Objective To study the biological significance of the common mutant preC/C gene in clinical HBV in China. Methods Site directed mutagenesis based on the unique enzyme site elimination was used to construct eukaryocyte expression vector with mutant HBV/C gene(V60、G87、L97). Expression vectors with wild and mutant preC/C gene were transferred into HepG2 cell. Culture supernatant was detected by ELISA for HBeAg. Results Result of DNA sequencing showed that the constructed mutant HBV preC/C gene had only one specific site variation compared with the wild type sequence. Goal DNA fragment was detected positive in the HepG2 cells transferred with wild and mutant preC/C gene. A value of HBeAg in the supernatant of the cells harboring L97 variant was higher than that of the wild and other variant strains( P

To study the characteristics of EBV transformed human peripheral blood B cell lines from hepatitis B patients and to provide a basis for further studies on the cellular immune function of hepatitis B patients.High titer of EBV was produced by B95 8 cell by induction with sodium n butyrate,and peripheral mononuclear cells from two different groups of hepatitis B patients were harvested and infected with EBV. The results showed that 4～8 weeks after infection 19 EBV transformed B cell lines from hepatitis B patients were established. It was found that colony formation of cells from A group appeared 2～3 weeks later than B group. Cells grew healthily and had good activity after 4 weeks of freezing.CD19 and CD20 were detected in 88 34% and 37 48% of the immortalized B cell membrane respectively. HBV DNA could not be detected in a11 19 immortalized B cell lines.It suggested that the time needed for the establishment of B cell lines was related with the immune state of the patients.HBV DNA could not exist persistently, and it would disappear finally in the immortalized B cell lines.

Objective In order to study the biological significance of HBV/C gene variant in vitro.Methods Retroviral vector pXT1 was used to construct the HBV/C gene expression vector and the recombinant plasmid pXT1-HBV/C was used to transfect immortalized human peripheral blood B cell lines to express HBcAg steadily in the host cells.Results plasmid pXT1-HBV/C was detected positive by PCR as well as enzyme digestion with Bgl Ⅱ and Xho Ⅰ.Meanwhile.transfected cells was detected to contain HBV/C gene by PCR and HBcAg was expressed in 47.4%of the ceils by means of flow cytometry.Conclusion Retroviral expression vector with HBV/C gene can transfeet into eucaryotic cells effectively and express the goal gene steadily.The recombinant cells may be used in the systematic studies on the biological significance of HBV/C gene variant.

Objective To observe the analgesic effects of tramadol combined with atropine during the oocyte retrieval operation in assisted reproduction treatment(IVF/ICSI-ET)? Methods Three hundred patients(four hundred cycles of infertility totally)in the Center for Reproductive Medicine were equally assigned into treatment group and control group according to the different analgesia: the former were treated with intramuscular injection of 100 mg tramadol combined with 0?5 mg atropine for analgesia before the operation and the latter with 50 mg pethidine hydrochloride? The two groups were compared in terms of blood pressure,pulse,degree of pains, rate of fertility,rate of cleavage and rate of transplantable embryo? Result There were no differences between them in terms of blood pressure,pulse,pain degree,rate of fertility and rate of cleavage and rate of transplantable embryo(all P > 0?05),but the incidence of adverse effect in the treatment group was significantly lower than that in the control group(P < 0?05)? Conclusion The application of tramadol hydrochloride combined with atropine sulphatev for analgesia during oocyte retrieval operation of IVF-ET is advantageous for its safety,lower incidence of advers effect and stable vital signs?

Objective To investigate the expression and genetic alterations of KLF6 in hepatocellular carcinoma (HCC) and explore their functional mechanisms in the oncogenesis and development of HCC. Methods Real-time quantitative-PCR, direct sequencing and LOH approaches were used to detect KLF6 genetic abnormalities in HCC. The experiment had 2 groups, an experimental group and a control group. In the experimental group, the transfected plasmid pcDNA3.0 was recombined with KLF6 and tranfected into HCC HepG2 cells. MTT, flow cytometry and Western blotting were used to observe the effect of anti-oncogene wild type KLF6 on HepG2 cells by transgenic method for 48 h.Results Expression levels of KLF6 were significantly downregulated in HCCs(P＜0. 01), as detected by qRT-PCR. LOH occurred in 11 (52%) of the 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 29%, and sequence changes located in activation domain of KLF6. Meanwhile, MTT assay showed a significant antiproliferative effect of the transfected wtKLF6 on HepG2 cells(42.7%, P＜0.05). Fluorescence-activated cell sorting analysis revealed that KLF6 induced apoptosis. Conclusion The deregulation of KLF6 together with genetic abnormalities of allelic imbalance and mutations may play an important role in HCC pathogenesis.

OBJECTIVETo investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system.

METHODSHepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis.

RESULTSCloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L.

CONCLUSION25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.

Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis B e Antigens ; genetics ; isolation & purification ; metabolism ; Plasmids ; genetics ; Protein Precursors ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; isolation & purification ; metabolism

In recent years, Chinese hamster ovary (CHO) production vessel volume has reached more than 1 000 L in Chinese biopharms, and 10 000 L in foreign big biopharms, such as Lonza and Genetech. In general, there are some steps seed bioreactor for seed expansion, which decreases the efficiency of production process. In this work, a perfusion-based process was developed to drastically increase the split ratio during the scale-up of CHO cell cultures. Fed-batch cultures were inoculated with cells propagated in either batch or perfusion cultures that grown in disposable Cellbags using the WAVE Bioreactor system. The higher cell concentration of 2 x 10(7) cells/mL with 95% viability allowed to increase the split ratio to about 1:50-1:100 for inoculum propagated in perfusion culture. The method described here could reduce the number of required expansion steps and eliminate two or three bioreactors. Disposable perfusion bioreactor with only a few liters working volume have the potential to directly inoculate volumes of up to 1 000 liters. This would allow to shorten process time in these bioreactors, which often are the bottleneck in plant throughput.
Animals ; Bioreactors ; CHO Cells ; cytology ; Cell Culture Techniques ; instrumentation ; methods ; Cricetinae ; Cricetulus

Objective To study the effective dose and precaution time of the irradiation of the close contact from the radiators who underwent implantation of radioactive 125Ⅰ seeds so as to guide scientifically people how to avoid radiation damage.Methods Twenty patients with different types of cancer underwent implantation of radioactive 125Ⅰ seeds with the median value of implantation depth of 2.16 cm.Within 24hs after the operations the dose rates 30 cm and 100 cm from the skin were measured with pocket-size radiometer so as to imitate the situations of the close contacts.The effective doses and precaution times of different persons were calculated according to relevant formula.Results The dose rate a person received at the same time points (1,54,78,and 109 d,respectively) decreased along with the increase of the distance from the skin (t =5.962,5.961,5.961,5.962,P ＜ 0.05).and the dose rate a person received at the same distance from the skin decreased along with the extension of time (30 cm:t =6.236,6.236,6.235,P＜0.05;100 cm:t=7.310,7.315,7.314,P＜0.05).At different time points,the dose rates at 30 cm distance point were all significant higher than those at the 100 cm point (P ＜0.05).The adult living together,minors and pregnant women sharing the room,colleagues,adults who slept together with the patients began to reach the 50％ dose constraint values 0,54,78 and 109 days after the operation.Conclusions After their precaution time,it's safe to contact with the patients for the groups;otherwise,it's necessary to take some protect works within the precaution time.

Objective To investigate the application effect of the Beckman Coulter PK 7300 automatic blood group analyzer and the microplate method in ABO blood group screening .Methods A total of 12000 EDTA anticoagulation whole blood samples from January to May 2015 were collected from voluntary blood donors .The Beckman Coulter Pk7300 automatic blood group analyzer and STAR sampling microplate manual colorimetric method were to conduct the detection analysis .Results The accuracy for detecting ABO blood group had no statistically significant difference between the two methods (P>0 .05) .In the ABO blood group screening , the detection rates of ABO subtype and antibody weakening in the Beckman Coulter Pk 7300 automatic blood group analyzer were higher than those in the micro plate method .3 cases of ABO blood group typing and reverse typing were consistent in the detection by the Beckman Coulter Pk7300 automatic blood group analyzer ,but was inconsistent in the detection by the microplate method .4 cases of ABO blood group typing and reverse typing were inconsistent in the the detection by the Beckman Coulter Pk 7300 automat-ic blood group analyzer ,but was consistent in the detection by the microplate method .Conclusion The Beckman Coulter Pk7300 automatic blood group analyzer can safely and effectively conduct the ABO blood group screening in blood donors .The samples of suspicious detection results still need to conduct the manual interpretation by combining with the test tube method .

Objective: To explore the characteristics of pain sensitivity of autism spectrum disorder (ASD), and to provide reference for clinical comprehensive intervention of ASD. Methods: A case-control study was conducted to investigate the characteristics of pain sensitivity in 142 ASD children and 142 normal children using the items related to pain sensitivity in the Autism Treatment Evaluation Checklist (ATEC). In addition, two recognized ASD model mice (BTBR mice and model mice induced by VPA) were selected as experimental group. The thermal pain threshold and mechanical pain threshold of BTBR mice were measured by electroshock seizure threshold and Von Frey filament test, and the differences of pain characteristics between BTBR mice and control mice were compared, the thermal pain threshold of model mice induced by VPA (VPA rats) was measured by electroshock seizure threshold, and the differences between BTBR mice and control mice (Con) were compared. Results: There was significant difference in pain sensitivity between ASD group and control group (χ 2=0.81，P<0.05), and the sensitivity of ASD children to pain was significantly lower than that of normal control children. The pain sensitivity of BTBR mice and C57BL/6 mice on the 42 nd day after birth was measured. The T-test analysis showed that the time taken for BTBR and C57BL/6 mice to retract their feet on the 42 nd day after birth (3.62±0.38,3.02±0.33)s (t=3.28,P<0.01), and the mechanical pain threshold (9.75±3.58,0.55±0.93)s (t=7.44,P<0.01). The detection of thermal stinging pain in VPA rats and con rats on the 9 th, 11 th, 13 th and 15 th day after birth was detected. The results of t test were as follows:P9(11.34±1.38,9.81±1.64)g, P11(11.37±1.98,9.36±1.11)g, P13(11.53±1.38,9.51±1.01)g and P15(12.05±2.91,8.74±1.60)g (t=-2.79,-2.25,3.95,3.95,P<0.05). Conclusion Compared with normal control children, ASD children show insensitivity to pain which is further supported by two types of animal models for ASD.