Objective To develop a method for monoclonal screening of CHO engineered cell lines using Verified In-situ Plate Seeding(VIPS). Methods Cell pool 1 stably transfected with target gene was inoculated by using monoclonal inoculation/imaging equipment(VIPS)in 96-well plates containing culture medium of different combinations(18 combinations,numbered 1～18)and volumes(100 and 200 μL/well). Monoclonal origin tracing pictures were taken,according to which,the monoclonal inoculation rate,clone formation rate and monoclonal proliferation rate were calculated and the image effects of monoclonal origin were evaluated. The applicability of this method was verified by cell pool 2,cell pool 3 and cell pool 4 stably transfected with target gene. Results The optimum monoclonal medium was Medium Ⅱ,and the volume of medium was 100 μL/well. Under the optimum process conditions,the monoclonal inoculation rate of Cell pool 1 was 80%,the clone formation rate was 83%,the proliferation rate was fast,and the monoclonal origin image was clear. While for Cell pool2,Cell pool 3 and Cell pool 4,the average rate of monoclonal inoculation was 78%,the average rate of clone formation was67%,the proliferation rate was slightly different and the image of cell division process was clear under the optimum process conditions. Conclusion In this study,the monoclonal screening method of CHO engineered cell lines developed using VIPS can improve the clone formation rate of CHO engineered cell lines and provide sufficient proof of monoclonal origin.

ObjectiveTo select a host cell line with traceable monoclonal origin and good growth status by serum-free suspension domestication culture of CHO-K1 cells,and verify its expression.MethodsAdherent CHO-K1 cells were domesticated into suspension cells suitable for serum-free medium by gradually decreasing serum concentration. Monoclonal cells were selected by cell seeding and imaging system Verified In-Situ Plate Seeding(VIPS system for short),and the parameters such as doubling time,agglomeration rate and cell diameter of monoclonal cells were comprehensively evaluated in continuous culture to determine the candidate clones. The candidate clones were transfected with plasmids contained green and red fluorescent proteins respectively,and one clone was identified as the final host cell of the engineering cell line according to the expression of fluorescent proteins.ResultsThe obtained CHO-K1 cell lines were adapted to serum-free suspension culture,and the doubling time was 20-24 h. The monoclonal cell line identified by monoclonal screening and yield evaluation had clear monoclonal origin and could be traced back. At an inoculation density of 0. 5 × 10~6cells/mL,the maximum growth density reached 8. 27 × 10~6 cells/mL through continuous cultivation of 7 d,the viability was not lower than 80% with the mean doubling time of 20. 31 h,and the exogenous gene was effectively expressed.ConclusionThrough serum-free domestication culture,CHO-K1 cell line with traceable monoclonal origin,good growth status and high protein expression was successfully obtained.

Objective To insert VEGF-R3(FIT-4)gene fragment into cloning vector and identify it.Methods By T-A cloning,the VEGF-R3 gene fragment amplified by RT-PCR was cloned on the PGEM-T Easy plasmid vector and transfected into E.coli JM109.the recombinant clones were screened by"white-blue plaque selection" and tested by the methods of single restrictional enzyme.Results The VEGF-R3 gene fragment amplified by RT-PCR showed a smear after the electrophoresis on agarose Gel and obtained white clones by"white-blue plaque selection".The electrophoresis result of the recombinant clones tested by the single restrictional enzyme was accordant with expectant result.Conclusion The recombinant clones through the methods T-A may be used in the further gene expression and gene therapy studies.

High-throughput screening is a regular approach available for identitying new lead compounds for the growing validated drug targets in drug screening. However, it has also introduced a large number of peculiar molecules which interfere drug screening. Pan assay interference compounds (PAINS) interfere with the progress of drug screening in various ways, such as interfering with a biochemical assay, modifying the protein, aggregate-based inhibitors and so on. So it is of vital significance to remove them. This paper has consulted the concept, category of PAINS and reviewed the way of PAINS interfering and the countermeasures to cope with them to direct the approach of high through screening and improve the hits percent.

Objective To study the relationship between the inhibitory effects that myelin membrane proteins exerted on nerve regeneration and the changes of intracellular cAMP levels in neurons. Methods To observe the effects of myelin membrane proteins extracted from central nervous system(CNS) by density centrifugation on the outgrowth of neurite of cerebella granule cells in culture and detect the changes of intracellular cAMP levels of the neurons with radioimmunoassay. Results 1\^Myelin membrane proteins of CNS inhibited neurite outgrowth of cultured cerebella granule cells. 2\^The cAMP level in neurons decreased in 5 minutes after contacting myelin membrane proteins and reached to the lowest level in 12 hours of contacting. Conclusion The inhibitory effects of myelin membrane proteins on the outgrowth of neurites may be related to the inhibitory factors which cause the decrease of cAMP level in neuron through the passway of signal transduction pathway. [

This paper introduces a solution to the development of next-generation telemedicine system based on Ipv6 with the analyses of present situation and related problems of telemedicine system.The devices,advantage of new system and a successful program based on Ipv6 are also mentioned.

Objective To explore an effective and safe therapeutic strategy in the treatment of coronoid process fracture com‐bined with ipsilateral zygomatic arch fracture .Methods Through the semi coronoid scalp incision ,`an open reduction and internal fixation of zygomatic arch fracture was done .The operation was modified that the area of zygomatic arch fracture was exposed ade‐quately ,and then the fracture fragments of zygomatic arch were turned up .The coronoid process fracture pieces were isolated and removed along with the direction of muscle fibers in the temporalis muscle .Results All Cases of the surgical incisions were healed by primary intention .After 3 - 24 months following up ,the function of mouth opening and closing ,and the other movements of mandible became normal .Conclusion Through the semi coronoid scalp incision ,zygomatic arch fracture reduction and internal fixa‐tion and the coronoid process fracture pieces removing can be done simultaneously .In this way ,an effective and safe therapeutic strategy for treating coronoid process fracture combined with ipsilateral zygomatic arch fracture .

Hypoxia is very common in solid tumor.It results in a series of changes of biological behavior in tumor cells.Hypoxia could facilitate malignant cells metastasis,in which the hypoxia inducible factor-1 plays an important role.The hypothesis for the tumor cells to metastasis is the degradation of the extracellular matrix(ECM).The ECM can be degraded by the matrix metalloproteinase,which is a critical factor in the process of tumor-metastasis.So far,it is not clear whether hypoxia have a role in the regulation of MMPs in tumor.So this review summarize the advanced knowledge on the influence of HIF-1 and MMPs in tumor-metastasis and the interaction between two genes.

Objective To explore the effects of different pressures of carbon dioxide pneumoperitoneum under laparoscopic surgery on IL-1β, IL-6, and TNF-α. Methods Ninety patients with ASA Ⅰor Ⅱwho were scheduled to elective operation under laparoscopic surgery from October 2010 to June 2012 were randomly divided into three groups .After endotracheal intubation , different carbon dioxide pressures , 10 mm Hg, 12 mm Hg, and 15 mm Hg, were orderly given to group 1, 2, and 3 to build pneumoperitoneum .The serum levels of IL-1β, IL-6, and TNF-α, as well as hemodynamic parameters , were assessed at the time after anesthesia ( T0 ) , after pneumoperitoneum development ( T1 ) , of position placement before operation ( T2 ) , after dismissing pneumoperitoneum (T3), and 24 hours after operation (T4), respectively. Results The measures of MAP, HR, and PETCO2 had no significant differences between each other of the three groups ( P >0.05), and the MAP and HR results showed significant differences among the three groups at different time points (P<0.05) whereas no difference for PETCO2 values (P>0.05).There were statistical significances between the three groups in levels of IL-1β, IL-6, and TNF-αat time points of T1 , T2 , T3 , and T4 , respectively (P<0.05).As compared with T0, there were significant differences in levels of IL-1βat the time points of T1 and T2 in the three groups (P<0.05).There were no significant differences in IL-6 levels between the group 1 and group 2 at every time points (P>0.05).In the group 3, the levels of IL-6 were significantly higher at time points of T 1, T2, and T3 than at T0(P<0.05).In the group 2 and group 3, the levels of TNF-αat T1, T2, and T3 were significantly different from at T0(P<0.05), whereas in the group 1, significant difference was seen only between the time points of T 0 and T1(P<0.05). Conclusion Low pneumoperitoneum pressure leads to minor stress effects .A 10 mm Hg carbon dioxide pneumoperitoneum is recommended .

Objective: To observe therapeutic effect of the method for tonifying the kidney and soothing the liver on impotence. Methods: 80 cases of impotence were randomly divided into a treatment group treated by Bushen Shugan Pills and a control group treated by Jingui Shenqi Pills. They were treated for 3 months and the International Erection Index Functional (IIEF-5)was determined at the end of 0,1,2,3 months after administration. Results:The total effective rate was 77. 5% in the treatment group and 52. 5% in the control group with a significant difference between the two groups (P