This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.
Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; pharmacology ; Transfection

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells.

METHODSApoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit.

RESULTSAfter 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05).

CONCLUSIONThe hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.

Animals ; Apoptosis ; drug effects ; HL-60 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; genetics ; Transfection ; Xenograft Model Antitumor Assays

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.

AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS:The effects of cladribine at different concentrations on the cell viability were detected by CCK -8 assay.Apop-tosis and cell cycle distribution were examined by flow cytometry.The protein expression levels were determined by Western blot.The levels of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA.The nitric oxide(NO)production was measured by Gries method.RESULTS:Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners.The IC50was about 3.644 μmol/L.The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L,and 77.23 % cells in S phase when the concentra-tion of cladribine was 1 μmol/L.The apoptosis was not induced by cladribine at 0.4~10 μmol/L.The protein expression of Bax and caspase-3 did not change.The expression of p21 increased and the p53 decreased(P<0.05).The levels of TNF-αand TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h.The levels of VEGF and NO decreased.CONCLUSION:Cladribine obviously inhibits the viability of EA.hy926 cells.The mechanism is related to the cell cycle arrest.Cladribine promotes the secretion of TNF-αand TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.

OBJECTIVETo retrospectively analyzed the clinical features and epidemiology of children with severe hand-foot-and-mouth disease during 2009 and 2010 in Shanghai to investigate some risk factors with fatal cases.

METHODAll the clinical records and laboratory results of serious patients were collected. A retrospective study was performed.

RESULTA total of 748 serious patients were enrolled into this study, and the ratio of male to female was about 1.7:1; 724 patients were categorized into stage 2 with 254 patients in 2009 and 470 in 2010; 24 patients were categorized into stage 3 with 17 in 2009 and 7 in 2010. The rate of severity in 2010 (1.5%) was lower than in 2009 (6.3%) (χ2=12.836, P<0.01). Seven patients of stage 3 died, with the fatality 29.2%, which was higher than in stage 2 (P<0.01). The children aged between 3 months 10 days to 12 years 9 months with onset median age of 25 months. Among them, 77.1% patients aged between 1 and 4 years which also accounted for 79.2% of the fatal cases (19/24). But there was no significant difference between the age and the severity (χ2=0.804, P>0.05). Fever (100%), vomiting (57.0%) and myoclonus jerk (62.3%) were the most frequent symptoms occurred in those serious cases. The average period of fever in children of stage 2 and 3 was (4.10±1.40) d and (5.05±1.05) d, respectively, which indicated significant difference between the two groups (t=3.173, P<0.05). The average values of white-blood-cell counts and blood glucose in fatal patients were (14.8±6.25)×10(9)/L and (8.63±3.51) mmol/L. They were higher when compared to those in stage 2 with the white-blood-cell counts of (11.8±4.23)×10(9)/L and blood glucose of (5.51±2.14) mmol/L (P<0.05). But there was no significant difference in C-reactive protein or cerebrospinal fluid white-blood-cell counts; A total of 182 patients were enrolled for MRI study during the acute stage with 37 (37/182, 20.3%) presented abnormal findings. Among them, most frequent findings were hyperintense lesions seen in brain stem (11 cases). A stage 3 case who died presented brain edema on MRI examination.

CONCLUSIONThe epidemic of HFMD has some correlation with the area, season, health condition of the family and gender of the children. Children under 4 years of age especially those who lived in rural areas were susceptible to the HFMD. Frequent vomiting or myoclonus jerk may indicate the central nervous system involvement. But persistent high fever may indicate tendency to deteriorate. Some laboratory examinations can help find the fatal cases at an early time.

Adolescent ; Child ; Child, Hospitalized ; Child, Preschool ; China ; epidemiology ; Enterovirus A, Human ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; physiopathology ; virology ; Humans ; Infant ; Male ; Retrospective Studies ; Risk Factors

Objective To provide data as age-gender dependent mean,standard deviation and percentile on height,weight.waist circumference (WC),hip circumference (HC).body mass index (BMI),waist hip ratio (WHR),waist to height ratio (WHtR) among 7-16 year-olds Chinese children and adolescents,towards setting up diagnostic criteria on metabolic syndrome for them.Methods A representative sample involvng 22 197 children and adolescence aged 7 to 16 years were randomly surveyed and they were from 6 representative geographical areas,including Beijing.Tianjin? Hangzhou,Shanghai,Chongqing and Nanning.A total of 21858 had available data,with male/female ratio as:11460/10 398.Using the standard methods,we measured height,weight.WC,HC,BMI.WHtR and other data in all age groups.Physique indexes among different geographic regions (North.Mid-west and East) were compared.Results (1) Both male and female showed an inaeasing trend of height.weight,waist circumference,hip circumference and BMI along with the increase of age.WHR of girls decreased gradually from 0.84 to 0.76 went from 7 to 16 years old while WHR of boys changed from 0.87 to 0.81 accordingly.(2) WHtR was rarely affected by age.It fluctuated between 0.42-0.43 in all girls and 0.44-0.45 in boys less than 11 years.WHtR of boys older than 12 years showed a slight decline from 0.45 to 0.42 of WHtR.(3) The average height,weight,BMI of children and adolescents from the northem regions (Beijing,Tianjin) were significantly higher than that of the mid-western (Chongqing,Nanning) and the eastem regions (Shanghai,Hangzhou) (P＜0.001 ),while those from the mid-western region were slightly higher than that of the eastern region (P＜0.05) in each of the age group.Conclusion Reference values and percentile curves for WC and WHtR of Chinese children and adolescents were provided.For the assessment of central obesity.WHtR had the advantages of relative stability and small degree of variation and rarely affected by age and gender,when compared with WC.and could be used as an simple index to reflect the central obesity of children and adolescents.

In this study, the molecular marker technology of SRAP and ISSR were applied in rapid identification of seeds from eight species of Brassica oleracea L. Firstly, using the genomic DNA of cabbage as template, SRAP and ISSR reaction systems were optimized through testing every factor, respectively, that affects PCR amplification. Then, using the optimized reaction systems, 30 SRAP primer pairs and 15 ISSR primers were applied to amplify genomic DNA of cabbage, savoy, purple cabbage, borecole, cauliflower, broccoli, Brussels sprouts, and kohlrabi The results showed that high polymorphisms were exhibited among the eight species of Brassica oleracea L. by SRAP primer pairs of M3-E5 and M4-E5, as well as ISSR primers of 844 and 888, especially primer 844 which can identify all eight materials efficiently.
Brassica ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Polymorphism, Genetic ; Repetitive Sequences, Nucleic Acid ; Seeds ; genetics

OBJECTIVETo determine the prevalence of Streptococcus suis and major pathogenic serotypes in middle part of Jiangsu province.

METHODSTonsillar specimens from 303 slaughtered pigs aged 6 to 8 months were investigated for the presence of Streptococcus suis and major pathogenic serotypes by polymerase chain reaction (PCR) method. Bacteriological examination compared with molecular genetics identification for three Streptococcus suis isolates were also done.

RESULTSThe overall carrier rate of Streptococcus suis was up to 88.0%, with the percentages of serotype 1(14), 2(1/2), 7 and 9 were 9.6%, 8.5%, 11.3% and 29.5% respectively in 2005. While in 2006, the prevalence of Streptococcus suis was 82.5%, with capsular types 1 (14), 2 (1/2), 7 and 9 were accounted for 17.6%, 2.4%, 25.8% and 20.0% of all the specimens. All the three isolates belonged to Streptococcus suis serotype 2,named 2a, 2f and 14e, which exhibiting the virulent phenotype cps2+/gdh+/mrp-/lepf-/sly-/fbps+/orf2+/89k-, cps2+/lgdh+/mrp-/epf-/sly-/fbps-/orf2-/89k- and cps2+/gdh+/mrp-/epf-/sly-/fbps/orf2-/ respectively. These isolates were all susceptible to amoxicillin, ampicillin, penicillin and resistant to amikacin and tetraycline. Clinical signs were not noted in BALB/c mice and rabbit.

CONCLUSIONPrevalence of the Streptococcus suis among the healthy herds in the areas was very high, with various capsule types of Streptococcus suis involved in the same herds, and the virulent phenotype of these 3 isolates were very different from those prevalent Streptococcus suis serotype 2 virulent isolates frequently discovered from the epidemic areas.

Amikacin ; therapeutic use ; Amoxicillin ; therapeutic use ; Ampicillin ; therapeutic use ; Animals ; China ; epidemiology ; Mice ; Mice, Inbred BALB C ; Molecular Epidemiology ; methods ; Penicillins ; therapeutic use ; Polymerase Chain Reaction ; Streptococcal Infections ; drug therapy ; epidemiology ; microbiology ; Streptococcus suis ; classification ; drug effects ; genetics ; pathogenicity ; Tetracycline ; therapeutic use ; Virulence

OBJECTIVETo study the effect of gamma linolenic acid (GLA) on atherogenesis in rats.

METHODSixty healthy male rats were randomly divided into 6 groups: normal contro 1, fed by normal feed; atherogenesis mode 1, fed by high lipid diet; positive control group 0.9 mg x kg(-1) x d(-1) of lovastatin and group IV 250 mg x kg(-1) x d(-1) duoxikang; high dose of 375 mg x kg(-1) x d(-1) GLA; low dose of 187.5 mg x kg(-1) x d(-1) GLA. After the model group received atherogenic diet for six weeks, serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were detected by enzyme method to confirm the formation of atherogenic. After fed for another five weeks, morphologic atherosclerosis of aorta in rats was observed by HE staining methods. The blood samples were collected and serum TC, TG, HDL-C, LDL-C, T-AOC, HL, LPL, NO, NOS, MDA and GSH were determined.

RESULTGLA attenuated the formation of atherosclerotic plaques, inhibited the level of serum TC, TG, MDA, OX-LDL, NO, NOS, HL, LPL and LDL-C and increased the level of T-AOC.

CONCLUSIONGLA might significantly attenuate the development of atherosclerosis in rats fed with high lipid diet through improving the antioxidation capacity of the body.

Animals ; Atherosclerosis ; blood ; drug therapy ; metabolism ; Cholesterol ; metabolism ; Diet, Atherogenic ; Lipids ; blood ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; gamma-Linolenic Acid

Objective To investigate the lipid levels of Han ethnicity Chinese children at school-age,to provide objective data for the formulation of prevention and management strategy regarding dyslipidemia among children and adolescents.Methods 20 191 children (with 10 669 boys and 9522 girls) aged 7 to 16 years old from 6 representative geographical areas,including Beijing,Tianjin,Hangzhou,Shanghai,Chongqing and Nanning,were surveyed in a randomly selected clustered sample in China.Data on fasting blood triglyceride (TG),total cholesterol (TC),lowdensity lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were measured.Non-high-density lipoprotein cholesterol (non-HDL-C) levels were calculated with data collection,entry,and collation were under the same criteria.Results (1) In the 7-16 year-old group,TG (P95) fluctuated between 1.26 mmol/L and 1.88 mmol/L,while TC (P95) was between 4.80 mmol/L and 5.46 mmol/L.LDL-C(P95) was between 2.67 mmol/L and 3.27 mmol/L while non-HDL-C (P95) was between 3.36 mmol/L and 3.91mmol/L,sugesting that age did not seem to be an affecting factor for the lipid level (P＞0.05).The level of HDL-C (P5) fluctuated bctwcen 1.08 mmol/L and 0.83 mmol/L,and the dependability analysis on HDL-C and age showed statistically significant difference (P＜0.01,r=-0.274).(2) In the 7-9 year-old group,the levels ofTG,TC,LDL-C and non-HDL-C of boys were lower but the HDL-C level was higher than in girls.However,in the 10-16 year-old group,the levels of five lipids of boys were all lower than in girls,with all the differences statistically significant (P＜0.05).(3) The levels of TG,TC,LDL-C and non-HDL-C in the obese group were significantly higher than those in non-obesity group,as HDL-C was significantly lower than in non-obese group(P＜0.01).Incidence rates of single and multiple dyslipidemia in obese group were significantly higher than in non-obese group (P＜0.01).(4) Grouped by region,the abnormal rates of TG were descending,with the ranking as North (10.4％),Midwest (9.7％) and East (8.3％),while the abnormal rates of TC were descending with the ranking as Midwest (6.0％),North (5.2％)and East (4.8％).The abnormal rates of LDL-C were descending as the ranking of North (3.1％),East (2.6％) and Midwest (0.9％),with the abnormal rates of non-HDL-C were descending as Midwest (6.5％),North(4.2％)and East (3.6％).The abnormal rates of HDL-C were descending as Midwess (14.2％),North(5.7％) and East(5.5％).All the differences in the above-said items were statistically significant (P＜0.05).(5) According to the standards of hyperlipidemia formulated by the American Academy of Pediatrics,the incidence rates of abnormal TG,TC,LDL-C,non-HDL-C,HDL-C were 9.4％,5.4％,2.2％,4.8％,8.6％ respectively.Conclusion (1) Levels of lipids were affected by many factors,but age was not one of them in children and adolescents.However,HDL-C was declining along with the increase of age,to some extent.(2)Girls had a relatively protective tendency through the increasing HDL-C level when they entered the puberty years.(3)Lipids levels in non-obese group were significantly better than the obese group.(4)The lipids levels of children and adolescents in the Eastern region of the country were better than that in the northern and mid-western areas.