Effects of nicotine on proliferation and chemosensitivity of A549 cells / 中国生化药物杂志

Objective To investigate the effect of nicotine on proliferation and chemosensitivity of A549 cells in vitro. Methods A549 cells was assessed by MTT assay to measure cell proliferation and was assessed by RT-PCR tomeasure chemosensitivity. Results 0.01~100μmol/L nicotine could promote the proliferation of A549 cells, the most marked proliferation at 1μmol/L, compared with the control group, the activity of A549 cells was increased by 1.85 times (P<0.01). When the concentration of nicotine above 1μmol/L, the proliferation of A549 cells had an decreasing tendency. When the concentration above 1000μmol/L, the proliferation of A549 cells can be inhibited. Nicotine can also reduce chemosensitivity of A549 cells to 5-FU, with the addition of nicotine, A549 cells survival rate increased significantly, the most marked at 1μmol/L, compared with the control group, the inhibitory rate of A549 cells was 9 % (P< 0.01). Nicotine significantly increased the expression level of α7 nAChR in A549 cells and decreased the expression of PTEN , in a concentration dependent manner. Compared with the control group, 1μmol/L of nicotine could increase the expression levels of α7 nAChR by 3.4 fold, and decrease the expression levels of PTEN by 60.36 % (P< 0.01). Conclusion Nicotinecan promote the growth of A549 cells and reduce chemosensitivity of A549 cells to 5-FU.

Effects of nicotine on proliferation and chemosensitivity of A549 cells / 中国生化药物杂志

Objective To investigate the effect of nicotine on proliferation and chemosensitivity of A549 cells in vitro. Methods A549 cells was assessed by MTT assay to measure cell proliferation and was assessed by RT-PCR tomeasure chemosensitivity. Results 0.01~100μmol/L nicotine could promote the proliferation of A549 cells, the most marked proliferation at 1μmol/L, compared with the control group, the activity of A549 cells was increased by 1.85 times (P<0.01). When the concentration of nicotine above 1μmol/L, the proliferation of A549 cells had an decreasing tendency. When the concentration above 1000μmol/L, the proliferation of A549 cells can be inhibited. Nicotine can also reduce chemosensitivity of A549 cells to 5-FU, with the addition of nicotine, A549 cells survival rate increased significantly, the most marked at 1μmol/L, compared with the control group, the inhibitory rate of A549 cells was 9 % (P< 0.01). Nicotine significantly increased the expression level of α7 nAChR in A549 cells and decreased the expression of PTEN , in a concentration dependent manner. Compared with the control group, 1μmol/L of nicotine could increase the expression levels of α7 nAChR by 3.4 fold, and decrease the expression levels of PTEN by 60.36 % (P< 0.01). Conclusion Nicotinecan promote the growth of A549 cells and reduce chemosensitivity of A549 cells to 5-FU.

Preparation of polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 / 中华烧伤杂志

<p><b>OBJECTIVE</b>To prepare the polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), and to determine the expression of EOLA1 in human umbilical vein endothelial cell (HUVEC).</p><p><b>METHODS</b>The protein samples (sample 1 and 2) expressing EOLA1 were purified and renatured. The protein concentrations were determined with bicinchoninic acid assay. The protein samples were identified with peptide mass fingerprinting (PMF) analysis. Protein sample with higher coincidence rate of amino acid sequence with theoretic protein was used to inoculate 4 mice; another 4 mice inoculated with adjuvant were used as control. Serum was isolated from collected mice blood. Polyclonal antibody of EOLA1 was purified with saturated ammonium sulfate precipitation, and was determined with ELISA for the titer (data were denoted by absorbance value). The expression of EOLA1 in HUVEC was determined with Western blot.</p><p><b>RESULTS</b>The concentration of protein sample 1 and 2 was respectively 0.124 16 mg/mL and 0.132 15 mg/mL. According to PMF analysis, the coincidence rate of amino acid sequence between protein samples and theoretic protein were 32% (protein sample 1) and 24% (protein sample 2). The polyclonal antibody of EOLA1 with titer more than 1:10 000 was obtained from mice inoculated with protein sample 1. The expression of EOLA1 protein in HUVEC was determined with polyclonal antibody of EOLA1.</p><p><b>CONCLUSIONS</b>The polyclonal antibody of EOLA1 can be prepared by inoculating mice with EOLA1 prokaryotic expressing protein, which can be used for determination of EOLA1 protein.</p>

Inhibition of integrin beta1 expression in human keratinocyte stem cells by vector-1 based interfering RNA strategy / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of integrin beta1 on the proliferation and differentiation of human keratinocyte stem cells (KSCs).</p><p><b>METHODS</b>DNA oligonucleotides targeting integrin beta1 at different locations were synthesized and inserted into BamHI-1 HindIII linearized p Silencer 3.1/H1 plasmids. The inserted sequences were verified by DNA sequencing. The KSCs were divided into control (without transfection), T1 (with transfection of vacant vector), T2 (with transfection of si integrin beta(1-1) vector), T3 (with transfection of si integrin beta(1-1) vector), and T4 (with transfection of si Negative vector) groups. The change in the expression of integrin beta1, was determined with Western blotting. The positive vector with the highest expression of integrin beta1 was selected and named as integrin beta1, and semi-quantitative RT-PCR was employed to detect the change in the expression of integrin beta1 mRNA.</p><p><b>RESULTS</b>The protein expression of integrin beta1, was not suppressed in control and T1 group, but it was suppressed in T2 and T3 groups, especially in T3 group (the suppression rate was 60%-70%, which was named si integrin beta1). The expression of integrin beta1 mRNA was obviously decreased by integrin beta1, transfection (the suppression rate was 70%).</p><p><b>CONCLUSION</b>The expression of integrin beta1, mRNA and protein could be down-regulated with recombinant si integrin P, vector transfection.</p>

Establishment of human endothelial-overexpressed lipopolysaccharide-associated factor 1 compelling expression model and its effects on the proliferation of ECV304 cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To design and construct the inducible expression vector of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), in order to establish EOLA1 compelling expression model, and to observe the effects of EOLA1 compelling expression on the proliferation of ECV304 cells.</p><p><b>METHODS</b>Inducible overexpression vector pOPRSV I-EOLA1 was constructed by amplifying the open reading fragment of EOLA1 and subcloning it into the Not I site and Xho I site of pOPRSV I vector. After sequencing, the pOPRSV I-EOLA1 recombinant vector and pCMVLac I vector were co-transfected into ECV304 cells. The cells resistant to G418 and hygromycin were screened by G418 and hygromycin, so that stable transfected cell strain was obtained. The growth curve of cells with or without isopropyl-beta-D-thiogalactoside (IPTG) induction were graphed with cell counting.</p><p><b>RESULTS</b>The inducible overexpressed EOLA1 vector was constructed successfully. The proliferation of the cells with EOLA1 compelling expression after induction of IPTG (44 +/- 17) x 10(4) was significantly higher than that without IPTG induction (27 +/- 11) x 10(4), (P < 0.01).</p><p><b>CONCLUSION</b>Compelling expression of EOLA1 protein can enhance the proliferation of ECV304 cell.</p>

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646).</p><p><b>METHODS</b>Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells.</p><p><b>RESULTS</b>A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatic analysis found that the EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons. EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P < 0.05).</p><p><b>CONCLUSION</b>The findings suggest that EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.</p>

Study on the injurious effect of a self designed micro-skin machine on the epithelia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the injury on micro-skin induced by a self designed micro-skin machine.</p><p><b>METHODS</b>Micro-skin was produced either with the machine or by hand. Cells at the edge of micro-skin were observed by transmission electron microscope. succinic dehydrogenase activity in supernatant of cultivated cells was analyzed, and the cell proliferation of micro-skin was assessed by (3)H-TdR. Twenty patients were enrolled in the study for the observation of the wound healing time between the two groups of micro-skin after being grafted.</p><p><b>RESULTS</b>Transmission electron microscope examination revealed that the cellular injury at the edge of the micro-skin in machine-made group was mild compared with that in man-made group. (3)H-TdR rate was elevated but the activity of succinic dehydrogenase in the supernatant of cultured cells decreased in supernatant of cultured cells of machine produced micro-skin. Wound healing time was shortened in machine made group. (P < 0.05).</p><p><b>CONCLUSION</b>The cellular injury at the edge of micro-skin in the machine made group was mild when compared with that in the man-made group with cell proliferation accelerated and wound healing time shortened.</p>