Objective To knock down hemolysin hlyA gene and insert green fluorescence protein gene of vibrio cholerae vaccine candidate NFYY101. Methods Amplified fragments of hlyA gene upstream (hlyAup) and downstream (hlyAdown),lacz-GFPuv,and hlyAup-laczGFPuv-hlyAdown, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442-hlyAup-laczGFPuv-hlyAdown. Following the construction of the recombinant suicide plasmids ,a vaccine candidate was constructed by homologous recombination ,while SM10λpir carrying pCVD442- hlyAup-laczGFPuv-hlyAdown was utilized as the donor strain to transfect NFYY101. Results Construction of recombinant suicide plasmids pCVD442- hlyAup-laczGFPuv-hlyAdown was satisfactory that hemolysin hlyA gene was knocked out and green fluorescence protein gene was successfully inserted of vibrio cholerae vaccine candidate NFYY101. Conclusion Construction of the recombinant suicide plasmid pCVD442-hlyAup-laczGFPuv-hlyAdown successfully can be used for knocked out the hlyA gene and added green fluorescence protein gene as genetic marker into the chromosomal DNA of vibrio cholerae vaccine candidate.

BACKGROUND:Adipose-derived stem cels are regarded as the potential seed cels for tissue engineering. Colagenase digestion is used to isolate adipose-derived stem cels from fat pads currently. However, there are some problems, such as cumbersome operation and high cost.
OBJECTIVE: To study the basic biological characteristics of adipose-derived stem cels by tissue explants culture and to explore the differentiation potential into osteoblasts, adipocytes and endothelial progenitor cels in vitro.
METHODS:Adipose-derived stem cels were isolated by tissue explants technique from the bilateral groin fat pads of rats under aseptic conditions, and cultured in vitro. Cel counting kit-8 was used to detect the proliferative activity, and flow cytometry was employed to analyze the expression of cel surface markers. Passage 4 adipose-derived stem cels were cultured in osteogenic medium, adipogenic medium and endothelial progenitor cel medium for 2-3 weeks, and then the cels were identified.
RESULTS AND CONCLUSION:Adipose-derived stem cels that were isolated by tissue explants culture were easily cultured, and after subculture, cels were mainly spindle-shaped and grew in clone-like manner with swirling arrangement. Cels that experienced repeated subcultures stil kept stronger proliferative ability and the cel growth curve was shaped as a parabola. Immunochemical staining analysis revealed that adipose-derived stem cels were positive for CD44, CD90 and CD29, but negative for CD31, CD45. After adipogenic/osteogenic induction, the cels were respectively positive for oil red O staining and alizarin red staining. Induced endothelial progenitor cels were identified with CD34 and the ability to uptake Dil-ac-LDL and FITC-UEA. These findings indicate using the using tissue explants culture, high-purity adipose-derived stem cels easy to proliferate can be harvested, highly express stem cels-related antigens, and have the ability to differentiate into osteoblasts, adipocytes and endothelial progenitor cels, which meet the needs of seed cels in tissue engineering research.

Objective:To analyze the clinical features and genetic factors of neonatal 17β-hydroxysteroid dehydrogenase type10 (HSD10) deficiency.Methods:The clinical characteristics and genetic test results of a child with HSD10 deficiency coming from Children′s Hospital of Nanjing Medical University in April 2019 were retrospectively analyzed.The keywords" 17β-hydroxysteroid dehydrogenase type 10 deficiency" or " 2-Methyl3-Hydroxybutyryl-CoA dehydrogenase deficiency" or " HSD10" , etc.were searched in various databases, including CNKI, Wanfang, Weipu, Embase and PubMed to review the cases collected from all published data until May 31, 2020.Results:The patient was a newborn male who developed symptoms on the first day after birth.The main signs were metabolic acidosis, increased blood ammonia and lactate, and hypotonia.Trio whole exom sequencing in the patient and his parents identified hemizygous NM001037811: c.650G>A, p.R217Q in the HSD17B10 gene that is inherited from the mother.Since the child died on the third day after birth, no further central nervous system examination was performed.The mother of the child has intellectual disability, the sibling sister is normal and the HSD17B10 locus is wild type.By lite-rature reviewing, 5 newborn cases with clear medical records and genetic test results were listed.All patients were male, and had onset of HSD10 deficiency within 1 week after birth.The main phenotypes include metabolic acidosis (increased blood ammonia and lactate), hypoglycemia, hypotonia, and convulsions.All 6 children died in early infancy.The corresponsive HSD17B10 variants were c. 740A>G/p.N247S, c.677G>A/p.R226Q, c.257A>G/p.D86G and c. 650G>A/p.R217Q, which did not indicate the hot spots of mutation. Conclusions:HSD10 deficiency in the neonatal period is relatively rare.The clinical diagnosis is difficult due to the serious condition and short course of the disease.Severe metabolic acidosis, hypotonia, and convulsions in neonatal patients are the main reasons for the poor prognosis, which can be attributed to the hemizygous variation and heterogeneity of the mutation site in male patients.c.650G>A may be closely associated with severe neonatal HSD10 deficiency, but the molecular biological mechanism needs to be further clarified.HSD10 deficiency has a poor prognosis and lacks effective treatment.

Objective To explore the clinical effect of anteromedial thigh perforator flap for repairing soft tissue defect in lower extremity.Methods From January 2009 to December 2012,12 patients with soft tissue defect of lower extremity were treated with free anteromedial thigh perforator flap.Among them,there were 5 cases of wound in front of tibia,4 cases of dorsal foot wound,3 cases of heel wound;the wound size was 3.0 cm ×3.5 cm-7.0 cm × 9.5 cm;3 cases were repaired with free perforator flap of descending genicular artery,2 cases with free anteromedial thigh perforator flap,and 7 cases with medial vastus muscle perforator flap.Results All the transplants survived.Among them,1 case of anteromedial thigh perforator flap and 1 case of descending genicular artery perforator flap appeared vascular crisis postoperative.After anticoagulation and antispasmodic treatment aggressively,the flaps survived.One case of descending genicular artery perforator flap necrosis in edge,and then were cured by changing dressing.Twelve cases were followed up 6-12 months after operation (average 8.6 months),and the texture of flaps were good,the appearance appropriate ;7 sensation of 5 free flaps after nerve anastomosis came back to S; the donor sites in all cases were sutured directly and healed,leaving only the linear scar,and no effect on function.Conclusion The anatomy of anteromedial thigh perforator flap is constant,and the operation is convenient.The clinical result of repairing soft tissue defect of lower extremity is satisfying.

Objective To investigate the clinical effect of transplantation of the free anterolateral thigh flap and iliotibial band for the repair of soft tissue defects at foot and ankle and functional reconstruction.Methods From January,2008 to January,2013,25 pediatric patients with soft tissue defects at foot and ankle were transplantation of the free anterolateral thigh flap and iliotibial band.Of the 25 cases,there were 12 cases at heel,8 cases at dorsum of foot,3 cases at medial malleolus and 2 cases at external malleolus.All the cases were accompanied with different degree of fracture or dislocation,and with soft tissue defect such as achilles tendon,extensor tendon of dorsal,collateral ligament of ankle.The length of soft tissue defect which were reconstructed with iliotibial band were 3 cm to 16 cm.The causes of injury:19 cases were crushed by motorcycle or bicycle,4 cases were crushed machines,2 cases were injuryed in traffic accidents.The areas of free anterolateral thigh flaps were 8 cm × 5 cm to 18 cm × 12 cm.All the patients begined to early rehabilitative exercise under the protection of orthosis after 2 weeks of the operation.Results All cases were followed up from 6 to 24 months,averaged of 14 months.All the flaps survived,and only 2 cases with necrosis of small area in distal,and which were healed by dressing.The healing time were 12 to 24 days,and the average of 15.1 days; The surgery function were assessed according to Thermann's scale,and the results was 14 cases for excellent,9 cases was good,the general was 2 cases,and the excellent and good rate was 92％.Conclusion The transplantation of the free anterolateral thigh flap and iliotibial band for the repair of soft tissue defect at foot and ankle and functional reconstruction is a safe and effective strategy,and it has the advantages such as covering the wound at foot and ankle approvingly,reconstruction of power device once,the flap and iliotibial band were in the same wound,the trauma of doner site is small invasive,early recovery functional exercise,shorten the treatment cycle,and relieve the suffering of children.

Objective To investigate the clinical effect of primary internal fixation combined with flap transfer for Gustilo Ⅲ B open fracture of lower extremity with severe soft tissue defct.Methods From January,2008 to January,2013,15 patients with Gustilo Ⅲ B open fracture of lower extremity and severe soft tissue defect were treated with primary internal fixation combined with flap transfer.Among them,there were 2 cases of foot and ankle fracture,9 of lower-tibia and fibula,3 of upper-tibia and fibula and 1 of distal femur fracture.The areas of the flaps were 10 cm × 8 cm-28 cm × 16 cm.Three cases were treated by bone grafting because of bone defect.Results All patients were followed up for 6-24 months.All flaps were survived,and only small area of 2 cases with the distal necrosis were cured by changing the dressing; In 1 case,the wound was infection and healed after anti-infective and drainage treatment; 13 cases had bone union in primary stage,and the other 2 cases were achieved delayed union; the average healing time was 7.2 months,and no case of osteomyelitis was noted; the time of wound healing was 12-36 days,with an average of 18.1 days.According to lower extremity function evaluation form,the excellent and good rate was 86.6％.Conclusion It is a safe and effective strategy to treat the Gustilo Ⅲ B open fractures of lower extremity and severe soft tissue defect with primary internal fixation combined with flap transfer,which has advantages of reliable fixation,covering the wounds with satisfaction,recovering exercise early,shorten the treatment period,alleviating the suffering of patients and so on.

Objective To investigate the applications of antibacterial agents to outpatients in primary hos -pitals in Hefei City of Anhui Province,and to provide reference for rational use of antibacterial agents.Methods In 2011, fourty-five primary hospitals in Hefei City were selected randomly ,including urban community health service centers (Group A) and township hospitals(Group B),and thirty or fourty outpatient prescriptions were analyzed monthly . Results In Group A, the percentage and intensity of antimicrobial usage , the proportion of the combination and injectable formulation were ( 45.36 ±20.02 )%, ( 89.73 ±25.50 ) DDDs · ( 100 cases ) -1 · d-1 , 13.34%, 23.16%,respectively,and the data in Group B were (61.36 ±17.18)%,(108.46 ±32.27)DDDs· (100 cases) -1 · d-1,29.13%,46.39%,respectively,which the former were significantly lower than the latter.Conclusion In primary hospitals,the applications of antibacterial agents to outpatiants are not rataional ,including high percentages of usage and unreasonable selection of species ,and more supervision and training need to be given to the medical staff , especially in township hospitals .

Objective To investigate the clinical effect of the free fibula composite tissue flap transplantation to repair the first metatarsal bone with soft tissue defect on foot.Methods From August, 2008 to August, 2013, 6 patients with the first metatarsal bone and soft tissue defect on foot were treated with transplantation of free fibula composite tissue flap.The causes: 2 cases in traffic accident injury, 4 cases in machine injury;3 cases with traumatic defect, and septic defect in 3 patients.Of the 6 cases, the fibular length with transplantation was 6 cm to 12 cm, and the flap area was 8 cm × 5 cm-18 cm × 16 cm;All the cases were followed-up in 3, 6, 12 months postoperatively to observe the fracture healing, and to assess injured limb function in 1 year postoperatively.Results All cases were followed up 12-24 months, and average of 14 months;All the flaps survived, and the metatarsal bone and fibula healing was good visibly in half a year, The surgery function were assessed according to Maryland's scale, and the excellent were 2 and the good were 4.Conclusion The transplantation of free fibula composite tissue flap to repair the first metatarsal bone with soft tissue defect on foot is a safe and effective strategy, and it has the advantages such as covering the wound at foot approvingly, one-time rebuild repair foot weight bearing area and the surrounding soft tissue defect, shorten the treatment cycle, for small area damage in donor area, and the function postoperative is good, etc.

BACKGROUND: Mechanical ventilation is a double-edged sword to acute respiratory distress syndrome (ARDS) including lung injury, and systemic inflammatory response high tidal volumes are thought to increase mortality. The objective of this study is to evaluate the effects of dynamic ventilatory factors on ventilator induced lung injury in a dog model of ARDS induced by hydrochloric acid instillation under volume controlled ventilation and to investigate the relationship between the dynamic factors and ventilator-induced lung injuries (VILI) and to explore its potential mechanisms. METHODS: Thirty-six healthy dogs were randomly divided into a control group and an experimental group. Subjects in the experimental group were then further divided into four groups by different inspiratory stages of flow. Two mL of alveolar fluid was aspirated for detection of IL-8 and TNF-α. Lung tissue specimens were also extracted for total RNA, IL-8 by western blot and observed under an electronic microscope. RESULTS: IL-8 protein expression was significantly higher in group B than in groups A and D. Although the IL-8 protein expression was decreased in group C compared with group B, the difference was not statistically significant. The TNF-α ray degree of group B was significantly higher than that in the other groups (P<0.01), especially in group C (P>0.05). The alveolar volume of subjects in group B was significantly smaller, and cavity infiltration and cell autolysis were marked with a significant thicker alveolar septa, disorder of interval structures, and blurring of collagenous and elastic fiber structures. A large number of necrotic debris tissue was observed in group B. CONCLUSION: Mechanical ventilation with a large tidal volume, a high inspiratory flow and a high ventilation frequency can cause significant damage to lung tissue structure. It can significantly increase the expression of TNF-α and IL-8 as well as their mRNA expression. Furthermore, the results of our study showed that small tidal ventilation significantly reduces the release of pro-inflammatory media. This finding suggests that greater deterioration in lung injury during ARDS is associated with high inspiratory flow and high ventilation rate.

Objective To investigate the predictive value of plasma cystatin C (CysC) in patients with acute coronary syndrome (ACS) after pereutaneous coronary intervention (PCI).Methods A total of 660 patients with ACS admitted to cardiovascular department were enrolled in this study from January 2009 to June 2010.The enrollment criteria were:(1) the stenosis degree was above 75％ in at least one coronary artery checked by coronary angiography and successful PCI; (2) normal renal function or mild dysfunction with glomerular filtration rate (GFR) ＞ 60 ml/ ( min · 1.73 m2 ).Exclusion criteria were severe liver and renal insufficiency,malignancies and valvular heart diseases.The plasma CysC levels were examined by the latex enhanced immune turbidity method within 24 hours after admission.The relevant clinical data were recorded.The patients were followed up by out-patient interview or telephone from March to June 2011 and adverse cardiovascular events were recorded.The patients were divided into four groups according to CysC level:Q1 (CysC＜1.02 mg/L),Q2 (1.02 mg/L≤＜CysC ＜1.17 mg/ L),Q3 (1.17 mg/L ≤ CysC ＜1.35 mg/L) and Q4 (CysC ≥ 1.35 mg/L).Univariate and multivariate Cox hazards regressions were established to analyze the factors related to prognosis.The proportion differences between four groups were tested by x2.The survival ratio was estimated using the Kaplan-Meier method.Statistical significance was established at a P value of less than 0.05.Results ① A total of 606 ( 91.7％ ) patients successfully accepted follow-up.Mean follow-up time was ( 14.3 + 1.7 ) months.Of them,95 patients were subjected to adverse cardiovascular events ( 15.7％ ).②The incidences of adverse cardiovascular events in Q2,Q3,Q4 were significantly higher than those in Q1 ( P ＜ 0.001 ).The rates of mortality,nonfatal myocardial infarction and target lesion revascularization in Q4 were higher than those in Q1 ( P ＜ 0.05 ).The incidences of heart failure in Q3 and Q4 were higher than that in Q1 ( P ＜ 0.05 ).③Univariate analysis demonstrated that CysC,creatinine,LVEF,age,history of PCI and NYHA grade ≥3 were the risk factors of poor prognosis (P ＜ 0.05 ).④ Multivarite cox hazards regression revealed that the elevation of CysC level remained an independent predictor of adverse cardiovascular events.The relative risk of Q3 and Q4 were 3.930 (95％ CI 1.306-11.829,P =0.015 ) and 6.380 (95％ CI 2.171-18.751,P =0.001 ) compared with Q1.⑤　The cumulative rates of survival without adverse cardiovascular events in Q2,Q3 and Q4 decreased compared with Q1 (P ＜ 0.001 ).Conclusions High plasma CysC concentration is an independent predictor of adverse cardiovascular events in patients with ACS after PCI.