained into the internal jugular vein and minorly drained into the external jugular vein; the posterior branch was joined by the posterior auricular vein and became the external jugular vein. Another type of retromandibular vein without the anterior and posterior branches directly connected with the posterior auricular vein and entered the external jugular vein. The retromandibular vein had the following branches (appearance frequency): facial transverse vein (88%), mandibular articular vein (98%), parotid veins (95%),anterior auricular veins (58%), styloid process vein (43%). 88% of the retromandibular vein was located in the superior and inferior trunks of the facial nerve and in the medial branches. The retromandibular vein connected with facial nerve trunk or major branches in a decussated fashion. 75% of the arc-shaped structures formed by the retromandibular vein and its branches coursed close to the inferior and posterior border of the mandibular angle, and 25% of the arc-shaped structures were not close to the lowest point of the mandibular angle. The thin periosteum between the venous wall and cortical bone was just the position of ostectomy line of mandibular angle. The retromandibular vein was easily injured no matter in vein dissection or ostectomy.

Objective To analyze the clinical characters,therapeutic effect of gastrointestinal stromal tumors (GIST),the effects of surgical approach and imatinib adjuvant therapy on it.Methods From January 2004 to April 2010,the clinical data of 214 patients with primary GIST were retrospectively analyzed.The effects of surgical approach and imatinib on the survival of the GIST patients were compared.Count data were analyzed by chi-square test,and the survival rates were analyzed with the life table method and Kaplan-Meier curve.Results A total of 214 GIST patients'survival rates of 1-year,3-year,5-year were 93.0％,87.0％ and 80.0％.According to the risk classification of National Institutes of Health,the differences of overall survival rates after surgery were statistically significant (x2 =22.058,P＜0.05).The differences of survival rate among different nuclear division number were statistically significant (x2=26.599,P＜0.05).The survival rate of pathological nuclear division number over 10/50 high power field (HPF) was the lowest.The overall survival rate of patients with gastrointestinal GIST was higher than patients with extragastrointestinal GIST,and the difference was statistically significant (x2 =68.139,P ＜ 0.05).After complete resection of the tumor,the survival rate of patients with local recurrence was higher than those with widely recurrence,and the difference was statistically significant (x2 =4.409,P＜ 0.05).After complete resection of the tumor,the survival rate of patients taking imatinib was higher than that of patients not taking imatinib.Conclusions The surgical treatment was the main therapy for GIST.Complete resection of the tumor and taking imatinib after surgery could improve the prognosis and the survival rate of the GIST patients.

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 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.

Objective To investigate the clinical and laboratory characteristics of hematological malignancies with t(1 1;19)(q23;p13.1).Methods Chromosome specimens of bone marrow cell from a patient with hematological malignancy were collected.After short-term culture,and chromosome karyotype analysis was carried out by R banding technique.Results The chromosome karyotype of the patient diagnosed with AML M4+ was t (11;19)(q23;p13.1).The patient did not obtain complete remission after application of MA regimen chemotherapy.Conclusions t(11;19)(q23;p13.1) translocation is a rare and recurring chromosome abnormality,which is related with a specific type of AML.The prognosis of the AML patients with this chromosome abnormality is poor.

Objective To explore serum Cystain C (Cys C) level in chronic heart failure (CHF) patients and its relation?ship with cardiac function and ventricular remodeling. Methods Patients with heart failure (n=75) and healthy adult (n=35) were enrolled in this study. According to the NYHA classification,CHF patients is divided into NYHA Ⅱ, NYHAⅢand NYHAⅣ;CHF patients can also be divided into 2 groups base on their level of serum Cys C:Cys C>0.95 mg/L is defined as elevated serum Cys C group, serum Cys C≤0.95 mg/L is defined as normal serum Cys C group;According to left ventricu?lar ejection fraction (LVEF), CHF patients can also be divided into 2 groups:LVEF≥0.50 is defined as ejection fraction re?served group, LVEF<0.50 is defined as the ejection fraction reduced group. Cys C, N terminal B-type natriuretic peptide (NT-proBNP), serum creatinine(Scr),UREA and Echocardiography were analyzed in all groups and their correlation was studied. Results Serum Cys C level in CHF patients was obviously higher than that in healthy controls, and it increased with NYHA heart function classification;compared with normal Cys C group,Cys C elevated group presented higher NT-proBNP level but lower eGFR and LVEF (P<0.05);compared with ejection fraction reduced group, ejection fraction reserved group demonstrated lower NT-proBNP, LVEDD, LVMI and Cys C levels (all P<0.05);Correlation analysis revealed that Cys C is correlated with age, NT-proBNP, LVEF, LVEDD, Scr and UREA (r=0.411, 0.658, 0.465, 0.310, 0.552, 0.486, P<0.01). Conclusion Serum Cys C can be used to evaluate cardiac function of heart failure, and is associated with ventricular remodeling.

Objective To observe the NTBC dependence of Fah-knockout mice and study the biological characteristics in order to use the model more effectively.Methods Examine the progressive changes in body weight, survival time, liver pathology and serological markers after the NTBC withdrawal.Results After removing of NTBC, Fah-knockout mice lost their body weight gradually, and finally died in 5 to 7 weeks, along with increased serum ALT, AST levels and deformation of the hepatocytes.Conclusions Fah-knockout mice have a strong drug dependence of NTBC and could be the ideal model to hereditary tyrosinemia type I and other liver injury.