AIM: To develop a reliable primary cultured hepatocytes model in vitro for liver metastasis research. METHODS: Hepatocytes were isolated by a modification of the two-step collagenase perfusion method. The apoptosis and cell cycle of hepatocytes were measured with flow cytometry. The proliferation of hepatocytes was detected by SRB method. RESULTS: The viability and purity of hepatocytes were 90% and 95%,respectively. The result of flow cytometry analysis showed that there was little apoptosis in hepatocytes and most of hepatocytes were in G_0/G_1 phase. The proliferation and albumin-secreting function of hepatocyte cultured by low glucose DMEM and high glucose DMEM were higher than that of cultured by RPMI1640 during 1 to 6 day, but there was no significant different between low glucose DMEM group and high glucose DMEM group. CONCLUSION: Hepatocytes have higher purity and viability with the normal biological activity for about 6 days by this method and it may be a cell model for the study of liver metastasis in vitro.

Objective To explore the relationship among single nucleotide polymorphism (SNP) of excision repair cross-complementing 1 (ERCC1) gene,chemotherapy sensitivity and clinical outcomes of epithelial ovarian cancer (EOC) patients treated with platinum.Methods Six tag single nucleotide polymorphisms (tagSNP;rs11615,rs3212986,rs735482,rs3212955,rs12610134 and rs3212958) were chose from ERCC1 gene.The genotypes of 6 tagSNP were determined by Snapshot method in 220 EOC patients.Primary clinical outcomes parameter contained EOC patients'responses to platinum-based chemotherapy,progression-free survival (PFS) and overall survival (OS) were analysed.Results The rs11615 C/T SNP of ERCC1,CC,CT and TT genotype frequencies were 53.1％,45.6％,1.4％ in responders to platinum-based chemotherapy,while 52.0％,35.6％,12.3％ in non-responders,respectively,in which there was significant difference between the two groups(P =0.002).Compared with the patients with CC genotype,the patients carrying TT genotype had a significantly poor response to platinum-based chemotherapy (OR =6.22,95％ CI:1.12-34.42).Similarly,the genotypes frequencies distribution of rs11615 C/T SNP of ERCC1 was different between the recurrence and non-recurrence group,death and survival group (all P ＜ 0.05).Kaplan-Meier survival analysis showed that the genotypes frequencies distribution of rs11615 C/T SNP of ERCC1 was associated with PFS and OS(P ＜ 0.01) of EOC patients.Cox's multivariate analysis suggested that patients with TT genotype had a shorter PFS (HR =2.19,95 ％ CI:1.14-4.22,P =0.009) and OS (HR =2.22,95 ％ CI:1.06-4.64,P =0.021) compared with those carrying CC genotype [adjusting for age,International Federation of Gynecology and Obstetrics (FIGO) stage,pathological type,grade and tumor residual size].The genotypes frequencies distribution of rs3212986,rs735482,rs3212955,rs12610134 and rs3212958 SNP of ERCC1 did not show the significant difference between the responders to platinum-based chemotherapy and non-responders.The other 5 tagSNP may not be associated with the PFS and OS of EOC patients (all P ＞ 0.05).Conclusion The rs 11615 SNP of ERCC1 may become a valuable prognostic biomarker for EOC patients treated with platinum-based chemotherapy.

AIM: To study the influence of injection agent of pidotimod on function of the immune-system in normal and immune-depressed mice, and to investigate the mechanisms of the immunomodulating action of pidotimod. METHODS: The expressions of TNF-? and IL-6 in mice spleen was detected by RT-PCR method. The effects of pidotimod on phagocytosis functions of mice peritoneal exudate cells was investigated by neutral red phagocytosis. The lymphocyte proliferations induced by Con A or LPS were detected by MTT method. Adopt the serum haemolysis to measure the production of serum antibody. RESULTS: The normal and immune-depressed mice were treated for 14 days with different dosages of pidotimod by injection. Pidotimod can significantly increase the expressions of TNF-? and IL-6 of spleen, potentiate phagocytosis of the peritoneal exudate cells, potentiate the lymphocyte proliferations ability induced by Con A or LPS. CONCLUSION: Pidotimod can potentiate amelioration normal and immune-depressed mice immunesystem function and increase expressions of TNF-? and IL-6 of spleen.

Objective To study possible clinical relationships of myeloperoxidase (MPO) and carotid atherosclerosis in diabetic patients on maintenance hemodialysis(MHD).Methods Levels of plasma MPO and serum elastase (ELT) of diabetic patients on MHD were determined by enzyme-linked immunosorbent assay.Carotid intima media thickness(CIMT),quantity and area of carotid plaque were measured by B-mode ultrasonography.Results Plasma MPO was significantly higher after hemodialysis(HD) than before HD(414.6 (198.9,671.5) μg/L vs 176.4 (69.9,243.3) μg/L,Z =-4.51,P ＜ 0.01).There was no significant difference of serum ELT before and after HD(198.0(146.9,270.5) μg/L vs 230.9(40.2,308.0) μg/L,Z =-1.87,P ＞ 0.05).There was positive correlation between the age and CIMT,quantity or area of carotid plaqueor(correlation coefficient:0.764,0.416 and 0.446 respectively,P ＜ 0.01 or P ＜ 0.05),There was positive correlation between MPO level before HD and age,Levels of serum ELT before HD,or CIMT(correlation coefficient:0.592,0.476 and 0.810 respectively,P ＜ 0.01).There was positive correlation between MPO level after HD and levels of serum ELT after HD (correlation coefficient:0.364,P ＜ 0.05).There was no correlation between the levels of plasma MPO after HD and CIMT,quantity or area of carotid plaqueor (P ＞ 0.05).Conclusion (1) Age and MPO level before HD contribute to carotid atherosclerosis in patients on MHD.(2)There was no relation between higher MPO level after HD and carotid atherosclerosis.

Objective To investigate the association between the six single nucleotide polymorphisms ( SNP),named as rs179247,rsl2101261,rs2284722,rs4903964,rs2300525,rsl7111394 in the intron 1 of thyroid stimulating hormone receptor gene (TSHR) and Graves' disease (GD).MethodsThe genotypes of the six SNPs were genotyped by Taqman probe technique on Fluidigm EP1 platform in 618 GD patients and 646 control subjects.Meanwhile,TSH receptor antibodies (TRAb) of the patients were determined.ResultsAmong the six SNPs,five S NPs were strongly associated with GD,with the most signals at rs179247_G,rs12101261_C,rs4903964 _G (P=2.85×10-10,OR=1.73,95％CI1.46-2.05;P=1.74×10-10,OR=1.73,95％CI 1.46-2.05;P=2.24×10-10,OR=1.69,95％ CI 1.44-1.99 ).The results of logistic regression analysis indicated that rs12101261 and rs4903964 were main susceptibility loci of GD in the intron 1 of TSHR.rs179247_G,rs1210126 1_C,and rs4903964_G were associated with subset of the GD patients with positive TRAb (P=4.24× 10-13,p=5.48× 10-13,P =3.89×10-12 ).Conclusionrs179247,rs12101261,and rs4903964 in TSHR intron 1 were significantly associated with GD in the Chinese Han population from Bengbu city.rs12101261 and rs4903964 were the major susceptibility SNPs associated with GD.TSHR gene may play a main role of susceptibility gene in the subset of GD patients with persistent positive TRAb.

OBJECTIVEThe aim of this experiment is to find proper cell carrier for skin tissue engineering.

METHODSVarious concentration of fibroblasts were seeded onto HA-ECM and cultured in vitro. The performance of cells' growth, array, adhesion and collagen secretion on HA-ECM was observed with light microscope and transmission electron microscope.

RESULTSThe fusiform fibroblasts oriented radiantly or longitudinally and closely packed onto the HA-ECM, they attached firmly and proliferated to confluence on the stromal surface of HA-ECM.

CONCLUSIONThe optimal cell concentration is 3.5 x 10(6)/ml, HA-ECM is ideal carrier for fibroblasts because of its excellent scaffold and diffusion characteristics. Futhermore, it has the bioactive molecules such as fibronectin and laminin which play an important role in fibroblasts attachment and proliferation on HA-ECM.

Amnion ; cytology ; Animals ; Cell Culture Techniques ; methods ; Cells, Cultured ; Extracellular Matrix ; Female ; Fibroblasts ; cytology ; Hematoxylin ; Humans ; Microscopy, Electron, Scanning ; methods ; Rabbits

To investigate the clinical features, genetic diagnosis, and treatment of a patient with Prader-Willi syndrome(PWS). For a case with clinically suspected PWS, methylation specific PCR(MSPCR)amplification was applied to CpG islands of SNRPN(exon α)gene locus in the 15q11-q13. Furthermore, the diagnosis was comfirmed by the method of bisulfite sequencing PCR(BSPCR). Metabolic status before and after the operation of sleeve gastrectomy were compared. Absence of amplification of paternal allele on chromosome 15q11-q13 was detected in the case by MSPCR, different from the normal control. Results of BSPCR further proved a full methylation of CpG islands in the SNRPN gene locus. Four months after sleeve gastrectomy, systemic metabolic status and ventricular function were improved. MSPCR and BSPCR were both consistent with genetic diagnosis of PWS. Weight loss surgery is expected to be a major therapy of this disease.

OBJECTIVETo repair rabbit tendon defects with tissue engineering method.

METHODSThe third passage of fetal skin fibroblast cells was labeled with 5-bromo-2' deoxyuridine (Brdu) and then seeded on human amnion extracellular matrix (HA-ECM). Using 1 cm-long-Achilles tendon defects as repairing models in the experimental group, tendon defects were core bridged with polydioxanone (PDS) and then capsulated with the complex of fibroblasts-HA-ECM. In the control group I, defective tendons were sutured with PDS following the former procedure and capsulated with HA-ECM (without fibroblasts). In the control group II, only PDS was applied to connect the defective tendons. Gross examination, light microscopy, scanning electronmicroscopy and biomechanical measurement of the repaired tendons were respectively performed at postoperative 1, 2, 3 month as well as immunohistochemical examination.

RESULTSThe optimal cell concentration for seeding fibroblasts was 3.5 x 10(6) cells/ml. Cells grew well and radiated or paralleled on HA-ECM. Immunohistochemistry showed that the labeled seed fibroblasts played an important role in tendonization. The results of light microscopy, electron microscopy, and biomechanical assessment suggested that the rate and quality of tendonization in the experimental group was superior to those of the control group I and II. The tensile strength in the experimental group was the greatest, the next was in the control group I, and the worst in the control group II (P<0.05).

CONCLUSIONSHA-ECM is the excellent carrier for fibroblasts. Fibroblasts-HA-ECM complex has the capability to repair tendon defect and to tendonize with rapid rate and good performance three months after operation. Its tensile strength is 81.8% of that of normal tendon.

Amnion ; transplantation ; Animals ; Cells, Cultured ; Extracellular Matrix ; Fibroblasts ; cytology ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Polydioxanone ; Rabbits ; Suture Techniques ; Tendons ; surgery ; ultrastructure ; Tensile Strength ; Wound Healing ; physiology

Objective To investigate the association between single nucleotide polymorphisms in the intron 1 of thyroid stimnulating hormone receptor gene (TSHR) and Graves' disease (GD) in the Chinese Han population from Linyi city,Shandong Province.Methods A total of 1759 GD patients and 1740 control subjects were recruited for genotyping in TSHR intron 1 with genome-wide association study (GWAS) and Taqman probe technique.At the same time,serum thyroid hormone and TSH receptor antibody (TRAb) levels of patients were determined.Results Five SNPs were selected for further replication.The rs12101261 _T was significantly associated with GD risk ( OR=1.257,95％CI 1.137-1.390,P =8.23 × 10-6 ). Logistic regression identified that rs12101261 was an independent susceptibility locus of GD ( P=1.61 × 10-3 ).Furthermore,rs12101261 _T was strongly associated with GD ( OR =1.317,95％ CI 1.171-1.481,P=4.14× 10-4 ) in TRAb positive patients,but no association in TRAb negative patients ( OR=1.056,95％ CI 0.892-1.251,P=0.524 ).Serum TRAb concentration showed remarkable difference among three genotype groups of rs12101261.Conclusions Five SNPs in TSHR intron 1 are associated with GD.rs12101261 contributes to increased GD risk independently and is associated with serum TRAb level.

Objective The aim of this study is to explore the hypoglycemic effect of active components of Anoectochilus roxburghii on zebrafish models. Methods Anoectochilus roxburghii components were extracted and separated into three groups: the alcohol extraction group, macromolecular polysaccharide group (≥ 5 ×103) and small molecular polysaccharide group (＜5×103). Zebrafish embryos were exposed to 2% glucose solution (2% Glu) at 24 h to imitate acute hyperglycemia phenotype, and then treated with the three Anoectochilus roxburghii components. Based on this high-glucose model, the zebrafish embryos at 72 h were collected to detect the whole tissue glucose value. Furthermore, semi-quantitative PCR and whole mount in situ hybridization were performed to detect the expression of mRNA levels of glycometabolism-related genes. Results An acute diabetic zebrafish model was induced by high glucose stress. In this model, some key factors during glycometabolism such as insulin, pck-1 and pdx-1 were significantly affected, while the alcohol extracts of Anoectochilus roxburghii obviously reversed these abnormalities induced by high glucose stress, even to normal levels. Conclusions The alcohol extracts of Anoectochilus roxburghii has obvious hypoglycemic effect on diabetic zebrafish model. Our result suggest that Anoectochilus roxburghii has a potential application in the hypoglycemic drug screening.