ABSTRACT
A method of identification of C=C location and relative quantitation of unsaturated phosphatidylcholine ( PC ) isomers in breast cells by online photochemical reaction-pulsed directed current electrospray-tandem mass spectrometry ( PB-pulsed-dc-ESI-MS/MS) was established with benzophenone ( BP) as a photochemical reactant. The three-phase extraction method was used to extract the lipids in the cells, and then the C=C in the unsaturated PC and the carbonyl in BP were specifically cycled under the irradiation of 254 nm ultraviolet light (Paternò-Büchi, PB reaction). The PB products were ionized and mass-isolated for low-energy collision dissociation through the non-contact pulsed-dc-ESI ionization method. The double bond position and the relative content of the location isomers were obtained from the resulting ions in the MS/MS spectrum. The C=C location of 8 kinds of unsaturated PCs in MCF-7 and MCF-10A was detected, and the relative contents of 4 kinds of C=C location isoforms were analyzed. It was found that the relative abundance of △9 isomer in PC 16:018:1 was not significantly different between the two cells. The relative abundance of△9 isomers in PC 18:018:1 and PC 18:118:1 was slightly different. However, there is a big difference of △9 in LPC 18:1 between the cancer cell and normal cell (56. 0% ± 1. 3% vs. 71. 7% ± 6. 8%). The establishment of such a rapid and easy mass spectrometry method can analyze the C=C location and the relative content of location isomers, and it is expected to be a powerful tool to identify different cell states and different disease states.
ABSTRACT
<p><b>OBJECTIVE</b>To study the effects of nuclear factor (NF)-kappa B p65 ASODN on transforming growth factor beta-1 (TGF beta 1) and intercellular adhesion molecule-1 (ICAM-1) of rat hepatic stellate cells (HSC) and the mechanisms of NF-kappa B p65 ASODN in treating liver fibrosis.</p><p><b>METHODS</b>Type IV collagen enzyme digestion and density centrifugation methods were used to separate rat hepatic stellate cells. NF-kappa B p65 ASODN was manually synthesized and completely phosphorothioate-modified. The changes of TGF beta 1 and ICAM-1 mRNA were detected by RT-PCR and albumen of TGF beta 1 and ICAM-1 were detected by ELISA. The changes of NF-kappa B activity were determined by ELISA.</p><p><b>RESULTS</b>NF-kappa B activity and the expressions of ICAM-1 and TGF beta 1 increased after the HSC were treated by TNF alpha. NF-kappa B activity weakened after being treated with NF-kappa B p65 ASODN (0.001-1.000 micromol/L), P less than 0.05 in a dose dependent manner. Transferring NF-kappa B p65 ASODN (0.001-1.000 micromol/L) also weakened the expression of ICAM-1 and TGF beta 1 mRNA and the protein induced by TNF alpha in HSC. It was also in a dose dependent manner, P less than 0.05.</p><p><b>CONCLUSIONS</b>After transferring NF-kappa B p65 ASODN into HSC, their NF-kappa B activity decreased, and their mRNA and protein expressions of ICAM-1 and TGF beta 1 also decreased. This may serve as a new way in treating hepatic fibrosis.</p>