Thrombospondin-2 Couples Pressure-Promoted Chondrogenesis through NF-jB Signaling

METHODS@#Rat BMSCs were isolated, cultured and identified. The time-dependent expressions of TSP-2 and Sox9 in BMSCs under a dynamic mechanical pressure of 0–120 kPa at 0.1 Hz for 1 h were tested by qPCR and Western blotting. The role of TSP-2 in chondrogenic differentiation of BMSCs under mechanical pressure was validated by using small interfering RNA. The impact of TSP-2 and mechanical pressure on chondrogenesis were detected and the downstream signaling molecules were explored using Western blotting. @*RESULTS@#Mechanical pressure stimulation of 0–120 kPa for 1 h significantly upregulated the expression of TSP-2 in BMSCs. The expression of the chondrogenesis markers Sox9, Aggrecan, and Col-II were all upregulated under dynamic mechanical pressure or TSP-2 stimulation. Additional exogenous TSP-2 may potentiate the chondrogenic effect of mechanical stimulation. After knock down TSP-2, the upregulation of Sox9, Aggrecan and Col-II under mechanical pressure was inhibited. The NF-jB signaling pathway responded to both dynamic pressure and TSP-2 stimulation, and the cartilage-promoting effect was blocked by an NF-jB signaling inhibitor. @*CONCLUSION@#TSP-2 plays an essential role in the chondrogenic differentiation of BMSCs under mechanical pressure. NF-jB signaling is involved in the mechano-chemical coupling of TSP-2 and mechanical pressure for the chondrogenic differentiation of BMSCs.

Optimization and purification of extraction of polysaccharides from Anoecto-chilus roxburghii / 药学实践杂志

Objective To optimize the process of ultrasonic extraction of polysaccharide in Anoectochilus roxburghii and to investigate the method of protein removal. Methods The extraction rate of polysaccharide was used as the detection index. On the basis of single factor investigation, Box-Behnken experimental design and response surface method were used to optimize the three factors of material-liquid ratio, ultrasonic time and ultrasonic extraction temperature. The five deproteinization methods including Sevage reagent method, TCA method, salt method (NaOH-CaCl2 and NaOH-NaCl) and hydrochloric acid method were investigated with the retention rate of polysaccharide and protein removal rate. Results The optimal extraction conditions of polysaccharide from Anoectochilus roxburghii were as follows: liquid-to-solid ratio was 10∶1, extraction temperature was 48 ℃ and extraction time was 36 min with extraction 2 times, ultrasonic power was 300 W, the extraction rate was 13.13%. NaOH-CaCl2 deproteinized methods∶ the loss rate of polysaccharide was 18.74%, and the removal rate of protein was 95.62%. Conclusion Ultrasonic extraction is easy to operate, and the optimized extraction method can achieve a high extraction rate. NaOH-CaCl2 deproteinization methods can get high protein removal rate and polysaccharide retention rate. This method is suitable for the research and development of the active components of the polysaccharides from Anoectochilus roxburghii.

Relationship between plasma copeptin level and prognosis in patients with severe acute pancreatitis / 临床外科杂志

Objective To analyze the relationship between plasma copeptin level and prognosis in patients with severe acute pancreatitis(SAP).Methods The clinical data of 48 SAP patients from January 2010 to October 2014 were collected and another 48 patients who accepted healthexamination dur-ing the same period were selected as controls.Difference in plasma copeptin level was compared between groups.The results of APACHEⅡ,Ranson and modified Marshall scores were also collected to evaluate the severity of disease and the existence of MODS.The prevalence of pancreas and peripancreatic tissues effusion,necrosis,pancreas and peripancreatic abscess,pancreatic pseudocyst and mortality of the experi-mental group were recorded to analyze the relationship among plasma copeptin,complications and mortali-ty.Results The plasma level of copeptin of the experimental group was 0.83 ~5.49ng/ml,with a mean of(3.48 ±1.32)ng/ml;while in the control group,it was 0.09 ~1.46ng/ml,with a mean of(0.23 ± 0.06)ng/ml.There was significant difference between the groups(P 4.02 ng/mL predicted in-hospital mortality of patients,with a sensitivity of 72.7%and a specificity of 81.0%(AUC =0.831,95%CI =1.051 ~1.679,P <0.001).Conclusion SAP patients have elevated levels of plasma copeptin,and higher copeptin levels predict high risks of complications and mortality.

Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification / 分析化学

Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. While in the presence of target protein, the target molecules bound specifically with aptamer probe, inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon ( the detection probes) , resulting in a significant fluorescence signal. The effects of length of complementary DNA ( CDNA ) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0 . 067-32 . 4 nmol/L with a detection limit of 0 . 03 nmol/L ( approximately 90 amol target molecules). Moreover, the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.