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Objective To investigate the association between the osteogenic differentiation in the soft tissue lump and the clinicopathological characteristics of osteosarcoma patients. Methods We retrospectively reviewed the clinical data of conventional osteosarcoma patients with soft tissue lumps, including Enneking stages, chemotherapy sensitivity, overall survival and post-metastatic survival time. The ossification level in soft tissue lumps was assessed by imaging and the proportion of osteoid matrix was assessed by pathological examination. Results A total of 189 cases were included in this study. In patients with Enneking IIIB, non-osteoblastic, partially osteoblastic and osteoblastic types accounted for 30.2%, 9.6% and 6.3%, respectively. Non-osteoblastic osteosarcoma patients had a higher rate of initial metastasis (P < 0.05); Chemotherapy efficiency of non-osteoblastic, partially osteoblastic and osteoblastic types were 60.5%, 59.6% and 31.3%, respectively. The osteoblastic osteosarcoma held the worst rate of chemotherapy sensitivity (P < 0.05). The overall survival of non-osteoblastic osteosarcoma was shorter than those of partially osteoblastic and osteoblastic types (P < 0.05). Post-metastatic survival time of osteoblastic osteosarcoma was longer than that of non-osteoblastic osteosarcoma (P=0.078). Conclusion For conventional osteosarcoma, the osteogenesis level in soft tissue lumps is related to the surgical stage, chemotherapy sensitivity and prognosis of tumors, which may provide guidance for the individual decision regarding chemotherapy and surgery timing on patients.
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OBJECTIVE:To provide reference for improving the rationality of discharged prescriptions. METHODS:According to the prescriptions that were often judged as forbidden or not recommended (such as irrational dosage and course of treatment , off-label drug use ,drug interaction ,medication suitability ,dosage form ) in the pre-audit system of discharged prescriptions (hereinafter referred to as the “audit system ”)of Union Hospital Affiliated to Fujian Medical University (hereinafter referred to as the“our hospital ”),combined with actual case analysis ,the clinical pharmacists looked for evidence-based evidence and evaluate it,communicated with the clinician for feedback ,maintained the content of misjudgment in the intercepted prescription in real time,set up the relevant rules of audit in the audit system ,evaluated the effect and put forward suggestions to improve the scientificity and accuracy of the audit results of the discharged prescription. RESULTS & CONCLUSIONS :For prescriptions that were judged to be forbidden or not recommended by the system ,such as the limited daily dose of Morphine hydrochloride tablet in patients with cancer pain ,the daily maximum dose of calcium sulfate D 3 after thyoidetomy ,hyper-indication use of megestrol acetate after chemotherapy for rectal cancer ,off-label usage of vitamin B 6 after postpartum delivery ,the combined use of clopidogrel bisulfate and oxcarbazepine after intracranial infection ,clinical contraindication of enteral nutrition powder after intestinal obstruction ,unsuitable dose of Dabigatran ester capsule after atrial fibrillation surgery and so on ,the clinical pharmacist looked for evidence-based evidence ,evaluated and communicated with the clinician . The relevant rules were updated to the audit system in real time. On the whole ,the audit system improves the work efficiency and the scientificity and accuracy of the audit results,but it is necessary to improve the professional knowledge level of the pharmacists ,the ability to find and use evidence-based evidence ,the accuracy of the audit system and the acceptance and recognition of the audit results by doctors ,so as to enhance the rationality of the discharged prescription ,reduce medication security risks.
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Objective:To investigate the 2 years’ efficacy of intravesical instillation of domestic BCG versus epirubicin in the prevention of recurrence of intermediate-risk or high-risk non-muscular invasive bladder cancer and predictive factors of BCG instillation.Methods:From July 2015 to June 2020, 18-75 years old patients with moderate to high-risk non muscle invasive bladder cancer (NMIBC) confirmed by pathological examination were involved. The ECOG score was 0-2. Exclusion criteria included ①immune deficiency or impairment (such as AIDS), using immunosuppressive drugs or radiotherapy, suspected allergic to BCG or epirubicin or excipients of the two drugs, fever or acute infectious diseases including active tuberculosis or receiving anti tuberculosis treatment, with severe chronic cardiovascular and cerebrovascular diseases or chronic kidney disease; ②combined with other urogenital system tumors or other organ tumors; ③combined with muscle invasive bladder urothelial carcinoma (≥T 2); ④undergoing chemotherapy, radiotherapy or immunotherapy within 4 weeks (immediate instillation after surgery not included); ⑤ pregnant or lactating women; ⑥ comfirmed or suspected bladder perforation; ⑦gross hematuria; ⑧cystitis with severe bladder irritation that may affect the evaluation; ⑨participat in other clinical trials within 3 months; ⑩alcohol or drug addiction; ?any risk factors that may increasing the risk of patients. Epirubicin 50 mg was irrigated immediately after the operation(TURBT or laser resection). The patients were randomly divided into BCG15 group, BCG19 group and epirubicin group by the ratio of 2∶2∶1, and the patients were maintained intravescical instillation for 1 year. The recurrence and adverse events of the three groups were compared. Univariate and multivariate analysis was performed to predict the risk factors of BCG irrigated therapy failure. Result:By June 15, 2020, the median follow-up duration was 22.1 months(12.1, 32.3), and there was no statistical difference between the groups ( P=0.9024). There were 274 patients enrolled in BCG19 group, 277 patients enrolled in BCG15 group and 130 patients enrolled in the epirubicin group. The drop-off rate was 16.6%(113 cases)and made no difference between groups( P=0.6222). There were no significant difference in age, gender, BMI, or ECOG score( P>0.05). During the follow-up, 116 cases was detected recurrence or progression. The recurrence rate of the three groups was 14.2% and 14.8% in BCG19 group and BCG15 group, and 27.7% in the epirubicin group. There was no difference in recurrence rate between BCG19 and BCG15 group( P=0.9464). The recurrence rate of BCG19 group was lower than that of the epirubicin group ( P=0.0017). The recurrence rate of BCG15 group was lower than that of the epirubicin group ( P=0.0020). There was no difference in the cumulative recurrence free survival rate between BCG19 and BCG15 group (95% CI0.57-1.46, P=0.7173). The cumulative recurrence free survival rate of BCG 19 group was better than that of the epirubicin group( HR=0.439, 95% CI0.26-0.74, P=0.0006), and the cumulative recurrence free survival rate of BCG15 group was better than that of the epirubicin group ( HR=0.448, 95% CI0.29-0.80, P=0.0021). The total incidence of adverse events in 19 BCG19, BCG15 and epirubicin group were 74.5%, 72.6% and 69.8% respectively. There was no difference in the incidence of adverse events between BCG19 and BCG15 group( P=0.6153). The incidence of adverse events in epirubicin group was lower than that of BCG19( P=0.0051) and BCG15( P=0.0167) groups.There was no significant difference in the incidence of serious adverse events (SAE) among the three groups ( P=0.5064). Log rank test univariate analysis and Cox risk regression model multivariate analysis showed that the history of bladder cancer recurrence( HR=6.397, 95% CI1.95-20.94, P=0.0001)was independent risk factor for BCG irrigation failure. Conclusions:The 2 years’ efficacy of intravesical instillation of domestic BCG is better than than of epirubicin with good tolerance and safety. There is no difference between BCG19 and BCG15 group. BCG doesn’t increase SAE compared with epirubicin. Recurrence status was an independent prognostic factor regarding recurrence-free survival.
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Objective To investigate the efficacy and safety of intravesical instillation of BCG vaccine in the prevention of early recurrence of middle and high risk non-muscle invasive bladder cancer.Methods From July 2015,patients with non-muscle invasive bladder cancer aged 18-75 years with informed consent were screened and underwent transurethral resection of bladder tumor (TURBT).Immediately intravesical instillation of epirubicin 50 mg was given postoperatively.After pathology was comfirmed,patients was enrolled in group 1 (BCG15) or group 2 (BCG 19) or the control group (epirubicin 18) randomly with SAS 9.3 software.Data of follow-up and Adverse event was collected and analyzed.Results By May 31,2019,531 patients were enrolled in the study.The drop-off rate was 20.1%.167 patients (143 males and 24 females)in group 1,172 patients (141 males and 31 females)in group2 and 84(75 males and 9 females) in the control group with follow-up data were analyzed.There were no significant differences in age,gender,BMI,ECOG score,risk stratification between the three groups (P =0.8641,P =0.2906,P =0.9384,P =0.6126).The median follow-up time makes no statistical difference between the groups (P =0.9251),12.0 (6.0,22.5) months,13.0 (6.0,22.3) months,and 13.0 (7.0,22.3) months.The median recurrence time of the three groups was 4.0 (3.0,6.0) months,4.5 (3.0,9.8) months,4.5 (3.0,8.8) months.There was no statistical difference between the three groups (P =0.2852).Risk stratification in the patients got no significant difference between the three groups (P > 0.05).The 1-year recurrence-free survival rates were 80.0% in the group 1 and 88.3% in the group 2 and 73.7% in the control group.The group 2 was superior to the group 1 and the control group (P =0.0281,P =0.0031).There was no significant difference between group 1 and control group (P =0.2951).There was no significant difference in the cumulative recurrence-free survival between the experimental group 1 and the experimental group 2,(95% CI 0.80-2.43,P =0.2433).The cumulative recurrence-free survival in the group 1 and the group 2 was better than the control group (95 % CI 0.31-0.92,P =0.0266;95 % CI 0.20-0.65,P =0.0008).All the cases underwent instillation were analyzed for adverse events.The incidence of overall AE(adverse events) in group 1 was 68.5% (152/222),the incidence of grade Ⅰ-Ⅱ AE was 53.2% (118/222),the incidence of grade Ⅲ-Ⅳ AE was 15.3% (32/222).The incidence of overall AE in the group 2 was 71.8% (160/223),the incidence of grade Ⅰ-Ⅱ AE was 60.1% (134/223),and the incidence of grade Ⅲ-Ⅳ AE was 11.7% (26/223).The overall AE rate in the control group was 53.2% (59/111),of which the incidence of grade Ⅰ-Ⅱ AE was 42.4% (47/111),and the incidence of grade Ⅲ-Ⅳ AE was 10.8% (12/111).There was no difference in the incidence of overall AE between the group 1 and the group 2 (P =0.4497).The incidence of AE in the two experimental groups was higher than that in the control group (P =0.0062,P =0.0008).There was no difference in the incidence of grade Ⅲ-Ⅳ AE between the three groups (P =0.3902).Conclusions BCG(19 instillation schedule) has a better effect on preventing recurrence after 1 year of bladder surgery,which is superior to epirubicin group.The long-term efficacy of BCG in preventing recurrence and the efficacy of different schedules need to be further followed up.The lower urinary tract symptoms,which are mainly urinary frequency,are one of the causes of case fallout and should be fouced in future.Compared with epirubicin,BCG perfusion does not increase the incidence of grade Ⅲ-Ⅳ adverse reactions,and is safe to use.
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Based on the intermolecular interaction, a molecularly imprinted polymer electrochemical sensor with specific identification of cavity on gold electrode surface was proposed with o-aminophenol as functional monomers and morin as template molecule. The performance and effect of MIP were investigated by cyclic voltammetry ( CV) and differential pulse voltammetry ( DPV) . Factors affecting the properties of sensor, such as polymeric membrane ratio, scanning cycles, elution time and adsorption time were investigated. Substances which had a similar structure to morin was used to compared the selective response. The result showed that the sensor had a good selectivity to morin. Under the optimized experiment conditions, the current response of the imprinted sensor was linear to the concentration of morin in the range of 0 . 05-1 . 7 μmol/L with linear equation as follows: I(μA)= 1. 0800lgc(mol/L)+ 9. 3599(R=0. 9934), and the detection limit of 0. 1 μmol/L. The sensor was used to determine the content of morin in black tea samples, and the recoveries of standard addition were between 104 . 0% and 108 . 0%.
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BACKGROUND:Inactivated autologous replantation in repair of bone defects after bone tumor resection has obvious advantages. Boiling, alcohol soaking, cryogenic freezing, microwave, radiation and other methods have been used for inactivation; however, they al have shortcomings. OBJECTIVE:To study the effect and feasibility of high temperature and high pressure inactivated autologous bone in repair of large segmental bone defects. METHODS: Bone defect models of bilateral distal radius were established in New Zealand white rabbits. Bone defect at the right side was repaired by high temperature high pressure inactivated autologous bone via in situ replantation, as experimental group. Bone defect at the left side was repaired byin situbone replantation, as control group. The general observation of bilateral radius, X-ray detection, bone radionuclide scan test and histological examination were conducted at the 6th, 12th and 24th weeks after surgery. RESULTS AND CONCLUSION: At the 24th week after surgery, X-ray films showed normal bone healing in these two groups. At the 24th week after surgery, bone radionuclide scan test showed that in the experimental group, the radiation on the repaired bone segments was uneven; the concentrations of nuclear elements were stil slightly higher on both ends, but decreased in the middle area; and the concentration in the control group was closed to normal. At the 24th week after surgery, histological observation showed that there were a majority of trabecular bone tissues in the bone defect area of the experimental group, and some woven bone tissues were immature, which was similar to the performance of the control group at the 12th week after surgery; and normal bone was visible in the control group. These results demonstrate that high temperature and high pressure inactivated autologous bone can be used to repair long segmental bone defects, but can result in delayed bone healing.
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Inorganic layered materials with the open structure of low dimension and good biological compatibility and stability are considered as one of the most promising materials for immobilizing biomolecules, which can not only adjust the spacing of layers according to guest molecular size to prevent leakage of guest molecule, but also reduce the effects of surrounding elements on immobilized molecules significantly. Moreover, they can promote electron transfer between enzyme and the electrode. Therefore, the inorganic layered materials have great application potential in electrochemical sensors. This paper reviews the ways of immobilizing guest molecules of inorganic layered materials and the applications and development prospects of the different types of inorganic layered materials including cationic, anionic and nonionic inorganic layered materials in electrochemical sensor.
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Objective:This study aims to investigate the protein expression of CD133, CD117, and Ki-67 in human osteosarcoma tissues and explore their relationships with the clinico-pathological features and risk of osteosarcoma. Methods:Immunohistochemical method was used to examine the protein expression of CD133, CD117, and Ki-67 gene in the paraffin specimens of 55 and 20 cases of osteosarcoma and osteochondroma, respectively. SPSS17.0 statistical software was used to explore the relationships among the expressions of CD133, CD117, and Ki-67 gene and the biological behavior and prognosis of osteosarcoma. Test criterion:P<0.05 was considered statistically significant. Results:The positive expression rates of CD133, CD117, and Ki-67 were significantly higher in the osteosarcoma tissues than in the benign osteochondroma tissues, and the differences were significant (P=0.016, P=0.008, and P<0.001, respectively). The mean survival and metastasis time were shorter in the CD133 or Ki-67 positively expressed osteosarcoma patients than in those with CD133 or Ki-67 negatively expressed osteosarcoma. The differences were significant (P<0.05). The expression of CD133 and Ki-67, surgical staging, and distal metastasis were relevant to the prognosis of osteosarcoma patients. The expression of CD133 and distal metastasis are the independent risk factors that affect the prognosis of these patients. Conclusion:The positive expressions of CD133 and Ki-67 closely correlated with the occurrence and progression of osteosarcoma, and may be used as an indicator for prognosis of the cancer.
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Objective To investigate the clinical distribution and drug resistance of multidrug-resistant (MDR)Pseudomonas aeruginosa to provide reference for clinical rational use of antibacterial drugs.Methods The strains were originated from bacterial culture in the samples of sputum,urine and wound secretion collected from the inpatients admitted in our hospital from January 2009 to December 2013.Results 200 strains of Pseudomonas aeruginosa were isolated during this period,in which 51 strains were MDR,accounting for 25.5%.The isolated MDR Pseudomonas aeruginosa strains were mainly distributed in the the respiratory de-partment(80.4%)and the surgical department(17.9%).MDR strains isolated from the sputum specimens accounted for 64.7%, followed by the wound secretion(19.6%)and urine(11.8%).Pseudomonas aeruginosa was seriously resistant to ampicillin-sulbac-tam with the resistance rate of was 100.0%,but had the low resistance rate to amikacin,imipenem,meropenem,piperacillin/tazobactam,ciprofloxacin,levofloxacin,polymyxin E,which were 28.9%,29.4%,25.0%,30.4%,34.5%,34.5% and 8.3%;the resistance rate to commonly used antibacterial drugs was 39.0%-58.3%.14 strains were pan-drug resistant Pseudomonas aerugi-nosa(PDR-PA);The detection rate was 7.0%.Conclusion The drug resistance of MDR Pseudomonas aeruginosa is more serious, the clinical application of anibiotics should be perfromed based on the result of drug susceptibility test.
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Objective:To determine the expression of miR-17-92 cluster in osteosarcoma tissue samples and explore its associa-tion with clinical significance. Methods: Quantitative polymerase chain reactiom analysis was used to examine the expression of miR-17-92 cluster in osteosarcoma tissues. Normal bone tissues from 63 patients were matched, and the relationships between the ex-pression of miR-17-92 cluster and the clinicopathological features and prognosis of osteosarcoma were explored. Results:The relative expression of miR-17-92 cluster in osteosarcoma tissues was significantly higher than those in adjacent normal tissues (P<0.05). The high expression of miR-17-92 had a significant correlation with reduced survival (P=0.027). Conclusion:The expression of miR-17-92 cluster closely correlates with the occurrence and progress of osteosarcoma and may be used as an indicator for osteosarcoma prognosis.
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Objective:To improve the sensitivity and the linear range of electrochemical immunosensor to detect Schistosoma japonicum (S.japonicum) antibody.Methods:Carbon inks and silver/silver chloride inks were printed on a polyethylene terephthalate (PET) board to make a two-electrode test strip,where carbon was the working electrode and S.japonicum soluble egg antigen (SEA) was fixed at one end of working electrode by different methods; silver/silver chloride electrode was used as control.We tested the valency of the antibody by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in an electrochemistry workstation,and conducted comparison with the results of ELISA.Two new immunosensing electrodes have been developed,based on glutaraldehyde cross-linked (GA) or chitosan-glutaraldehyde cross-linked (Chit-GA) transducer fixing S.japonicum antigen.We tested the titer of the antibody by means of CV and DPV.Results:Our experimental S.japonicum antigen (50 μg/L) is the optimal test concentration for the GA sensor,and 10 μg/L for Chit-GA sensors.The immune reaction time of both electrodes is all essentially complete in 1 minute.The linear range for S.japonicura antibody in human positive serum sample detection by the glutaraldehyde cross-linked immunosensor is 1∶1000 to 1∶400,and by the chitosan-glutaraldehyde cross-linked immunosensor is 1∶1000 to 1∶500.As the concentration of dilution ratio of S.japonicum antibody in human positive serum sample increased,the test value of DPV increased proportionally.Conclusion:GA sensor and Chit-GA cross-linked S.japonicum sensors have high sensitivity and broad linear range response,and both exhibited a good linear relationship between the DPV signal and the test antibody titer.
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BACKGROUND: Pilose antler polypeptides (PAP) have been proved to promote the proliferation of condrocytes cultured in vitro and expressions of glycosaminoglycan (GAG), type Ⅱ collagen and Aggrecan protein in the extracellular matrix.OBJECTIVE: To investigate the feasibility of chondrogenic phenotype differentiation of rabbit bone marrow-derived mensenchymal stem cells (BMSCs) in a defined medium and then to study the effect of PAP on chondrogenic differentiation of BMSCs in vitro.METHODS: The third passage BMSCs from rabbits were randomly divided into control group cultured in ordinary medium, induced group cultured in defined medium, and PAP group cultured in defined medium containing 10 mg/L PAP. An equal volume of articular chondrocytes were selected from rabbits as articular cartilage group. The cellular morphological and functional characteristics were observed after 1, 2, 3 weeks in centrifuge tubes by histological, biochemical and reverse transcription-polymerase chain reaction (RT-PCR) technique. RESULTS AND CONCLUSION: Cell masses in the control group gradually crumbled after 2 weeks, and hematoxylin-eosin staining could not be done. Cell masses in the induced and PAP groups were semitransparent, but slightly contracted. A part of these cells were round or oval with a high density distribution at the surface. The content of GAG and mRNA expression of type Ⅱ collagen in the induced and PAP groups were increased with culture time, and higher than those in the control group at different time points (P < 0.05). The content of GAG and mRNA expression of type Ⅱ collagen in the PAP group were higher than those in the induced group, but lower than those in the articular cartilage group (P < 0.05). The results indicated that BMSCs can differentiate into chondrogenic phenotype in the defined medium, and PAP can significantly enhance chondrogenic phenotype differentiation of BMSCs. But the quality of cultured cartilage tissue is poorer than that of the articular cartilage.
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Objective To investigate the characteristics and prevention of deep fungal infection (DFI) complicated by severe acute pancreatitis (SAP). Methods Clinical data of 135 SAP cases admitted from Oct. 1998 to Oct. 2009 were studied, the prevention and management of SAP with DFI ( 32cases) were investigated, among them there were 19 males and 13 females. Results Seven patients (21.9%) died of these 32 cases. The SAP patients with DFI were of more critical condition, longer course and having to stay in ICU about (21±6) d, most needed surgical debridement, on long-term indwelling catheters, intubation and mechanical ventilation, parenteral nutrition, long-term use of broad-spectrum antibiotics. These patients remained in the hospital for about (78 ± 14) d. Conclusions DFI is a common complication in SAP cases causing a significant mortality hence necessitating comprehensive prevention and control measures such as proper handling of the original disease, aseptic operation, prophylactic antibiotics and antifungal agents, which can reduce the incidence and mortality of SAP with DFI.
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Objective: TO explore the effect of VEGF-C gene transfection on the expression of VEGF-C in human cervical carcinoma HeLa cells and the mechanisms of its anti-apoptosis effect. Methods: The con-structed pcDNA3.1(+)NEGF-C vector was transformed into human cervical cancer HeLa cells and was select-ed by G418. The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. HeLa cells with overexpression of VEGF-C were named as HeLa/S1. The expression level of NF-KB and bcl-2 mRNA was determined by RT-PCR in transfected cells. Results: After transfection by liposome, the VEGF-C mRNA level and the expression of VEGF-C protein in transfected cells were higher than those in the control groups. HeLa/S1 cell line was successfully established. In HeLa/S1 cells, the expression of NF-κB (2.06±0.09 vs 1.35±0.02 vs 1.38±0.02 P<0.05) and bcl-2 gene mRNA (2.02± 0.67 vs 0.41±0.06 vs 0.37±0.06, P<0.05) level were higher than those in the control groups. Conclusion: VEGF-C gene transfection by liposome can increase the expression of VEGF-C in human cervical cancer HeLa cells. NF-κB is stimulated and induces the overexpression of bcl-2 gene in HeLa/S1 cells.
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@#ObjectiveTo explore the effect of different properties and hemisphere of stroke on trunk control ability and the relationships between the trunk control and cognition, activities of daily living (ADL).Methods75 inpatient stroke patients were divided into the group A (right hemorrhages, n=15), group B (right infarcts, n=25), group C (left hemorrhages, n=14) and group D (left infarcts, n=21). All cases in four groups were assessed by Mini-Mental Status Examination (MMSE), Sheikh Trunk Control Evaluation and Modified Brathel Index (MBI) before and after rehabilitation therapy.ResultsThe scores of Sheikh and MBI of the group A and group C were significantly lower than that of the group B and group D before treatment ( P<0.05~0.001). After treatment, the scores of MMSE, Sheikh Trunk Control Evaluation and MBI of the patients in four groups all increased, but the scores of the patients in the group A and group C increased more than other groups. There was no significant difference in Sheikh scores between right and left hemisphere stroke. All groups had high relationship between Sheikh Trunk Control Evaluation and MBI before and after treatment. There was no relationship between Sheikh Trunk Control Evaluation and MMSE before treatment, but having part relationship after treatment.ConclusionThe trunk control of hemorrhage patients are damaged more severe than that of the infarct cases, but they resume also faster. There are no different between right and left hemisphere stroke in trunk control impairment. The ability of trunk control has high relationship with ADL before and after treatment, it has no relationship with cognize before treatment but has relationship after treatment.
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BACKGROUND:Gene therapy is the direction of spinal cord injury(SCI) therapy,the key of which is construction of targeting gene and vector. OBJECTIVE:To construct the recombinant adenovirus vector carrying human brain-derived neurotrophic factor(hBDNF) marked enhanced green fluorescent protein(EGFP). DESIGN,TIME AND SETTING:A single sample observation was completed in the First Affiliated Hospital of Fujian Medical University from September 2007 to June 2008. MATERIALS:Competent E. coli DH-5? was obtained from the American Stratagene Company. Plasmid pDC316-hBDNF,pDC316-mCMV-EGFP,pBHGlox_E1,3Cre and package system AdMax and 293 package cell strain were purchased from the Canadian Mixcrobix-Biosystems Company. METHODS:The hBDNF gene was constructed by PCR with plasmid pDC316-BDNF as template. With enzyme digestion,the hBDNF gene was inserted into the vector pDC316-mCMV-EGFP and the shuttle plasmid pDC316-hBDNF-mCMV-EGFP was constructed,which was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad-hBDNF-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method,then the virus particles were counted and the purity and titer were determined. MAIN OUTCOME MEASURES:①PCR identification of plasmid pDC316-hBDNF. ②Construction and identification of the shuttle plasmid pDC316-hBDNF-mCMV-EGFP. ③Packing,amplification and purification of recombinant adenovirus vector Ad-hBDNF-EGFP. ④PCR identification of the recombinant adenovirus. ⑤Titer of recombinant adenovirus. RESULTS:PCR amplification,restriction analysis and sequencing identified that both recombinant shuttle plasmid pDC316-hBDNF-mCMV-EGFP and recombinant adenovirus vector Ad-hBDNF-EGFP were correctly constructed. After amplification and purification,the virus particle count,A260/A280 and titer of recombinant adenovirus were 2.4?1011 VP/mL,2.0 and 0.8?1010 CCID50/mL,respectively. CONCLUSION:Recombinant adenovirus vector Ad-hBDNF-EGFP is successfully constructed,which laid a foundation for further study regarding gene function and therapy.
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Objective:To investigate the inhibitory effects of endostatin (ES) on the growth and metastasis of transplanted osteosarcoma UMR106 cells in nude mice. Methods: pBudCE4.1/ES was amplified and transfected into osteosarcoma cell line UMR106 through lipidosome; the non-transfected UMR106 cells were eliminated by Zeocin. The proliferation of ES-UMR106 cells was examined by MTT assay and the production of ES by ES-UMR106 cells was investigated by ELISA. The effects of ES on the in vitro proliferation of vascular endothelial cells were observed by MTT assay. Sixteen nude mice were randomly divided into 2 groups: one group was transplanted with UMR106 cells and the other with ES-UMR106 cells. The tumor size, pathological observation, tumor angiogenesis, and the pulmonary metastasis were observed. Results: The proliferation of UMR106 cells was not affected by ES transfection. ES-UMR106 cells expressed bioactive ES, and the ES level in the medium was higher than 350 ng/ml 48 h after transfection. ES produced by ES-UMR106 cells significantly inhibited the proliferation of vascular endothelial cells. Compared with UMR106 transplanted tumor, ES-UMR106 transplanted tumor grew slowly in nude mice, and the formed tumors was well-margined and had less angiogenesis and mass necrosis. Two of the 8 mice transplanted with UMP106 cells had pulmonary metastasis and no metastasis was found in ES-UMR106 transplanted group. Conclusion: Transfection with recombinant endostatin plasmid can inhibit the growth and metastasis of transplanted osteosarcoma in nude mice by inhibiting vascular endothelial cell proliferation.