This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology

Objective To evaluate the influence of Fuzhenghuayu decoction on fibrotic liver tissue and angiotensin-converting enzyme-angiotensin Ⅱ-angiotensin Ⅱ 1 receptor (ACE-Ang Ⅱ-AT1R) axis using a nonalcoholic fatty liver fibrosis rat model system.Methods Forty male Sprague-Dawley (SD) rats were randomly divided into the following groups:normal control group,liver fibrosis model group,and liver fibrosis model Fuzhenghuayu drug intervention at low-dose [0.75g/(kg.d)] group and high-dose [1.5g/(kg.d)] group.Except the normal control group,the other three groups were fed high-fat diet for 24 weeks to induce nonalcoholic hepatic fibrosis model.The drug intervention was administered via oral-gastric irrigation once daily for 6 times per week over a 6-week period.The rats were sacrificed at the end of 6 weeks for serum and liver tissue collection.The levels of serum total cholesterol (TC),triglycerides (TG),alanine aminotransferase (ALT),aspartate aminotransferase (AST) were measured by standard biochemical assays.The Ang Ⅱ contents of plasma and liver tissue were surveyed and evaluated by the radioimmunoassay method.Liver pathology was detected using HE staining and Masson trichrome staining.The mRNA and protein expressions of ACE,AT1R,α-smooth muscle actin (α-SMA) in the liver tissue were evaluated with real time-PCR,immunohistochemical staining,respectively.Results Compared with the model group,the levels of serum ALT and AST in the low-dose group and high-dose group decreased conspicuously,especially in the high-dose group,with a statistically significant difference (P＜0.05);While the difference in the levels of serum TC and TG between the three groups was not statistically significant.Compared with the normal control group,Ang Ⅱ levels in plasma and liver tissue significantly increased in the other three groups;Further more,there was no significant difference in the plasma Ang Ⅱ level between the three groups (P＞0.05);While the level of liver tissue Ang Ⅱ decreased significantly in the low-dose group and high-dose group than that in model group (P＜0.05).Compared with the model group,the extent of pathological changes in hepatic tissues ameliorated after Fuzhenghuayu intervention according to HE and Masson staining,especially in the high-dose group.According to real time-PCR and immunohistochemical staining,the mRNA and protein expressions of ACE,α-SMA and AT1R decreased significantly in low-dose group and high-dose group than that in model group (P＜0.05),and the high-dose group showed the most robust decrease.Conclusions The Fuzhenghuayu decoction reduces nonalcoholic fatty hepatic fibrosis effectively,thereby leading to down-regulated the expressions of ACE-Ang Ⅱ-AT1R axis.These effects may represent the mechanism by which this drug suppresses hepatic fibrosis.

The traditional methods of skeletal age estimation mainly include Numeration, Atlas, and Counting scores. In recent years, other new methods were proposed by several scholars. Utilizing image logical characteristics of X-ray film to extrapolate skeletal age is a key means by present forensic medicine workers in evaluating skeletal age. However, there exist some variations when we present the conclusion of skeletal age as an "evidence" directly to the Justice Trial Authority. In order to enhance the accuracy of skeletal age determination, further investigation for appropriate methodology should be undertaken. After a collective study of pertinent domestic and international literatures, we present this review of the research and advancement on skeletal age evaluation methods of X-ray film.
Age Determination by Skeleton/trends* ; Carpal Bones/diagnostic imaging* ; Elbow/diagnostic imaging* ; Female ; Forensic Medicine/methods* ; Hand Bones/diagnostic imaging* ; Humans ; Image Processing, Computer-Assisted/methods* ; Male ; Radius/diagnostic imaging* ; Tomography, X-Ray Computed/methods* ; X-Ray Film