Objective To investigate the role of ECRG2 gene in migration and invasion of human fibrosarcoma HT1080 cells. Methods Tet-on system was used to set up ECRG2-inducible cell clones.Knockdown of ECRG2 gene was done by stably expressing short hairpin RNA (shRNA) specific for ECRG2 mRNA in a highly invasive fibrosarcoma cell line HT1080. The expressions of ECRG2 proteins were detected by Western blotting assay. Wound-healing scrape assay and transwell invasion assay were performed to assess the effect of ECRG2 gene on the invasive properties of HT1080 in vitro. Results The most effective ECRG2siRNA interference and inducible was selected by Western blotting assay. Knockdown of ECRG2 gene significantly promoted the migration and invasion of HT1080 cells while expression of ECRG2 impaired the migration and invasion of HT1080 cells in vitro. Conclusion ECRG2 gene is correlated with the invasive and metastatic potential of fibrosarcoma carcinoma, and expression of ECRG2 induces the inhibition of migration and invasion of HT1080 cell line.

Objective To investigate the role of ECRG2,a novel tumor suppressor gene,in spindle assembly checkpoint. Methods Using siRNA approach to deplete the expression of ECRG2, using immunofluorescence to test the distribution of ECRG2,using Western blotting to examine the expression cell cycle proteins.Results ECRG2 localized to centrosomes during interphase and kinetochores during mitosis.Further analysis revealed that ECRG2 participates in the spindle assembly checkpoint.Depletion of ECRG2 abolished the spindle assembly checkpoint.Conclusion Our results indicated that ECRG2 is important for ensuring spindle assembly checkpoint,accurate chromosome segregation,and its depletion may contribute to chmmosome instability and aneuploidy in human cancers.

Objective: To examine the effects of ? carotene and ? tocopherol on LDL oxidation by monocyte derived macrophages from SD rats (in DMEM medium). [WT5FZ]Methods: Macrophages were incubated with LDL in DMEM in which freshly dissoved VE (40,100,200 ?mol/L) and ?C (0.5,1.0,2.0 ?mol/L) had been added. After incubating for 24 h, the mediums were centrifuged to determine TBARS and lipofusin and dienes. [WT5FZ]Results: Supplementation of ? tocopherol significantly decreased TBARS, lipofusin and conjugated dienes production and electrophoretic mobility. Supplementation of ? carotene at 0.5 ?mol/L significantly decreased TBARS, lipofusin and conjugated dienes production and electrophoretic mobility, but other levels showed no effect. Increasing ? carotene concentration to 2.0 ?mol/L in the system increased the lipofusin production compared with control group. ? tocopherol at 200 ?mol/L and ? carotene at 0.5 ?mol/L were the most effective in all dose groups. [WT5FZ]Conclusion: Cell mediated oxidation of LDL can be regulated by antioxidants such as ? tocopherol and ? carotene.

Objective:To investigate the effect of VE and VC at different dietary levels on plasma lipid metabolism, lipid peroxidation and mRNA of scavenger receptor (SR). Methods:Male SD rats, fed purified and cholesterol enriched diet, were divided into five groups(12 rats per group). The experimental groups were fed diets with either VE(150 or 350 mg/kg diet) or VC (1 000 or 2 000 mg/kg diet) respectively. The experiment lasted 56 days. Results:Compared with control group, both VE and low dosage VC could significantly lower the concentrations of total cholesterol(TC), low density lipoprotein cholesterol(LDL C), atherogenic index (AI),Apo B and lipoperoxide, and increase the levels of high density lipoprotein cholesterol (HDL C), and inhibit the expression of SR mRNA..Conclusion:VE and VC can decrease the concentrations of serum TC and LDL C,restrain formation of Ox LDL and expression of SR mRNA.

Objective To establish a single nucleotide polymorphisms genotyping (SNP) method for a convenient, accurate, and routine analysis of clinical samples. Methods Based on the design of oligonucleotide probe, the assay was performed through three steps:the conjunction of the detection probe, universal amplification, labeling and ELISA reaction. The genotype of each SNP was revealed by reading signals of each set of reaction tubes. This assay was applied to detect sixty-two plasma samples of lung cancer for circulating DNA for three SNPs of EGFR, c.2573T＞G(L858R), EGFR, c.2582T＞G＞T(G719C). Results were compared with those obtained by direct sequencing. Results The heterozygote mutation was identified for L858R by both methods, although no mutation was detected for L861Q and G719C. Six samples were identified as heterozygotes with the new method, and only two samples were unambiguously identified as heterozygotes by the direct sequencing. Two additional samples could not be identified as heterozygotes because the peak of mutant allele was very low compared with that of wild allele. Conclusion The developed method enabled accurate identification of SNP in a convenient manner, and which is adapted to routine analysis from heterogeneous samples unambiguously.

Objective:To investigate the efficacy of single time intra-articular different concentration of allogeneic bone marrow mesenchymal stem cells ( BM-MSCs ) injection in the treatment of Sprague-Dawley ( SD) rat model of osteoarthritis ( OA) .Methods: In the study, 32 SD rats were equally ran-domized into 4 groups:control group, high concentration group (1 ×107/mL BM-MSCs), low concentra-tion group (5 ×106/mL BM-MSCs) and high vs.low concentration group.The two knees of each rat were set up to a pair.The induction of OA was performed surgically randomly at one side in model group, and bilaterally in the other groups, which were through anterior cruciate ligament transaction ( ACLT) and medial meniscus excising.After the operation, the SD rats were allowed free movement.Four weeks later, different concentrations of allogeneic BM-MSCs isolated from the SD rats, expanded in vitro and suspended in phosphate buffered solution( PBS) were delivered in the articular cavity of both knees;PBS was used as the control.After injection, we excised the femoral nerve and sciatic nerve to disuse the low limb.The cartilage histological sections of knees were scored by Mankin scoring system to assess the se-verity of the pathology.mRNA of collagen Ⅱwas detected by real time polymerase chain reaction ( RT-PCR) .eGFP was detected by fluorescence microscope.Assessments were carried out 4 weeks after the operation in model group, and 3 weeks after injection in the other groups.Results:Mankin scores of the BM-MSCs side and control side were 6.60 ±0.40 vs.10.00 ±0.32 in low concentration group ( P<0.05), and 5.40 ±0.51 vs.9.60 ±0.51 in high concentration group (P<0.05).Mankin scores of high sv.low concentration group were 6.40 ±0.51 vs.7.60 ±0.75 (P>0.05).mRNA expression of collagen Ⅱ of the BM-MSCs side in low concentration group was 106%±1%in contrast to the control side.As in high concentration group it was 108%±1%, and 102%±1%in high vs. low concentra-tion group.Labeled BM-MSCs were detected unexpectedly in the synovial membrane but not in cartilage tissue three weeks from injection.Conclusion:BM-MSCs could promote cartilage repair and inhibit OA progression through a trophic mechanism.There was no difference between the two concentrations.

Objective To retrospectively study the risk factors of aortic arch calcificationand its influence on the survival prognosis of maintenance peritoneal dialysis patients. Methods One hundred seventy-seven cases of maintenance peritoneal dialysis patients were enrolled, including 66 cases of aortic arch calcification cases. Their general dialysis data were collected for the evaluation of dialysis adequacy and residual renal function, and their chest X-rays were recorded to assess the degree of aortic arch calcification. The two variables Logistics regression was used to analyze independent risk factors of aortic arch calcification; Kaplan-Meier analysis was used to analyze the influence on prognosis of dialysis patients; and multivariate COX regression was employed to analyze independent risk factors of death in dialysis patients. Results Among the 177 selected cases of peritoneal dialysis patients, 66 cases (37.29%) presented with aortic arch calcification. Elevated serum phosphorus was an independent risk factor of aortic arch calcification (OR=54.69 ,95%CI:10.01-298.65, P<0.01). The probability of survival in patients with mild and moderate (severe) calcification of aortic arch was less than those without calcification. Moderate (severe) calcification of aortic arch was the independent risk factor of all-cause mortality and cardiovascular disease mortality, whose hazard ratios in patients with calcification were 3.779 times and 5.636 times of those in patients without calcification respectively. Conclusions Hyperphosphatemia is an independent risk factor promoting the development of calcification of aortic arch. The probability of survival in patients with mild and moderate (severe) calcification of aortic arch is less than those without calcification; moderate (severe) calcification of aortic arch is the independent risk factor of all-cause mortality and cardiovascular disease mortality.

Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.

Objective To compare the effectiveness of nano-hydroxyapatite/collagen (nHAC)and autologous mesenchymal stem cells (AMSCs) for the repair of femoral defect in a rabbit model with femoral defect under the monitoring of the synchrotron radiation hard X-ray. Methods The rabbit models of traumatic bone defect were established and completely randomized into three groups. The femoral defects filled with nothing were used as control group (Group A) , the femoral defects filled with nHAC as Group B and the femoral defects filled with nHAC + AMSCs as Group C. Phase-contrast imaging with synchrotron radiation hard X-ray was applied to detect the degradation and repair process of each group at postoperative weeks 4, 8 and 12, respectively. Results Phase-contrast imaging with synchrotron radiation hard X-ray could display the reparative process. Four weeks after operation, there was collapse in some defect areas in Group A, and the degradation of nHAC and new bone formation were observed in Groups B and C. Eight weeks after operation, fibrous tissues were observed in the defect area in Group A, while osteogenesis and nHAC degradation were more obvious in Groups B and C. Twelve weeks after operation, the defect areas were still unhealed and were substituted by fibrous tissues in Group A, tissue densities of defect areas in Group C were identical with periphery areas, and trabecular bones were formed in Group C. There were statistical differences in the osteogenesis between Group A and Groups B and C,with Group C the best. Conclusion Phase-contrast imaging with synchrotron X-ray can detect the reparative process at a micro-level and plays an important role in the development of tissue engineering.

BACKGROUND: Endoplasmic reticulum (ER) stress has been proved to be related to the occurrence of diabetes, dilated cardiomyopathy and neurodegenerative diseases. Indeed, it is closely associated with osteoarthritis. OBJECTIVE: To explore the effect of ER stress on the chondrocyte viability as well as the occurrence and development of osteoarthritis in rats. METHODS: Rat chondrocytes were isolated and cultured, and the ER stress in the rat chondrocytes was by 10 mg/L tunicamycin. The expression levels of ER stress markers C/EBP-homologous protein and 78 kDa glucose-regulated protein were detected by western blot assay, and the proliferation and apoptosis of chondrocytes were detected by cell counting kit-8 assay and AnnexinV-FITC flow cytometry, respectively. In the in vivo experiment, 15 Sprague-Dawley rats were selected and subjected to anterior cruciate ligament transection and medial meniscectomy to establish an animal model of osteoarthritis. Tunicamycin, tauroursodeoxycholic acid and PBS (blank control group) were respectively injected into the articular cavity, and then the progression of osteoarthritis was assessed by hematoxylin-eosin staining at 4 weeks after treatment. RESULTS AND CONCLUSION: After addition of tunicamycin, the expression levels of C/EBP-homologous protein and 78 kDa glucose-regulated protein were significantly upregulated, the viability of chondrocytes was decreased gradually, while the apoptotic rate was increased significantly. Results from gross observation and hematoxylin-eosin staining suggested that tunicamycin promoted the progression of osteoarthritis and tauroursodeoxycholic acid delayed the deterioration of cartilage in the rats. These findings indicate that ER stress results in the decreased chondrocyte viability and increased apoptosis, which may be an important pathogenesis of osteoarthritis. Additionally, tauroursodeoxycholic acid can effectively alleviate osteoarthritis induced by ER stress.