The partial segment of Marek′s disease virus (MDV) glycoprotein B (gB) gene was amplified by PCR. The segment was cloned into pET-28a vector to obtain the recombinant pET-gB plasmid. The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level as inclusion body after induced with 1.0mmol/L IPTG. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His?Bind affinity chromatography. Mice were immunized i.p. by the purified protein to make the polyclonal antibody. The titer of the antibody by indirect ELISA was 1?10~ -5 . Moreover, the analysis by western blot proved that antibody was specific to the recombinant protein. These works lay a favorable foundation for the study of the immune response by MDV gB.

Animals ; Bufo bufo ; Caspase 3 ; genetics ; metabolism ; Female ; Immunohistochemistry ; Male ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

Dental Bonding ; methods ; Dentin ; Dentin-Bonding Agents ; chemistry ; Electricity ; Humans

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Objective To study the clinical efficacy of Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced non-small cell lung cancer (NSCLC) and its influence on the levels of immune function in patients. Methods Totally 94 elderly patients with NSCLC were divided into observation group and control group, with 47 cases in each group. Both groups were given targeted cryoablation therapy and under CT review after surgery to observe lesions condition. Observation group was given Qingfei Sanjie Pills, once a bottle, twice a day, orally, for three months. Recent efficacy of the two groups were evaluated. The levels of IgG, IgA, CD3+, CD4+, CD8+, and complications in the two groups were detected. The survival rate of 1 year, 2 years and 3 years in the two groups were observed. Results Two cases in the observation group and three cases in the control group were invalid. The total effective rate was 84.4% (38/45) in observation group and 65.9% (29/44) in the control group, with statistical significance (P<0.05). After the treatment, the levels of IgG, IgA, CD3+, CD4+and CD8+in the observation group were significantly higher than the control group (P<0.05). After treatment, the incidence rates of increased cough, postoperative pain, bloody sputum, fever and nausea and vomiting in the observation group were lower than the control group. The 1 year, 2 years and 3 years survival rates were 91.1%, 75.6% and 64.4% in observation group, and 79.5%,63.6% and 52.3% in the control group,with statistical significance(P<0.05).Conclusion Qingfei Sanjie Pills combined with targeted cryoablation therapy on elderly patients with advanced NSCLC has good clinical efficacy, which can improve immune system of patients and increase survival rate.

Amounts of researches show that EPO is characterized with neurotrophic and neuroprotective manner, especially in brain stroke, which attracts a large numbers of researchers to study it. With the accumulating researches on its neuroprotection, many related mechanisms were revealed, such as antioxidant, anti-apoptosis, angiogenesis, anti-inflammatory, which suggests a multiple targets role of EPO on brain stroke. However, because of the high risk of thromboembolism in clinical administration of rhEPO and its analogs, the herbs are potential to be a replacer for its less side effects. Many researchers suggested that a larger of herbs were founded having the action of increasing the endogenous EPO in the model of anemia and cerebral ischemia. At the same time, there herbs were also proved that they had the action of against cerebral ischemia while some without considering the role of EPO in the reports. Considering of the action of promoting EPO of these herbs and the neural protection of EPO, this essay mainly summarizes the studies of herbs promoting EPO in the cerebral ischemia and discusses the mechanism of regulating the EPO of these herbs, for the aim of finding the potential drugs against cerebral ischemia.
Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Erythropoietin ; genetics ; metabolism ; Humans ; Neuroprotection ; drug effects ; Plants, Medicinal ; chemistry

Ac-Phe-Lys-PABC-DOX (PDOX) is a smart doxorubicin (DOX) prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. This study was aimed at elucidating the effectiveness and toxicities of DOX and PDOX in patient-derived MCF-7 breast cancer cells in vitro. The MCF-7 cells were exposed to both PDOX and DOX, and cytotoxicities, cell cycle and P53/P21 signaling alterations were studied. Abundant cathepsin B was found in the MCF-7 cells, and treatment with PDOX and DOX triggered dose- and time-dependent cytotoxicity and resulted in a significant reduction in cell viability. The IC50 of PDOX and DOX was 3.91 and 0.94 μmol/L, respectively. Both PDOX and DOX caused an up-regulation of the P53/P21-related signal pathway, and PDOX significantly increased expression of P53 and caspase 3, and arrested the cell cycle at the G1/G2 phase. As compared with DOX, PDOX reduced toxicities, and it may have different action mechanisms on breast cancer cells.

To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(=6.397,=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(=8.241,=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(=2.916,=0.0434;=3.452,=0.0260). In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.
DNA Damage ; DNA Replication ; Human papillomavirus 16 ; Mitosis ; Oncogene Proteins, Viral

OBJECTIVETo establish discriminant functions of diarrhea-predominant irritable bowel syndrome (IBS-D) by studying it from quantitative diagnosis angle, hoping to reduce interference of subjective factors in diagnosing and differentially diagnosing Chinese medical syndromes of IBS-D.

METHODSA Chinese medical clinical epidemiological survey was carried out in 439 IBS-D patients using Clinical Information Collection Table of IBS. Initial syndromes were obtained by cluster analysis. They were analyzed using step-by-step discrimination by taking information of four Chinese medical diagnostic methods and serum brain-gut peptides (BGP) as variables.

RESULTSClustering results were Gan stagnation Pi deficiency syndrome (GSPDS), Pi-Wei weakness syndrome (PWWS), Gan stagnation qi stasis syndrome (GSQSS), Pi-Shen yang deficiency syndrome (PSYDS), Pi-Wei damp-heat syndrome (PWDHS), cold-damp disturbing Pi syndrome (CDDPS). Of them, GSPDS was mostly often seen with effective percentage of 34. 2%, while CDDPS was the least often seen with effective percentage of 5.5%. A total of 5 discriminant functions for GSPDS, PWWS, GSQSS, PSYDS, and PWDHS were obtained by step-by-step dis- crimination method. The retrospective misjudgment rate was 4.1% (16/390), while the cross-validation misjudgment rate was 15.4% (60/390).

CONCLUSIONThe establishment of discriminant functions is of value in objectively diagnosing and differentially diagnosing Chinese medical syndromes of IBS-D.

Alarmins ; Brain ; Cluster Analysis ; Diarrhea ; classification ; diagnosis ; Hot Temperature ; Humans ; Irritable Bowel Syndrome ; classification ; diagnosis ; Medicine, Chinese Traditional ; Qi ; Retrospective Studies ; Surveys and Questionnaires ; Yang Deficiency

OBJECTIVETo determine vacA dominant genotypes of Helicobacter pylori in patients with peptic ulcer (PU) or chronic gastritis (CG).

METHODSH.pylori strains were isolated from mucosa samples of gastric antrum and corpus of patients suffering from PU (n=29) or CG (n=34), 126 strains of H.pylori were selected for PCR to detect s and m regions in vacA gene of the isolates. Parts of the amplification products were sequenced after T A cloning. The correlation between infection or coinfection with different vacA genotypes of H.pylori and different gastroduodenal diseases was further analyzed.

RESULTSThe positive amplification products of vacA gene, s and m regions, were found in the DNA samples of all the isolates. In these products, s1a/m1, s1a/m2, s1a/m1b and s1a/m1b m2 genotypes of vacA gene were detected and s1b and s2m1a genotypes absent. Proportions of the s1a/m1, s1a/m2, s1a/m1b and s1a/m1b-m2 genotypes were 7.1% (9/126), 61.9% (78/126), 29.4% (37/126) and 1.6% (2/126), respectively. 17.5% (11/63) of the patients were confirmed to be coinfected with different genotype H.pylori strains. No statistical differences were found in the distribution of different genotype H.pylori strain infection in the gastric diseases (P>0.05). In comparison with the reported sequences of H.pylori strain 60190 with s1a genotype and strain 87-203 with m2 genotype, homologies of the nucleotide sequences of s1a PCR products from 6 strains of H.pylori isolates and m2 PCR products from 4 strains of H.pylori isolates were 93.15% approximate, equals 94.86%and 93.63% approximate, equals 97.61%, respectively.

CONCLUSIONH.pylori with s1a/m2 or s1a/m1b are the dominant genotypes in the PU or GC patients in Zhejiang area. The nucleotide sequences of partial amplification products from the vacA dominant genotypes of H.pylori show high homology compared with the reported sequences. Part of the patients may be coinfected with different vacA genotypes of H.pylori.

Adolescent ; Adult ; Aged ; Bacterial Proteins ; genetics ; Base Sequence ; Child ; Female ; Genotype ; Helicobacter pylori ; genetics ; Humans ; Male ; Middle Aged