Knockdown of human telomerase reverse transcriptase induces apoptosis in cervical cancer cell line

Background & objectives: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase enzyme that maintains telomere ends by the addition of telomeric repeats to the ends of chromosomal DNA, and that may generate immortal cancer cells. Hence, the activity of telomerase is raised in cancer cells including cervical cancer. The present study aimed to validate the unique siRNA loaded chitosan coated poly-lactic-co-glycolic acid (PLGA) nanoparticle targeting hTERT mRNA to knock down the expression of hTERT in HeLa cells. Methods: The siRNA loaded chitosan coated polylactic-co-glycolic acid (PLGA) nanoparticles were synthesized by double emulsion solvent diffusion method. The characterization of nano-formulation was done to determine efficient siRNA delivery. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate silencing efficiency of nano-formulation. Results: Size, zeta potential and encapsulation efficiency of nanoparticles were 249.2 nm, 12.4 mV and 80.5 per cent, respectively. Sustained release of siRNA from prepared nanoparticle was studied for 72 h by ultraviolet method. Staining assays were performed to confirm senescence and apoptosis. Silencing of hTERT mRNA and protein expression were analyzed in HeLa cells by RT-PCR and Western blot. Interpretation & conclusions: The findings showed that biodegradable chitosan coated PLGA nanoparticles possessed an ability for efficient and successful siRNA delivery. The siRNA-loaded PLGA nanoparticles induced apoptosis in HeLa cells. Further studies need to be done with animal model.

Human telomerase reverse transcriptase and cutaneous malignant tumor / 国际肿瘤学杂志

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is closely related to immortalization of cells, tumorigenesis and senescence of cells. hTERT has a great relationship with the occurrence and development of skin malignant tumors (melanoma, squamous cell carcinoma, basal cell carcinoma, cutaneous lymphoma, etc.). Abnormal expression of hTERT has important clinical application value in early diagnosis, individualized comprehensive treatment and prognosis evaluation of cutaneous malignant tumors.

Construction of human telomerase reverse transcriptase periodontal ligament cell line mediated by adenovirus / 华西口腔医学杂志

OBJECTIVE@#This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase (hTERT) gene by using the adenovirus method.@*METHODS@#Polymerase chain reaction (PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT. Packaged adenovirus particles were used for infection of human periodontal ligament cells. The expression levels of hTERT and osteogenic genes, such as alkaline phosphatase, Runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen Ⅰ mRNA, were detected by quantitative real-time PCR (qRT-PCR). The ability of osteogenic differentiation was observed by alizarin red staining, and the cell proliferation was determined by CCK-8.@*RESULTS@#Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells. The infected cells were similar to normal periodontal ligament cells. The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses. Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability. CCK-8 showed that the periodontal ligament cell line had strong proliferation capability.@*CONCLUSIONS@#The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method, thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.

Human telomerase reverse transcriptase and cutaneous malignant tumor / 国际肿瘤学杂志

Human telomerase reverse transcriptase (hTERT)is a catalytic subunit of telomerase and is closely related to immortalization of cells,tumorigenesis and senescence of cells. hTERT has a great relation-ship with the occurrence and development of skin malignant tumors (melanoma,squamous cell carcinoma, basal cell carcinoma,cutaneous lymphoma,etc. ). Abnormal expression of hTERT has important clinical appli-cation value in early diagnosis,individualized comprehensive treatment and prognosis evaluation of cutaneous malignant tumors.

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP / 吉林大学学报(医学版)

Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Research progress of telomere,telomerase and human telomerase reverse transcriptase in tumors / 肿瘤研究与临床

Telomere and telomerase maintain the stability of chromosome in structure and function.Studies have found that they are closely related with the development of human tumors.Telomeres are deoxyribonucleic acid in the terminal of chromossote of eukaryotes to maintain the integrity of chromosone.Telomerase is a kind of special structure of nucleoprotein,it prolongs the telomere and maintains the telomere' s stability,and it is directly related to the unlimited proliferation and canceration of cells.Human telomerase reverse transcriptase (hTERT) is both the important component and speed limit subunit of telomerase,it directly determines the telomerase activity.Further studies on structure,function and interaction of telomere,telomerase and hTERT help understand the developing mechanisms of tumors,they have important significance to the tumor diagnosis and treatment,and can provide new targets for cancer treatment.

Construction of human mucosa oral epithelial cell lines overexpressing telomerase reverse transcriptase gene mediated by lentivirus / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct a cell line of oral mucosa epithelial cells that stably express human telomerase reverse transcriptase (hTERT) by lentiviral vectors, approaches for the establishment of stable and efficient immortalized oral mucosa epithelial cell lines were explored.</p><p><b>METHODS</b>Whole RNA was extracted from 293T cells. The hTERT gene was amplified by polymerase chain reaction (PCR) and cloned into the lentiviral vector as pLVX-puro-hTERT. The lentivirus particles were successfully packaged and used to infect primary oral epithelial cells. The positive cell clones were selected by puromycin. Finally, the expression of hTERT was examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot analysis.</p><p><b>RESULTS</b>The sequencing results confirmed the construction of the recombinant lentivirus pLVX-puro-hTERT. The morphology of infected cells was similar to that of normal oral mucosal epithelial cells, with a cobble stone-like appearance. The qRT-PCR and Western blot results showed that hTERT was overexpressed in infected cells compared with the normal group (P<0.05).</p><p><b>CONCLUSIONS</b>The oral epithelial cell line with stable expression of hTERT was successfully established by the lentivirus, which provides an experimental basis for the establishment of a highly efficient and stable oral epithelial immortalized cell line.</p>

Expression and significance of human telomerase reverse transcriptase mRNA in different subtypes of melanoma / 肿瘤研究与临床

Objective To detect the expression of human telomerase reverse transcriptase (hTERT) mRNA in the melanoma, and to analyze the relationship between the expression and subtypes and clinicopathological features of melanoma. Methods Expression of hTERT mRNA was detected by real-time quantitative PCR in 64 cases of melanoma and 30 cases of nevus. SPSS 17.0 software was used to analyze the relationship between hTERT mRNA expression and clinical pathological features of melanoma. Results The relative expression of hTERT mRNA in melanoma tissues was higher than that in nevus tissues [(52.43±5.42) vs (21.38±3.73), t= 4.72, P= 0.000]. The expression of hTERT mRNA in melanoma had no significant correlation with age, gender, ethnicity (all P> 0.05), but had relationship with subtypes, lymph node metastasis, Clark classification (all P 0.05). Conclusions The expression of hTERT mRNA in melanoma is high, especially in mucosal melanoma. hTERT may play an important role in the occurrence and development of melanoma.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

Visfatin effects on telomerase gene expression in AGS gastric cancer cell line.

AIM: The aim of this study was to assess visfatin expression and its effect on human telomerase gene expression in AGS gastric cancer cell line. MATERIALS AND METHODS: In this study, human gastric cancer (AGS) cell line was established as an in vitro model. Reverse transcription polymerase chain reaction (RT‑PCR) and enzyme‑linked immunosorbent assay was performed to show that visfatin expression in messenger ribonucleic acid (mRNA) and protein level respectively. After stimulating with increasing concentrations of visfatin for times of 24 h and 48 h, cell proliferation was assessed by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay. In order to investigate telomerase gene expression affected by visfatin in AGS cell line, total RNA was extracted and complementary deoxyribonucleic acid was synthesized buy using commercially available kits. Expression of human telomerase reverse transcriptase (hTERT) mRNA was carried out by real‑time RT‑PCR. RESULTS: After visfatin treatment gastric cell line proliferation was enhanced and was increased the expression of hTERT. CONCLUSIONS: The obtained data showed that visfatin induces endogenously gastric cancer cell proliferation and increases telomerase (hTERT) gene expression, as a cancer gene. Based on this study, it is suggested that expression of this adipocytokine protein in real samples could be biomarker for gastric cancer.

The expression of hTERT and telomerase in human fetal gastric fibroblasts / 重庆医学

Objective To detect the expression of human telomerase reverse transcriptase (hTERT ) and telomerase in hu‐man fetal gastric fibroblasts .Methods Human fetal gastric fibroblasts were isolated and cultured ,epithelial cells were exduded by CK‐18 immune staining .hTERT protein was determined by indirect immunofluorescence .Telomerase activity was analyzed using telomeric repeat amplification protocol assay (TRAP)while the same analysis in adult gastric fibroblast ,which as to be positive con‐trol .Results Cultured fibroblasts were CK‐18 negative .The positive hTERT immunostaining was detected in both cellular cyto‐plasm and nuclear compartments .Amplified telomeric repeats (about 170 bp) in human fetal gastric fibroblasts were longer than those (160 bp) in adult gastric fibroblasts .Conclusion hTERT and telomerase were expressed in human fetal gastric fibroblasts .

Ubiquitin and its role in the tumor / 国际肿瘤学杂志

Ubiquitin-proteasome pathway is one of the primary pathway in intracellular protein degrada-tion,therefore the ubiquitin conjugating enzyme D3(UbE2D3)which involved in the ubiquitin mineralization process can affect the biological effects accordingly to affect some protein and nucleic acid content and activity. The function that participate in modification and degradation of some cancer factors is vital in tumor cells,and followed by tumor biological behavior changing. Researches show that UbE2D3 correlates with human telomer-ase reverse transcriptase( hTERT),radiation sensitivity,aggressive,etc in breast cancer.

Study on proliferation capability and human telomerase reverse transcriptase expression of bone marrow-derived mesenchymal stem cells from rheumatoid arthritis patients / 中华风湿病学杂志

Objective By comparing the proliferation and the human telomerase reverse transcriptase expression of bone marrow mesenchymal stem cells (BMSCs) from rheumatoid arthritis (RA) patients and normal human to explore the existence of BMSCs defects or variation in RA patients,and whether these cells could be used in autologous transplantation.Methods BMSCs were obtained from bone marrow of eight RA patients and healthy volunteers.The cells were isolated andcultured by gradient centrifugation and adherence cultivation,and the surface markers of BMSCs were identitfied by flow cytometertry.Firstly,BMSCs reproductive activity of the two groups were compared:the auxodrome and cell cycle of BMSCs were measured respectively,then the generation doubling time of cell colony and proliferation index were calculated in order to see whether there was a difference of the proliferation capability between the two groups.Furthermore,the hTERT were measured.Results BMSCs were no significant differences between morphology of RA patients and healthy control group.The cells were obtained proof MSCs by flow cytometry.The difference was not statistically significant (P＞0.05),RA group and the healthy control group P3-generation cell population doubling time (4.7±0.3) d,(4.8±0.3) d.The difference was statistically significant (P＜0.01),P6 generation of healthy control group cell population has doubling time (5.7±0.4) d,RA group (6.3±0.3) d,buut the group RA BMSCs population doubling time is longer than the healthy control group.The difference was not statistically significant (P＞0.05),on one hand of the healthy control group P3 BMSCs proliferation index (17.9±2.1)％,on the other hand RA group is (16.9±2.1)％.The difference was statistically significant (P＜0.01).P6-generation BMSCs proliferation index in healthy control group (10.7±1.8)％,RA group (6.2±2.2)％,RA group proliferation index than the control group.The difference was also significant (P＜0.01).The healthy control group P6 generation of MSCs hTERT expression was (10.6±1.5),RA group is (3.1±1.0),hTERT RA patients expression is lower compared with normal control group.Conclusion There is no significant difference in the proliferative capacity of the two groups at P3,but the BMSCs proliferation at P6 lags behind in the normal group in RA patients,which inferred that the senescences of the BMSCs of RA patients are earlier than healthy donors.The determination results of hTERT have shown that the difference between the two groups is statistically significant,and it proves that the premature aging may be associated with hTERT expression.

Association between hTERT Gene Amplification and High-Risk HPV Infection in Cervical Lesions / 天津医药

Objective To investigate the relationship between the amplification of human telomerase reverse tran-scriptase (hTERT) gene and high-risk human papillomavirus (HR-HPV) infections in cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Methods The cervical epithelial cells were collected from 34 samples of normal cervical epithelium, 31 samples of CIN (gradeⅠ), 33 samples of CIN (gradeⅡ), 34 samples of CIN (gradeⅢ) and 20 samples of cer-vical carcinoma. HPV DNA was detected by polymerase chain reaction-reverse dot blot hybridization (PCR-RDB) and the amplification of hTERT gene was detected by fluorescence in situ hybridization (FISH). Results Twenty subtypes of HR-HPV were detected including HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 73 and 82. The inci-dence of HR-HPV infection was higher in CINⅡgroup (72.73%), CINⅢgroup (85.29%) and cervical carcinoma group (90.00%) than that of normal cervical epithelium group (20.59%). There was no significant difference in the positive rate of HR-HPV DNA between CINⅠ group (54.84%) and normal cervical epithelium group (P < 0.005). The positive rate of hTERT gene amplification was higher in cervical carcinoma group (80.00%) than that of normal cervical epithelium group (0). There were no significant differences in the positive rates of hTERT gene amplification between CINⅠgroup ( 3.22%), CIN Ⅱ group (18.18%), cervical carcinoma group and CIN Ⅲ group (41.18%). There was positive correlation between hTERT gene amplification and HR-HPV infection (r=0.238, P<0.05). Conclusion The incidence of HR-HPV infection was positively correlated with hTERT gene amplification in cervical lesions. HR-HPV infection may be an early event of ab-normal amplification of hTERT gene. The detection of HPV-DNA and hTERT gene can be used in the clinical diagnosis of early cervical lesions.

Anti-tumor effects of enterovirus 71 on human malignant glioma cell line U251 in vitro / 中华实用儿科临床杂志

Objective To investigate the effects of enterovirus 71 (EV71) on growth,apoptosis and human telomerase reverse transcriptase (hTERT) expression of human malignant glioma cell line U251 in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In the experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously for 72 hours with U251 cells,but in the control group,EV71 were not put.The morphological features and growth of cells were observed under inverted microscope at 0 hour,24 hours,48 hours and 72 hours of EV71 treatment,respectively.At the same time,cell apoptosis was measured by flow cytometry using Annexin V/PI double staining method.hTERT expression was detected with semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR).Results The growth of U251 cells was inhibited significantly at 24 h of EV71 treatment under inverted microscope in experimental group when compared with control group,and at 48 h and 72 h,the morphological features of these cells became significantly shrunken,smaller and irregular shape in experimental group,but which were uniform size in control group.At 24 h,48 h,72 h of EV71 treatment,flow cytometry showed the apoptosis rates were significantly higher in experimental group than that in control group [24 h:(12.55 ± 2.38) ％ vs (1.42 ± 0.21) ％,48 h:(65.60 ± 8.48)％ vs (1.42 ±0.17)％,72 h:(87.52 ±3.05)％ vs (1.41 ±0.16)％,all P =0.000],and there was upward trend day by day,but no change in control group.At the same time,hTERT mRNA expression levels of U251 cells were significantly lower in experimental group than control group [24 h:(0.58 ± 0.05) vs (0.89 ± 0.05),48 h:(0.23 ± 0.04) vs (0.89 ± 0.03),72 h:(0.10 ± 0.03) vs (0.90 ± 0.06),all P =0.000],and the downward trend was observed day by day,but there was no this trend in control group.Conclusions EV71 can effectively inhibit the growth of U251 cells,induce the cell apoptosis,and has the potential anti-tumor effect.Its mechanisms may be partly related to down regulation the hTERT expression of U251 cells.

Peroxisome proliferator-activated receptor gamma agonist suppresses human telomerase reverse transcriptase expression and aromatase activity in eutopic endometrial stromal cells from endometriosis / 대한생식의학회지

OBJECTIVE: To investigate the effect of peroxisome proliferator activated receptor gamma (PPARgamma) agonist on the cell proliferation properties and expression of human telomerase reverse transcriptase (hTERT) and aromatase in cultured endometrial stromal cell (ESC) from patients with endometriosis. METHODS: Human endometrial tissues were obtained from women with endometriosis and healthy women (controls) using endometrial biopsy. Isolated ESCs were cultured and the cell proliferation was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay and expression of hTERT, aromatase, and cyclooxygenase (COX)-2 by western blotting according to the addition of rosiglitazone (PPARgamma agonist). RESULTS: We demonstrate that the cultured ESCs of endometriosis showed hTERT protein overexpression and increased cellular proliferation, which was inhibited by rosiglitazone, in a dose-dependent manner. At the same time, PPARgamma agonist also inhibited aromatase and COX-2 expression, resulting in decreased prostaglandin E2 production in the ESCs of endometriosis. CONCLUSION: This study suggests that PPARgamma agonist plays an inhibitory role in the proliferative properties of eutopic endometrium with endometriosis by down-regulation of hTERT and COX-2 expression; this could be a new treatment target for endometriosis.

Human telomerase reverse transcriptase protects human embryonic cortical neurons / 中国组织工程研究

BACKGROUND: Telomerase can maintain the telomere length and avoid cel replicative senescence and apoptosis in somatic cells. Its catalytic subunit cal ed telomerase reverse transcriptase has roles in mediating cellsurvival and anti-apoptotic functions. OBJECTIVE: To evaluate the effects of human telomerase reverse transcriptase on amyloid β1-40-induced human embryonic cortical neurons injury. METHODS: Human cortical neurons derived from 12-16 weeks old aborted fetuses were transfected with recombinant adenovirus vector encoding human telomerase reverse transcriptase. Expression of human telomerase reverse transcriptase was evaluated by immunocytochemical staining. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol enzyme-linked immunosorbent assay kit. Human embryonic cortical neurons were treated with 10 μmol/L ol/L amyloid β1-40 after transfected for 3 days. Cel viability, reactive oxygen species levels and glutathione contents in human embryonic cortical neurons were respectively detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and chromatometry. RESULTS AND CONCLUSION: Expression of human telomerase reverse transcriptase reached peak at 3 days after transfection, and the telomerase activity was rebuilt; 10 μmol/L amyloid β1-40 could significantly reduce the cel viability of neurons and glutathione content (P < 0.05 and P < 0.01), and increase the reactive oxygen species levels (P < 0.05). The neurons transfected with human telomerase reverse transcriptase gene could be significantly against the toxicity of amyloid β1-40 and increase the cel viability and glutathione content (P < 0.05 and P < 0.01), and decrease the reactive oxygen species levels (P < 0.05). The results indicate that human telomerase reverse transcriptase can protect amyloid β1-40-induced human embryonic cortical neurons injury

Construction of series reporter plasmids with truncated and deleted human telomerase reverse transcriptase promoter / 中华微生物学和免疫学杂志

Objective To construct series of reporter plasmids with truncated and deleted hTERT promoter.Methods Gene fragments of hTERT promoter was amplified by PCR and cloned into pGL3-Basic to construct luciferase reporter vectors.Dual luciferase assays were performed with cell lysates of HepG2 and COS-7 cells cotransfected with hTERT promoter reporter plasmids and pRL-TK.Results Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed and respectively named pGL3B-895,pGL3B-371,pGL3B-DELS2,pGL3B-349,pGL3B-329,pGL3B-318,pGL3B-306.Dual luciferase reporter assays showed that all the reporter vectors have promoter activity both in HepG2 and COS-7.Conclusion Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed,and their promoter activity were verified.These plasmids provide necessary experimental naterials for further investigation of regulation of hTERT during hepatocarcinoma development.

Expressions of CD90 and hTERT in hepatocellular carcinoma and Their Clinical Significance / 中华肝胆外科杂志

ObjectiveTo study the expressions of CD90 and hTERT in hepatocellular carcinoma (HCC),and their relationships to progression of tumor.MethodsThe expressions of CD90 and hTERT in hepatocellular carcinoma were detected by S-P immunohistochemical staining.Twenty patients with hemangiomas of liver were used as control.ResultsCompared with the control group,the positive rates of CD90 and hTERT in HCC were significantly higher (63.9％ and 47.2％ vs 0％ and 0％).The positive rates of CD90 and hTERT were significantly higher in patients with tumors at UICC Ⅲ-Ⅳ stage than at UICC stage Ⅰ -Ⅱ (79.1％ and 62.5％ vs 33.3％ and 16.6％).The CD90 expression correlated with hTERT positively.There were significant differences in survival between patients with CD90+ and CD90- or hTERT+ and hTERT-.The median postoperative survivals for patients with CD90+ and hTERT+,CD90- and hTERT- were 85 d and 76 d,505 d and 463 d,respectively.ConclusionsCD90 expression correlated positively with progression of HCC.It has the potential to serve as a prognostic marker for HCC.

Construction and identification of a double-regulated replicative adenovirus AdTPHre-hEndo / 中华肝胆外科杂志

Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.