To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

Objective To explore changes of brain function among patients with partial epilepsy in resting state by using the blood oxygenation level dependent functional magnetic resonance imaging (fMRI) techniques.Methods fMRI scanning was performed in resting state among 60 patients with partial epilepsy and 60 gender,age and educational levels matched normal controls.The functional connectivity analysis was applied to calculate the default mode network ( DMN ) functional connectivity in resting-state fMRI.SPM5 was used to analyze differences in functional connectivity between the two groups( P ＜0.001,cluster ＞50).Results Left precuneus and adjacent posterior cingulate cortex ( Pcu/PCC),angular gyrus,and cingulate gyrus were involved in the DMN of epileptic patients.By contrast,the DMN of controls included left Pcu/PCC,right angular gyrus,bilateral medial frontal lobe and temporal lobe.Compared with normal controls,patients with partial epilepsy showed a significantly decrease in functional connectivity of DMN region such as left inferior parietal lobule,supramarginal gyrus,parahippocampa gyrus and superior temporal gyrus,and bilateral uncus,while no regions were found increased functional connectivity in patients group.Conclusions Patients with partial epilepsy show abnormal changes in functional connectivity of DMN in resting state by fMR],which may associate with the potential pathophysiological mechanisms of epilepsy.The findings demonstrate that the resting-state fMRI might detect the extensive changes of brain function in partial epilepsy with negative results of conventional MRI,suggestive of fMRI as an effective and non-invasive method to explore brain function in epilepsy.

AIM:To study the effects of noninvasive delayed limb ischemia preconditioning ( NDLIP) on ani-mal cardiac function , myocardial morphology and myocardial apoptosis after myocardial infarction ( MI ) .METHODS:Healthy SD male rats [n=45, weighing (250 ±10) g] were randomly divided into 3 groups:MI group:the animal model of MI was established by surgical ligation of left anterior descending artery ( LAD) after 2 weeks;NDLIP group:after the success of the MI animal model , NDLIP was carried out every other day until the 4th, 6th and 8th weeks;sham group:as the negative control group , the animals were taken heart LAD threading but no ligation .All rats were fed conventionally .At the end of the 4th, 6th and 8th weeks, all rats were made ventricular intubation , and then the hemodynamic parameters were recorded .The blood samples were withdrawn from the abdominal aorta and the serum was separated via centrifugation . The serum contents of Bcl-2 and Bax were measured by ELISA .Left ventricular anterior wall was homogenized .The mito-chondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲand Ⅳin the myocardial tissues were detected by ELISA .RESULTS:At the end of the 4th, 6th and 8th weeks, compared with MI group, left ventricular systolic pressure in NDLIP group was significantly increased , while left ventricular end-diastolic pressure in NDLIP group was significantly decreased ( both P<0.05).Mitochondrial respiratory chain complexesⅠ, Ⅱ, Ⅲ and Ⅳ in NDLIP group were significantly increased (P<0.05).The serum level of Bcl-2 in NDLIP group was significantly increased and Bax level was reduced remarkably (both P<0.01) .CONCLUSION:NDLIP improves the hemodynamic indexes , promotes the mitochondrial respiratory function and inhibits cell apoptosis , thus improving the prognosis of MI .

Aim To study the preventative effects of noninvasive delayed limb ischemic preconditioning ( NDLIP) on sudden cardiac death in rats with myocar-dial infarction. Methods Thirty healthy SD male rats weighting ( 250 ± 10 ) g were randomly divided into 3 groups:① myocardial infarction ( MI ) group: animal model of MI was established by making surgical ligation of animal LAD. ② MI plus NDLIP group: after the success of the animal model of MI, NDLIP was carried out every other day until 4th week. ③Sham group:as the negative control group, animals were taken heart LAD threading but no ligation. All rats were fed con-ventionally. At the end of 4 weeks, three groups of rats were administered with metaraminol ( 0. 2 mg · min-1 ) . ECG, drug cumulant of sudden death and death onset time were recorded. After sudden death, blood samples were withdrawn from abdominal aorta and serum was separated via centrifugation. ELISA method was used to measure serum caspase-3 , HSP70 and SOD concentration. Results While metaraminol led animal cardiac sudden death, the rats heart rate ( HR) kept declining with the increase of dosage of metaraminol during the administration period. Rat HR of MI+NDLIP group [ ( 479 ± 8 ) vs ( 416 ± 19 ) beat ·min-1 , ( 446 ± 32 ) vs ( 370 ± 20 ) beat · min-1 , (376 ± 53) vs (305 ± 29) beat·min-1, (307 ± 63) vs (244 ± 33) beat·min-1, (283 ± 45) vs (121 ± 35 ) beat · min-1 , P <0. 01 ] was markedly higher than that of MI group at 0 , 5 , 10 , 30 , 50 min before death. Compared with MI group, drugs cumulant to sudden death and death onset time of MI + NDLIP group [ ( 14. 58 ± 3. 03 ) vs ( 10. 76 ± 2. 73 ) mg, (72. 9 ± 15. 2 ) vs ( 53. 8 ± 13. 6 ) min, P <0. 01 ] were significantly increased. Compared with MI group, serum caspase-3 content of MI+NDLIP group was sig-nificantly reduced [ ( 2. 01 ± 0. 52 ) vs ( 2. 34 ± 0. 38 )μg·L-1 , P<0. 01 ]; HSP70 levels were remarkably increased [ ( 3. 01 ± 0. 58 ) vs ( 2. 70 ± 0. 43 ) μg · L-1 , P <0. 05 ]; SOD levels were significantly im-proved [(1. 99 ± 0. 65) vs (1. 70 ± 0. 58) mg·L-1, P<0. 01 ] . Conclusion NDLIP can prevent sudden cardiac death after myocardial infarction in rats, which may be mediated by reducing the myocardial cell apop-tosis, increasing protective protein expression and en-hancing antioxidant capacity.

The Flanders virus (FLAV) is a number of family Rhabdoviridae ,contains a single‐stranded ,negative‐sense vi‐ral RNA .Here we describe a molecular detection method developed for fast measurement of FLAV based on Taqman RT‐PCR method .In this study ,FLAV specific primers and probe were designed based on the FLAV L gene sequences published in GeneBank .Quantitative standard curve of FLAV TaqMan PCR was also successfully established .The specificity and stability test showed that the system is specific and the coefficient variables were all less than 1 .7% .Quantitative standard curve based on the genomic copy was drawn ,and the lowest detectable limit (LOD) of system was 100 copies/PCR ,with higher sensitivity and stability than that of the conventional RT‐PCR assay targeting the same gene .

Objective To analyze the growth phenotype and blood biochemical parameters of chromosome 1 substi-tution mouse strain(CSS1), and investigate their potential of QTL mapping .Methods Body weight, body length, tail length, organ weight of the CCS1 mice were measured at different days to create a growth curve while blood biochemical in -dexes were measured at about the 80th day.Results The CCS1 mice were different from C57BL/6 mice in several inde-xes.Compared with the C57BL/6 mice during different developmental stages , six strains including B6-Chr1KM mice were significantly different in body weight .There were five strains including B6-Chr1CM mice significantly different with C57BL/6 mice in body length, and all of the CSS1 mice were significantly different from C57BL/6 mice in tail length.Part of CCS1 mice were significantly different from C57BL/6 mice in the weight of liver, spleen, kidney and brain.The ALT of female B6-Chr1CM mice was significantly higher than that in the C 57BL/6 mice.The ALP of female B6-Chr1HZ mice was signifi-cantly higher than that in the male C57BL/6 and B6-Chr1KM mice, and was significantly lower than that in the C57BL/6 mice.The TB of male B6-Chr1CM, B6-Chr1SMX and B6-Chr1HZ mice was significantly higher than that of the C 57BL/6 mice.The TG of male B6-Chr1SMX mice and male B6-Chr1TW mice was significantly higher than that in the C 57BL/6 mice. Conclusions The phenotype of Chr1 CSS mice is quite different from commonly used inbred strain C 57BL/6 mice.CCS1 mice show great potential in QTL mapping for their characteristic growth phenotype and blood biochemical indexes .

Objective:To investigate whether caffeic acid phenethyl ester (CAPE) would improve peritoneal dialysis (PD)-associated peritoneal fibrosis by alleviating oxidative stress through activating nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway.Methods:Thirty-two male Sprague-Dawley rats were randomly divided into four groups by the random number table: control (CON) group (0.9% normal saline 20 ml/d intraperitoneal injection), CAPE group (0.9% normal saline 20 ml/d+CAPE 10 mg·kg -1·d -1 intraperitoneal injection), PD group [4.25% glucose peritoneal dialysis fluid (PDF) 20 ml/d intraperitoneal injection with lipopolysaccharide 0.6 mg/kg intraperitoneal injection at day 1, 3, 5 and 7], and PD+CAPE group (CAPE 10 mg·kg -1·d -1 intraperitoneal injection in addition to PD group), with 8 rats per group. On day 28, rats were euthanized after peritoneal equilibration test, and then the parietal peritoneum and omentum were collected for follow-up tests. To further investigate the mechanism, primary peritoneal mesothelial cells (PMCs) of rats were isolated and cultured. The PMCs were stimulated with 2.5% glucose PDF and added with 5 μmol/L CAPE intervention. The Nrf2 inhibitor (ML385) was used to identify whether CAPE protected PMCs from PDF by activating the Nrf2/HO-1 pathway. Histopathological staining was used to detect structural changes of the peritoneum, and immunohistochemical analysis was performed on cleaved caspase-3, Bax, α-smooth muscle actin (α-SMA), fibronectin (FN), and typeⅠ collagen (Col-Ⅰ) protein. Western blotting was used to detect the protein expression of α-SMA, FN, transforming growth factor-β1 (TGF-β1), HO-1 and nuclear Nrf2 (N-Nrf2). The apoptosis detection kit was used to detect apoptosis and flow cytometry was used to detect reactive oxygen species (ROS) in PMCs. The malondialdehyde (MDA) and superoxide dismutase (SOD) activity detection kit were used to detect MDA content and SOD activity. Cell immunofluorescence was used to analyze the protein expression of Nrf2 in PMCs. Results:Compared with the CON group, the PD group had thicker peritoneum, and the expression levels of cleaved caspase-3, Bax, α-SMA, FN, Col-Ⅰand MDA in peritoneum were significantly higher, while HO-1, N-Nrf2 protein expression and SOD activity were lower (all P<0.05). Compared with the PD group, the parietal peritoneum morphology of CAPE+PD group was improved, accompanied by reduced cleaved caspase-3, Bax, α-SMA, FN, Col-Ⅰ protein expression, and MDA content, while N-Nrf2, HO-1 protein expression, and SOD activity were higher (all P<0.05). Compared with the CON group, the PD group had significantly lower ultrafiltration volume and higher peritoneal permeability (both P<0.05). After CAPE intervention, the peritoneal transport function of the rats was significantly improved ( P<0.05). In cultured PMCs, PDF inhibited nuclear translocation of Nrf2 and protein expression of HO-1, and upregulated intracellular ROS level. In addition, PDF increased cell apoptosis and the protein expression levels of α-SMA, TGF-β1 and FN (all P<0.05). CAPE activated nuclear translocation of Nrf2, increased HO-1 protein expression, downregulated intracellular ROS level, and partially reversed PDF-induced cell apoptosis and epithelial- mesenchymal transition (all P<0.05). The protective effects of CAPE on PMCs were partially abolished by ML385 (all P<0.05). Conclusions:CAPE can reduce PD-induced PMCs apoptosis and epithelial-mesenchymal transition by attenuating oxidative stress, and significantly improve peritoneal fibrosis and ultrafiltration function. The beneficial effects of CAPE on peritoneum are related to activation of Nrf2/HO-1 pathway.

OBJECTIVETo study the effect of lentiviral vector mediated herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) on graft- versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo- BMT) in mice.

METHODSDonor splenic lymphocytes from C57BL/6 which were infected by lentiviral vectors carrying HSV-TK were transplanted into 60Co gamma ray irradiated recipient mice with donor bone marrow cells. GCV 25 mg x kg(-1) x d(-1) was administered in 3 groups on day 0, +7, +12 respectively after transplant for 7 days by intraperitoneal injection. Survival time, severity of GVHD, incidence of GVHD, T lymphocytes immune reconstruction and of allogeneic chimerism ratio were detected after allo-BMT.

RESULTSThe average survival times for GCV 0 day, +7 day and +12 day group were (30. 10 +/- 5.21) d, (36.40 +/- 5.28) d and (28.20 +/- 4.82) d respectively, being significantly longer than that in the control group [(15.10 +/- 0.43) d] (P < 0.05). The 50 d-survival rate for TK/GCV + 7 day group was 60%. While for 0 day and +12 day group was 40% and 30% respectively. The incidence of grade III approximately IV GVHD in the control group was 100%, and the dead mice in experimental groups showed pathological changes of II approximately III GVHD. Long-term alive recipient mice only developed grade I approximately II GVHD after allo-BMT. The number of CD4+ lymphocytes in experimental groups was higher than that in control group (P <0.05), but CD8+ lymphocytes was lower on day +5, +10, +15 day (P <0.05). Allogeneic chimerism rate of recipient mice on +30 d was 100%.

CONCLUSIONSHSV-TK/GCV induced by the lentiviral vectors has a definite effect in prevention of GVHD after allo-BMT. GCV administrated from 7 days post-transplantation showed the best effects.

Animals ; Bone Marrow Transplantation ; immunology ; Ganciclovir ; pharmacology ; Genetic Vectors ; Graft vs Host Disease ; prevention & control ; Lentivirus ; genetics ; Lymphocyte Transfusion ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Transfection ; Transplantation, Homologous

OBJECTIVETo summarize the effectiveness of Chinese and Western integrative medicine in treating medium and advanced lung cancer, and to provide guidelines for clinical application.

METHODSFor this metaanalysis, a comparative search of Chinese medicine data in Chinese National Knowledge Infrastructure (CNKI) and Chinese BioMedical Literature Database (CBM) was undertaken to identify articles related to randomized comparative research of Chinese and Western integrative medicine in treating medium and advanced lung cancer between 1996 to 2006. Quality of life (QOL) was estimated using RevMan 4.2 software for data processing, adopting the odd ratio (OR) and the 95% confidence interval (CI).

RESULTSThrough meta-analysis of 10 qualified articles, the results were as follows: the merging effectiveness of QOL [OR=3.80, 95% CI (2.65, 5.47)]; the rate of survival [OR=3.44, 95% CI (2.04, 5.80)]; the tumor response rate [OR=1.88, 95% CI (1.37, 2.58)]; the tumor developing rate [OR=0.33, 95% CI (0.23, 0.48)]. Significant differences existed between the Chinese and Western integrative medicine treatment group and the Western treatment group (P<0.01).

CONCLUSIONSChinese and Western integrative medicine treatment of medium and advanced lung cancer has shown to improve patients' QOL and survival rate; it also can control tumor development in the short term.

Carcinoma ; epidemiology ; pathology ; therapy ; Combined Modality Therapy ; Disease Progression ; Humans ; Integrative Medicine ; methods ; Lung Neoplasms ; epidemiology ; pathology ; therapy ; Medicine, Chinese Traditional ; methods ; Neoplasm Staging ; Publication Bias ; statistics & numerical data ; Randomized Controlled Trials as Topic ; statistics & numerical data ; Treatment Outcome ; Western World

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.
Adenoviridae ; genetics ; metabolism ; physiology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genes, p53 ; Genetic Therapy ; Genetic Vectors ; Humans ; Neoplasms ; metabolism ; pathology ; virology ; Oncolytic Viruses ; genetics ; metabolism ; physiology ; Quality Control ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Virus Replication