RASSF1,a putative tumor suppressor gene identified at 3p21.3 has been frequently observed.Many reports suggested that those was no RASSF1 expression or was at abnormally low level in many carcinomas.It is possible that aberrant DNA methylation and loss of heterozygosity might be involved in the loss of RASSF1 expression.Other results demonstrate a relationship between EBV infection and aberrant methylation.

ObjectiveTo study the expression and the role of α-smooth muscle aetin (α-SMA) and collagen Ⅰ ( Col Ⅰ ) in lung fibroblasts of newborn rats with hyperoxia-induced lung injuries.Methods Thirty full-term newborn Wistar rats were randomly assigned to hyperoxia group (90％ oxygen exposure,n =15 ) and air group (room air exposure,n =15) within 12 h after birth.Then lung fibroblasts were isolated and primary cultured from rat lungs on postnatal 3 d,7 d,and 14 d.The distribution of α-SMA protein were measured by immunohistochemistry.The levels of Col Ⅰ were detected by ELISA.ResultsThere were no significant differences in the levels of α-SMA and Col Ⅰ expressions between the two groups at 3 d ( P＞0.05 ).While the expression of α-SMA ( 112.60 ± 4.61 vs 94.69 ± 2.38,200.30 ± 3.97 vs 103.04 ± 1.91,P＜0.01 ) and Col Ⅰ protein [ ( 28.66 ± 1.15 ) μ.g/L vs ( 24.62 ± 3.15 ) μg/L,( 30.60 ± 0.65 ) μg/L vs (27.46 ± 1.68 ) μg/L,P ＜ 0.05 ] in lung fibroblasts caused by hyperoxia were significantly higher than those in air-exposed group on postnatal 7 d and 14 d.There was positive correlation between α-SMA and Col Ⅰ protein ( r =0.72,P＜0.01 ).ConclusionHyperoxia promotes differentiation of lung fibroblasts into myofibroblasts,and synthesis of type Ⅰ collegen in neonatal rats,which leads to lung fibrosis finally.

Objective To investigate dynamic changes of extracellular signal regulated protein kinase (ERK) 1/2 in lung fibroblast of newborn rats with chronic lung disease (CLD) caused by hyperoxia.Methods Full-term newborn rats were randomly divided into two groups:air-exposed group and hyperoxia - exposed with 90％ oxygen group.Rats were sacrificed separately 3 d,7 d and 14 days after exposure to air or 90％ oxygen. Then lung fibroblasts of rats were isolated and primarily cultured. By using Immunocystochemistry,Western-blot and RT-PCR methods,the levels of ERK1/2 protein and expressions of ERK1/2 mRNA were measured. Results The levels of p-ERK1/2 protein in lung fibroblast in the hyperoxia group were significant higher on the 7th day and 14th day after exposure to 90％ oxygen compared with those in the air-exposed group (P ＜0.01 ).And the levels of total ERK1/2 protein and expressions of ERK1/2 mRNA did not change noticeably and were not significantly different between two groups (P ＞0.05 ).Conclusions The activation of phosphorated ERK1/2 may lead to lung fibrosis caused by hyperoxia in newborn rats.

ObjectiveTo investigate the relationship between metastasis-associated gene 1 ( MTA1 )expression and invasive and metastatic ability of cervical cancer cell. MethodsThree kinds of plasmids pcDNA3( control group), pcDNA3-MTA1 ( MTA1 group) and pSilencer3. 1-MTA1-siRNA ( MTA1-siRNAgroup) were transfected into human cervical cancer cell line CaSki cells. Reverse transcription (RT)-PCR and western blot were used to detected MTA1 mRNA and protein expressions. The effects of MTA1 expression on CaSki cell growth and proliferation, cell migration, adhesion and invasion, and cell cycles were tested by methyl thiazolyl tetrazolium (MTT), clone formation experiment, wound-healing assay, transwell assay, adhesion assay and flow cytometry, respectively. In animal experiment, three groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability. ResultsCompared with control group, MTA1 mRNA and protein were significantly overexpressed in MTA1 group, while MTA1-siRNA group showed lower MTA1 expression. Compared with control group, MTA1 group showed significantly accelerated cell growth; while MTA1-siRNA group showed decreased cell growth since the second day (P＜0. 05). Clone formation number in control, MTA1 and MTA1-siRNA group were 133 ±6, 169 ± 10 and 57 ±5,respectively. MTA1 group showed accelerated cell formation, while MTA1-siRNA group showed the reverse effect compared with that in control group(P ＜ 0. 05 ). At 24, 48 and 72 hours after wounding, the healing ability of MTA1-siRNA group significantly lagged behind that in the control group, while MTA1 group showed accelerated cell healing ability. The adhesion rate of control, MTA1 and MTA1-siRNA group were (69. 3 ± 3. 6) ％, ( 80. 4 ± 5. 6 ) ％ and ( 39. 2 ± 7.4 ) ％ separately at 90 minutes after cell seeding. In contrast with control group, MTA1 group promoted the adhesion of CaSki cell to matrigel matrix, while MTA1-siRNA group inhibited the adhesion process (P ＜0. 05 ). In the migration assay, the number of cells migrated to the bottom side of the membrane in control,MTA1 and MTA1-siRNA group were 153 ± 17,247 ± 38 and 82 ± 10, respectively. The number of cells in the invasion assay were 231 ± 19,354 ± 36 and 76 ± 7, respectively. Compared with the control group, MTA1 group significantly increased the migration and invasion ability, while MTA 1-siRNA group showed lower cell migration and invasion ability (P ＜ 0. 05 ). In cell cycle experiment, no significant differences of cell proportions including G1, S and G2 stage were found among three groups (P ＞ 0.05).In animal experiment, compared with control group,MTA1 group showed accelersted tumor formation and growth,whilethe MTA1-siRNA group showed the reverse effect ( P ＜ 0. 05 ). ConclusionsMTA1 may play its roles to promote cervical cancer cell invasion, migration, adhesion, as well as cell growth and colony formation, while RNA interference against MTA1 may decrease the malignant phenotypes. This study shows that it will be an effective beginning to explore metastasis mechanisms and cancer gene therapy strategy targeting MTA1 in cervical cancer.

Using the cyclodextrin bonded polysiloxane (bikis〔（2,6-di-O-pentyl-3-O-hex-6-O-enyl)-pentakis(2,6-di-O-pentyl-3-O-methyl)-β-cyclodextrm-polysiloxane〕) as gas chromatographic stationary phase, α-hydroxy acetones were separated and the values of the enantiomeric excess(e.e) of 3-hydroxy-2-butanone were determined. The results showed that the high resolution gas chromatography (HRGC) using the chiral gas chromatographic stationary phase could determine the productive rate of the asymmetric hydrogenaton reaction and evaluate the enantoselectivity of the catalyst system.

OBJECTIVE:To establish a method for simultaneous determination of 10 flavonoids in Astragalus membranaceus. METHODS:HPLC method was adopted. The determination was performed on Agilent SB-C18 column with mobile phase consisted of acetonitrile-0.3% formic acid (gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength was set at 254 nm, and the column temperature was 35 ℃. The sample size was 10 μL. RESULTS:The linear ranges of calycosin-7-O-glucoside,iso-quercitrin,genistin,ononin,calycosin,quercetin,genistein,kaempferol,isorhamnetin and formononetion were 0.03029-1.5145μg (r=0.9994),0.01500-0.7500 μg(r=0.9995),0.00739-0.3695 μg(r=0.9991),0.12011-6.0055 μg(r=0.9998),0.03836-1.918 μg (r=0.9999),0.02989-1.4945 μg(r=0.9995),0.00704-0.352 μg(r=0.9994),0.01683-0.8415 μg(r=0.9995),0.00454-0.227μg(r=0.9999),0.01336-0.668 μg(r=0.9999),respectively. RSDs of precision,stability and reproducibility tests were all lower than 2.0% . The recoveries were 99.55% -100.45%(RSD=0.36% ,n=6) ,99.34% -101.00%(RSD=0.59% ,n=6) , 98.05%-100.36%(RSD=1.27%,n=6),99.73%-100.13%(RSD=0.18%,n=6),99.70%-100.30%(RSD=0.22%,n=6), 99.67%-103.27%(RSD=1.37%,n=6),98.13%-104.41%(RSD=2.37%,n=6),96.35%-100.06%(RSD=1.46%,n=6), 99.47%-101.13%(RSD=0.60%,n=6),99.70%-100.06%(RSD=0.15%,n=6),respectively. CONCLUSIONS:This method is convenient,sensitive,stable and reproducible,can be used for simultaneous determination of 10 flavonoids in A. membranaceus.

OBJECTIVETo evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cells in vitro and in vivo.

METHODSCells were transfected with Ad-p53 (100 MOI), and PCNA-ASO (1.6 micro mol/L) was then introduced into the cells using a cationic lipid (lipofectamine, 20 micro l/ml). In vitro and in vivo antitumor effects of combining PCNA-ASO with Ad-p53 were measured using the MTT assay, flow cytometry, clone formation, and a nude mice model.

RESULTSThe combination of PCNA-ASO and Ad-p53 inhibited cell viability in both the EJ (89.3%) and BIU-87 (78.6%) cell lines. The ability of the cells to form foci was also reduced by 74.8% in EJ cells and by 67.5% in BIU-87 cells (P < 0.01). A significant decrease of cells in the S phase (11.4% in EJ cells, 14.6% in BIU-87 cells) and a significant increase of cells in G1 phase (62.2% in EJ, 56.8% in BIU-87) were noted. The mean tumor volume after 7 days of treatment with PCNA-ASO or Ad-p53 in combination decreased to 47.6% or 36.4% of the initial tumor size in the two cell lines respectively.

CONCLUSIONThese results indicate that combined PCNA-ASO and Ad-p53 in the treatment of bladder cancer with mutant p53 has important therapeutic potential, significantly suppressing the growth of human bladder cancer both in vitro and in vivo.

Adenoviridae ; Animals ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Nude ; Oligonucleotides, Antisense ; administration & dosage ; Proliferating Cell Nuclear Antigen ; administration & dosage ; Recombinant Proteins ; Transfection ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; administration & dosage ; Urinary Bladder Neoplasms ; therapy

Objective To investigate the expression and clinical significance of ATAD2 in patients with hilar cholangiocarcinoma (HC) Methods The qRT-PCR,Western blottig and immunohistochemistry were used to detect the expression of ATAD2 in HC and normal bile duct tissues,and the relationship between ATAD2 and the clinicopathological factors,prognosis was analyzed.Results The positive expression rate of ATAD2 in HC tissues was 70％ (57/81),significantly higher than that of normal bile duct tissues 7％ (2/30) (x2 =35.678,P ＜0.01),and ATAD2 expression was associated with lymph node metastasis and TNM staging (respectively x2 =28.619,31.612,all P ＜ 0.01).The 1'-,3'-,and 5-year survival rates of ATAD2 negative HC patients were 87.5％,54.1％,and 33.3％,respectively.The 1'-,3'-,and 5-year survival rates of ATAD2 positive HC patients were 40.3％,14.0％ and 1.7％,respectively (x2 =14.162,P ＜ 0.01).Univariate analysis and multivariate COX regression model analysis showed that microvascular invasion,lymph node metastasis and ATAD2 expression were independent factors influencing the prognosis of patients with HC (respectively F =4.703,4.961,5.013,all P ＜ 0.05).Conclusion The expression of ATAD2 in HC increased,and the overexpression of ATAD2 was closely related to the occurrence,development,invasion,metastasis and poor prognosis of HC.

To evaluate the effects of adenovirus (Ad)-mediated transfer of p53 and p16 on human bladder cancer cells EJ, EJ were transfected with Ad-p53 and Ad-p16. Cell growth, morphological change, cell cycle, apoptosis were measured using MTT assay, flow cytometry, cloning formation, immunocytochemical assays. Ad-p16 or Ad-p53 alone could inhibit the proliferating activity of EJ cells in vitro. Ad-p53 could induce apoptosis of partial EJ cells. G1 arrest was observed 72 h after infection with Ad-p16, but apoptosis was not obvious. The transfer of Ad-p16 and Ad-p53 could significantly inhibit the growth of EJ cells, decrease the cloning formation rate and induce apoptosis of large number of EJ cells. The occurrence time of subcutaneous tumor was delayed and the tumor volume in 4 weeks was diminished by using Ad-p53 combined with Ad-p16 and the difference was significant compared with using Ad-p53 or Ad-p16 alone. It was suggested that the transfer of wild-type p53 and p16 could significantly inhibit the growth of human bladder cancer in vitro and in vivo.
Adenoviridae ; genetics ; Animals ; Cell Division ; Genes, p16 ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Transfection ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; genetics ; pathology ; therapy