Hypoxia specific SDF-1 expression by retinal pigment epithelium initiates bone marrow-derived cells to participate in Choroidal neovascularization in a laser-induced mouse model.

PURPOSE: Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age-related macular degeneration (AMD). Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) plays a critical role in homing of bone marrow-derived cells (BMCs) to choroidal neovascularization (CNV). In this study, we investigated the contribution of hypoxia specific HIF-1α-induced SDF-1 expression in retinal pigment epithelium (RPE) cells and the potential role of SDF-1 in CNV formation. MATERIALS AND METHODS: Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells of gfp(+/+) transgenic mice to sublethally irradiated C57BL/6J mice. CNV was induced by laser photocoagulation. Ocular tissue was processed for immunofluorescence to detect HIF-1α and SDF-1 expression, and cell surface markers such as CXCR4, CD34 and CD31 and so on during CNV formation. In vitro, adult human RPE (hRPE) cells were cultured under conditions of chemical hypoxia using CoCl2 administration. And RNAi technique was used to knock down HIF-1α gene to observe the expression of HIF-1α and SDF-1 in hRPE cells. RESULTS: BMCs trafficked around laser lesion adjacent to RPE layer 4 h after laser photocoagulation, where SDF-1 expression was relatively higher. With increasing expression of SDF-1, more BMCs were infiltrated into laser lesion to participate in CNV, and both reached peak at 3 d (p < 0.05). About 81% BMCs involved in CNV were CXCR4+. Many of them acquired the surface marker of endothelial precursor cells (CD34+) and endothelial cells (CD31+). The constituent ratio of CD34+ and CD31+ BMCs increased with SDF-1 expression. In vitro, we proved that hypoxia specific-HIF-1α influenced SDF-1 expression in hRPE cells. CONCLUSIONS: These findings suggested that hypoxia-induced SDF-1 expression in RPE might be a critical initiator for recruitment of BMCs in CNV. SDF-1 might be another important factor in BMCs' differentiation into endothelial cells to participate in the CNV.

[Construction of Tet-on inducible CXCR1 eukaryotic expression plasmid and identification of the expression character in NIH3T3 cells].

AIM: To construct tetracycline (Tet)-controlled inducible vector CXCR1-pTREhyg, and then detect the expression character of CXCR1 under the regulation of Dox in NIH3T3 cells. METHODS: A full length cDNA of human CXCR1 was cloned from sample of fibroblast like synovium (FLS) in rheumatoid arthritis (RA) patient by RT-PCR and then sub-cloned into the pTREhyg plasmid after sequence analysis. CXCR1-pTREhyg and pTet-on was co-transfected with Lipofect2000 to NIH3T3 cells, and the expression of IL-8RA was detected by Western blot after given different concentration of Dox. RESULTS: Western blot showed that CXCR1 could be induced by Dox in NIH3T3 cells, and the phosphorylated Erk-1/2 level was significantly increased after IL-8 stimulation. CONCLUSION: Tet inducible recombinant vector of CXCR1-pTREhyg was successfully constructed, and it could be expressed in NIH3T3 cells. The stimulation of IL-8 obviously changed the activity of Erk-1/2 in the transfected NIH3T3 cells. This work has laid foundations for further study on the relationship between CXCR1 and RA disease.

Advances and Perspectives on Genetic Modification of Hevea brasiliensis / 中国生物工程杂志

As the major commercial source of natural rubber,Hevea brasiliensis attracts much attention.However,the heterozygous nature,long breeding cycle are strong limitations for conventional breeding.While genetic engineering,which can be used to widen the germplasm base and produce desirable agronomic traits quickly and efficiently,offers a viable alternative approach to complement traditional breeding.Comprehensive analysis indicates that in the past two decades,with calli derived from immature anther or integumental tissues of immature fruit as receptors,both biolistic and Agrobacterium-mediated transformation methods were employed for developping rubber genotypes with improved latex yield,tolerance to tapping panel dryness syndrome,producing high-value recombinant proteins,etc.Being recalcitrant to tissue culture,the transformation efficiency of Hevea is comparatively low,and the procedures are still needed to optimize.Finally,breeding objectives and strategies to improve transformation efficiency were also proposed in the review.

[Expression and antibody preparation of a novel serine protease ESP30].

AIM: To express ESP30, a novel serine protease, in Escherichia coli and to prepare anti-ESP30 antibody. METHODS: ESP30 gene was amplified by PCR from the genome of aeromonas hydrophila, and cloned into the expression vector pDH2. The ESP30 expression was carried out under thermal induction. The antiserum was prepared by immunizing rabbit with ESP30. The titer and specificity of the antibody were detected by ELISA and Western blot respectively. RESULTS: The ESP30 non-fusion protein with relative molecular mass (M(r)) of 66,000 was highly expressed in E. coli. The rabbit antibody against ESP30 was obtained. The ELISA titer of antiserum against ESP30 was about 1:128,000. Western blot analysis showed that the antiserum could bind to the expressed ESP30 specifically. CONCLUSION: The rabbit antibody against ESP30 has been successfully prepared, which lays the foundation for further studying the structure and function of the novel protease ESP30.

[Expression of GBD gene of Streptococcus mutans glucan binding protein A in mammalian cells].

OBJECTIVE: To evaluate the expression of recombinant plasmid pcDNA3.1/GBD of glucan binding protein of Streptococcus mutans in mammalian cells COS-7. METHODS: Eukaryotic plasmid carrying encoding gene of GBD of Streptococcus mutans gbpA was constructed and the plasmid was introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein in COS-7 cells was detected by immunochemistry technique. RESULTS: The positive expression was detected in plasma of the cells which were transfected with recombinant plasmid pcDNA3.1/GBD. The cells which were transfected with pcDNA3.1 were negative. CONCLUSION: GBD can translate and express in COS-7 cells after transfected with recombinant plasmid pcDNA3.1/GBD. The expressed protein locates in the plasma and the protein is able to combine with anti-GbpA antibody. The expressed protein has the antigenicity and is a candidate gene vaccine.

Effects of fibronectin, VEGF and angiostatin on the expression of MMPs through different signaling pathways in the JEG-3 cells.

PROBLEM: The objective of this study was to evaluate the possible signal pathway of fibronectin (FN), vascular endothelial growth factor (VEGF) and angiostatin (AS) on the expression of matrix metalloproteinases (MMPs) in JEG-3 cells. METHODS OF STUDY: JEG-3 cells were cultured and were examined for the effect of FN, VEGF and AS on the expression of MMPs by immunocytochemistry, gelatin zymography, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We found that up-regulation of the expression of MMPs was induced by FN and VEGF through the focal adhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) and Flt-1/p38SAPK/MAPKAPK2 signaling pathways, respectively. Furthermore, AS down-regulated the expression of MMPs through the integrin alphaVbeta3/FAK signaling pathway independent of the integrin-binding motif Arg-Gly-Asp (RGD). CONCLUSION: These data indicate that the expression of MMPs is regulated by many independent factors (such as FN, VEGF and AS) through different signaling pathways which influence the behavior of trophoblast cells.

[Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937].

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.