Recombinant human epidermal growth factor combined with vacuum sealing drainage for wound healing in Bama pigs.

BACKGROUND: Vacuum sealing drainage (VSD) and epidermal growth factor (EGF) both play an important role in the treatment of wounds. This study aims to explore the effects of the combination of VSD and EGF on wound healing and the optimal concentration and time of EGF. METHODS: We tested the proliferation and migration capacity of HaCaT and L929 cells at different EGF concentrations (0, 1, 5, 10, and 100 ng/ml) and different EGF action times (2, 10, and 30 min). A full-thickness skin defect model was established using male, 30-week-old Bama pigs. The experiment included groups as follows: routine dressing change after covering with sterile auxiliary material (Control), continuous negative pressure drainage of the wound (VSD), continuous negative pressure drainage of the wound and injection of EGF 10 min followed by removal by continuous lavage (V + E 10 min), and continuous negative pressure drainage of the wound and injection of EGF 30 min followed by removal by continuous lavage (V + E 30 min). The wound healing rate, histological repair effect and collagen deposition were compared among the four groups. RESULTS: An EGF concentration of 10 ng/ml and an action time of 10 min had optimal effects on the proliferation and migration capacities of HaCaT and L929 cells. The drug dispersion effect was better than drug infusion after bolus injection effect, and the contact surface was wider. Compared with other groups, the V + E 10 min group promoted wound healing to the greatest extent and obtained the best histological score. CONCLUSIONS: A recombinant human epidermal growth factor (rhEGF) concentration of 10 ng/ml can promote the proliferation and migration of epithelial cells and fibroblasts to the greatest extent in vitro. VSD combined with rhEGF kept in place for 10 min and then washed, can promote wound healing better than the other treatments in vivo.

A new pyrazoline-based probe of quenched fluorescent reversible recognition for Cu2+ and its application in cells.

A new pyrazoline-based probe D was designed and synthesized, which can be used as a highly sensitive, selective and reversible recognizing fluorescent to detect Cu2+. The recognition properties of this compound was investigated by UV-vis absorption and fluorescence spectrophotometry. The results showed that the probe D forms a 1:1 complex with Cu2+ and displayed a linear fluorescence response to Cu2+ with a detection limit of 1.94×10-7M. In addition, the probe have a good biocompatibility in living cells.

Protection mechanism of deacetylase inhibitor on spleen of rats with severe hemorrhagic shock.

OBJECTIVE: To explore the protection and molecular mechanism of histone deacetylase inhibitors (HDACIs) on the spleen of rats with hemorrhagic shock. METHODS: A total of 60 SPF male SD rats were selected for the modeling of severe hemorrhagic shock using the method of arterial and venous cannulation with the time-divided bleeding. The measurement of mean arterial blood pressure and blood lactic acid was used to verify the modeling. The modeled rats were randomly divided into shock group, shock + suberoylanilide hydroxamic acid (SAHA) group, shock + autogenous transfusion group and shock + SAHA + autogenous transfusion group. Three hours after the treatment, the spleen of rats was collected and TUNEL method was employed to detect the apoptosis of spleen cells in each group. The statistical analysis was performed. Afterwards, real-time PCR and western blot were employed to detect the expression of BCL-2, BAX and caspass3 in the spleen of rats in each group. RESULTS: A total of 53 rats had successful modeling of severe hemorrhagic shock, with success rate of 88%. Cell apoptosis in the severe hemorrhagic model group was the most serious. After the intervention of HDACIs and the autogenous transfusion, the tissue injury was a bit recovered. Cell apoptosis was least in the shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BCL-2 was significantly increased (P < 0.05), with highest relative expression of BCL-2 in shock + SAHA + autogenous transfusion group (P < 0.05). After the intervention of HDACIs and the autogenous transfusion, the relative expression of BAX was significantly decreased (P < 0.05), with lowest relative expression of BAX in the intervention group of single HDACIs. The change in the expression of caspass3 was similar to BAX, namely the relative expression of caspass3 was significantly decreased after the intervention of HDACIs and the autogenous transfusion (P < 0.05). CONCLUSIONS: HDACIs and autogenous transfusion can all protect the spleen injury because of the severe hemorrhagic shock. Its molecular mechanism may be related to the regulation on the expression of BCL-2/BAX and caspass3, which may affect the apoptosis process of cells.

Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: A vaccine for enterovirus 71 (EV71) is needed to address the high burden of disease associated with infection. We assessed the efficacy, safety, immunogenicity, antibody persistence, and immunological correlates of an inactivated alum-adjuvant EV71 vaccine. METHODS: We did a randomised, double-blind, placebo-controlled, phase 3 trial. Healthy children aged 6-35 months from four centres in China were randomly assigned (1:1) to receive vaccine or alum-adjuvant placebo at day 0 and 28, according to a randomisation list (block size 30) generated by an independent statistician. Investigators and participants and their guardians were masked to the assignment. Primary endpoints were EV71-associated hand, foot, and mouth disease (HFMD) and EV71-associated disease during the surveillance period from day 56 to month 14, analysed in the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01508247. FINDINGS: 10,245 participants were enrolled and assigned: 5120 to vaccine versus 5125 to placebo. 4907 (with three cases of EV71-associated HFMD and eight cases of EV71-associated disease) versus 4939 (with 30 cases of EV71-associated HFMD and 41 cases of EV71-associated disease) were included in the primary efficacy analysis. Vaccine efficacy was 90·0% (95% CI 67·1-96·9) against EV71-associated HFMD (p=0·0001) and 80·4% (95% CI 58·2-90·8) against EV71-associated disease (p<0·0001). Serious adverse events were reported by 62 of 5117 (1·2%) participants in the vaccine group versus 75 of 5123 (1·5%) in the placebo group (p=0·27). Adverse events occurred in 3644 (71·2%) versus 3603 (70·3%; p=0·33). INTERPRETATION: EV71 vaccine provides high efficacy, satisfactory safety, and sustained immunogenicity. FUNDING: China's 12-5 National Major Infectious Disease Program, Beijing Vigoo Biological.

Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial.

BACKGROUND: Enterovirus 71 (EV71) outbreaks are a socioeconomic burden, especially in the western Pacific region. Results of phase 1 clinical trials suggest an EV71 vaccine has a clinically acceptable safety profile and immunogenicity. We aimed to assess the best possible dose and formulation, immunogenicity, and safety profile of this EV71 vaccine in healthy Chinese children. METHODS: This randomised, double-blind, placebo-controlled, phase 2 trial was undertaken at one site in Donghai County, Jiangsu Province, China. Eligible participants were healthy boys or girls aged 6­36 months. Participants were randomly assigned (1:1:1:1:1) to receive either 160 U, 320 U, or 640 U alum-adjuvant EV71 vaccine, 640 U adjuvant-free EV71 vaccine, or a placebo (containing alum adjuvant only), according to a blocked randomisation list generated by SAS 9.1. Participants and investigators were masked to the assignment. The primary endpoint was anti-EV71 neutralising antibody geometric mean titres (GMTs) at day 56, analysed according to protocol. The study is registered with ClinicalTrials.gov, number NCT01399853. FINDINGS: We randomly assigned 1200 participants, 240 (120 aged 6­11 months [infants] and 120 aged 12­36 months [children]) of whom were assigned to each dose. 1106 participants completed the study and were included in the according-to-protocol analysis. The main reasons for dropout were withdrawal of consent and refusal to donate a blood sample. Infants who received the 640 U adjuvant vaccine had the highest GMTs on day 56 (742·2 [95% CI 577·3­954·3]), followed by those who received the 320 U formulation (497·9 [383·1­647·0]). For children, those who received the 320 U formulation had the highest GMTs on day 56 (1383·2 [1037·3­1844·5]). Participants who received the vaccine had significantly higher GMTs than did who received placebo (p<0·0001). For the subgroup of participants who were seronegative at baseline, both infants and children who received the 640 U adjuvant vaccine had the highest GMTs on day 56 (522·8 [403·9­676·6] in infants and 708·4 [524·1­957·6] in children), followed by those who received the 320 U adjuvant vaccine (358·2 [280·5­457·5] in infants and 498·0 [383·4­646·9] in children). 549 (45·8%) of 1200 participants (95 CI 42·9­48·6%) reported at least one injection-site or systemic adverse reaction, but the incidence of adverse reactions did not differ significantly between groups (p=0·36). The 640 U alum-adjuvant vaccine group had a significantly higher incidence of induration than did the 640 U adjuvant-free group (p=0·001). INTERPRETATION: Taking immunogenicity, safety, and production capacity into account, the 320 U alum-adjuvant formulation of the EV71 vaccine is probably the best possible formulation for phase 3 trials. FUNDING: The National Science and Technology Major Project (2011ZX10004-902) of the Chinese Ministry of Science and Technology, China's 12­5 National Major Infectious Disease Program (2012ZX10002-001), and Beijing Vigoo Biological.

[Effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells to osteoblasts].

OBJECTIVE: To investigate the effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells (MSC) to osteoblasts. METHODS: Six male Beagle dogs weighed 10-15 kg each were divided into three groups, group A: medicine serum group, group B: non-medicine serum group and group C: bovine serum group. The serum of group A was obtained from the femoral artery of 2 Beagle dogs drinking equivalent dose of traditional Chinese medicine according to body surface area for 7 continuous days. The serum of group B was collected from the femoral artery of 2 Beagle dogs fed with equal volume of normal saline for 7 days. The serum of group C was fetal bovine serum. The tibia marrow was harvested from another 2 Beagle dogs and MSC were isolated and purified by density gradient centrifugation. MSC were cultured in DMEM solution with fetal bovine serum. After MSC were digested by trypsin, MSC were cultured in DMEM solution with the osteogeneic inducer, which contained dexamethasone, antiscorbutic and beta-glycerophosphate. Morphological and histological changes of the MSC were observed under an inverted microscope. Alizarin monosulfonate and nitric acid argentum staining was performed to observe the calcium deposition. MSC were curtured in DMED solution with medicine serum (group A), non-medicine serum (group B) and bovine serum (group C) respectively. The growth curve was detected by the methyl thiazolyl tetrazolium (MTT). The alkaline phosphatase (ALP) activities were detected to observe the differentiation of MSC. RESULTS: The original MSC were observed as fibroblast-like cell shapes. After the osteogeneic inducer was added, MSC were polygon cells with a few polyprocess. Calcium deposition appeared during 10-14 days and alizarin monosulfonate and Von Kossa staining presented positive. MTT results showed that the number of adherent cells of group A was more than that of group B and that of group C significantly after 6 days (P < 0.05). ALP detection proved that ALP activity of group A was more than that of group B and that of group C significantly after 5 days (P < 0.05). CONCLUSIONS: The traditional Chinese medicine promotes the differentiation of MSC to osteoblasts and osteogenesis.

[wt-p53 gene repair for p53 mutations of salivary pleomorphic adenoma cells].

OBJECTIVE: To analyze the original mutated codon of p53 gene of salivary pleomorphic adenoma (SPA) and to evaluate the repair effects of wt-p53 on SPA cells. METHODS: Four cases of SPA were obtained from clinical fresh samples and SPA cells were separated and cultured, and then the cells were transduced by Ad-wt-p53. The cells and the corresponding tumor tissue DNA were extracted, PCR and single strand conformational polymorphism (SSCP) and DNA sequencing analysis were performed. RESULTS: PCR-SSCP analysis showed 3 out of 4 SPA with abnormal exon 8 and abnormal exon 6. DNA sequencing analysis showed that exon 6 point mutation was found at codon 203 (GTG-->GCG), poly-codon mutations were found in exon 8 at codon 272 (GTG-->GT square), 275 (TGT-->T square T), 276 (GCC--> square CC) and at codon 290 (CGC-->CGCC). After transduced by Ad-wt-p53, all of the mutated codons were repaired. CONCLUSIONS: p53 gene mutation involved many codons that occurred frequently in the tumorigenesis of SPA. Exogenous wt-p53 could compensate and repair all the mutated p53 codons of SPA cells. SPA cells transduced by Ad-wt-p53 showed the typical apoptosis.

[Joint toxicity on multi-component mixtures of SDS and substituted aromatic compounds].

The toxicity to Photobacterium phosphoreum of different mixture systems of sodium dodecyl sulfate (SDS) and three substituted aromatic compounds according to equiconcentration ratio of 1 : 1, as well as individual toxicity, were determined respectively. The joint toxicity was evaluated by toxicity unit (TU), additive index (AI), similarity parameter (lambda), and mixtures toxicity index (MTI). Four evaluating methods were compared and the modes of joint action of different systems were analyzed primarily. The coherent results were obtained from four different evaluating methods. Conclusions can be drawn that the binary joint effects of phenol and toluene mixed with SDS showed weak synergistic, while the other joint effects of SDS and substituted aromatic compounds were antagonistic. Of the evaluating methods, Toxic Unit is most sensitive with higher value of its parameter. Mechanisms of joint toxicity action could be discussed according to luminescence principle of Photobacterium phosphoreum and molecule structures of the mixture components.

[Study of p16INK4/CDKN2 gene homozygous deletions and point mutation in squamous cell carcinoma of buccal mucosa].

OBJECTIVE: To explore the correlation between homozygous deletions and mutation of p16 gene and the carcinogenesis and progression of squamous cell carcinoma of buccal mucosa. METHODS: Thirty buccal cancers, 10 leukoplakias and 8 buccal mucosas were involved. DNA was extracted from the tissues. PCR was used to analyses homozygous deletion of p16 gene. PCR-SSCP-DNA sequencing was performed to detect the point mutation of p16 gene. Immunohistochemical techniques were used to detect the expression of P16 protein. RESULTS: Gene deletions and point mutations were not found in leukoplakia and normal buccal mucosa. Gene deletions were found in 7 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (23.3%), while point mutations were found in 5 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (16.7%). Sequencing analysis showed that 5 cases point mutations were missense mutations, occurred on exon 2. Three cases occurred in the same point, codon 99 (GAT --> AAT). The result of immunohistochemical stains showed that 11 out of 12 cases gene inactivation did not expressed P16 protein. CONCLUSION: Homozygous deletion and point mutation of p16 were the main pattern of gene inactivation in squamous cell carcinoma of buccal mucosa. There was a closely correlation between p16 gene inactivation and the carcinogenesis of squamous cell carcinoma of buccal mucosa.

[The relationship between methylation of p16 INK 4/CDKN2 gene in promoter region and progress of squamous cell carcinoma of buccal mucosa].

OBJECTIVE: To determine the correlation between methylation of p16 gene in promoter region and the carcinogenesis and progression of squamous cell carcinoma (SCC) of buccal mucosa. METHODS: Methylation of pl6 gene in SCC and leukoplakia of buccal mucosa was investigated by MSP and pl6 protein was analyzed by Western blot. RESULTS: The methylation of p16 gene was found in 15 of 30 cases SCC and 1 of 10 cases of leukoplakia of buccal mucosa (P < 0.05). Methylation of p16 gene in SCC of buccal mucosa was not related with age, sex, cell differentiation and clinical stage. But methylation of p16 in the cases with lymph node-metastasis was higher than that in the cases without lymph node-metastasis protein (P < 0.05). Meanwhile Methylation of p16 gene was positively correlated with no-expression of p16 protein (P < 0.01). CONCLUSIONS: The methylation of p16 gene leaded to the inactivation of p16 gene and was related with the carcinogenesis and progress of SCC of buccal mucosa.

[The effects of HSV-tk suicide gene and wild-type p53 gene on pleomorphic adenoma cells of salivary gland].

OBJECTIVE: To study the therapeutic effects of combined gene therapy of wild type p53 (wt-p53) and herpes simplex virus thymidine kinase (HSV-tk) gene on pleomorphic adenoma cells of salivary gland. METHODS: Wild type p53 and HSV-tk gene were transfected into human pleomorphic adenoma cells of salivary gland by using recombinant adenovirus vector. The efficiency of transfection was checked and gene was expressed by RT-PCR methods. The cell inhibition after transfected was verified by light microscope and MTT. RESULTS: The proliferation of the pleomorphic adenoma cells transfected wt-p53 and HSV-tk gene was inhibited and the cell survival rate decreased to 54% and 38% respectively in 5 days. However, when wt-p53 gene combined with HSV-tk/GCV system, the killing effects was significantly stronger (P < 0.05) and the cell survival rate decreased to 20%. CONCLUSION: Combining p53 gene with HSV-tk/GCV system for gene therapy in pleomorphic adenoma cells of salivary gland is a valuable method.