This study was aimed to investigate the impact of specific siRNA on survivin gene in transfected lymphoma cell line and provide experimental evidences for future treatment of mantle cell lymphoma. The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into Jeko-1 that showed high survivin expression in mRNA level. The levels of survivin mRNA and protein expression were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. The apoptosis effect was examined by calculating the ratio of Annexin V-FITC/PI positive cells using flow cytometry. The inhibition of cell proliferation was assayed with CCK-8 reagent after transfection. The results showed that expression of survivin mRNA was markedly suppressed by the siRNA. The relative expression levels were 0.49 ± 0.03, 0.38 ± 0.02 and 0.17 ± 0.02 at time points of 24, 48 and 72 h respectively, compared with the control group; the inhibitive rates of cell proliferation were (31.2 ± 2.1)%, (43.3 ± 3.4)% and (52.6 ± 2.5)%; the apoptotic rates of cells were (6.3 ± 0.5)%, (13.5 ± 1.1)% and (23.6 ± 1.6)% respectively; survivin protein expression levels were gradually reduced. It is concluded that the siRNA targeting survivin down-regulates the expressions of survivin mRNA and protein evidently. The siRNA of survivin displays the potent ability to inhibit the proliferation of lymphoma cell line Jeko-1; survivin may become a potential molecular target for the therapy of lymphoma in the future.
Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the apoptosis effect of diffuse large B-cell lymphoma cell line (DLBCL) SUDHL-4 induced by IL-21 and its related mechanism.

METHODSSUDHL-4 cells were treated with IL-21 at different concentration (1000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) for 24 h, 48 h, 72 h, respectively. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The cell growth curves were drawn and half inhibitory concentration (IC(50)) values were calculated. The cell apoptosis were detected by flow cytometry (FCM), the expression of the caspase-9, caspase-3, cleaved caspase-3, Bcl-2, Bcl-XL, Bid, Bax and c-myc protein in SUDHL-4 cells treated with IL-21 by western blot, the mRNA expression of Bcl-2, Bcl-XL, Bid, Bax, c-myc by Survivin gene with RT-PCR.

RESULTSIL-21 markedly inhibited SUDHL-4 cell growth in a time- and dose-dependent manner. The 48 hIC(50) was 140.9ng/ml; The FCM showed that the apoptosis proportion of SUDHL-4 cells treated with 100 ng/ml of IL-21 apoptosis (AnnexinV-FITC(+) positive cells) gradually increased (48 h: 19.7 ± 2.3%). The protein expression of caspase-9, caspase-3, Bcl-2 and Bcl-XL decreased in a time-dependent manner. The Bax and c-myc protein markedly increased, but the Bid protein level did not change. IL-21 up regulated c-myc and Bax gene expression, however down regulated Bcl-2 and BCL-XL gene expression, but the gene expression of Bid and Survivin hadn't been changed significantly.

CONCLUSIONSIL-21 can inhibit proliferation and induce apoptosis of SUDHL-4 cell. The mechanism may involve in endogenous mitochondrial pathway mediated by the c-myc and the Bcl-2 genes.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Humans ; Interleukins ; administration & dosage ; pharmacology ; Lymphoma, Large B-Cell, Diffuse ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-X Protein ; metabolism

BACKGROUND: Cancer stem cells have bean a hot topic in life science research. EpCAMhigh/CD44+ colorectal cancer stem cells are beneficial to observe morphological characteristics and distribution of cancer stem cells.OBJECTIVE: To investigate the quantity, location, distribution and hematoxylin-easin staining morphologic features of colorectal cancer stem cells (Co-CSCs).DESIGN, "rIME AND SETrlNG: The observational study was performed at the Department of Pathology, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from March to August 2008.MATERIALS: A total of 67 colorectal cancer paraffin embedding samples were collected from 200512007 archive at the Department of Pathology, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA, comprising 27 males and 40 females, aged 29 90 years.METHODS: EpCAM and CD44 was used to label cancer stem cells in paraffin embedding colorectal cancer samples. The locations of EpCAM and CD44 double positive cells were detected by SP(streptavidin HRP) immunohistochemical staining and double immunohistochemical staining. The morphologic features of them were investigated on hematoxylin-easin staining at the same position.MAIN OUTCOME MEASURES: Number, location, distribution of EpCAM and CD44-posltive cells, and morphology following hematoxylin-eosin staining.RESULTS: The number of double-positive cells accounted for 0.1% to 30.0% of all tumor cells, and the cells were scattered or distributed focally along the basement of glandular-like structure. The cells with scarcely cytoplasm were cube or oval, and its nucleus was oval or high cylindrical, deep stained and homogeneous; The quantity of double-positive cells were negatively correlated with the differentiation of colorectal cancer.CONCLUSION: Cancer stem cells are the causes of tumor development, metastasis and recurrence and drug resistance. To observe the number, location, distribution and morphology of Co-CSCs and to analyze the relationship between Co-CSCs and pathological parameters will provide guidance for the diagnosis, staging, prognostic evaluation and clinical prognosis of cotorectal carcinoma.

BackgroundOsteopontin is highly expressed in many metastatic tumors,but its expression in uveal melanoma and the relationship of osteopontin with metastasis is still under investigation.Objective This study was to investigate the relationship between osteopontin expression and uveal melanoma metastatic potential.Methods Fifty samples of uveal melanoma were collected in Beijing Tongren Hospital.The expressions of osteopontin in the samples were detected using immunohistochemistry,and expressions of osteopontin mRNA in different metastatic potential uveal melanoma cell lines (MU M-2B,C918,OCM-1A) were analyzed using quantitative polymerase chain reaction (Q-PCR).ResultsOsteopontin was positively expressed in 10 samples of 13 patients with metastasis and 14 samples of 37 patients without metastasis,showing a significant difference between them (x2=5.888,P=0.000).There were 11 samples and 13 samples exhibiting the positive expression of osteopontin in 15 epithelial type and 35 non-epithelial type of uveal melanoma respectively with the significant difference (x2=5.510,P =0.000).The positive expressing numbers of osteopontin were 20 tumors of 30 affected tumors and 4 tumors of 20unaffected ciliary tumors with the significant difference (x2 =10.470,P =0.000 ).No significant differences in the expression of osteopontige were found between different gender,age,eyes,largest basal diameters and invasion of scleral canal( P =0.536,0.256,0.802,0.848,0.555 ).The relative expression value of osteopontin mRNA was in turn 1.00±0.04,0.91 ±0.03,0.08±0.01 in MUM-2B,C918,OCM-1A with the significant difference among three cell lines( F=33.135,P＜0.05).The osteopontige mRNA expression was significant enhanced in high metastatic potentialcell lines( MUM-2B,C918 ) compared with low metastatic potential cell lines( OCM-1 A) (P＜0.05 ),but no significant difference was seen between two high metastatic potential cell lines(MUM-2B,C918) (P=0.804).Conclusions Osteopontige expression is associated with the uveal melanoma cell line invasion and metastasis potential.These results imply that osteopontige expression maybe an indicator of uveal melanoma invasion and metastasis ability.

Objective To discuss clinical effects and indications of minimally invasive tension band technique in treatment of transverse patella fractures.Methods Thirty-eight patients with transverse patella fracture (close or slightly polluted open ones) were treated with minimally invasive tension band technique from July 1997 to June 2005 in our department.Twenty-six were fixated with percutaneous Kirschner wire tension hand and 12 with percutaneous cannulated screw tension band.The reduction,movement range and function of the knee were analyzed.Results Surgeries went on uneventfully.All the cases were followed up from six months to 6.5 years (average,32 months).Fracture healing time lasted from 6 to 12 weeks,averaging 7.7 weeks.No such compli- cations as traumatic arthritis occurred.Reduction was excellent in 21 cases,good in 11 and fair in six,with the good-to-excellent rate being 84.2%.All the affected knee joints could move in normal range.The functional re- covery was assessed as excellent in 34 cases and good in four.Conclusions Internal fixation with minimally invasive tension band technique is good for treatment of transverse patella fractures.It is chiefly fit for close or slightly polluted open fractures with limited wound,and can also be used for the comminuted fractures if the bone fragments are no more than three and the fracture site is suitable.

BACKGROUND:Pelvic and acetabular fractures in elderly are frequently observed in the clinic, and the incidence gradual y increased. Moreover, it is difficult to conduct reduction and fixation due to their physical status and osteoporosis to different degrees. Locking plate is ideal fixation material in surgical treatment of acetabular fractures in elderly due to the special functions and good molding of screw and locking plate.
OBJECTIVE:To analyze the advantages and disadvantages of locking reconstruction plate used in the acetabular fracture for the aged patients and to improve the clinical effect of acetabular fractures in elderly.
METHODS:From January 2010 to February 2013, 21 aged patients with acetabular fracture were treated by open reduction and internal fixation with locking reconstruction plates made of titanium and 00Cr 18 Ni 14 Mo 3 stainless steel in the Department of Orthopedics Affiliated Hospital of Jining Medical University.
RESULTS AND CONCLUSION:Of 21 patients, 20 cases were fol owed up for 12-18 months (mean 14 months), but one dropped out. Al the fractures were healed within 4-6 months and no failure cases. According to Matta evaluation criteria, the satisfactory rate was 90%. According to the D’Aubigne scoring system, the excellent and good rate was 95%. These data indicated that locking reconstruction titanium plate is reliable, has low failure rate and satisfactory clinical effects for acetabular fracture in aged patients, and is ideal fixation material in treatment of acetabular fractures in elderly.

Objective To assess the effectiveness and safety of non-insufflation intra-abdominal compression as a prehospital hemostatic technique in treatment of liver injury.Methods Twenty-nine male Wistar rats were enrolled and randomly assigned into four experimental groups:IAP0 group ( n =8 ),IAP5 group (n =8),IAP10 group (n =8) and IAP15 group (n =5).Then,the anticoagulated rat models of severe liver injury were established with different intra-abdominal pressures ( 0,5,10 and 15 mm Hg) by using a non-expansible insufflated gas bag.Once the mean artery pressure (MAP) was less than 95 mm Hg,Lactated Ringer' s solution was infused through the right jugular vein at speed of 3.3 ml · min-1 · kg-1 until MAP reached 100 mm Hg.Thirty minutes later,the animals were killed by intravenous injection of 0.2 ml saturated potassium chloride solution to measure the intraperitoneal total blood loss,liver weight,volume of infused solution and MAP.Results No death was observed in IAP0,lAP5 and IAP10 groups and four rats (4/5) died at 10-15 minutes following exposure to the 15 mm Hg intra-abdominal pressure in IAP15 group.Total blood loss was reduced to ( 54.20 ±11.30)ml/kg in IAP0 group,(43.98 ±9.2) ml/kg in IAP5 group,(32.49 ±7.40) ml/kg in IAP10group and (25.77 ± 14.16) ml/kg in IAP15group ( P ＜0.01 ).The infused volume of resuscitation solution was (31.06 ± 3.14) ml in IAP10 group,which was higher than (24.94 ±6.67) ml in IAP0 group,(23.06 ± 7.98) ml in IAP5 group and (16.50 ±7.27) ml in IAP15 group (P ＜0.05).Liver weight was ( 11.18 ± 1.45) g in IAP5 group,( 12.13 ± 0.96) g in IAP10 group and ( 11.41 ± 1.20) g in IAP15 group,which was significantly higher than (10.03 ±0.58) g in IAP0 group (P＜0.05).The MAP was (64.81 ± 19.65) mm Hg in IAP5 group and (65.80 ± 15.36) mm Hg in IAP10 group ( insignificant difference between groups,P ＞ 0.05 ),which was higher than (41.22 ± 10.00) mm Hg in IAP0 group and (44.50 ±28.60) mm Hg in IAP15 group (P ＜0.05).Conclusions Non-pneumatic intra-abdominal compression can effectively control bleeding in rat models of severe liver injury,while the adverse effect of intra-abdominal hypertension should be avoided.

Abstract] Objective To analyze the molecular characteristics of the full-length genome of a new-ly isolated genotypeⅤJapanese encephalitis virus (JEV) strain (XZ0934) in China and the first genotypeⅤJEV strain ( Muar) isolated in Malaya 60 years ago.Methods Several softwares including ClustalX 2.0.9, DNAStar 7.1, Bioedit 7.2.5 and MEGA6.06 were used to conduct sequence alignments and phylo-genetic analysis.Results The full-length genomes of XZ0934 strain (isolated in Tibet, China in 2009) and Muar strain (isolated in Malaya in 1952) were composed of 10 983 and 10 988 nucleotides, respective-ly.The XZ0934 strain was highly similar with the Muar strain showing the homology of 90.6%in nucleotide (nt) and 98.3%in amino acid (aa).The open reading frame (ORF) of the two genotype Ⅴ JEV strains encoded 3433 aa while the ORF of other four genotypes (Ⅰ-Ⅳ) (10 299 nt) encoded 3432 aa.Compared with JEV strains of other genotypes, a serine were inserted into the NS4A gene of JEV strains genotype Ⅴand 10-14 nucleotides were inserted into the downstream of the ORF stop codon in 3′-untranslated region. Phylogenetic analysis of E sequences of all JEV strains genotypedⅠ-Ⅴrevealed that in the cluster of geno-typeⅤ, XZ0934 and 10-1827 ( isolated from mosquitoes in South Korea, 2010) stains formed a branch and were divergent from that of Muar strain indicating that there were molecular genetic differences among geno-typeⅤJEV strains after a 60 years hiatus.Conclusion The two genotypeⅤJEV strains showed high lev-els of identity in nucleotide sequences and amino acid sequences with serine insertion in the NS4A gene. However, there were molecular genetic differences between genotypeⅤJEV strains isolated after a 60 years hiatus.

Objective To study the effect of fluoride on bone metabolism in rats, and to understand the mechanism of pathogenesis of skeletal fluorosis. Methods A total of 80 Wistar rats were randomly divided into 4 groups that included control group (distilled water), low-dose group(NaF, 50 mg/L), medium-dose group (NaF,100 mg/L) and high-dose group(NaF, 150 mg/L), respectively. After being bred for 12 weeks, the rats were put to death (etherization). Incidence of dental fluorosis was estimated, and serum was collected. Radioimmunoassay was employed to detect the levels of osteocalcin (BGP), parathyroid hormone (PTH) and calcitonin (CT), respectively.Colorimetry method was employed to determine the levels of alkaline phosphatase (ALP) and acid phosphatase (ACP). Results Incidence of dental fluorosis between the four groups was significantly different statistically(x2 =82.81 ,P ＜ 0.01 ). The incidence was significantly different(x2 = 22.67, 40.00, 40.00, all P＜ 0.01 ) between low-dose ( 80%, 16/20), medium-dose ( 100%, 20/20), high-dose groups ( 100%, 20/20) and control group (0,0/20),respectively. Serum levels of BGP, PTH, CT were significantly different between the groups(F = 38.614, 20.778,3.023, P ＜ 0.01 or ＜ 0.05). There was no significant difference between the four.groups of ALP and ACP in serum (F = 0.609,2.895, all P ＞ 0.05 ). Serum BGP in low-dose, medium-dose and high-dose groups[ ( 19.60 ± 12.79),(33.41 ± 10.81 ), (39.46 ± 9.51 )mg/L, respectively] was significantly higher than that of the control group[ (7.35 ± 3.22)mg/L, all P ＜ 0.01 ]. Serum PTH in low-dose, medium-dose and high-dose groups[ (72.27 ± 25.38), (67.80 ± 12.01), (106.52 ± 36.37)pmol/L] was significantly higher than that of the control group[(47.08 ± 9.22)pmol/L,all P ＜ 0.01 ]. Serum PTH of the high-dose group was significantly higher than that of the low-dose and the mediumdose groups(all P ＜ 0.01 ). Serum CT in medium-dose and high-dose groups[ ( 13.39 ± 2.07), ( 15.05 ± 4.77)pmol/L ] was significantly lower than that of the control group[ (26.06 ± 28.31 ) pmol/L, all P ＜ 0.05 ] and also significantly lower than that of the low-dose group [ (24.49 ± 14. 10) pmol/L, all P ＜ 0.05 ]. Conclusions Fluoride affects bone metabolism in rats, BGP, PTH and CT play a key role in the pathogenesis of skeletal fluorosis.

To establish a LC-MS/MS method to determine the concentrations of liquiritin, glycyrrhizin, glycyrrhetinic acid, amygdalin, amygdalin prunasin, ephedrine, pseudoephedrine and methylephedrine of Maxing Shigan decoction in rat plasma, and study the differences on their pharmacokinetic process in normal rats and RSV pneumonia model rats. After normal rats and RSV pneumonia model rats were orally administered with Maxing Shigan decoction, the blood was collected from retinal vein plexus of different time points. Specifically, tetrahydropalmatine was taken as internal standard for determining ephedrine, while chloramphenicol was taken as internal standard for determining other components. After plasma samples were pre-treated as the above, the supernatant was dried with nitrogen blowing concentrator and then redissolved with methylalcohol. The chromatography was eluted with mobile phase consisted of acetonitrile and 0.1% formic acid solution in a gradient manner. ESI sources were adopted to scan ingredients in ephedra in a positive ion scanning mode and other ingredientsin a negative ion scanning mode. The multiple-reaction monitoring (MRM) method was developed the plasma concentration of each active component. The pharmacokinetic parameters of each group were calculated by using Win-Nonlin 4.1 software and put into the statistical analysis. The result showed the plasma concentration of the eight active ingredients, i.e., liquiritin, glycyrrhizin, glycyrrhetinic acid, amygdalin, amygdalin prunasin, ephedrine, pseudoephedrine and methylephedrine within the ranges of 1.04-1040, 1.04-1040, 0.89-445, 1.05-4200, 1.25-2490, 0.3-480, 0.3-480, 0.3-480 microg x L(-1), with a good linearity and satisfactory precision, recovery and stability in the above ingredients. After modeling, except for glycyrrhetinic acid whose pharmacokinetic parameters were lacked due to the data missing, all of the rest components showed significant higher Cmax, AUC(0-1) and lower clearance rate (CL) than that of the normal group, indicating the increase in absorption in rats in the pathological state by reducing the clearance rate. The method is accurate and sensitive and so can be used to determine the plasma concentrations of the eight active ingredients in Maxing Shigan decoction. RSV pneumonia-infected rats absorbed more ingredients in Maxing Shigan decoction.
Animals ; Chromatography, High Pressure Liquid ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacokinetics ; Male ; Pneumonia, Viral ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Respiratory Syncytial Virus Infections ; drug therapy ; metabolism ; Tandem Mass Spectrometry