Objective:In order to study the effect of forkhead box c2 (Foxc2) on the development of skeleton and aorta Methods:Rat monoclonal anti mouse Foxc2 antibody was prepared by using recombinant fusion protein GST Foxc2, and used it to detect the expression of Foxc2 protein in C2C12 myoblasts and embryo of mouse Results:The titer of rat anti mouse Foxc2 antibody was 1: 5 000 This monoclonal antibody can specifically recognized Foxc2 protein producing in mouse L cells and human bladder carcinoma HTB9 cells transfected with CX Foxc2 plasmid Endogenous Foxc2 protein was detected in the mouse myoblast C2C12 cells and increased significantly in C2C12 cells after treatment with bone morphogenetic protein 2(BMP 2) Endogenous Foxc2 protein level was lowered markedly by transfecting C2C12 cells with antisense Foxc2 sequence Foxc2 protein in 11 5 dpc embryo was localized in the developing selerotome and mesenchymal tissue around the aorta Conclusion:Rat anti mouse Foxc2 monoclonal antibody was successfully prepared by using recombinant fusion protein GST Foxc2 The BMP 2 induced Foxc2 protein may play an important role in regulating the osteoblastic differentiation of C2C12 myoblasts and in the formation of axial skeleton and aortic arch

Objective To analyze nucleophosmin (NPM1) gene mutations in exon 12 in patients with acute myeloid leukemia (AML) and evaluate the clinical appliance of three methods which are frequently used for detecting gene mutation. Methods Genomic DNA from bone marrow of 54 AML patients was detected by PCR for NPM1 exon 12 and screened by PCR-capillary electrophoresis, denature high-performance liquid chromatography (DHPLC) and direct sequencing separately. FLT3-ITD (FMS-like tyrosine kinease internal tandem duplication) was detected by agarose gel electrophoresis and PCR-capillary electrophoresis. Results Seven AML sample harbored NPM1 gene mutations. Five of them were the most common mutation, known as type A (an insertion of a TCTG tetranucleotide at position 960 bp). One of them was type D (an insertion of a CCTG tetranuclectide at position 960 bp). The new variant was a deletion of a TGGCAGTG sequence at 958 bp and insertion of a GCCCGCGGTTTA sequence instead. The detection ratio of the three methods was all 100% and capillary electrophoresis was more rapid, reliable and easier than the other two methods. Moreover it could detect FLT3-ITD simultaneously. The resolving power of DHPLC was affected by many factors. The direct sequencing method was tedious and the heterozygous sequence might be misread. Conclusions There is a new mutation at position 958 bp with a 12-nucleotide insertion and substitution. PCR-capillary electrophoresis is convenient to screen NPM1 mutations of AML in clinical practice.

Objective To study the human lymphangiogenesis in the early stage of embryo and the expression of forkhead box C2(FOXC2) in human lymphangiogenesis.Methods Lymphatic vessel endothelial haluronic acid receptor-1(LYVE-1) was used as the special marker of lymphatic vessels.Immunohistochemical and double immunofluorescence staining were used to detect the expressions of FOXC2 and LYVE-1 in lymphatic vessels of 85 human embryos of 5 to 11 weeks pregnancy and analyze the features of lymphatic vessel genesis and development.Results LYVE-1 was initially detected in lymphatic vessels in 7-week human embryos.The lymphatic vessel endothelial cells presented brown staining for LYVE-1 in jugular and thoracic region.LYVE-1 was also found to express in human embryonic mesentery lymphatic vessels on the 70th day of pregnancy.The expression of FOXC2 in human embryo was detected prior to that of LYVE-1.At the beginning of the 6th week of pregnancy,FOXC2 was obviously seen in human embryonic mesoderm mesenchyme.FOXC2 expressed not only in human embryonic lymphatic vessels,but also in the vertebral body,cardiovascular wall and other tissues.The expressions of FOXC2 and LYVE-1 were still seen in human embryonic lymphatic vessel endothelial cells after 80 days of pregnancy.Conclusion Lymphangiogenesis of human embryo begins between the 7th and 8th weeks of pregnancy.FOXC2 and LYVE-1 could have some relation with the human embryonic lymphatic vessel genesis and development.

A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.
Animals ; Chemokine CXCL13 ; Chemokines, CXC ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sequence Analysis, DNA