Inhibition of malignant phenotype of HeLa cells by RNA interference of the telomerase activity / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subunit (hTERT) gene by RNA interference in vitro.</p><p><b>METHODS</b>Four shRNAs (A, B, C and D) targeting hTERT gene were designed and prepared by in-vitro transcription. The expression of hTERT gene was evaluated by immunofluorescent staining and telomeric repeat amplification protocol (TRAP) ELISA (TRAP-ELISA), after transient transfection of shRNAs by lipid formulation. Through the initial selection, shRNA (B) was noticed as the most efficient one in down-regulating hTERT gene and therefore was chosen as the ultimate shRNA used in the experimental groups. Those transfected by non-silencing RNAi were chosen as the control groups. Cell spreading and migration were studied by microscopy and cell adhesion to fibronectin (FN) was assayed by cell counting kit-8 (CCK-8). Cell invasion was assessed by Boyden chamber assay.</p><p><b>RESULTS</b>Cell spreading study revealed that the rates of spreading cells in the experimental groups were (5.6 +/- 2.3)% at 30 min, and (26.3 +/- 6.1)% 2 h after the inoculation, respectively, whereas the rates of spreading cells in the control groups were (31.3 +/- 7.9)% and (79.4 +/- 4.8)%, respectively. There were significant differences between the two groups (P < 0.01). However, most of the cells in both groups became spreading after 24 h. Cell adhesion assay demonstrated that the rate of adhesion cells on FN in experimental groups was (67.2 +/- 2.8)%, less than that in control groups (83.7 +/- 5.4)% (P < 0.05). The relative migration distance was (27.1 +/- 6.2)% in the experimental group, lower than that of the control group (58.7 +/- 15.0)%. The invasion assay revealed that the invading cells were 75.7 +/- 14.5 in the experimental group, in contrast to 165.1 +/- 11.0 in the control group after 4 h incubation on matrigel. The difference between these two groups was significant (P < 0.05).</p><p><b>CONCLUSION</b>In vitro shRNA silencing of hTERT gene can down-regulate the telomerase activity, leading to an inhibition of the malignant phenotype of HeLa cells, including decreased ability of cell spreading and adhesion, reduction of cell migration, and declined invasive ability through Matrige assay.</p>

Telomerase activity,PI3K/AKT signaling pathway and cellular biological behavior in HeLa cell line / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific PI3K/AKT signaling pathway inhibitor, LY294002.</p><p><b>METHODS</b>CCK-8 assay was used to determine IC50 of LY294002. The expressions of total AKT and phosphorylation AKT (P-AKT) were determined using Western blot. Telomerase activity of cell was measured by TRAP-ELISA assay. Cell growth curve, flow cytometry technique and Hoechst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration was determined by cell wound healing assay.</p><p><b>RESULTS</b>IC50 value of LY294002 of treated HeLa cells was 1.73 mg/L. Western blot showed that LY294002 enabled to decrease P-AKT activity in the presence of same total AKT protein. The cell telomerase activity was decreased to 36.72% in contrast to 98.61% of the control. LY294002 decreased the telomerase activity in HeLa cells, and the growth capacity of the cells was significantly suppressed. The number of cells at G0/G1 phases increased to 66.88% compared with that of the control cells (47.36%). The apoptosis rate also increased from 2.4% to 14.9%. The relative migration distance decreased to 24.6% compared with that of control (62.57%).</p><p><b>CONCLUSION</b>LY294002 inhibition of PI3K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to PI3K/AKT signaling pathway.</p>

Biologic characteristics of rat bone marrow mesenchymal stem cells cultured in vitro / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate biological characteristics of rat bone marrow mesenchymal stem cells (MSC) cultured in vitro and to explore their potential applications.</p><p><b>METHODS</b>MSC were isolated from rat bone marrow by density gradient centrifugation and were induced to differentiation. Flow cytometry was used to characterize their surface antigen expression, cell cycle status and cell growth parameters. Telomerase activity was determined by TRAP-ELISA assay.</p><p><b>RESULTS</b>Fusiform MSC became larger and flattener with increasing passages of culture. After the fourth passage, the MSC showed an immunophenotype of CD29 (94.75% +/- 3.68%), CD71 (95.43% +/- 2.23%), and CD90 (98.08% +/- 3.88%). After the seventh passage, MSC with such immunophenotype decreased with CD29: 50.00% +/- 3.35%, CD71: 50.70% +/- 2.43%, and CD90: 48.60% +/- 2.83%. Cells with such immunoprofile completely disappeared after passage 9. Overall, MSC grew faster during the first 5 passages. The number of MSC in S and G(2)/M phases were 38.36% +/- 2.01% and those in G(0)/G(1) phase were 61.64% +/- 2.13% after 3 passages. The cell growth decreased after passage 7. Percentage of MSCs in S and G(2)/M phases was 10.83% +/- 1.63% and that in G(0)/G(1) was 89.17% +/- 1.96% after passage 12, after which the cells failed to further divide. After passage 9, MSCs lost their ability to differentiate to Von Kossa and oil red O positive staining cells. In addition, telomerase activity of MSC also gradually decreased with the prolonged passages, from the original 52.7% +/- 0.78% to no telomerase activity.</p><p><b>CONCLUSION</b>The biological and immunophenotypical characteristics of cultured MSC showed obvious alterations with increasing numbers of passage of culture.</p>

Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.</p><p><b>METHODS</b>RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.</p><p><b>RESULTS</b>1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.</p><p><b>CONCLUSION</b>Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.</p>