【Abstract】Objective To explore the relation between the expression of telomerase and DNA ploidy with biliarypancreatic system cancer, so as to find a better way to diagnose and distinguish jaundice between malignance and benign disease.Methods Endoscopic retrograde cholangiopancreatography (ERCP) were performed before operation in patients with obstructive jaundice. The bile and pancreatice juice were collected before ERCP. Biopsy specimens from part of patients were obtained during ERCP. All cancer specimens were possessed once again during operation and were assessed by the activity of telomerase and DNA ploidy. Results ① Telomerase positive rate 〔87.50%(56/64)〕 of tissue specimens in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕，P=0.000. ② Telomerase positive rate〔71.88%(46/64)〕of Bile and pancreatice juice in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕， P=0.000, tissue specimens obtained by endoscopy with malignant obstructive jaundice had detectable telomerase activity, positive rate was 83.33%（20/24）. ③ The rate of DNA heteroploid with malignant obstructive jaundice was 62.50%（40/64）, that of diploid can be seen in all patients with benign obstructive jaundice, the difference was statistically significant (P=0.000). ④ The rate of telomerase positive and DNA heteroploid in high differentiation tumor were significantly lower than in middlelow differentiation tumor （P=0.028，P=0.001）.Conclusion Applying the duodenoscope we collected the bile and pancreatic fluid before operation and obtain biopsy specimens whose telomerase activity and DNA ploid were detected. This is simple, safe, quick method which can identify the malignant and benign obstructive jaundice.
【Abstract】ObjectiveTo investigate the expressions of hTERT mRNA and BRCA1 protein and to analyze the correlation between these two factors in breast cancer. MethodsThe expression of hTERT mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). The expression of BRCA1 protein was examined by immunohistochemistry. ResultsThe positive rates of hTERT mRNA and BRCA1 protein were 72.1%（31/43） and 34.9%（15/43） in breast cancer tissue, were 5.0%(2/40) and 77.5%(31/40) in paracancerous breast tissue respectively. Significant difference existed between breast cancer tissue and paracancerous breast tissue (P＜0.05). Significant negative correlation existed between the expression of BRCA1 protein and expression of hTERT mRNA （r=-0.995, P＜0.01）. ConclusionThe expression of hTERT mRNA is upregulated in breast cancer, and expression of BRCA1 protein is downregulated in breast cancer. BRCA1 protein expression may be associated with expression of hTERT mRNA in breast cancer, which may be involved in the carcinogenesis of breast cancer.
Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from humankeloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection. Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group.At the same time of high expression of wt-P53 protein, the telomeraseactivity of KFBs in transfection group was significantly lower than that in theuntransfection group(P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.
OBJECTIVE: To analysis the proliferation properties and telomerase activity of human embryonic tendon cells transformed by ptsA58H plasmid cultured in vitro continuously. METHODS: The 40th, 70th, and 75th passages of transformed human embryonic tendon cells (THETC) were adopted. The collagen secretion of THETC was detected by immunohistochemical methods, the growth curve of different passages of THETC was compared, and chromosome karyotype was analyzed. Total RNA of THETC were extracted to detect human telomerase reverse transcriptase (hTERT) mRNA expression by RT-PCR technique. RESULTS: When THETC were subcultured to 70 passages, the morphological characteristics of cells changed and began replicative senescence. THETC still could secret type I collagen normally. The chromosome of THETC was heteroploid (2n = 94). There were no hTERT mRNA expression. CONCLUSION: SV40 transfection can not make human embryonic tendon cells immortalization, on the other hand, human embryonic tendon cells transformed by ptsA58H plasmid has no tendency of malignant transformation.
Objective To review the research process of telomerase reverse transcriptase (TERT) in the restoration of neurological diseases. Methods The related l iterature on TERT in the restoration of neurological diseases was extensively reviewed and comprehensively analyzed. Results TERT was the significant component of telomerase and the critical regulator of telomerase activity. It played an important role in the pathomechanism of neurological diseases including tumors,neurodevelopmental deficits, and nerve injury. TERT was becoming a research focus in the reparative therapy of neurological diseases. Conclusion TERT has manifested its great academic significance and appl ication prospects in the reparative therapy of neurological diseases, which deserves a further investigation.
【Abstract】Objective To study the difference of telomerase activity in the common thyroid lesions . Methods The telomerase activity was detected in 19 patients with thyroid carcinomas, 15 samples adjacent to thyroid carcinomas，21 specimens of thyromas, 17 cases of nodular goiters and 13 pieces of normal thyroid tissues by telomeric repeat amplification protocol（TRAP). Results Eighteen of 19 samples of thyroid carcinoma, 1 of 15 samples adjacent to the cancer and 1 of 21 adenoma of the thyroid specimens showed positive telomerase activity, all 17 cases of nodular goiters and 18 samples of normal thyroid tissues exhibited negative telomerase activity, and the rate of positive telomerase activity of thyroid carcinomas was significantly higher than that of the other tissues (P＜0.0001). Conclusion The telomerase is an important qualitative marker of thyroid carcinoma and a useful index in differential diagnosis of thyroid lesions.
Objective To explore the values of telomerase in the diagnosis, therapy and prognostic parameter of colorectal cancer. Methods Telomerase activity in colorectal cancer, peri-cancerous and normal mucosa was detected by PCRTRAP-ELISA assay. Results The positive rates of telomerase in colorectal cancer, peri-cancerous and normal mucosa were 84.8%, 20.0% and 0% respectively. 66.7% of the early stage colorectal cancer expressed telomerase. Telomerase activity was reversely correlated with tumor differentiation.Conclusion Telomerase may be an earlier event of malignant progression in colorectal cancer. It might be a parameter for diagnosis of colorectal cancer.
Objective Telomerase reverse transcriptase (TERT) is the key factor to determine cell growth and l ifespan. Meanwhile, it is tightly related to resistance of cell to stress and apoptosis. However, up till now l ittle is known about the role TERT plays in nervous system. To investigate the effect of conditioned medium from astrocytes (AS) transfected with TERT on neurons subjected to hypoxia-ischemia-reperfusion (HI-RP) through construction of in vitro HI-RP model of neurons. Methods An eukaryote expression plasmids containing rat full length TERT gene was constructed as pcDNA3-TERT. Twenty newborn rats at age of 3 days were sacrificed and their cerebral cortex were collected for isolation and cultivationof AS. Then AS were transfected with pcDNA3-TERT through l iposomes mediation, and positive clones were selected by G418 and expanded for continuous culture to establ ish the plamid pcDNA3-TERT transfection group. Meanwhile, the empty plasmid pcDNA3 transfection group and the non-transfection group were establ ished as control. The expression of gl ial fibrillary acidic protein (GFAP), which was the specific marker of the AS, was detected by immunocytochemistry, as well as the expression of TERT. Astrocyte conditioned medium (ACM) of the plamid pcDNA3-TERT transfection group was collected as TERT-ACM, while the ACM of the empty plasmid pcDNA3 transfection group and the non-transfection group were collected respectively as p-ACM and ACM. Next, 60 rats at age of 1 day were sacrificed and their cerebral cortex were collected for isolation and cultivation of neurons. The neurons were randomly divided into experimental group and normal group, the experimental group were further divided into 4 groups including control group, ACM group, p-ACM group, and TERT-ACM group. The neurons of control group were subjected to HI damage in serum-free DMEM, and the neurons of ACM group, p-ACM group, and TERTACM group were subjected to HI damage in different medium which contained ACM, p-ACM, and TERT-ACM, respectively. After duration of HI for 3 hours under the environment with 5%CO2, 1%O2, and 94%N2; the neurons of experimental groups were placed in CO2 incubator to imitate RP for 3, 6, 18, 24, and 36 hours in vitro. The neurons of normal group were not subjected to HI and RP treatment. During the treatment of HI-RP, the survival ratio of neurons was detected by means of MTT, the lactate dehydrogenase (LDH) activity of neuron medium with LDH detection kit, and the neuronal apoptosis by means of TUNEL. Results The percentages of GFAP positive cells were 98%, 99%, and 98% in non-transfection group, plasmid pcDNA3-TERT transfection group, and plasmid pcDNA3 transfection group, respectively. There was no expression of TERT in no-transfection group and plasmid pcDNA3 transfection group, and the percentage of TERT positive cells in plasmid pcDNA3- TERT transfection group was 98%. Compared with normal group, the survival ratio of ......（余见正文）
Objective To investigate the variety of telomerase activity in the course of liver cancer development, and the possibility of using telomerase as a marker of HCC. Methods Human liver specimens, comprising 22 HCC and adjacent peritumoral tissues, 12 liver cirrhosistissues, 6 nodulat regenerative hyperplasia (NRH) tissues and 10 normal liver tissues, were examined for telomerase activity by TRAP assay based on PCR. Results Twenty of 22 HCC and 14 of 22 adjacent tissue specimens were positive for telomerase activity with a positive rate of 90.9% and 63.6% respectively. Ten of 12 liver cirrhosis tissues were positive with a positive rate of 83.3%. 5 of 6 NRH were positive with a positive rate of 83.3%. Telomerase activity was negative in 10 normal liver tissues. Conclusion Telomerase may occur in the progress of hepatocarcinogenesis. Telomerase can be used as a tumor marker of HCC.
【Abstract】Objective To design the hammerhead ribozyme gene according to the hTR sequence in the gallbladder cancer cell, and build it into the eukaryon expression vector pTriEx-4. Methods According to the hTR cDNA sequence, the authors designed the primers and take the hTR template area gene from the gallbladder cancer cells by RT-PCR.The hammerhead ribozyme gene was synthesize according to the result of sequencing, and combine them with eukaryon expressing vector. Identified the exactitude of recombine vector by digestion.Results The 68 bp sequence extracted from the cell through the RT-PCR had the same template sequence comparing with the hTR cDNA. The recombinant plasmid with the hammerhead ribozyme gene was correct by digestion identification. Conclusion The RT-PCR method can extract the gallbladder cancer cell’s hTR gene. We construct the eukaryon expression vector containing the hammerhead ribozyme gene successfully which is the foundation for gene therapy of gallbladder cancer.