Objective To study the effect of endothelial progenitor cells (EPCs) transplantation on CCl4 induced hepatic cirrhosis in rats. Methods Eight male SD rats were used as normal control. Thirty rats were induced liver cirrhosis by feeding with 25% CCl4/olive oil for 12 weeks, and then were subdivided into cirrhosis group (n = 10), EPCs transplanted group (n = 10) and saline control group (n = 10). EPCs were transplanted into the portal vein for 4 weeks in EPCs transplanted group. Rats in EPCs nontransplanted group were sacrificed at the beginning of the 12th week. Rats in EPCs transplanted group and saline control group were killed at the beginning of 16th week. Serum biochemical parameters were examined. The degree of liver cirrhosis was evaluated by Masson staining and by detecting the expression of α-SMA, Collagen Ⅲ and Ki67. Results The volumes of liver in cirrhosis group were twice as much as that in normal rats. 12 weeks after CCl4 administration, compared with saline control group, in EPCs transplanted group, hepatic activity index (HAI) ( F = 75. 062, P < 0. 01 ), the levels of ALT( F = 29. 942, P<0.05), AST(F=16.618,P<0.05) and TBIL(F=9.911 ,P<0.05) in serum decreased, the level of Alb ( F = 4. 944, P < 0. 05 ) and Ki67 ( F = 45. 966, P < 0. 01 ) was increased, the expression of α-SM A ( F = 7.86,P<0.05) and collagen Ⅲ (F = 135.787,P <0.01) decreased (P <0.05). Compared with untransplanted group, in EPCs transplanted group, the levels of ALT, AST and TBIL in serum were lower; In saline control group, the levels of ALT, AST and TBIL in serum were higher, the level of Alb and Ki67was lower, the expression of α-SMA and collagen Ⅲ were higher( P < 0. 05 ). Compared with normal rats, in saline control group, the levels of INR were higher (P < 0. 05 ). Conclusion EPCs transplantation improves hepatocye regeneration and ameliorates established hepatic cirrhosis.

Objective To establish and assess the utility of 99Tcm-sulfur colloid (SC) salivary imaging in the routine evaluation of pulmonary aspiration in adult patients with respiratory tract diseases.Methods Eight patients (7 men,1 woman; age range 68 to 80 years,mean age (76 ± 4) years) with respiratory tract disease and history of aspiration by clinical assessment were evaluated prospectively by 99Tcm-SC salivary imaging from April to July 2012.A dose of 74.0 MBq 99Tcm-SC was added to 20 ml saline,mixed well,and administered orally to patients.Dynamic imaging was acquired with posterior projection for 30 min at a rate of 30 s per frame.Two experienced physicians assessed all examination results and reached consensus for final diagnosis.Radioactivity detected at either the bronchi or within the lung fields was reported as positive for aspiration.This study was approved by the institutional review board of Hospital Ethical Committee,and the written informed consent was obtained from patients or their guardians.Results All patients were positive for aspiration by 99Tcm-SC salivary imaging (8/8).Aspiration into bilateral main bronchus was seen in 2 cases,right main bronchus and branch in 4 cases,and left main bronchus and branch in 2 cases.Aspirated tracer could be visualized as early as 3 min,latest at 24 min,and the median was 19 min.Conclusion 99Tcm-SC salivary imaging is useful for the detection of aspiration in adult patients with respiratory tract diseases.

Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.

Objective To detect the expression of HERG (human ether-à-go-go-related gene ) potassium channel in human osteosarcoma,and explore the effects of silencing HERG by small interfering RNA (siRNA)on the proliferation and apoptosis of osteosarcoma cells and the mechanisms responsible for HERG regulation.Methods The expressions of HERG in osteosarcoma MG-63 cells and tissues were detected by reverse transcription polymerase chain reaction (RT-PCR),Western blotting and immunohistochemistry.Next, osteosarcoma cells were divided into three groups:HERG-siRNA group,control-siRNA group and blank group. CCK-8,colony formation,flow cytometry and Tunel assay were used to measure the proliferation and apoptosis of the osteosarcoma cells.Finally,Western blotting analysis was performed to detect the expression of nuclear factor-κB (NF-κB)pathway in osteosarcoma cells treated with HERG siRNA.Results Osteosarcoma cells and tissues were found to highly express HERG.Inhibition of HERG in the osteosarcoma cells significantly inhibited the cell proliferation and induced cell apoptosis.Compared to control-siRNA group or blank group,HERG-siRNA could inhibit the proliferation of MG-63 cells significantly [HERG-siRNA group:(75.34 ± 4.45)%;compared to control-siRNA group:(100.60 ±5.31)%;t =3.64,P =0.007;compared to blank group:(100.00 ±5.66)%;t =3.43,P =0.009].The similar results were obtained from colony formation assay (HERG-siRNA group:134.30 ±11.82;compared to control-siRNA group:225.30 ±11.56;t =5.51, P =0.002;compared to blank group:232.80 ±12.21;t =5.80,P =0.001).HERG-siRNA transfected MG-63 cells demonstrated a significant increase of apoptotic rate compared to control-siRNA transfected cells or untreated cells [HERG-siRNA group:(28.10 ±2.21 )%;compared to control-siRNA group:(9.36 ± 2.42)%;t =5.72,P =0.005;compared to blank group:(10.92 ±2.51)%;t =5.14,P =0.007].This resultwas further confirmed by Tunel assay.The cells transfected with HERG-siRNA (31.57 ±2.08)% dem-onstrated extensive apoptosis,compared with the control-siRNA group [(10.35 ±1.82)%;t =7.69,P =0.002)]or blank group [(7.96 ±0.88)%;t =10.48,P =0.001].Silencing HERG gene down-regulated the cIAP-1,XIAP,Bcl-2,Survivin,P-IκBαand NF-κB p65 expression,compared to the control groups. Conclusion HERG is highly expressed in osteosarcoma.HERG silencing can suppress osteosarcoma progres-sion through NF-κB pathway and suggest that HERG may be a novel molecular target for osteosarcoma therapy and diagnosis.

Objective To explore the effects of nerve growth factor (NGF) and low frequency electricity stimulus on the configurations of skeletal muscle cells and the change of muscle fibre types in the denervated skeletal muscle separately.Methods The denervated rat model was established and model animals were injected with the NGF and given the stimulus (frequency=2 Hz) about 30 days. The configurations and the change of muscle fibre types were observed by immunohistochemistry and image analysis.Results The muscle fibre was in chaos and the boundary was not obvious among cells in the denervated rats; the muscle fibre of the denervated rats with NGF injection and low frequency electricity stimulus was more regular and the boundary of cells was clearer, the cells number was more than those of the denervated rats. Compared to normal rats, the proportion of Ⅰ muscle fibre in the denervated rats increased ( P<0.05), whereas the proportion of Ⅱ muscle fibre decreased ( P<0.05); it had no significant differences of the two types of muscle fibre between the denervated rats with NGF injection, low frequency electricity stimulus and the denervated rats ( P<0.05).Conclusion NGF injection and low frequency electricity stimulus can make the configurations of denervated muscle to better.

Objective To detect the influence of miRNA-34a (miR-34a) on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation.Methods The osteoblastic cell line MG-63 and Saos-2,human osteoblastic cell line hFOB 1.19,10 osteosarcoma tissues and 10 normal bone tissues were selected.The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction (qPCR).Next,a eukaryotic expression vector named pcDNA/miR-34a was constructed.Then,osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8,colony formation and xenograft model of nude mice.Finally,Western blot analysis was used to detect the expression of ether-à-go-go 1 (Eag1) gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2'-O-Methyl antisense oligoribonucleotide (miR-34a-2'-O-Me).Results Compared with normal bone tissues and osteoblastic cell line,miR-34a was down-regulated in osteosarcoma cell lines and tissues.Compared with the blank group and the control group,the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h:blank group (40.05±4.82) ％,control group (36.88± 4.66) ％,miRNA-34a group (26.24±6.22) ％;MG-63 96 h:blank group (83.55±5.95) ％,control group (80.13± 4.48) ％,miRNA-34a group (30.21±7.26) ％;Saos-2 72 h:blank group (46.45±8.15) ％,control group (43.33± 6.89) ％,miRNA-34a group (26.81±3.17) ％;Saos-2 96 h:blank group (84.79±4.10) ％,control group (80.14± 3.11) ％,miRNA-34a group (31.77±5.17) ％].The similar results were obtained from colony formation assay (MG-63:blank group 83.40±3.29,control group 80.00±3.06,miR-34a group 24.40±2.71;Saos-2:blank group 85.00±3.32,control group 80.60±3.29,miR-34a group 30.40±4.94).The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment (all P ＜ 0.001).Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1 (P ＜ 0.001).Conclusion MiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1.Moreover,it may be a novel target for osteosarcoma therapy.

Objective:To investigate the influence of all-trans-retinoic acid (ATRA) on the proliferation and invasion of osteosarcoma 143B cells and its possible regulatory mechanism.Methods:Different concentrations of ATRA were used to treat human osteosarcoma 143B cells, and the optimal concentration and treatment time those affected cell proliferation were selected. The MTS method, Transwell migration and invasion experiments were used to detect the changes in the proliferation, migration and invasion of 143B cells after ATRA treatment. The real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression changes of miRNA-34a (miR-34a), E2F1 and Eag1 in osteosarcoma 143B cells after ATRA treatment. Then miR-34a was interfered and E2F1 was overexpressed, and the abilities of cell proliferation, invasion and migration abilities as well as the expression changes of miR-34a, E2F1 and Eag1 in 143B cells were detected.Results:The proliferation inhibition of 143B cells was most obvious when 143B cells were treated with 10 μmol/L ATRA for 72 h. The cell migration and invasion numbers when 143B cells were treated with 10 μmol/L ATRA for 72 h were lower than those in the negative control group [(73±3) cells vs. (182±5) cells, t = 21.46, P<0.01; (94±3) cells vs. (203±7) cells, t = 13.70, P<0.01]. 10 μmol/L ATRA could promote the expression of miR-34a in 143B cells and inhibit the expressions of Eag1 and E2F1 (all P<0.01). Compared with ATRA group, the ability of cell proliferation in ATRA+miR-34a interference group was restored after 72 h of treatment [cell survival rate (41.0±2.2)% vs. (25.0±3.6)%, t = 108.68, P<0.01]. Compared with ATRA group, the abilities of cell migration and invasion in ATRA+miR-34a interference group were restored [(122±14) cells vs. (64±10) cells, t = 21.06, P<0.01; (103±10) cells vs. (59±8) cells, t = 24.27, P<0.01), and the mRNA and protein expressions of Eag1 and E2F1 in cells were promoted (both P<0.01). Compared with ATRA group, the ability of cell proliferation in ATRA+E2F1 overexpression group was restored [cell survival rate (40.0±3.4)% vs. (24.0±3.1)%, t = 108.74, P<0.01]; the abilities of cell migration and invasion in ATRA+E2F1 overexpression group were restored [(78±12) cells vs. (29±8) cells, t = 13.52, P<0.01; (75±12) cells vs. (49±10) cells, t = 6.28, P<0.01], and the mRNA and protein expressions of Eag1 and E2F1 in cells were promoted (both P<0.01). Conclusion:ATRA inhibits the proliferation and invasion of osteosarcoma cells via regulating miR-34a-E2F1-Eag1 signaling pathway, and it may become one of the effective treatment drugs for osteosarcoma.

Objective To observe the characteristics of hepatic progenitor cells(HPCs)activation in liver tissues of patients with hepatitis B cirrhosis,and to investigate the relationship between the number of HPCs and HBV infection.Methods Cytokeratin 7(CK7)-was stained immunohistochemically in liver tissues of 16 patients with hepatitis B cirrhosis.HPCs and duetular reactions were quantitively analyzed.The expression of HBsAg and HBcAg were also detected to evaluate its relationship with HPCs activation.Results HPCs were extensively activated and marked duetular reactions can be observed in cirrhotic liver tissues.Tlle expression of HBsAg was positively correlated with HPCs activation.Conclusions HPCs are extensively activated in cirrhotic liver tissues,and HBV infection may facilitate its activation.

Objective To construct the lentiviral vector (pSIH-H1) for E6AP small-interfering RNA(siRNA) and to detect its effect on breast cancer ZR 75-1cell growth.Methods E6AP siRNA was designed and constructed based on hu-man papillomavirus E6-associated protein ( E6AP) cDNA sequence.The expression of E6AP was examined by real-time quantitative PCR(qRT-PCR) and Western blotting.The effect of E6AP on ZR75-1 cell growth was determined by cck-8 kit.Results DNA sequencing indicated that E 6AP siRNA expression vector was constructed successfully .qRT-PCR and Western blotting experiments showed that pSIH-H1-E6AP siRNA could suppress the E6AP gene expression.Suppression of E6AP could markedly inhibit the growth of ZR 75-1.Conclusion A lentivirus RNA interference ( RNAi) vector targeting E6AP gene is successfully constructed ,which inhibits the cell growth of ZR 75-1.

Objective To detect the effect of E6AP on gastric cancer cell proliferation and migration.Methods The expression of E6AP in different gastric cancer cell lines and normal gastric mucosa epithelial cell lines was detected by Western blotting.Gastric cancer cells BGC-823 stably expressing E6AP short hairpan RNA(shRNA) were obtained by lentiviral vector of E6AP.The effect of E6AP on BGC-823 cell growth and migration was determined by CCK-8 kit, Tran-swell and wound healing assay.Results Gastric cancer cell line BGC-823 in which E6AP was stably knocked down was established.Knockdown of E6AP inhibited the proliferation and migration of BGC-823 cells.Conclusion E6AP plays a key role in gastric cancer proliferation and migration.