Objective To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. Methods The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3. 1 ( + )-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1 ( + )group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. Results ( 1 ) The mRNA expression of beclin 1 and caspase-9: pcDNA3. 1 ( + )-beclin 1 group were 994.72 ±468.76 and 12. 88 ±2. 71, pSUPER-beclin 1 group were 0. 18 ± 0. 63 and 0. 11 ± 0. 08, pcDNA3. 1 ( + ) group were 0. 57 ± 0. 12 and 4. 28 ± 3. 25,pSUPER group were 0. 67 ± 0. 29 and 2. 77 ± 1.27, and HeLa group were 0. 74 ± 0. 25 and 3.67 ± 3.78,respectively. The eukaryotic expression vector pcDNA3. 1 ( + ) -beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells( P ＜0. 05 ), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9 ( P ＜ 0.05 ). (2) The cell proliferations: pcDNA3.1 ( + ) -beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells(P ＜0. 05). (3) The rate of apoptosis: pcDNA3.1 ( + )-beclin 1 group was (28.2 ±2.3)%, pcDNA3. 1( + ) group was(14.6 ±4.6)%,pSUPER-beclin 1 group was(5.7 ±2. 0) %, pSUPER group was( 16. 2 ± 3.1 ) %, and HeLa group was( 11.2 ± 3. 0) %. The pcDNA3. 1 ( + ) -beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate(P ＜0. 05 ). (4)The activity of autophagy: more autophagy cells were identified in pcDNA3.1( + )-beclin 1 group; the rate of autophagy of five group were( 10. 3 ± 1.5)% in pcDNA3. 1 ( + ) -beclin 1 group, ( 3.6 ± 0. 8 ) % in pcDNA3. 1 ( + ) group, ( 1.2 ± 0. 3 ) % in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1. 1)% in HeLa group, there was statistical significances between test groups and control groups( P ＜ . 05 ). (5)Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1 ( + )-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups( P ＜ 0. 05 ). The tumor size was smaller from 21st day after injection in pcDNA3.1( + )-beclin 1 group than in control groups(P ＜0. 05). From 28th day after injection,the tumor weigh was (0. 52 ± 0. 08 )g in pSUPER-beclin 1 group, apparently more than HeLa group (0. 37 ±0. 12) g and pSUPER group (0. 34 ± 0. 24 ) g ( P ＜ 0. 05 ). While in pcDNA3. 1 ( + )-beclin 1 group the tumor weighed (0. 18 ±0. 12) g, which was lower than HeLa group and pcDNA3. 1 ( + ) group (0. 34 ± 0. 18 ) g ( P ＜ 0. 05 ) . Conclusions Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.

Objective To assess the changes in blood coagulation after infusion of hydroxyethyl starch 45/ 0.7(molecular weight = 450000 Dalton, 70% of glucose units have been substituted) ,200/0.62, 200/0.5 and 130/0.4.Methods Forty rabbits weighing (2.6 ?0.5)kg were randomly divided into 5 groups of 8 animals each: group I HES450/0.7; group II HES 200/0.62; group III HES200/0.5; group IV HES 130/0.4; group V 0.9% NaCl. The animals were anesthetized with intramuscular seconal and ketamine. Hydroxyethyl starch or normal saline was infused at 10 ml?kg-1?h-1 for 3 h. Blood samples were taken before and 1,2,3 h after staring the infusion for determination of prothrombin time ( PT), activated partial thromboplastin time (APTT) and fibrinogen level (FIb) and thromboelastograph examination (TEG) .Results (1) After infusion of 30 ml?kg-1 HES 450/0.7 or 200/0.62 (in group I and II ) R time (representing the rate of initial fibrin formation) and K time (coagulation time) were significantly prolonged (P

Objective To assess the effects of gelatine and 6% hydroxyethyl starch 200/0.5 (HES 200 / 0.5) on phagocytic activity of human neutrophils and monocytes using flow cytometry.Methods Thirty-three ASA Ⅰ - Ⅱ patients aged 18-70 years scheduled for urological minor surgery were randomly divided into three equal groups of eleven patients :group I gelatine;group II HES 200 / 0.5 and group Ⅲ lactated Ringer's solution (LR) . 10 ml?kg-1 of gelatine, HES or LR was infused over 60 min and venous blood samples were taken before infusion and 1 h after the start of infusion for determination of phagocytes with ingested FITC-labeled E coli by flow cytometry. Results In gelatine group the percentage of neutrophils and monocytes with phagocytic activity decreased significantly after infusion ( P

Objective To study the subchronic toxicity of fipronil in rats and to protect and improve the environment and human health. Methods 80 SD rats were randomly divided into four groups,20 in each groups (10 males and 10 females). The rats were treated with fipronil by gavage,in doses of 25,10,4,0 mg/kg,once a day,10 ml/(kg?d),for 90 consecutive days. The test method was according to GB15670—1995. Results The acute toxicity symptom of rats in high dose showed that cachexia,dull,hair floppy. No acute toxicity symptoms were found in the moderate and low dose groups. No significant difference was found in the serum and urine routine test indexes. Liver index increased significantly in the high dose(25 mg/kg)and moderate dose(10 mg/kg) exposed rats. The activity of aspartateaminotransferase (AST) in the serum increased only in male rats. No significant changes in indicators were found in the low exposed rats. The histopathologic examination showed that in the high and moderate dose groups,in the liver,spot and focus necrosis,the hepatocytes around central veins became bigger and cloudy swelling,some hepatocytes presented ballooning degeneration. No obvious pathological change was seen in the low dose group. Conclusion Fipronil has a toxicity to the liver,based on 90 day oral toxic test in rats,no-observed adverse effect level (NOAEL) is 4 mg/(kg?d).

Objective To establish Phage Amplified Biologically Assay (PhaB) detecting isoniazid(INH) resistance rapidly and evaluate PhaB assay for drug susceptibility testing of isoniazid(INH) in clinical isolates of Mycobacterium tuberculosis(MTB). Methods Detecting the INH resistance of 167 clinical isolates of MTB by PhaB assay,comparing the results of PhaB with that of Bactec-960 system and analyzing the sensitivity, specificity and accuracy of PhaB assay. Results When the mixture of 0.2 ?g /ml INH and MTB was incubated in 37℃ for 48 h, the accurate results were obtained rapidly by calculate the reduce of plaque of PhaB assay. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, PPV, NPV and accuracy of PhaB assay was 96.4%, 96.4%, 93.1%, 98.2% and 96.4% respectively. Conclusions The PhaB assay with highly sensitivity specificity are highly consistent with Bactec-960 system. Not only it takes only three days to detect drug susceptibility of INH in clinical isolates of MTB but also it is easily to operate. We believe that this low-cost assay may be a good rapid screening of INH resistance in MTB isolates.

Objective Evaluating possibility of phage amplified biologically(PhaB) assay in detecting susceptibility of Mycobacterium tuberculosis(MTB) to four first-anti-tuberculosis-drugs on the same time and of 139 clinical isolates to streptomycin(S), isoniazid(H), rifampin(R) and ethambatal(E) at the same time, comparing with the results of Bactec-960 and determining the minimal inhibitory concentrations(MIC) of isolates which results were not consistent. Results Concordance rates of the susceptibility to S, H, R and E in 139 clinical isolates detected by PhaB and Bactec-960 are 97.1%, 99.3%, 95.7% and 95.0% respectively. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, positive and negative predictive value (PPV and NPV) as well as accuracy of susceptibility test to S detected by PhaB assay was 90.0%, 99.1%, 96.4%, 98.2% and 97.1% respectively, to H 97.9%, 98.9%, 97.9%, 98.9% and 95.0% , to R 86.2%, 97.3%, 89.3%, 96.4% and 95.0%, to E 81.0%, 97.5%, 85.0%, 96.6% and 95.0%. There are 19 inconsistent results of 13 isolates in comparing PhaB with Bactec-960. 18 results of 12 isolates by MIC are identical with the results of PhaB assay. 1 result of 1 isolate is identical with Bactec-960. Conclusions[KG1]The results of susceptibility to S, H, R and E detected by PhaB were highly concordance rate with the results of Bactec-960. PhaB assay can be used for rapid screening of susceptibility test for MTB.

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19- T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.

Objective To study the immunogenicity,immune modulatory effects and mechanisms of mesenthymal stem ceils (MSCs) derived from the bone marrow of patients with multiple myeloma (MM).Methods The mononuclear cells from the bone marrow of MM patients were obtained and cultured.Immunophenotypes were investigated by using FACS.The levels of cytokines were determined by using enzyme linked immunosorbant assay (ELISA).The endocytosis of monocyte-derived dendritic cells (DCs) was investigated by using FACS.Moreover,the immunoregulatory ability of DCs on proliferation of T lymphocytes was detected by using mixed lymphocyte culture assay.Results MM-derived MSCs had no expression of HLA-DR and costirnulatory molecules (CD40,CD80,CD83 and CD86).MM-derived MSCs could significantly suppress proliferation of T lymphocytes in a dose-dependent manner,which could be reversed by antiTGF-β1 and anti-HGF antibodies.MM-derived MSCs inhibit the endocytosis of DCs.MM-derived MSCs could inhibit the secretion of IL-12 and significantly inhibit the function of DCs on proliferation of T lymphocytes.Conclusion MM-derived MSCs harbored low immunogenicity and immunoregulatory effect in vitro,and this effect was achieved through cytokines.

BACKGROUND:Bone marrow mesenchymal stem cells derived from multiple myeloma patients are characterized by pluripotential differentiation, immunoregulation and supporting hematopoiesis, but whether these features are affected after cryopreservation is unclear.
OBJECTIVE:To study the biological characteristics of cryopreserved bone marrow mesenthymal stem cellderived from multiple myeloma patients.
METHODS:Bone marrow mesenchymal stem cells derived from multiple myeloma patients were cryopreserved in-196 ℃ liquid nitrogen for 1 month (short term) and 12 months (long term) with Iscove’s modified Dulbecco’s medium containing 10%dimethyl sulfoxide and 40%fetal bovine serum as cryoprotectant. The viability and proliferation ability of thawed bone marrow mesenchymal stem cells were investigated. Hematopoiesis support of thawed bone marrow mesenchymal stem cells in vitro was detected by long-term bone marrow culture and the methylcellulose progenitor assay. The immunoregulatory ability of thawed mesenchymal stem cells was detected by mixed lymphocyte culture assay.
RESULTS AND CONCLUSION:The cellviability was (92.9±7.5)%and (86.7±9.2)%for mesenchymal stem cells cryopreserved as long as 1 month or 12 months, respectively. Furthermore, thawed bone marrow mesenchymal stem cells possessed the ability of supporting colony forming and could significantly suppress proliferation of T lymphocytes. At last, there were no changes detected as compared with pre-cryopreserved mesenchymal stem cells in the abilities of proliferation, hematopoiesis support and immunoregulation. Cryopreservation can decrease the cellviability of bone marrow mesenchymal stem cells derived from the patients with multiple myeloma, but cannot affect the abilities of proliferation, hematopoiesis support and immunoregulation.

Objective To determine the contents of four effective constituents in Euphorbiae Semen decoction;To provide evidence for Euphorbiae Semen decoction into clinical application. Methods Established quantitative analysis multi-components by single marker method was used to determine the contents of diterpenoids constituents, such as euphorbia storoid, euphorbia factor L2, and euphorbia factor L3. HPLC method was used to determine the contents of aesculetin. Results Contents of euphorbia storoid, euphorbia factor L2, and euphorbia factor L3 in smashed Euphorbiae Semen decoction were 0.015 9%, 0.005 9% and 0.024 1%, respectively. However, the contents of the above three constituents could not be detected in whole Euphorbiae Semen decoction. The content of aesculetin (0.693 6%) in smashed Euphorbiae Semen decoction was more than that in whole Euphorbiae Semen decoction (0.288 2%). Conclusion Decoction digestion effect of diterpenoids constituents in Euphorbiae Semen decoction is not good. Decocting with water is not suitable for the clinical application of Euphorbiae Semen for purgation and diuresis. Aesculetin in smashed Euphorbiae Semen decoction has good decoction digestion effect, in which clinical use for antisepsis and anti-inflammation is effective.