This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

Objective To investigate the possibility of transforming growth factor β (TGF-p) inducing chondrogenesis of precartilaginous stem cells (PSCs) and discuss expression mechanism of extracel-luar matrix. Methods PSCs were induced into a chondrogenic pathway in alginate bead culture in the absence of serum and in the presence of TGF-β1, β2, or-β3. The temporal pattern of expression of cartilage-specific extracellular matrix during chondrogenesis were analyzed by immunocytochemistry, immunoflu-orescence, RT-PCR, immunoprecipitation, Western blot and spectrophotometer. Results Type Ⅱ collagen staining was positive at days 7, 14 and 21 in alginate bead culture, showing most intense staining in the TGF-p3-treated culture. Expression of type Ⅱ collagen was increased in TGF-β3 group. Immunocytochemi-cal analysis of a number of other extracellular matrix components showed widespread expressions of aggre-can, fibromodulin and COMP in alginate bead culture that presented TGF-p3 for 21 days. The expressions of Aggrcan, fibromodulin, type Ⅰ and ⅹ collagen, and COMP were detected by RT-PCR in TGF-β3 group within 8 days, while type Ⅱ collagen began expression at days 8-21. COMP or type X collagen was present in TGF-β3 group at days 7, 14 and 21 by immunoprecipitation or Western blot analysis respectively. The extracted glycosaminoglycan content or the glycosaminoglycan/DNA rate in TGF-βl group was significantly lower than those in TGF-β2 group or TGF-β3 group at days 14 and 21 (P <0.01). Conclusions TGF-β can evocate chondrogenesis of PSCs, when rapid deposition of cartilage-specific extracellular matrix is involved. The sequential events in this pathway leading from the undifferentiated stem cells to mature chon-drocytes can be investigated by analysis of key matrix elements.

Objective To explore the significance of using cytologic and RT-PCR methods to examine(peritoneal) washings and peritoneal tissues of gastric cancer patients in prediction of peritoneal micrometastasis.Methods The peritoneal washings of 38 patients with gastric cancer and 5 patients with benign gastric(lesions) were collected and,at the same time,a small amount of omentum and peritoneum were removed for control.CEAmRNA expression of free cells in peritoneal washings were detected by RT-PCR method and(also) cytology of the washings were performed.Results The CEAmRNA expression rate of peritoneal washings and peritoneal tissues were 36.8%(14/38) and 39.5%(15/38)respectively.Both were more(sensitive) than that of cytologic examination 26.3%(10/38).TNM staging,depth of invasion,lymph node metastasis,and serosal involvement were related to the expression rate of CEAmRNA.Conclusions mRNA of CEA is more sensitive and specific than cytologic examination for detecting free cancer cells in peritoneal cavity.It is an effective method for detecting peritoneal micrometastases in gastric cancer patient.

BACKGROUND:After arthroscopic knee surgery, deep vein thrombosis easily occurs. Currently, there were no specific clinical manifestations in deep vein thrombosis, so a fast, convenient and reliable risk assessment tool was needed to evaluate the clinical high-risk groups for prevention and intervention. The effectiveness of Caprini Risk Assessment Scale used in thrombosis risk assessment has been confirmed by a large number of researches, but the current domestic research is less.
OBJECTIVE:To verify the validity of Caprini risk assessment scale in evaluations of high deep venous thrombosis risk patients among knee arthroscopy patients, and to explore effective strategies for prevention of deep vein thrombosis in patients undergoing knee arthroscopic surgery.
METHODS: A case-control study design was used to colect 49 deep vein thrombosis patients admitted to the Department of Orthopedics, Renhe Hospital of Three Gorges University from January 2008 to June 2015 as case group, and randomly selected 98 patients admitted during the same period of non-deep vein thrombosis patients as control group. Caprini risk assessment scale was used to assess risk assessment and risk grading of deep venous thrombosis, and to explore the correlation between risk classification and risk of deep vein thrombosis.
RESULTS AND CONCLUSION: (1) Basic conditions comparison: application time of tourniquet, the proportion of smoking patients, and proportion of deep venous thrombosis and (or) the history of pulmonary thromboembolism were higher in the case group than in the control group (P < 0.05). (2) Caprini score was significantly higher in the case group than in the control group (P < 0.001). In the case group, the proportion of very high risk patients (53%) was highest, folowed by high risk (25%), totaly 78%. In the control group, the proportion of high risk patients (32%) was highest, folowed by low risk (29%). Significant differences in above risk degree analysis were identified between the two groups (P< 0.001). (3) Deep venous thrombosis and (or) the history of pulmonary thromboembolism was positively correlated with Caprini score in the case and control groups (P < 0.05). Caprini score was positively associated with application time of tourniquet in the case group (P< 0.05). (4) Logistic regression analysis of Caprini risk classification and the risk of deep vein thrombosis: with increased caprini risk classification, the risk of deep vein thrombosis increased significantly. The risk of deep venous thrombosis in patients with high risk and very high risk was 2.130 and 11.786 times of patients with low risk, respectively. (5) These results indicate that Caprini risk assessment model can effectively assess the risk of deep vein thrombosis among patients receiving knee arthroscopy.

Objective To prepare the Env-pseudotyped subtype B HIV-1 with enhanced green fluorescent protein ( EG-FP) gene,explore HIV-1 infection mechanisms and develop feasible methods of identification .Methods The Env-pseudo-typed viruses were packaged in HEK293T cells by cotransfection, and the reporter gene and P24 protein were detected by PCR, Western blot and ELISA .Reporter gene amplification , viral titration assay and a single round of infection assay were performed after the env-pseudotyped viruses infected HIV-1 permissive cell .Results and Conclusion A generation and identification method of the pseudotyped HIV-1 was established . The Env-pseudotyped subtype B HIV-1 has been prepared, which is able to infect SupT1 and TZM-bl cells through infection assay .

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts.hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3.Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system.pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines.The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM)components was detected after transfection by real-time quantitative PCR,ELISA,immunochemistry and Western blotting,respectively.The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing.Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs.Real-time quantitative PCR,immunochemistry and Western blotting showed that the cartilage-specific ECM markers,i.e.,cartilage oligomeric matrix protein (COMP),Aggrecan,collagen type Ⅹ and Ⅱ were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells.It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.