Airway epithelial cell-derived insulin-like growth factor-1 triggers skewed CD8 +T cell polarization / 中国药理学通报

Aim To investigate the effects of airway epithelial cell-derived insulin-like growth factor-1 (IGF1) on CD8 +T cell polarization. Methods Hu-man airway epithelial cell line, RPMI2650 cells, was cultured in the presence of a mice allergen, Der p1, for 72 h. IGF1 expression was checked with quantita-tive RT-PCR and Western blot. Der p1-primed RP-MI2650 cells, recombinant IGF1 and anti-IGF1 anti-body was cocultured respectively with CD8 + T cells, which were activated by anti-CD3/CD8 Ab. Apoptotic cells frequency was calculated with flow cytometry. The alteration of p53 gene hypermethylation in CD8 + T cells elicited by Der p1-primed airway epithelial cell and IGF1 was plotted. Results Both mRNA(23. 1%± 5. 2% vs 5. 2% ± 2. 3%, P < 0. 01 ) and protein (33. 4 ± 6. 4 vs 9. 2 ± 4. 6, P <0. 01 ) expression of IGF1 in RPMI2650 cells markedly increased after ex-posure to Der p1 . The increase of apoptotic CD3/CD28 Ab-activated CD8 + T cells was abolished by the pres-ence of Derp1-primed epithelial cells ( 41. 7% ± 8. 2%vs 5. 2% ± 1. 8%, P <0. 01 ) . The results were con-firmed by the addition of recombinant IGF1 . Anti-IGF1 antibody abolished the effect of the epithelial cells. Derp1-primed epithelial cells inhibited p53 gene mR-NA( 29. 1% ± 5. 9% vs 16. 2% ± 4. 3%, P <0. 01 ) and protein ( 63. 3 ± 8. 9 vs 26. 9 ± 5. 6 , P <0. 01 ) ex-pression. Anti-IGF1 antibody abolished the effect. Re-combinant IGF1 promoted CD8 + T cells′p53 gene hy-permethylation. Conclusion Der p1 induces RP-MI2650 cells to produce IGF1 , and this factor prevents CD8 + T cell apoptosis by inducing p53 gene hyperm-ethylation.

PLCE1 suppresses apoptosis of lung adenocarcinoma cells via modulating p53 expression / 中国病理生理杂志

AIM:To investigate the role of phospholipase C epsilon 1 ( PLCE1 ) in modulating the apoptotic mechanism in lung adenocarcinoma A 549 cells.METHODS:PLCE1 inhibitor U-73122 was used to suppress the expres-sion of PLCE1.The expression of PLCE1 and p53 in A549 cells was evaluated by quantitative real-time PCR and Western blotting.Apoptosis was assessed by flow cytometry .RESULTS:A549 cells expressed high level of PLCE1 and low level of p53.Inhibition of PLCE1 markedly increased the expression of p 53, and increased the apoptosis of A 549 cells.CON-CLUSION:PLCE1 suppresses apoptosis of A549 cells via inhibiting the expression of p53.

PLCE1 modulates p53 expression and apoptosis in esophageal cancer cells / 中国药理学通报

Aim To investigate the role of phospho-lipase C epsilon 1 ( PLCE1 ) in modulating the apoptot-ic mechanism in esophageal cancer ( Eca ) cells. Methods Eca cell lines, OE33 and CP-C cells were cultured to assess the expression of PLCE1 . siRNA suppress expression of PLCE1 . The expression of PLCE1 and p53 was evaluated by quantitative real time PCR and Western blot. Methylation analyses of p53 were performed by bisulfite conversion of genomic DNA. Apoptosis was assessed by flow cytometry. Results OE33 and CP-C cells expressed high levels of PLCE1 . Knockdown of PLCE1 markedly increased the expression and hypomethylation of p53 , and in-creased the frequency of apoptosis. Conclusion PLCE1 suppresses apoptosis of Eca cells via promoting p53 promoter methylation and inhibiting expression of p53 .

Activation of protease-activated receptor 2 inhibits apoptosis of lung cancer cells / 中国药理学通报

Aim To investigate the activation of prote-ase-activated receptor 2 ( PAR2 ) in regulation of the expression of epidermal growth factor receptor ( EGFR) and apoptosis of lung cancer ( LC) cells. Methods LC cells A549 and its EGFR-silenced counterpart were incubated in the medium added with tryptase. Quanti-tative RT-PCR and Western blotting were used to de-tect the expression of EGFR in A 5 4 9 cells . The apop-tosis and Bax/Bcl-xL of cells were also recorded. Re-sults Treating A549 cells with tryptase in the culture for 48 hrs resulted in a marked increase in the expres-sion of EGFR in A549 cells. Marked decreases in a tryptase dose-dependent manner in apoptotic A549 cells were detected in the presence of tryptase. Expo-sure to tryptase markedly decreased the levels of Bax and increased the levels of Bcl-xL in A549 cells, which were not shown in EGFR-deficient A549 cells. Conclusion Tryptase can increase the expression of EGFR in LC cell line, A549 cells, which protects A549 cells from apoptosis, increases Bcl-xL, and sup-presses Bax in A549 cells.

Experimental study on the Caspase-3 siRNA decreases apoptosis in stable cell lines of bone marrow mesenchymal stem cells / 器官移植

Objective ToexploreamethodofusingCaspase-3smallinterferenceribonucleicacid (siRNA)in vitro to decrease the apoptosis in stable cell lines of bone marrow mesenchymal stem cell (MSC)in ordertoestablishMSCcelllineswithstableexpression.Methods VirusparticlescontainingCaspase-3siRNA were generated in 293FT cells by retroviral packaging system,then were transfected into MSC of rats. And the transfected cells were screened and cultured to get the stable MSC lines. According to the characteristics of retrovirus,the stable expression of the cell lines was identified by Western blot and real-time fluorescent quantitative PCR (RT-QPCR). The apoptosis of stable cell lines and normal MSC were detected by terminal dexynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL ) and their differences were compared.Results Comparedwiththenormalcells,thecaspase-3proteinexpressionandmRNAcontentof stable MSC lines were significantly reduced. In the same condition in vitro,the apoptosis quantity of stable MSC linessignificantlydecreasedcomparedwiththenormalcells.Conclusions StablecelllinesofMSCwithstable expression of Caspase-3 siRNA can be obtained by retroviral packaging system. The apoptosis quantity of stable MSC Lines significantly decreased compared with the normal cells.

Cytotoxicity of T cells transduced with WT1 peptide-specific T-cell receptor gene against human lung cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells.</p><p><b>METHODS</b>HLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay.</p><p><b>RESULTS</b>The WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402.</p><p><b>CONCLUSION</b>These data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.</p>

Epidermal growth factor receptor-containing exosomes induce tumor-specific regulatory T cells / 中国药理学通报

Aim Toinvestigatewhetherepidermal growth factor receptor ( EGFR )-containing exosomes could induce tumor-specific regulatory T cells, and the effects of those T cells on tumor protein-specific CD8+Tcells.Methods TheexosomeswithEGFRwerepu-rified from NSCLC tumor, which modulated tolerogenic property of DCs. Then the induced TolDCs generated tumor-specific Tregs, with which the tumor protein-specific CD8 + T cells were suppressed. Results 80%exosomeswereEGFRpositivefromLCpatients while less than 2% exosomes were EGFR positive from control lung tissue. After exposed to the exosomes in the culture for 7 days, the IDO+ DCs proportion was much higher than that in control group ( 80. 8% ± 3. 2% vs 65. 6% ± 6. 4%, P <0. 05 ) . The induced Tregs was also higher ( 24. 1% ± 5. 2% vs 4. 2% ± 2. 3%,P<0. 01 ) , which suppressed the proliferation of CD8+ T cells(5. 4% ± 0. 2% vs 86. 7% ± 9. 3%, P<0.01).Conclusion Thepurifiedexosomesin-duce tolerogenic DCs. Coculture of the tolerogenic DCs and Th0 cells generates the tumor antigen-specific reg-ulatory T cells. The Tregs could suppress the tumor an-tigen specific CD8+ T cells.