BACKGROUND:Recently,in vitro nucleus gelatinosus and annulus fibrosus cell culture technology,especially cartilage tissue engineering development and primary success in autogenous intervertebral disc cell transplantation to repair nucleus pulposus defect,brings a hope for regeneration and reparation of degenerated intervertebral disc.OBJECTIVE:To explore the dose-dependent and time-dependent effects of insulin-like growth factor-Ⅰ(IGF-Ⅰ) on proliferation of anulus fibrosus cells in vitro.DESIGN,TIME AND SETTING:Single-sample observation was performed at the Laboratory of Parasite,Shanxi Medical University between September 2006 and January 2007.MATERIALS:Thirty 1-month-old Wistar rats,irrespective of gender,were selected.METHODS:Anulus fibrosus cells were isolated and cultured in vitro.In dose-dependent test,IGF-Ⅰ(0.1,1.0,10,and 100 ?g/L),prepared by volume fraction 0.01 or 0.1 calf serum HAMF-12,were added.Cells not with IGF-Ⅰ served as control.Cells were cultured for 72 hours.In time-dependent test,optimal dose of IGF-Ⅰ containing volume fraction 0.1 calf serum and F-12 solution was added,and cells not with IGF-Ⅰ served as control.The cells were cultured for 1,3,5,and 7 days.MAIN OUTCOME MEASURES:Cell biological features were detected using HE staining and toluidine blue immunohistochemical staining;Dose-dependent and time-dependent effects were examined by MTT assay.RESULTS:HE stained cells were fusiform-shaped with pseudopodia,round or oval nucleus.The cytoplasm was blue after stained with the toluidine blue.The immunohistochemical test revealed that there existed positive expression of typeⅠcollagen in the cells.In the presence of 10% calf serum,IGF-Ⅰ significantly improved cell proliferative activity in a dose-dependent manner within effective dose range.CONCLUSION:IGF-Ⅰ can stimulate cell proliferation of rat anulus fibrosus cells in vitro in a dose-dependent and time-dependent manner.

Objective:To investigate the expression of NKG2D in peripheral blood of patients with ovarian benign or malignant tumors, and the expression of the human MHC class Ⅰ chain-related gene A(MICA) on the correspondent tumor tissues. To analyze and discuss the function of NKG2D in anti-ovarian cancer mechanism and its relation with immune escaping of cancer.Methods:Flow cytometry analysis was used to detect NKG2D in the peripheral blood of 42 ovarian carcinoma patients, 23 ovarian tumor patients and 20 health persons. The expressions of MICA in part of the correspondent tissues were examined by means of reverse transcription-polymerase chain reaction (RT-PCR).Results:The expression of NKG2D were (94 23?6 02)%?(98 70?0 98)%?(98 61?1 59)% respectively, compared with the other two groups, the NKG2D expression in malignant group was significantly low; The rate of MICA mRNA expression in ovarian carcinomas was significantly higher than that in benign and normal tissues. No evident relation was found between the expression of MICA mRNA and the clinical factors.Conclusion:The activity of NK cell and the anti-cancer cellular immunity level reduce in patients with ovarian cancer; The decrease of the receptor NKG2D is a reason for the descend of the activity of NK cells; MICA mRNA expression is relative to malignant transformation; the immune-escape of ovarian cancer probably is relative to the down-regulation of NKG2D and the up-regulation of its ligand MICA.

Objective:To study the cooperated effect of Human Embryonic Lung Fibroblast(HELF) feeder layer and LIF in the culture of human Embryonic Stem(hES) cells and establish the method of identifying hES cells from Primordial Germ Cells(PGCs).Methods:Embryonic lungs were mechanically disaggregated,then cultured to establish HELF feeder layer.Gonadal ridges and mesenteries of embryos were mechanically disaggeregated,then cultured and passaged in vitro.Comparing the growth characters of hES cells in different conditions.To identify hES cells through their biological characteristics.Results:The coactions of HELF feeder layer and LIF play an important role in proliferation and undifferentiation of hES cells in vitro.High levels of AKP and telomerase activity are associated with hES cells. The cultrued cells have been continuously passaged for more than two months and found to be karyotypically normal and stable.When differentiating,they form embryoid bodies(EBs).Conclusion:To avoid indection of the heterogeneous protein,HELF feeder layer, in the presence of LIF,can be used in culture of hES cells;hES cells from PGCs can be identified through their biological characteristic. [

Objective To explore the expression and significance of chemokine CXC reeeptor (CXCR)3 and CXCR4 and their ligands(CXCL)at the early pregnancy decidua and villi.Methods Decidual mononuclear cells were isolated from the normal decidua of 5-8 weeks pregnant women by lymphocyte separation medium in vitro.CD56+natural killer(NK)cells were purified by dynabeads cell sorter kiL Purity and phenotype of CD56+decidua NK cells were analyzed by fluorescence-activated eell sorter (FACS).Gene expression of CXCR3 and CXCR4 in decidua NK cells and CXCL9,CXCL10 and CXCL12 in early pregnancy decidua and villi was assessed bv RT.PCIZ Protein expression of CXCL9,CXCL10 in normal endometrium and early pregnancy decidua was characterized and quantified by streptavidin-biotin pemxidase chain reaction(SP)immunohistochemistry and computered image analysis system.Correlations between the gray degree of CXCL9 and CXCL10 and the number of CD56+NK cells in upper tissue were analyzed by Spearman's correlation ceefficient rank tesL Results The phenotype of 98.7%decidua NK cells was CD56bright.The genes of CXCR3 and CXCR4 were expressed in decidua NK cells and that of CXCL9 and CXCL1O were expressed in early pregnancy decidua and CXCLI2 in early pregnancy villi.CXCL9 and CXCL10 were expressed in the cytoplasm of surface epithelia,glandular epithelia and stromal cells of early pregnancy deeidua and were not expressed in villi by immunohistochemistry.The gray degree of CXCL9 and CXCL10 in the secretory phase endometrium(56±43,59±47)was stronger than that in the proliferative phase(16±18,8±14,P<0.05)and reached the highest(143±35,158±29,P<0.05)in the early pregnancy decidua.The number of cD+56 NK cell in the secretory phase endometrium(60±20)was more than that in the proliferative phase endometrium(23±4,P<0.05)and was the most in the early pregnancy decidua(114±15,P<0.05).The gray degree of CXCL9 in upper tissue had a positive correlation with the number of CD+56 cells(r=0.88,P<0.05)and that of CXCL10 had a similar pattern to CXCL9(r=0.86,P<0.05).Condusion The interactions between CXCL9,CXCL10 and CXCL12 expressed in decidua and villi and CXCR3,CXCR4 expressed in CD+56 decidua NK cells may influence the CD+56 NK cell recruitment at the maternal-fetal interface.

Objective To evaluate the role of two-dimensional ultrasound combined with contrast-enhanced ultrasonography (CEUS) in the classification of liver nodules in cirrhotic patients.Methods Consecutively cirrhotic patients with intrahepatic nodules at Xixi Hospital of Hangzhou were included from November 2015 to December 2016.All (142 nodules in 109 patiens) presented as non-cancerous focal lesions on conventional magnetic resonance imaging and CT examination and had available information of liver biopsy.Each lesion was percutaneous biopsied under the guidance of two-dimensional ultrasound.Ultrasonographic parameters evaluated were as following:(1) sizes of nodules under US;(2) ultrasonographic characteristics of the nodular;(3) CEUS enhancement features of the nodules.Four types of hepatic nodule suggesting different histology were defined according to the ultrasonographicparameters.x2 test was used to compare the difference of hepatocellular carcinoma (HCC) incidence among liver nodules with varying sizes and nodules with different enhancement features under CEUS.As for the statistical differences of HCC and high-grade dysplastic nodule (HGDN) incidence between type Ⅲ & Ⅳ nodules and type Ⅰ & Ⅱ nodules,x2 test was also used for analysis.Results A total of 142 eligible nodules were detected in 109 patients with cirrhosis,including 16 HCCs,2 intrahepatic cholangiocellular carcinomas (ICC),41 HGDNs,40 low-grade dysplastic nodules (LGDN) and 43 regenerative nodules (RN).In terms of diameter,all (6/6) the nodules larger than 2.0 cm,20.0％ (8/40) of middle size nodules (1.5-2.0 cm),were HCCs.The remained 2 lesions of HCC came from two subgroups with even small size nodules [1.0-1.4 cm (n=93),and ＜ 1.0 cm (n=3),in diameter],respectively.Two lesions of ICC were attributed to nodules with a 1.0-1.4 cm diameter.About 28 nodules with a diameter of 1.5-2.0 cm,13 nodules with a diameter of 1.0-1.4 cm were HGDN.HCC incidences between these 4 groups were different significantly (x2=61.425,P ＜ 0.001).Asfor the CEUS,14 nodules exhibited a rapid enhancement feature in arterial phase,12 of which were HCC.In56 nodules with a slow enhancement feature,4 nodules were HCC.HCC incidences between these 3 groups were different significantly (x2=75.752,P ＜ 0.001).Under the combined ultrasonography,HCC incidences of type Ⅲ and type Ⅳ nodules were significantly higher than that of type Ⅰ and type Ⅱ lesions [21.9％ (16/73)vs 0 (0/65),x2=15.222,P ＜ 0.001],similar result was observed in the comparison of HGDN incidences between type Ⅲ & Ⅳ and type Ⅰ & Ⅱ nodules[53.4％ (39/73) vs 3.1％ (2/65),x2=38.842,P ＜ 0.001].Conclusion The classification presented by this study,combining the three ultrasonographic parameters,which is nodule size,nodular echo characteristics and enhancement features of the nodules under CEUS,could be helpful for the diagnosis of HCC in cirrhotic patients with ill-defined nodule on routine image examination.

AIMIn order to look for new bioactive compounds, investigation on the chemical constituents, especially on the typical polyacetylenes from the rhizomes of Cirsium japonicum DC. was carried out.

METHODSChromatographic techniques including silica column chromatography and preparative silica thin-layer chromatography were used to separate and purify the constituents. Their structures were elucidated by physicochemical properties and spectral analyses including UV, IR, 1HNMR, 13CNMR, HMQC, HMBC and HREIMS.

RESULTSTwelve compounds were isolated from the rhizomes of Cirsium japonicum DC., and their structures were identified as cis-8, 9-epoxy-heptadeca-1-ene-11, 13-diyne-10-ol (1), ciryneol A (2), 8,9,10-triacetoxyheptadeca-1-ene-11,13-diyne (3), ciryneone F (4), cireneol G (5), ciryneol H (6), ciryneol C (7), p-coumaric acid (8), syringin (9), linarin (10), beta-sitosterol (11) and daucosterol (12).

CONCLUSIONCompounds 4, 5 and 6 are new compounds, compound 3 is a new natural product and compound 8 was isolated from this plant for the first time.

Cirsium ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Molecular Conformation ; Molecular Structure ; Plants, Medicinal ; chemistry ; Rhizome ; chemistry

OBJECTIVETo investigate whole blood viscosity changes at different time points in rats model with lumbar vertebrae semidislocation, study Shi's theroy of qi and blood and "Gucuofeng and Jinchucao" [symbols: see text], also reveal pathological physiology characteristics of spinal disorder.

METHODSThirty-six SPF male rats weighted 350 to 450 g were randomly divided into rotatory fixation group (RF group), simple fixation group (SF group) and Sham group (Sham group), 12 rats in each group. Exterior vertebrae implanted through L4-L6 segments of lumbar vertebrae in RF and SF group were connected fixed device. In RF group, L5 spinous process were rotated to right, and caused L5 spinous process was non collinear with L4 and L6; in SF group, external fixed device were simple connected without rotation. At 1, 4, 8 and 12 weeks after fixation, whole blood viscosity changes were tested.

RESULTSAt 4 and 8 weeks after fixation, high (150/s), medium (60/s) and lower (10/s) shear rate in RF and SF group were higher than that of Sham group (P<0.05). At 1 and 12 weeks, there was no sigificant differences among three groups in whole blood viscosity (P>0.05).

CONCLUSION"Gucuofeng and Jinchucao" [symbols: see text] vertebrae could raise whole blood viscosity, increase degree of bloos stasis and induce or aggravate spinal disorder in further.

Animals ; Blood Viscosity ; Disease Models, Animal ; Joint Dislocations ; surgery ; Lumbar Vertebrae ; injuries ; surgery ; Male ; Rats ; Rats, Sprague-Dawley ; Time Factors

BackgroundThe immunotherapy for retinoblastoma(RB) is gradually concerned recent year.To seek relative immune-associated antigen is a basis of immunotherapy.NY-ESO-1 and NY-SAR-35 are two kinds of genes of cancer testis antigen( CTA ).To understand their expressions in RB tissue can offer index for relative study.ObjectiveThis study was to investigate the expressions of two CTA NY-ESO-1 and NY-SAR-35 in RB and explore the possibility of them as potentially promising targets for antigen-specific immunotherapy of RB.Methods The samples were obtained from 15 RB eyes,12 non-tumor retinopathy eyes and 22 normal eyes with other benign eye diseases.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expressions of NY-ESO-1 mRNA and NY-SAR-35 mRNA in the samples.Genes of positive PCR results were sequenced randomly.The relevance of the expression of the two cancer-testis antigen genes with the clinical characteristics such as tumor stage,tumor size and clinical stage were analyzed.This study was approved by Ethic Committee of Guangxi Medical University.Written informed consent was obtained from each patient before operation. Results NY-ESO-1 mRNA was positively expressed in 6 RB samples and NY-SAR-35 mRNA was expressed in 9 RB samples.In the non-tumor retinopathy samples and normal eye tissues,NY-ESO-1 mRNA and NY-SAR-35 mRNA were absent.No significant relevances were found between the expressions of the NY-ESO-1 mRNA and NY-SAR-35 mRNA with clinical characteristics such as age ( P =0.426,0.822 ),gender ( P =0.180,0.464 ),pathological classification ( P =0.744,0.582 ),tumor size ( P =0.760,0.790),and clinical stage ( P =0.868,0.707 ).Conclusions NY-ESO-1 and NY-SAR-35 have high expressing frequencies in RB tissue and their expressions in RB have specificity.These results offer a clue for the identification of targets antigen of RB.

Objective To investigate whether the proteasomes inhibitor MG262 exerts its anticancer function by inducing apoptosis in human ovarian cancer cells,and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.Method Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours.Cell viability was evaluated with 3-(4,5-dimethyhhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing.Flow cytometry was used to detect cell apoptosis rate.The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immtmosorbent assay (ELISA).Western blot was used to detect the expression of phosphorylated ERK(pERK) .Results The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion(P＜0.05).After 24 h incubation with MG262 at 1,10,20,40,60 and 80 nmol/L,the viability rates of SKOV3 were (94.6±3.1)%,(92.7±3.7)%,(89.5±7.7)%,(84.2±5.1)%,(82.0±7.4)%and(76.8±11.0) % respectively,and after 48 h incubation,those figures were further decreased to (91.3±10.1)%,(86.8±4.5)%,(74.6±4.2)%,(56.8±2.1)%,(49.3±4.5)% and (37.4±5.4) %,respectively(P＜0.05).Apoptosis rate of SKOV3 cells induced by MG262,PD98059 or their combination was (30.7±4.3)%,(26.8±8.6)% and (50.3±10.6)%,respectively,which were significantly different compared with controls (P＜0.05).In contrast to SKOV3 cells,apoptosis rate of 293T ceils induced by MG262,PD98059 or their combination was (14.5±5.3) %,(16.2±7.5) % and (10.8±7.3)%,respectively,which were not significantly different compared with controls (P＞0.05).pERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different.There was no significant difference between experimental and control 293T cells(P＜0.05).In addition,MG262 down-regulated VEGF secretion and expression in SKOV3 ceils (P＜0.05).Conclusions Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.