[Effects of porcine acellular cartilaginous matrix on the proliferation and differentiation of human adipose-derived stromal cells].

OBJECTIVE: This study aimed to evaluate the effects of porcine acellular cartilaginous matrix (pACM) on the proliferation and differentiation of human adipose-derived stromal cells (hADSCs). METHODS: pACM was prepared from porcine articular cartilage through decellularization treatment. hADSCs were isolated from human adipose tissues and cultured with different pACM concentrations. No pACM was used as the control group. The effect of pACM on hADSCs proliferation was detected by CCK-8 method. Moreover, the effect of pACM on hADSCs chondrogenic differentiation was analyzed through fluorescence quantitative polymerase chain reaction and Western blot. RESULTS: hADSCs proliferation rate in 0.5, 1.0, and 2.0 mg·mL⁻¹ pACM groups was not significantly different from that in the control group, whereas that in 4.0 and 8.0 mg·mL⁻¹ pACM group was lower than that in the control group (P<0.05). The expression levels of pACM chondrogenic genes, including SOX-9, collagen type Ⅱ alpha 1 chain (COL2A1), and aggrecan (ACAN) and cell adhesion-related gene LAMININ in 0.5, 1.0, and 2.0 mg·mL⁻¹ pACM group were higher than those of the control group (P<0.05), but that of a stemness-related gene Notch-1 was lower than that of the control group (P<0.05). No statistical difference was found in the expression of a lipogenesis-related gene peroxisome proliferator-activated receptor-γ (PPAr-γ) (P>0.05). The expression levels of chondrogenic proteins (SOX-9, COL2A1, and ACAN) were higher than those of the control group (P<0.05). CONCLUSIONS: Appropriate pACM con-centrations do not affect hADSCs proliferation but can induce hADSCs chondrogenic differentiation.

Overexpression of Hypo-Phosphorylated IκBß at Ser313 Protects the Heart against Sepsis.

IκBßis an inhibitor of nuclear factor kappa B(NF-κB) and participates in the cardiac response to sepsis. However, the role of the hypo-phosphorylated form of IκBß at Ser313, which can be detected during sepsis, is unknown. Here, we examined the effects of IκBß with a mutation at Ser313→Ala313 on cardiac damage induced by sepsis. Transgenic (Tg) mice were generated to overexpress IκBß, in which Ser-313 is replaced with alanine ubiquitously, in order to mimic the hypo-phosphorylated form of IκBß. Survival analysis showed that Tg mice exhibited decreased inflammatory cytokine levels and decreased rates of mortality in comparison to wild type (WT) mice, after sepsis in a cecal-ligation and puncture model (CLP). Compared to WT septic mice, sepsis in Tg mice resulted in improved cardiac functions, lower levels of troponin I and decreased rates of cardiomyocyte apoptosis, compared to WT mice. The increased formation of autophagicvacuoles detected with electron microscopy demonstrated the enhancement of cardiac autophagy. This phenomenon was further confirmed by the differential expression of genes related to autophagy, such as LC3, Atg5, Beclin-1, and p62. The increased expression of Cathepsin L(Ctsl), a specific marker for mitochondrial stress response, may be associated with the beneficial effects of the hypo-phosphorylated form of IκBß. Our observations suggest that the hypo-phosphorylated form of IκBß at Ser313 is beneficial to the heart in sepsis through inhibition of apoptosisand enhancement of autophagy in mutated IκBß transgenic mice.

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering.

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration.

A new method of wound treatment: targeted therapy of skin wounds with reactive oxygen species-responsive nanoparticles containing SDF-1α.

OBJECTIVE: To accelerate wound healing through promoting vascularization by using reactive oxygen species (ROS)-responsive nanoparticles loaded with stromal cell-derived factor-1α(SDF-1α). METHODS: The ROS-reactive nanomaterial poly-(1,4-phenyleneacetone dimethylene thioketal) was synthesized, and its physical and chemical properties were characterized. ROS-responsive nanoparticles containing SDF-1α were prepared through a multiple emulsion solvent evaporation method. The loading capacity, stability, activity of the encapsulated protein, toxicity, and in vivo distribution of these nanoparticles were determined. These nanoparticles were administered by intravenous infusion to mice with full-thickness skin defects to study their effects on the directed chemotaxis of bone marrow mesenchymal stem cells, wound vascularization, and wound healing. RESULTS: The synthesized ROS-reactive organic polymer poly-(1,4-phenyleneacetone dimethylene thioketal) possessed a molecular weight of approximately 11.5 kDa with a dispersity of 1.97. ROS-responsive nanoparticles containing SDF-1α were prepared with an average diameter of 110 nm and a drug loading capacity of 1.8%. The encapsulation process showed minimal effects on the activity of SDF-1α, and it could be effectively released from the nanoparticles in the presence of ROS. Encapsulated SDF-1α could exist for a long time in blood. In mice with full-thickness skin defects, SDF-1α was effectively released and targeted to the wounds, thus promoting the chemotaxis of bone marrow mesenchymal stem cells toward the wound and its periphery, inducing wound vascularization, and accelerating wound healing.

[Study and translation of systemic control technology for the burn and trauma related lung injuries].

Burns and traumas are common injuries during both peace time and wartime. Lung is the earliest organ subjected to dysfunction and the incidence is highest. The systemic protective technology for the burn and trauma related lung injuries is based on evidence-based medicine and translational medicine. It includes a series of effective measures, such as rescue and treatment scheme for massive burn casualties, prophylactic tracheostomy, protective ventilation strategy, sequential cell protection, and prevention and treatment of sequelae, which prevents aggravation of lung injuries caused by ischemia reperfusion, oxidative stress, and iatrogenic factors, as well as reduces the incidence of complications to ensure the recovery after burns and traumas.

[Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli].

Cloning and expression of gsiB was carried out for studying protein structure and function of glutathione transport system. The coding sequence of Escherichia coli gsiB that encodes the periplasmic solute-binding protein of a glutathione transporter was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring GFP reporter gene through the method Sequence and Ligation-Independent Cloning (SLIC). The resulting recombinant plasmid pWaldo-GFP-GsiB was transformed into different E. coli strains and the expression conditions were optimized. It was found that E. coli BL21(DE3) was the best strain for gsiB gene expression among the four strains tested. Induction at lower incubation temperature of 18 degrees C and 0.1 mmol/L of IPTG led to higher expression of gsiB in E. coli BL21(DE3). Western blotting analysis also showed the expression of gsiB and the molecular weight of expressed protein as expected.

[Effects of heroin and purine nucleotide compensation on neutral alpha-glucosidase in rat epididymides].

OBJECTIVE: To study the changes of neutral alpha-glucoside activity in the epididymis of heroin-dependent and heroin-withdrawal rats, and to investigate the effects of intervention with purine nucleotide (AMP and GMP). METHODS: Eighty Wistar rats were randomly divided into 8 groups of equal number, control, nucleotide, heroin, heroin + nucleotide, 3 d withdrawal, 9 d withdrawal, 3 d nucleotide (nucleotide administrated for 3 days after heroin withdrawal) and 9 d nucleotide (nucleotide administrated for 9 days after heroin withdrawal). Neutral alpha-glucosidase activity in the epididymis was detected in each group of rats. RESULTS: Compared with the control group, neutral alpha-glucoside activity was markedly decreased in the heroin group (P < 0.05), and also in the 3 d and 9 d withdrawal groups, although with no significant differences (P > 0.05). CONCLUSION: Heroin reduces neutral alpha-glucoside activity in the epididymis of rats, and this effect may continue for some time after drug withdrawal, while purine nucleotide can keep neutral alpha-glucosidase activity in a relatively stable state.

[Characteristics analysis on major genes and the encoded proteins of human G9P[8] rotaviruses LL52696 and LL52727].

Two Rotavirus G9P[8] strains (LL52696 and LL52727) were recognized during a sentinel-based survey in Lulong, China. Phylogenetic analysis of the VP7 gene showed that both strains isolated constituted a divergent genetic cluster distinct from the other G9 strains isolated in China. Analysis of VP4, VP6, and NSP4 genes revealed that these strains were closely related to Lulong strains. We hold that two strains were reassortant between G9 and Lulong predominant strains.

[A research on rotaviral diarrhea outbreak in Guangxi Province].

OBJECTIVE: To analyze epidemiological characters of an outbreak of rotavirus diarrhea in Daxing County, Guangxi Province. METHODS: Rotavirus-positive specimens were identified by ELISA kit. G/P typing assays were confirmed with multiplex seminested RT-PCR. Full-length VP7 genes of 4 positive specimens were amplified and analyzed. RESULTS: 30 cases of Rotavirus-positive were identified from 64 specimens. The attack rate was 46.9%, and G/P typing was G1P[8]. A change of VP7 amino acid residue is at positions 68. CONCLUSION: G1P[8] rotavirus is the etiologic agents of this diarrhea outbreak. In addition, adults were included in this outbreak.

Molecular characterization of unusual human G5P[6] rotaviruses identified in China.

BACKGROUND: We found an unusual human rotavirus, LL36755 of G5P[6] genotype, in a stool sample collected in Lulong County, Hebei Province, China. This is the first detection of rotavirus serotype G5 in Asia. OBJECTIVES: To identify and characterize G5 rotaviruses in 988 stool samples collected from children under 5 years old with acute gastroenteritis. STUDY DESIGN: We analyzed 459 rotavirus-positive samples with RT-PCR using G5 genotype-specific primers. The G5 strains were sequenced. RESULTS: Two additional G5-positive samples (LL3354 and LL4260) were identified. VP7, VP4, VP6 and NSP4 genes of LL3354, LL4260 and LL36755 strains were sequenced. The VP4 sequences formed a group with porcine P[6] strains. The VP6 sequences of strains LL3354 and LL36755 were phylogenetically close to the major clusters of SGI and SGII rotaviruses, respectively. The deduced VP6 protein of strain LL4260 had characteristics of both SGI and SGII strains, but best fit with a cluster of atypical SGI viruses. In addition, based on NSP4 sequences, the three G5 strains belonged to genogroup B and were closest to human strain Wa. CONCLUSION: These results indicate a dynamic interaction of human and porcine rotaviruses and suggest that reassortment could result in the stable introduction and successful spread of porcine gene alleles into human rotaviruses.

Novel human rotavirus of genotype G5P[6] identified in a stool specimen from a Chinese girl with diarrhea.

During a rotavirus surveillance conducted in Lulong County, Hebei Province, China, a total of 331 stool specimens collected in 2003 from children under 5 years old with diarrhea were screened. We identified a novel group A human rotavirus of genotype G5P[6]. Phylogenetic analysis confirmed that the VP7 protein of this newly identified strain, LL36755, was closely related to those of the G5 strains. As such, it has 95.4% homology with its counterparts in the porcine G5 strains C134 and CC117 at the amino acid sequence level. On the other hand, the VP4 protein of the LL36755 strain was 94.5% homologous to those of the porcine P[6] strains 134/04-10, 134/04-11, 221/04-7, and 221/04-13. Our findings indicate a dynamic interaction between human and porcine rotaviruses.

[Study on the expression of androgen receptor in testis, epididymis and prostate of adult rats with diabetes].

OBJECTIVE: To investigate if there is abnormal expression of androgen receptor (AR) in adult diabetic rats. METHODS: The diabetic model rats were induced by using streptozotocin (STZ), which were divided into three groups: control (group C), diabetes (group D) and diabetes with insulin replacement group (group ID). The mRNA and protein expressions of androgen receptor in testis, epididymis and prostate were detected by Northern blot and radioimmunoassay, respectively. RESULTS: Serum testosterone levels and AR mRNA in epididymis of group D or Group ID were lower than those of group C (P <0.05) , respectively, and the protein expression of AR in testis and prostate of group D was lower than that of group C (P <0.05). CONCLUSION: The expression of androgen receptor decreased in testis, epididymis and prostate of diabetic rats, which weakened the biological effects of AR, and it might be one of the causes that resulted in the sexual and productive dysfunction in diabetic rats.

[The effect of semen antisperm antibody on human sperm acrosin activity].

OBJECTIVES: To study the effect of antisperm antibody(AsAb) on human sperm acrosin activity. METHODS: AsAb and sperm acrosin activity were measured and analyzed in 3,432 infertile men and 65 fertile volunteers. RESULTS: AsAb positive rate was 10.20% in 3,432 case of male infertility, and 9.37% in 2,882 infertile males who received tests of sperm acrosin activity. Acrosin activity of infertility cases were lower than those of fertile cases(P < 0.001). The comparison between AsAb positive group and AsAb negative group infertility cases showed no significant differences of acrosin activity (P > 0.05). Between normal acrosin activity group and abnormal acrosin activity group, there was no significant difference of AsAb positive rate (P > 0.05). CONCLUSIONS: Antisperm antibody could not affect acrosin activity.

[Changes of 5 alpha-reductase type II activity in sexual gland of diabetic male rats].

OBJECTIVES: To study the changes of sexual gland 5 alpha-reductase type II activity in pubertal and adult rats with diabetes. METHODS: We selected 40 and 90 days old male Wistar rats as pubertal and adult animal model respectively, 30 rats in each group. The rats were randomly divided into three groups: control group (C), diabetic group (D) and diabetes with insulin replacement group (ID). The activity of 5 alpha-reductase type II was measured with thin layer chromatography in the epididymis, prostate and testis. RESULTS: 1. In all sexual glands of pubertal rats, the activity of 5 alpha-reductase type II in D group is significantly lower than that in C and ID groups. 2. In all sexual glands of adult rats. there is no difference in the activity of 5 alpha-reductase type II among these groups. CONCLUSIONS: The activity of 5 alpha-reductase type II is likely to be influenced by metabolic environment, hormonal levels and local specific factors in pubertal rats, but it is relatively stable in adult rats.