Aim To observe the timed analgesic effects by butabital (B), acetaminophen(A) and caffeine (C) in combination (BAC), in which the proportion was fixed as1. 25∶ 8. 1∶1. Method Three types of experimental methods, including the tailflicking method, the hot plate method and the pressurizing tail method, weretaken to determine indices at different times after the animals were adminstered(ig) high, median and low BAC dose. Results and conclusion BAC had a stronganalgesic effect in three types of experiments. The effect began 30 min after ad-ministration, arrived maximum at 1 h, decreased at 2 h and disappeared at 4 h.There was a dose-effect relationship between large and little BAC dose.

Objective To investigate and understand the reference value range of hematological parameters for peripheral blood routine in preschool children from Shanghai.Methods The Sysmex XS-800i automated hematology analyzer and the original rea-gents were used to measure the hematological parameters in peripheral blood samples collected from 7 692 healthy preschool chil-dren aged 2-6 years in Shanghai,including white blood cells(WBC),red blood cells(RBC),platelet(PLT)count,hemoglobin(Hb), hematocrit(HCT),mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH)and mean corpuscular hemoglobin con-centration(MCHC);the various parameters in different age groups were collected and statistically processed to establish the refer-ence intervals for each parameter.Results In the blood routine among preschool children in Shanghai,there were statistically signif-icant differences in all parameters except WBC and PLT count between sexes among preschool children in Shanghai(P <0.05).The reference intervals of hematological parameters obtained in this investigation were obviously differed from those offered by the man-ufacturer.However,compared with those from the related reports,the difference existed in partial parameters,the upper limit of WBC count was highest compared with the results from some areas,while the reference value ranges of MCV,MCH and MCHC were higher than those of other area study results.Conclusion The independent blood routine medical reference value range for preschool children in Shanghai should be established and the influence of the factors of gender,instrument and reagents also should be taken into consideration in establishing the reference value range.

AIM: To investigate the angiogenesis in the process of sarcoma 180 (S_(180)) tumor transplantation and changes of regulator factors, and explore the possible mechanism. METHODS: The S_(180) transplanted tumor in the Km mouse was used to detect the tumor angiogenesis by immunohistochemical examination of FⅧ. The levels of VEGF (V) and endostatin (E) in serum and the homogenate of tumor tissue were measured by ELISA and EIA, and the correlation between tumor weight and microvessel count (MVC) and morphology in tumor was also analyzed by multiple ANNOVA method. RESULTS: MVC, the relative count of total vessels and relative total vessel area increased with the development of transplanted S_ 180 . VEGF level in tumor tissue were higher at the 10th and 15th day than the 5th day after tumor transplantation. Endostatin in the tumor tissue and serum both reached the highest level at the 15th day, V/E ratio did not changed in this process. Furthermore, MVC, average vessel area and relative total area had a significant correlation with tumor weight. CONCLUSION: MVC increases in the development of S_(180) transplantation tumor and is related with the tumor weight; the positive regulator of angiogenesis in the tumor tissue is up-regulated during tumor growth, and the regulators in the tumor tissue maintains a relative balance.

Objective To investigate the clinical efficacy and significance of preoperative superselective endovascular embolization in treatment of patients with vascularized meningioma.Methods 3-9 days before operation, 98 patients with vascularized meningioma underwent DSA and preoperative superselective embilization with PVA particles, gelatin sponge, and silk. The surgical intervention was performed after embolization. Results The vascularized meningioma was mainly supplied by the middle meningeal artery, ascending pharyngeal artery, occipital artery, internal maxillary artery as well as submeningeal artery. After the supplying artery was embolized, tumors' staining in 42 cases disappeared completely and that of 56 cases disappeared totally or partly. Most patients were operated on 3-9 days after embolization. 64 tumors were removed completely, while other 34 cases were removed totally or partly. The average volume of bleeding during the operation was 950 ml in the former and 1 500 ml in the latter.Conclusions The best time for operation is 7～9 days after embolization. Preoperative embolization of the vascularized meningioma contributes to reduce the bleeding significantly during the operation, increase the safety of the operation and improve the total removal of the tumor. It is a safe and effective microinvasive method.

Objective To develop a high-resolution melting ( HRM ) assay for rapidly screening Gilbert syndrome ( GS) and Crigler-Najjar syndrome ( CNS) associated with UGT1A1 defects.Method Methodology was developed .Then, we applied the established method to analyze 61 clinical samples from neonatal patients with severe unexplained unconjugated hyperbilirubinemia .Neonates with known risk factors for developing hyperbilirubinemia , such as ABO hemolysis, G6PD deficiency, sepsis, hypoxic ischemic encephalopathy were excluded .Five pairs of PCR primers were designed to detect the five common mutations (G211A, C686A, C1091T, C1352T and T1456G) in Asia population.PCR and HRM Assay conditions were optimized.UGT1A1 genotyping in clinical samples was performed by using the established HRM analysis , and all results were subsequently confirmed by direct DNA sequencing .Results The mutants were readily differentiated by using HRM analysis .In this study, 42 neonates were identified with UGT1A1 mutation, and 4 different known variants were detected .Conclusion HRM analysis in this study was economical, convenient, rapid, effective for screening UGT1A1 gene mutations, which can serve as an reliable method for the clinical diagnosis of GS and CNS and the large-scale molecular epidemiological research of UGT1A1 gene-related diseases.

OBJECTIVETo research the mechanism of dopamine (DA) controlled memory in mice.

METHODSMice received i.p. injection of scopolamine (0.3 mg/kg, SCOP 0.3, and 3.0mg/kg, SCOP 3.0, respectively, n = 10) and saline (NS, n = 10) for 60 days in experiment 1. Memory of mice was detected by dark avoidance behavior in the 53" d and the 60"' d. Animals were sacrificed after the memory test; brain tissues were processed for Fos-ir and TH-ir by immunohistochemistry. Mice were divided into four groups according results of expri-ment 1, they received i.p. injection of apomorphine (0.1 mg/kg, APO 0.1, 0.5 mg/kg, APO 0.5, and 2.0 mg/kg, APO2.0 respectively, n = 10).

RESULTSMemory was inhibited in mice injected scopolamine 3.0 mg/kg. Latency was significantly less than in NS group, only 1/ 4 that of NS group (P > 0.05). The number of mistake of SCOP 3.0 group increased about four times than that of NS group (P > 0.05). But there was no difference of latency and number of mistake between SCOP 0.3 and NS group in expriment 1. Scopolamine-induced memory deficit was associated with decreased cellular activation, indicated by Fos immunoreactive (ir) staining, in NAcc CA1 and CA3 (P < 0.05), and also associated with decreases in the number of cells labeled for tyrosine hydroxylase (TH-ir), the rate limiting enzyme for dopamine conversion (P < 0.01) and the number of cells co-labeled for TH-ir/Fos-ir (P <0.01) in the ventral tegmental area(VTA), apomorphine lessened scopolamine-induced memory deficit in experiment 2. The number of cells co-labeled for TH-ir/Fos-ir (P <, 0.05) was increased in VTA after apomorphine treatment.

CONCLUSIONApomorphine lessened scopolamine-induced memory deficit in mice by increasing DA activities in VTA.

Animals ; Apomorphine ; pharmacology ; Disease Models, Animal ; Dopamine Agonists ; pharmacology ; Male ; Memory Disorders ; chemically induced ; drug therapy ; Mice ; Scopolamine Hydrobromide ; toxicity

OBJECTIVE: Investigate common deafness gene mutations in patients with severe and profound non-syndromic hearing loss in Liaoning in order to understand their hereditary etiologies and characteristics at the molecular level. METHOD: Peripheral blood samples were obtained and the DNA templates were extracted from 128 non-syndromic hearing loss patients who are sporadic in clinics. The deafness gene chip was applied to detect hot-spot deafness gene mutations including GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA. Deafness etiology questionnaires, pure tone audiometry, auditory brainstem response, tympanometry and temporal bone CT were also applied. RESULT: Various types of gene locus mutations were seen in 52 of the 128 patients (40.6%); (1) GJB2 gene mutations (n=22) included c. 235 del C homozygous mutation (n=10), c. 235 del C heterozygous mutation (n=5); c. 176_191 del 16 heterozygous mutation (n=l); c 35 del G heterozygous mutation (n=l); c. 235 del C/c. 299_300 del AT mutation (n=l), c. 235 del C/c. 176_191 del 16 mutation (n=l), c. 35 del G/c. 176_191 del 16 mutation (n=l); c. 299_300 del AT/c. 919-2 A>G mutation (n=l), c. 235 del C/c. 919-2 A>G mutation (n=l). (2) SLC26A4 gene mutations (n=30) included c. 919-2 A>G homozygous mutation (n=6), c. 919-2 A>G heterozygous mutation (n=17), c. 2168 A>G homozygous mutation (n=l), c. 2168 A>G heterozygous mutation (n=2), c. 2168 A>G/c. 919-2 A>G mutation (n=2), c. 919-2 A>G/GJB2 c. 235 del C mutation (n=2); (3) No GJB3 and mitochondrial 12S rRNA mutation. Genetic deafness was confirmed at the gene level in 24 cases (18.8%) and 28 patients (21.9%) were diagnosed as carriers of genetic deafness gene mutations. CONCLUSION Genetic deafness occupies a large population in deaf community in Liaoning. Molecular genetic screening for these mutations and genetic counseling are effective methods to prevent the occurrence of hereditary hearing loss and provide theoretical guidance.
Adolescent ; Child ; Child, Preschool ; China ; Connexins ; DNA Mutational Analysis ; Deafness ; genetics ; Female ; Genetic Testing ; Humans ; Infant ; Male ; Mutation

Objective To investigate the clinical significance and mechanism of WW domain containing oxidoreductase (WWOX) gene and p73 gene abnormal expression in acute lymphocytic leukemia (ALL).MethodsCase-control study was used in the research.Forty-eight cases of bone marrows from ALL patients were collected,including 32 cases newly diagnosed,11 cases with complete remission and 5 case with relapse.Thirty-one cases of bone marrows from non-leukemia patients were used as control group.All the samples were collected from First Affiliated Hospital of Guangxi Medical University from July 2010 to July 2011.The doctors punctured patients' bone marrows 3 milliliters from the left of posterior superior iliac spine.Samples were bottled up with EDTA anti-coagulation tube.1 milliliter bone marrow was used to extract genome RNA with purity from 1.8 to 2.0.And then,the level of WWOX and p73 gene transcripts were tested immediately using reverse transcriptase-polymerase chain reaction (RT-PCR).Meanwhile genome DNA was also extracted from the other 2 milliliter bone marrow with purity from 1.7 to 1.9,which was used to detect the promoter methylation of WWOX gene and the first exon methylation of p73 gene by methylation PCR (MS-PCP).x2 test and Fisher's exact test were used to compare tbe methylation status of WWOX and p73 gene.Results In 31 controls,expression of WWOX and p73 gene mRNA was 94.00％.The total expression frequency of WWOX gene mRNA in 48 ALL samples was 48.00％ (23/48),much lower than control (x2 =17.434,P =0.000 ).There was significant difference (x2 =10.471,P =0.001 ) between newly diagnosed cases 34.38％ ( 11/32),complcte remission cases (90.91％,10/11 ) and control.The total expression frequency of p73 gene mRNA in 48 ALL samples was 56.00％ (27/48),much lower than control (x2 =12.697,P =0.000).There was significant difference (P =0.012 ) between newly diagnosed cases 43.75％(14/32) and complete remission cases 90.91％(10/11).It was unmethylation in 31 controls.The total methylation frequency of WWOX gene promoter region in 48 ALL samples was 44.00％(21/48),much lower than control (x2 =18.473,P =0.000).There was significant difference (P =0.012) between newly diagnosed cases 56.25％ (18/32),complete remission cases 9.09％ (1/11 ) and control.The total methylation frequency of p73 gene the first exon region in 48 ALL samples was 35.00％(17/48),much lower than control (x2 =13.990,P =0.000).There was significant difference (P =0.033) between newly diagnosed cases 46.88％ (15/32),complete remission cases 9.09％ ( 1/11 ) and control.There was a negative correlation between the expression of WWOX gene mRNA and its methylation status(r =- 0.678,P =0.000),the same as p73 gene ( r =- 0.577,P =0.000).ConclusionsThe abnormal methylation of WWOX and p73 gene may be the major mechanism of gene silence in ALL,which leads to no expression of WWOX mRNA or p73 mRNA.And the abnormal methylation of WWOX and p73 gene may be relevant with the process of occurrence and development in ALL.It may be an effective and significant to detect methylation status of WWOX gene and p73 gene for the diagnosis and treatment of ALL patients.(Chin J Lab Med,2012,35:820-825)

10.3969/j.issn.2095-4344.2013.23.026

AIM: To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms.METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and "bystander effect" were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS: DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The "bystander effect" was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G 0-G 1 phase blockade. CONCLUSION: HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of "bystander effect" was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.