Fas／Fasl系统在再生障碍性贫血及骨髓增生异常综合征发病中作用的比较

目的：探讨Fas/Fasl系统与再生障碍性贫血（aplastic anemia,AA)及骨髓增生异常综合征（myelodysplastic syndrome,MDS)发生发展的关系。方法：分别用TUNEL方法和免疫组化标记方法检测凋亡细胞和Fas、Fasl表达细胞并计算其百分率，并以ELISA方法检测血清中的sFas的含量。结果：（1)AA组与MDS组BMMNC中的凋良细胞百分率。Fas表达细胞百分率、Fasl表达细胞百分率均比正常对照组高（P＜0．05）；（2）AA组血清sFas的含量比正常对照组低（P＜0．05），而MDS组比正常对照组高（P＜0．05）；（3）两组血清sFas的含量间比较有显著差异（P＜0．05）。结论：本实验结果表明Fas/Fasl系统介导的凋亡可能参与了AA与MDS骨髓造血异常的病理生理过程。但Fas/Fasl系统介导的凋亡在两种疾病发病中的作用机制可能不完全相同。

Correlation of free radical level and apoptosis after intracerebral hemorrhage in rats

Objective To investigate the correlation of perihematomal free radical level and neuronal apoptosis following the intracerebral hemorrhage （ICH）. Methods Animals were randomly divided into 4 groups： sham operation group, model group, 1 mg/kg edaravone group, and 3 mg/kg edaravone group. Each group was then divided into seven subgroups, in which the rats were correspondingly killed at 6 h, 12 h, 24 h, 48 h, 72 h, 7 d or 14 d （n = 1 in each subgroup of the sham group, and n = 6 in each subgroup of the other 3 groups）. By Horseley-Clarke technique, autoblood （80 μL） were administered into the left caudate putamen of SD rats in a double administration-withdrawal way. Rats in the sham group were needled in but not administered with autoblood. The ICH model was then evaluated by Bederson＇s scale. Around the hematoma, the levels of malonaldehyde （MDA） and hydroxyl radical were tested by spectrophotometer, and the process of apoptosis was tested by terminal deoxynucleofidyltransferase-mediated dUTP-biotin nick end labeling （TUNEL） method. Results （1） ICH significantly increased the levels of MDA and hydroxyl radicals. Significant differences in MDA and hydroxyl radical contents were observed among the four groups. （2） In the sham group, a small number of TUNEL-positive cells were found. In the other three groups, the TUNEL-positive cells were observed at 6 h, increased significantly at 24 h, and reached peak level at 3 d, then fell profoundly at 7 d, but remained detectable at 14 d. （3） The positive correlation existed between apoptosis and free radical level （r = 0.2003）, and existed between apoptosis and MDA content （r = 0.6563） in the brain. Conclusion Post-hemorrhagic apoptosis was related to the production of free radicals, indicating that the elevated free radicals following the ICH could induce neuron and glial cell apoptosis.

Trichostatin A Induces Apoptosis by Inhibiting Telomerase Activity and Expression of Telomerase Reverse Transcriptase in HL-60 Cells

Aim To investigate the effects of trichostatin A （TSA） on telomerase activity and the expression of human telomerase reverse transcriptase （hTERT） during apoptosis in vitro and the mechanisms in HL-60 cells. Methods The proliferative activity of HL-60 cells was assessed by MTT assay. Cell apoptosis was analyzed by flow cytometry. Telomerase activity was examined by TRAP-ELISA. The expression of telomerase subunits was analyzed by RT-PCR. Results A time- and dose-dependent inhibition was detected in HL-60 cells treated with TSA. After treatment with 600 nmol· L^-1 TSA for 48 h, the apoptosis rate in HL-60 cells was 42. 6% and telomerase activity decreased 1.95 ± 0.25, 1.73 ± 0. 12, and 1.52 ± 0. 09 for 12, 24, and 48 h, respectively. The expression of hTERTmRNA decreased. No significant changes were observed in the expression of hTRmRNA and hTPI mRNA. Condusion TSA inhibits telomerase activity and induces apoptosis in HL-60 cells. The underlying mechanism may be related to the down-regulation of hTERT transcription.

Inhibitory Effect of Oridonin on the Proliferation of NB4 Cells and Its Mechanism

Objective: To investigate the anti-proliferation e?ect of oridonin on leukemic NB4 cells and its mechanism. Methods: NB4 cells in culture medium in vitro were given di?erent concentrations of oridonin. The inhibitory rate of the cells were measured by MTT assay, cell apoptotic rate was detected by ?ow cytometry(FCM), morphology of cell apoptosis was observed by hoechst 33258 ?uorescence staining , and the activity of telomerase was detected using TRAP-PCR-ELISA before and after apoptosis occurred. Results: Oridonin (over 8 μmol/L) could decrease the telomerase activity, inhibit the growth of NB4 cells and induce apoptosis signi?cantly in a time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed by hoechst 33258 ?uorescence staining especially after the cells treated by oridonin for 48–60 h. Conclusion: Oridonin could inhibit the proliferation and induce the apoptosis of NB4 cells in vitro. One of the mechanisms may be the decrease of the telomerase activity of NB4 cells.

Inhibition of Telomerase with hTERT Antisense Increases Susceptibility of Leukemic Cells to CDDP-induced Apoptosis

Objective: To investigated the e?ect of inhibition of telomerase with hTERT antisense on leukemic cells (HL-60 and K562) to CDDP-induced apoptosis. Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and puri?ed. Telomerase activity was detected by Telomerase PCR ELASA kit and cell apoptosis was observed by morphological method and determined by ?owcytometry. Results: AS PS-ODN could signi?cantly inhibit telomerase activity by down regulat- ing the hTERT expression, and increase the susceptibility of leukemic cells to CDDP-induced apoptosis. Conclusion: Inhibition of telomerase with hTERT antisense can increases the susceptibility of leukemic cells to CDDP-induced apoptosis.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

A retrograde apoptotic signal originating in NGF-deprived distal axons of rat sympathetic neurons in compartmented cultures

Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glyco- gen synthase kinase 3 （GSK3） activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotro- phins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.

Study of the expressions of p53 and bcl-2 genes, the telomerase activity and apoptosis in GIST patients

AIM： To explore the relationship between clinicobiological behavior and the expression levels of telomerase activity, apoptosis, p53 gene and bcl-2 gene in gastrointestinal stromal tumors （GISTs）. METHODS： The intensity of telomerase activity, apoptosis, p53 and bcl-2 expression in GISTs were detected by telomeric repeat amplification protocol, in situ end-labeling technique, and immunohistochemistry, respectively. RESULTS： The positive rates of telomerase activity of malignant GIST, potential malignant GIST and benign GIST were 85% （17/20）, 22.8% （2/9） and 0 （0/9）, respectively. The apoptosis indices of malignant GIST, potential malignant GIST, and benign GIST were 11.7±5.4, 30.2±5.6 and 45.2 ±7.2, respectively. The intensity of telomerase activity and apoptosis were related to the biological characteristics of GISTs （85% vs 22.8%, 0, 0; P 〈 0.01 or 11.7±5.4 vs 30.2±5.6, 45.2±7.2, 72.1±9.3; P 〈 0.05）. The intensity of telomerase activity was negatively correlated with cellular apoptosis （22.9±8.4 vs 9.5±5.7, P 〈 0.01）. The intensity of telomerase activity was positively correlated with/753, bcl-2 expression （40.0% vs 78.9%, 40.0% vs 84.2%; P 〈 0.05）. CONCLUSION： The detection of telomerase activity, apoptosis and its control genes in GIST will be helpful for the discrimination of the malignant and benign GIST and evaluation of the prognosis.

Rainfall occurrence and its relation to flood damage in China from 2000 to 2015

China is highly susceptible to flood disasters and subjected to great damage every year.Furthermore, the flood frequency has exhibited an increasing trend in recent years. Most flood events,including flash floods and river flood, are induced by rainfall. This study investigates annual variations of rainfall occurrence over China during the period from 2000 to 2015 at the national and regional scale using daily rainfall data from the Tropical Rainfall Measuring Mission. The Mann-Kendall test is performed for trend detection, and statistical data of flood damage published by China's government,including destroyed crop area, damaged buildings,direct economic loss, percentage of GDP(gross domestic product), and death toll are correlatively analysed with rainfall occurrences. The results show that storm rain events show the greatest variation among three rainfall types(moderate rain, heavy rain and storm rain). The variation coefficients of rainfall over Northeast China, North China, and Northwest China are the highest, whereas that for Southwest China is the smallest. Moderate rain, heavy rain over Central China, and moderate rain over Southwest China exhibits decreasing trends, whereas the remaining exhibit increasing trends. The correlation between the rainfall occurrences and these flood damage indices at the national scale shows that only direct economic loss has a strong positive correlation with rainfall occurrences, and the other indices have weaker correlations. The correlation is strong in three north regions, except for death toll in Northwest China. In contrast, the correlation between flood damage and rainfall is weak in East China, Central China, Southwest China, and South China. Overall,death toll is strongly correlated with the number of damaged buildings, implying that flood fatalities in China are likely associated with building collapse, and are dominated by specific extreme events. This study can provide a scientific reference for flood management in China.

ANTI-PROLIFERATION EFFECT OF ORIDONIN ONHL-60 CELLS AND ITS MECHANISM

ObjectiveTo investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism. Methods HL-60 cells invitroin culture medium were given different concentrations of oridonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM),morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was det-ected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred. Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly. The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especi-ally after cells were treated 48-60 hours by oridonin. Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells invitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.

Expression of telomerase gene and apoptosis related genes in benign and malignant breast lesion

Objective:To analyse the expression of telomerase and apoptosis related protein,and explore the possible mechanism of breast cancer development.Methods:Immunohistochemistry method(SP)was used to detect the expression of hTERT,p53 and bcl-2 in the tissues of 48 cases of human breast cancer and 42 cases of benign lesions in breast.Results: The positive rates of expression of hTERT,p53 and bcl-2 in breast cancer were 87.50%,56.25%and 54.17%,respectively. Compared with the groups of adjacent noncancerous and benign lesions,there was a significant difference among three types of tissues(P<0.05).The positive rates of expression of p53 and bcl-2 in the group with positive expression of hTERT were 64.28%and 61.90%,respectively,and their difference was significant compared with the negative group(P<0.05). Conclusion:There is a correlation between the activation of telomerases and p53 gene mutation in the development of breast cancer,and they are perhaps relation to the down regulation of bcl-2.

Silencing Sp1 suppresses telomerase activity and promotes apoptosis of SW480 cells line in colorectal carcinoma

Objective:The aim of the study was to examine the effect of Sp1 on the expression of the human telomerase reverse transcriptase(hTERT) gene in human colorectal carcinoma SW480 cells.Methods:The Sp1 shRNA plasmid was transfected into colorectal carcinoma SW480 cells line by liposome mediation for transient expression.After Sp1 shRNA plasmid transfected SW480 cells,the exogenous Sp1 protein expression was determined by the method of Western blot.At same time,hTERT mRNA expression was detected by RT-PCR,telomerase activity was determined by the telomeric repeat amplification protocol(TRAP) assay,and the apoptotic rate of cells was also tested by flow cytometry.Results:The protein expressions of Sp1 gene could be reduce by transfecting of pGenesil-1-Sp1(+) recombinant plasmid into SW480 cells.The apoptotic rate was increased compared with pGenesil-1-Sp1(-)/SW480 and SW480(P < 0.05),which indicated that lowexpression of Sp1 gene could lead to low level of telomerase activity and induce apoptosis.Conclusion:Silencing Sp1 may suppress the activity of telomerase by inhabiting hTERT gene expression.

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA （siRNA） has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA （hTR） can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells （HRCCs）.METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined.RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed.CONCLUSION siRNA agains： the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.

An experimental research of Weining granule in treating gastric precancerous lesions

Objective： To investigate potential therapeutic effects and mechanism of Weining granule in the treatment of gastric precancerous lesions. Methods： Sixty rats were randomly assigned to a blank group or a model group or to receive retinoic acid or high-, medium- or low- dose of Weining granule. General conditions of the animals were observed before and after treatment. Changes in gastric mucosal pathohistology, telomerase activity, proliferation index （PI） and apoptosis index （AI） were measured. Results： General conditions, including activity and eating, were improved in all Weining-granule-treated groups with the numbers of rats having intestinal metaplasia （IM）, atypical hyperplasia （ATP） or positive telomerase activity being significantly lower than those in the model group （P 〈 0.05 or P 〈 0.01）. Compared with the model group, all doses of Weining granule significantly decreased PI （P 〈 0.01） and increased AI （P 〈 0.05）. Conclusion： Weining granule may provide a therapeutic benefit for the treatment of gastric precancerous lesions by inhibiting telomerase activity and proliferation of gastric cancer cells and by accelerating their apoptosis.

Decreasing Pin1 suppresses telomerase activity by NF-κB in HCT116 cells colorectal carcinoma

Objective： The aim of our study was to investigate the effect of Pin 1 on the telomerase activity in human colorectal carcinoma HCT116 cells. Methods： Firstly, we transfected plasmid pGenesil-l-Pinl （p-shRNA） using liposome （Lipofectamine 2000） into colorectal cancer HCT-116 cells to down-regulate the expression of Pin1. To detect the apoptotic rate of HCT116 cells was by cytometry （FCM）. The expression of Pin1 and hTERT at RNA levels in human colorectal cancer HCT116 cells were determined by RT-PCR. To evaluate the activity of telomerase was by TRAP-silver staining. The subcellular localization and accumulative level of p-NF-κB/p65 protein at the nuclear was detected by Immunofluorescence and Western blotting. The DNA-binding activity of NF-κB/p65 was detected by electrophoretic mobility shift assay （EMSA）. Results： Using liposome into colorectal cancer HCT-116 cells, and down-regulate the expression of Pin1 （0.392 ± 0.072-fold; P = 0.001）, and the apoptotic rate was increased （11.40% ± 1.54%; P 〈 0.05）. Compared with transfected p-CON cell group, in transfected p-shRNA cell group, the transcription of hTERT was lower （0.171 ±0.060-fold; P = 0.001） by quantitative real-time RT-PCR, and the results of TRAP-silver staining analysis suggested that the telomerase activity was significantly declined （0.384 ± 0.015-fold; P 〈 0.05）. Furthermore, it was demonstrated by Immunofluorescence that p-NF-κB/p65 had a nuclear localization, and the level of p-NF-κB/p65 protein at the nuclear was reduced with silencing the expression of Pin1 by Western blotting. Using EMSA, it was suggested that NF-κB/p65 was able to bind to hTERT promoter, and the direct interaction was declined with silencing the expression of Pin1. Conclusion： Taken together, silencing Pin1 may suppress activity of telomerase and the expression of hTERT by inhibiting NF-κB/p65 activity and reducing the combination of NF-κB/p65 and hTERT gene promoter.

PRMT5通过抑制NF-κB削弱DR4介导的趋化因子CCL20释放

构建表达DR4的质粒pCMV-DR4-HA(DR4)和表达PRMT5的质粒pCMV-PRMT5-Flag,共转染293T细胞,验证DR4和PRMT5的相互作用.将DR4和蛋白精氨酸N端甲基化转移酶5(protein arginine methyltransferase5,PRMT5)两种质粒分别或者共转染293T细胞,研究PRMT5对DR4引起的炎症因子释放的影响,探讨PRMT5抑制DR4(tumor necrosis factor-related apoptosis-inducing ligand receptor1)引起炎症因子释放的分子机制.分别通过RT-PCR和ELISA方法对炎症因子的表达进行定量检测.通过双荧光报告基因方法检测PRMT5对DR4引起NF-κB活性变化的影响,并且使用WesternBlot方法检测ERK的表达变化.DR4和PRMT5在293T细胞中能相互结合,PRMT5过表达降低了DR4引起的NF-κB活性和ERK的磷酸化,导致CCL20分泌减少.因此,PRMT5在293T细胞中与DR4结合,通过改变NF-κB和ERK激酶活性影响了CCL20的分泌,参与了DR4介导的细胞免疫调节.