Detection,cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

AIM:To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1.METHODS:We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein.RESULTS:The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins.CONCLUSIONS:The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.     

Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Effect of mutant β-catenin gene on the proliferation of hepatocytes

AIM:The β-catenin is a key molecule in the Wnt signal pathway, which plays a critical role in normal development and tumorigenesis. However, the mechanisms of the β-catenin on the cell growth control are still not completely defined. The aim of this study was to test the hypothesis that the mutant β-catenin may regulate the hepatocyte proliferation. METHODS: The immortalized murine hepatocyte cell line, AML12, was used for this study. A plasmid that contain mutant β-catenin S33Y was transfected into the AML12 cells and a stable cell line AML12S33Y was established. The cell growth property of this cell line and the parental cell were compared by flow cytometry analysis and direct cell count. The cells were also tested for the ability to form soft agar colonies, and the ability to form tumors in the severe immune deficient mice (SCID). RESULTS:1. The mutant β-catenin containing cell line AML12S33Y has higher proliferating index compared with the parental AML12 cells (P<0.01), suggesting that mutant β-catenin promotes cell growth. 2. The mutant β-catenin cells formed small colonies in soft agar after 4 weeks of culture, but did not generate tumor in SCID mice. CONCLUSION:The mutant β-catenin promotes liver cell growth.     

Rat urotensin-II-induced main pulmonary artery contractions are mediated by MAPK

AIM: To investigate rat Urotensin-II(rat U-II)-induced vasoconstriction of rat main pulmonary arteries and the role of mitogen-activated protein kinase(MAPK). METHODS: The main pulmonary artery was dissected from the male Sprague-Dawley rats and artery ring width was 3-4 mm. Concentration-response curves were generated to rat U-II(0.03 nmol/L-30 nmol/L).Inhibitor of MAPK, PD 98059(0.1 μmol/L-10 μmol/L) were added into the medium after rat U-II(30 nmol/L)induced vasoconstriction had reached plateau to construct the relaxant concentration-response curves and their EC₅₀ and E_max. RESULTS：Rat U-II was a potent vasoconstrictor of isolated rat main pulmonary arteries [EC₅₀=7.95±0.40, E_max=(14.28±6.34)% of the response to 60 mmol/L KCl]; PD 98059 caused concentration-dependent relaxations of rat U-II precontracted arteries [EC₅₀=5.91±0.45, E_max=(81.39±13.65)%]. CONCLUSION: Rat U-II was a potent vasoconstrictor of rat main pulmonary arteries and this response was mediated through MAPK.     

Effects of TGF-β1 and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells(HSPC)

AIM:To study the effect of TGF-β₁ and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells (HSPC). METHODS:CD₃₄⁺cells were purified from fresh umbilical cord blood by immunomagnetic beads, and mononuclear cells were purified from bone marrow by Ficoll-hypaque. The effects of TGF-β₁ and /or TNF-α antisense PS-ODNS on ex vivo expansion of CD₃₄⁺ cells、CFU-GEMM、CFU-GM、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems.RESULTS:TGF-β₁ antisense PS-ODNS cooperated with cytokines increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E, which was as 4, 2.6, 2.7, 1.8, 2.1 times as that of the control (the cytokines combination), respectively. TNF-α antisense PS-ODNS cooperated with cytokines respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 4, 2.9, 2.6, 1.7, 1.8 times as that of the control. The above two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5.3, 2.1, 2.7, 1.9, 1.8 times as that of the control.CONCLUSION:Inhibition of endogenous TGF-β₁ and TNF-α by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.     

Effect of tea-polyphenols on the activation of NF-κB and the expression of TGF-β1 mRNA in THP-1 cells incubated with VLDL,LDL or ox-LDL

AIM:To investigate the effect of tea-polyphenols (TP) on the activation of NF-κB and the expression of TGF-β₁ mRNA in THP-1 cells (a human acute monocytic leukemia cell line). METHODS:THP-1 cells were incubated with the different concentrations of TP, VLDL, LDL or ox-LDL. In the THP-1 cellls, the nuclear malposition rate of NF-κB was detected with immunohistochemistry technique, the positive index of the TGF-β₁ mRNA expression was detected by hybridization in situ, and accumulation of total cholesterol (TC) in cells incubated with 0.4-40 μg/L TP was determined with oxidase assay. RESULTS:The nuclear malposition rate of NF-κB, the positive index of the TGF-β₁ mRNA expression and TC in THP-1 cells incubated with 0.4-40 μg/L of TP were lower than those with 0 μg/L of TP in TP-V group, TP-L group and TP-O (P＜0.05). The differences of these markers in THP-1 cells incubated with more than 40 μg/L TP in TP-V group, TP-L group and TP-O were not statistically significant, compared with TP-C group (P＞0.05). CONCLUSION:TP inhibited the activation of NF-κB, the expression of TGF-β₁ mRNA and the foam cell formation in the mono-macrophage.     

Effect of fibronectin on proliferation and collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats

AIM:To investigate the effects of fibronectin (FN) on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb_SHR) and WKY (CFb_WKY). METHODS:CFb derived from 12-week-old spontaneously hypertensive rat (SHR) and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by cell number counting and[³H]-TdR incorporation using 24-well plates pre-coated with 5 μg/cm² of FN. Collagen synthesis was determined by [³H]-proline incorporation. RESULTS:As compared with control, the cell number of fibroblasts derived from SHR and WKY were significantly increased to 163.75% and 170.42% respectively after 72 h incubation with FN in the presence of 0.4% FCS from a intial cell density of 1×10⁴ cells/mL. DNA synthesis of CFb was markedly promoted by FN. FN induced an increased in [³H]-proline incorporation in both CFb_SHR and CFb_WKY. CONCLUSION:FN is able to promote cell proliferation and collagen synthesis of CFb derived both from SHR and WKY.     

Bt对小地考虑呼吸作用的影响

利用红外CO2分析仪测定小地老虎幼虫呼吸节律的变化结果显示：小地老虎幼虫呼吸节律属于不规则波动型，取食含Bt烟叶的幼虫呼出的CO2量显著低于对照组，且呼吸节律的波动性也明显增大，说明小地考虑幼虫取食Bt后，呼吸代谢受到一定干扰，出现呼吸水平降低及代谢紊乱现象。     

几种除草剂药液表面张力、叶面接触角与药效的相关性研究

在除草剂氟磺胺草醚、灭草松和精喹禾灵药液中添加不同浓度的喷雾助剂ABS(十二烷基苯磺酸钠)和JFC ,以温室盆栽法测得各处理抑制杂草的效果,结果表明,在多数情况下添加喷雾助剂可使3种供试除草剂的药效显著提高。     

新型取代氨基(或芳氧)磺酰基苯氧丙酸酯的合成及其除草活性

由α-苯氧基丙酸酯出发,合成了取代氨基(或芳氧)磺酰基苯氧丙酸酯,并测定了它们的除草活性。所有的化合物均经1H NMR和元素分析确证,初步的生测结果表明上述化合物具有一定的除草活性。