BACKGROUND: Akt gene plays an important role in cell survival, proliferation, metabolism, and apoptosis. Although mechanism of Akt gene still remains unclear, it, a major element of survival signal, has aroused much attention in the domain of molecular biological research. OBJECTIVE: To construct eukaryotic expression vector pDC316-Akt1 for Aktl gene and to observe the expression in 293 cells. DESIGN, TIME AND SETTING: An observational study was performed at Molecular Biological Laboratory of Academy of Military Medical Sciences of Chinese PLA between September and December 2006. MATERIALS: pCD316 plasmid was provided by Benyuan Zhengyang Company; DH5α and 293 cells were reserved in our laboratory. METHODS: Aktl DNA segment was cloned from total RNA of hepatic tissue using RT-PCR and inserted into eukaryotic expressing vector pDC316 between EcoRI and Hind Ⅲ sites after sequencing. And then, 293 cells were transfected with the recombinant by liposome. MAIN OUTCOME MEASURES: Aktl gene content in transfected 293 cells using Westem blotting. RESULTS: Sequencing test indicated that Aktl amino acid sequence was in accordance with wild Aktl sequence. Akt-pDC316 was transcripted in 293 cells, and Akt1 protein with relative molecular weight of 55 000 was highly expressed in 293 cells when assayed using Western blotting. CONCLUSION: Eukaryotic expression vector for Aktl gene highly expresses in 293 cells.

Objective: This study aimed to observe the long-term effect of Pseudomonas aeruginosa preparation used in peritoneal injection of advanced colorectal cancer patients during surgery. Methods: A total of 83 colorectal cancer patients who received surgery between September 2006 and March 2008 were enrolled in this study. The patients were divided into two groups. Palliative resection and a 10 ml P. aeruginosa peritoneal injection were performed in 30 of 83 patients in the treatment group. Simple palliative resection was conducted in the other 53 patients, which comprised the control group. Both groups were then treated by regular chemotherapy and radiotherapy. Results:The follow-up visit was completed in 79 of 83 patients, with a high follow-up rate of 95.2%. No significant difference was found in the five-year overall survival time between the two groups (P=0.403). However, the five-year median survival time in the control group was only 13.9 ± 2.14 months, whereas that in the treatment group was 17.2 ± 2.12 months. Conclusion: Within a short period, peritoneal injection of P. aeruginosa during surgery could confer certain survival advantages for advanced colorectal cancer patients. However, the long-term effect of this therapy remains unknown.

Objective: To investigate the relation of Treg/Th17 ratio with HBeAg loss in chronic HBV infection patients during enticarvir antiviral treatment. Methods: The sera and peripheral blood mononuclear cells (PBMCs) were obtained from the enrolled chronic HBV infection patients and healthy controls at different time points of enticarvir antiviral treatment. The HBV markers, HBV-DNA, serum alanine transaminase(ALT), the HBV-specific IL-17 levels, and the frequencies of Th17, Treg cells in PBMCs were determined. Results: HBV-DNA and Treg cell frequencies were decreased and the frequency of Th17 cells was rapidly increased in all patients; the secretion of IL-17 decreased upon specific stimulation with HBcAg. Therapy induced inhibition of HBV replication led to great decrease of Treg/Th17. The decrease of Treg/Th17 ratio was closely related to loss of serum HBeAg at the fourth week. Conclusion: Inhibition of viral replication can not only reduce the activity of Th17 cells, but also quickly decrease the Treg/Th17 ratio. The Treg/Th17 ratio at the fourth week can serve as a marker for the effect of entacavir treatment for chronic hepatitis B.

AIM: To evaluate the effects of 23G vs 20G pars plana vitrectomy ( PPV ) combined with internal limiting membrane peeling, phacoemulsification and intraocular lens implantation for macular epiretinal membrane with cataract. ·METHODS: Totally 45 eyes of 45 patients with macular epiretinal membrane and cataract were enrolled in this retrospective non-randomized controlled clinical study. All eyes were treated with PPV combined with internal limiting membrane peeling, phacoemulsification and intraocular lens implantation. There were 20 eyes in 23G PPV group, and 25 eyes in 20G PPV group. The best corrected visual acuity ( BCVA ) , intraocular pressure (IOP), counting of corneal endothelial cells ( CEC) and central retinal thickness ( CRT ) were examined before surgery. BCVA results were converted to the logarithm of the minimum angle of resolution ( LogMAR ) visual acuity. All operations were performed by the same doctor. Operation time for vitrectomy and membrane peeling, average ultrasound energy ( AVE) and effective phacoemulsification time ( EPT ) were recorded. BCVA and CRT were observed postoperatively at 30d and 90d, counting of CEC was observed postoperatively at 90d. IOP was observed postoperatively at 1d and 7d. ·RESULTS:The mean operation time for vitrectomy were 12. 57± 1. 35min in 23G group and 17. 30 ± 1. 19min in 20G group. The difference was statistically significant ( t =-12. 488, P<0. 01). There were no statistical significances in operation time for membrane peeling, AVE and EPT between 23G and 20G groups ( t=-0. 68,-1. 186,-0. 737, P=0. 500, 0. 242,0. 465). On 1d after surgery, IOP in 23G group was lower than that in 20G group, the difference was statistically significant (t= -2. 345, P=0. 024). The BCVA and CRT of the two groups both improved after operations. There were no statistically significant differences between two groups in terms of IOP, BCVA, and CRT ( F = 0. 465, 1. 895, 0. 689; P = 0. 499, 0. 176, 0. 411). IOP, BCVA and CRT were significant statistical different in different time-point within each group ( F=291. 245, 103. 06, 665. 402, P<0. 01 ). Different surgical methods of 23G and 20G had interactive effects on IOP with different time points ( F = 13. 245, P<0. 01 ), but different surgeries had no interactive effects on BCVA and CRT with different time points (F=1. 212, 2. 293;P=0. 283, 0. 129). The counting CEC in 23G group was more than that in 20G group postoperatively at 90d, the difference was statistically significant (t=2. 049, P=0. 048). ·CONCLUSION: The 23G PPV combined with internal limiting membrane peeling, phacoemulsification, intraocular lens implantation for macular epiretinal membrane with cataract is effective. Compared with 20G PPV, 23G PPV has advantages in operation time for vitrectomy and counting CEC. But lower IOP is likely in 23G PPV on 1d after surgery

OBJECTIVETo identify the Cynomorii Herba and its analogues species using DNA barcoding technique.

METHODTotal genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2 (ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees.

RESULTThe ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification.

CONCLUSIONThe ITS2 region is an efficient barcode for identification of Cynomorii Herba, which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.

Objective To study the structure of class 1 integrons in 90 strains of Pseudomonas aeruginosa isolated during two periods of 1992-1996 and 2003-2005,and to get information about the structure changing of class 1 integrons by comparing their structures in two different periods.Methods Routine PCR and long PCR were performed to amplify the class 1 integrons and the gene cassettes they carried, followed with sequencing and blast via GenBank.Results Thirteen out of 41 strians ioslated during the period of 1992-1996 were positive on class 1 intergrons.Long PCR showed that the class 1 integron was 1868 bp in length and contained 2 resistance genes averagely.Six types of resistance genes of qacEA1 (n=6), sull (n=14),aadA1 (n=2),aadB (n=1),PSE-1 (n=2) and tetA (n=1) were found in these integrons, which consisted of 5 patterns of resistance cassette arrangements.Nineteen strains were proved to carry class 1 integrons in 49 isolates from 2003-2005.The mean DNA sequence length of them was 3383 bp with 3.6 resistant genes in averagely,10 types of resistance genes,qacEA1 (n=18),sull (n=25),aadA1 (n=6), aadB (n=7),aacA4 (n=2),PSE-1 (n=3),VEB-1 (n=4),OXA10 (n=1),cm1 A (n=1) and tetA (n =2),were identified in these integrons,which were composed of 9 patterns of resistance cassette arrangements.Conclusion In terms of produce length and resistance cassettes carried in the integrons, greater complexity is found in the structure of class 1 integrons in strains isolated during 2003-2005 than those during 1992-1996.

Objective In view of being short of the mammalian model in neuroma-in-continuity,the experiment injured the part of peroneal nerve to the formation of the neuroma-in-continuity and was applied to the foundation of farther research.Methods Twelve New Zeland rabbits were selected as experimental sub- jects randomly.One lateral peroneal nerves of twelve rabbits were resected,the damaged nervous tissues' slice were showed to the typical pathological changes of neuroma by the stain of HE,luxol fast blue after six weeks. As compared with the health sides of six model rabbits,the methods of real-time PCR and Western blot were used to evaluate the expression of CNTF,CGRP mRNA and protein in injured nerves and L_7、S_1 dorsal root ganglions respectively.Results The injured nerve formed the typical pathological changes of neuroma at six weeks.Compared with hea|thg side the expression of CNTF mRNA and protein was down-graded at the lateral of neuroma(P＜0.05),and the expression of CGRP mRNA and protein was up-graded(P＜0.05).Con- clusion The method of partly injuring the peroneal nerve could effectively set up the model of the neuroma-in- continuity,furthermore,resulted to the expression changes of the CNTF,CGRP mRNA and protein.

Objective To develop a real-time polymerase chain reaction(PCR)system to determine transcriptional level of MexAB-OprM multidrug efflux pump gene and to investigate the impact of androgra- pholide on MexAB-OprM gene transcription in Pseudomonas aeruginosa.Methods The fragments of mexB gene of mexAB-oprM operon and 30S rRNA gene rpsL were amplified and cloned into two plas- mids respectively.These plasmids were used as external standards for real-time PCR.Real-time PCR was applied to measure the mRNA transcripition of mexB and rpsL gene in Pseudomonas aeruginosa growing in medium with different concentrations of andrographolide.Results The plasmids for standard curve were constructed successfully.The relative mexB mRNA expressions in 50,100,150 and 200?g/mL andrographolide were 0.04?0.03,0.06?0.07,0.09?0.03 and 0.04?0.03 respectively, which were significantly lower than that in the control(0.24?0.04,P0.05).Conclusion Andrographolide can reduce the transcriptional level of MexAB-OprM,which may he one mechanism for its anti-infection effect.

Objective To observe and analyze the MRI manifestations of Klippel-Trenaunay syndrome(KTS).Methods Thirty-one cases with diagnosed KTS underwent MRI on a 1.5 T MR system. MRI,MR venography(MRV),MR angiography(MRA)and X-ray venography(XRV)were performed.The pathological changes of the limbs and their veins were observed.Results MRI found soft tissue hemangiomas in 12 cases,soft tissue swelling of the extremities in 27 cases,superficial varicosities in 21 cases,and malformation of the veins in 27 cases.In twenty patients who underwent both MRV and XRV, superficial varicosities in 17 cases and persistent sciatic veins in 11 cases were found with both techniques. The increase of tributary veins was found in 10 cases with XRV,while found in 15 cases with MRV.The erratic venous course was found in 4 cases with MRV.The abnormalities of deep veins were found in 8 cases with MRV,while found in 7 cases with XRV.Conclusion MRI is an efficient and reliable imaging method for diagnosis of KTS.

Objective To explore the diagnostic merits of the average apparent diffusion coefficient(ADCav) for leukoencephalopathy in neonates and children.Methods One hundred and fifty-six neonates and children with central nervous system signs or symptoms were classified into 6 groups according to their ages(1 d-0.05).Contrast to the normal,the ADCav of leukoencephalopathy in neonates and children decreased.With increasing age,there showed a linear downtrend in each group.Conclusions The ADCav rises in neonates and children with leukoencephalopathy.The ADCav variation precedes changes in routine MRI.