It has been reported that angiogenic growth factors may be useful in the treatment of ischemic heart disease. In our experiment a recombinant deficient adenovirus vector coding for aFGF was intramyocardially administrated into swines with chronic ischemic myocardium to investigate its effect on the improvement of myocardial function. In the present study, experimental minipigs underwent thoracotomy and placement of an Ameroid constrictor on the left circumflex branch coronary artery. Four weeks later, Ad.aFGF( n =7), Ad.Null( n =5) or PBS( n =6), was administrated directly into the myocardium at 10 sites in the circumflex branch distribution area (10 9 pfu or 100?l). Another four weeks later, cardiac function was examined by echocardiography and the results showed significant improvement of myocardial function in Ad.aFGF animals compared with Ad.Null and PBS animals. So such a strategy of gene therapy can be used in patients with ischemic heart disease.

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective To evaluate the value of contrast-enhanced ultrasound (CEUS) in diagnosing ischemic-type biliary lesion (ITBL) and analyze the relation between the enhancement patterns of bile duct wall of ITBL and its outcome.Methods 36 patients confirmed with ITBL (24 cases),anastomotic biliary stricture (3 cases),cholangitis (4 cases),biliarysludge (1 cases),and acute rejection (4 cases),who underwent CEUS examination,were enrolled in this study.The images were retrospectively analyzed in consensus by 2 readers.After reviewing the images,the readers were asked to make a diagnosis of ITBL.The diagnostic standard was hypo-or non-enhancement of hilar bile duct wall in arterial phase on CEUS.Results The diagnostic sensitivity,specificity,accuracy,positive predictive value and negative predictive value were 66.7％,83.3％,72.2％,88.9％,and 55.6％ for reader 1;62.5％,83.3％,69.4％,88.2％,and 52.6 ％ for reader 2,respectively.The interobserver agreement was good (κ =0.83).In 24 ITBL patients,the ratio of mortality or retransplantation with non enhancing hilar bile duct wall in arterial phase was much higher than that with enhancing hilar bile duct wall (non-enhancement 54.5％,hypo-enhancement 20％,hyper-or iso-enhancement 12.5％).Conclusions CEUS had diagnostic value of ITBL.Non-enhancing hilar bile duct wall in arterial phase on CEUS predicated the poor outcome.

Objective To investigate the effect of growth differentiation factor 11 ( GDF11 ) on aorta in apolipoprotein E-Null( ApoE-/-) mice and its possible mechanisms. Methods Four-week-old healthy male ApoE-/-mice were fed with high-fat diet for 1 week and were then divided into 4 groups:vehicle group(n=10), GDF11 group (n=10),adeno-associated virus-green fluorescent protein group(AAV-GFP group, n=10), and AAV-GDF11 group ( n=10 ) . The mice received intraperitoneal injection with phosphate buffered saline, GDF11 protein, a single injection of purified AAV-GDF11 or AAV-GFP through the tail vein, respectively. After 4 weeks, serum GDF11/8 level and endothelium-dependent vasodilatation were detected. After 12 weeks, serum GDF11/8, interleukin-6 (IL-6), tumor necrosis factor-α( TNF-α), total cholesterol ( TC), triglycerides ( TG), oxidized low density lipoprotein(ox-LDL), and free fatty acids(FFA)levels were measured, the plaque areas in aortic enface and cross sections were measured by oil red O or HE staining, the macrophages/T lymphocytes infiltration in plaques were detected with immunohistochemistry, and the mRNA expressions of IL-6, TNF-α, and IL-10 were determined by real-time PCR. Results Compared with vehicle or AAV-GFP groups, GDF11 and AAV-GDF11 groups presented improved endothelium-dependent vasodilatation, decreased levels of blood inflammatory factors, blood lipid, reduced plaque on face area sections[Vehicle group : GDF11 group:(31. 23 ± 3. 12)% vs (17. 18 ± 2. 17) %;AAV-GFP group : AAV-GDF11 group:(38.01±4.43)% vs(14.54±2.86)%,P<0.05]andcrosssections[Vehiclegroup :GDF11 group:(19. 87 ± 2. 11)% vs (10. 32 ± 1. 47)%;AAV-GFP group : AAV-GDF11 group:(23. 02 ± 2. 76)%vs (9.06±1.63)%, P<0. 05]. There were less macrophages and T lymphocytes infiltration in plaques and lower mRNA expressions of inflammatory factors at aortic wall. Conclusion GDF11 reduces the area of atherosclerotic lesion in ApoE-/-mice, which may be involved in endothelial protection, such as to reduce inflammatory reaction, and to change cellular composition in plaques.

Objective: To explore the effect of irisin on atherosclerosis with possible mechanisms in diabetes mellitus (DM) mice. Methods: A total of 30 ApoE-/- mice were randomly divided into 2 groups: Control group, the mice received citrate buffer solution for modeling control,n=10. DM group, the mice received streptozotocin injection for DM modeling,n=20; the DM group was further divided into 2 subgroups as DM control (DM-C) group, the mice received normal saline injection for 12 weeks and DM + irisin group, the diabetic mice received irisin injection for 12 weeks.n=10 in each subgroup. With 4 weeks of irisin intervention, the endothelium-dependent vasodilatation was detected. With 12 weeks of intervention, the blood levels of tumor necrosis factor-α (TNF-α), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and oxidized low-density lipoprotein (ox-LDL) were examined by ELISA, the plaque areas in aortic en face and cross sections were measured by Oil red O or HE staining, the macrophages/T lymphocytes inifltration in plaques were detected with immunohistochemistry, and the mRNA expressions of IL-6, IL-10, TNF-α were determined by RT-PCR. Results: Compared with DM-C group, DM + irisin group presented improved endothelium-dependent vasodilatation, decreased levels of blood inlfammatory factors, reduced plaque on face area sections (22.57 ± 2.17) % vs (35.09 ± 2.38) % and cross sections (19.36 ± 1.85) % vs (25.53 ± 7.87) %,P < 0.05, less macrophages (30.5 ± 2.79) % vs (41.34 ± 9.13) % T and lymphocytes infiltration (28.11 ± 4.24) % vs (35.79 ± 9.11) % in plaques and lower mRNA expressions of inflammatory factors(IL-6: 1.76 ± 0.50 vs 3.78 ± 1.15; TNF-α: 1.05 ± 0.30 vs 2.11 ± 0.48; ICAM-1: 1.96 ± 0.69 vs 2.71 ± 0.72; VCAM-1: 0.87 ± 0.21vs 1.45±0.25; MCP-1: 1.34 ± 0.34 vs 1.77 ± 0.55) at aortic wall, P<0.05.Conclusion: Irisin may improve atherosclerosis condition in ApoE-/- DM mice, the endothelial protection and antiinflammatoryreaction were the important mechanisms. Irisin has the potential for preventing/treating atherosclerosis.

Objective To establish a clinical nursing pathway for elderly parturients. Methods The clinical nursing pathway draft for elderly parturients were designed though the method of case analysis and literature review. Delphi method was used by 10 experts engaged in clinical medical or nursing with 2 rounds. Results The recovery rates of the two Delphi method were 100%, the experts who put forward the proposal on the 2 rounds of expert conclusion were 60.00% (6/10) and 20.00% (2/10) respectively. The expert's familiarity with the correspondence is 0.90, the coefficient of judgment is 0.80 and the coefficient of authority is 0.85.The mean value and standard deviation of the 2 rounds of correspondence were 3.60～5.05 (standard deviation 0.34～0.94) and 3.91～4.13 (standard deviation 0.26～0.60)respectively. The coordination coefficients of the 2 rounds of experts consultation were 0.441 and 0.592 (all P＜0.001). The clinical nursing pathway for elderly parturients with pregnancy management were three stages: confirmed pregnancy to 13+6weeks, 14 weeks to 27+6days pregnant and 28 weeks pregnant to childbirth, including a total of 74 projects for 4 modules such as medical history collection, physical examination, clinical laboratory and other examination and nursing measures. Conclusions The practicable clinical nursing pathway for elderly parturients was constructed because its high degree of familiarity,authority,coordination and concentration. The statistical analysis results are reliable,it is beneficial to standardize the pregnancy management for elderly parturients and improve the quality of clinical nursing.

Objective: To establish real-time recombinase polymerase amplification（RPA）for the rapid detection of Vibrio parahaemolyticus（VP）. Methods: An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin（tlh）gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP. Results: A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method. Conclusion The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.

Objective To investigate the ultrasonograms of biliary sludge and its relationship with the prognosis of patients after liver transplantation.Methods Ultrasonograms of 36 patients with biliary sludge after liver transplantation were retrospectively analyzed.These patients were divided into two groups according to the different treatment outcomes:poor prognosis group and favorable prognosis group.The characteristics of initial ultrasonograms of biliary sludge when they were found at first time compared between two groups .Results Biliary sludge combined with ischemic-type biliary lesions accounted for 95% ( 19/20 ) in poor prognosis group while 13%(2/16)in favorable prognosis group.There was significant difference between two groups (P<0.05).In favorable prognosis group , biliary sludge was found locating in hepatic hilar region in all 16 cases (16/16) and the sludge had clear boundaries dissociated with bile duct walls were observed in 14 cases (14/16).There was no incrassation of bile duct walls in hepatic hilar region , nor dilation of intrahepatic bile ducts observed in 14 cases ( 14/16 ).In poor prognosis group , biliary sludge was observed widespread not only in hepatic hilar region but also in intrahepatic biliary ducts in 10 cases ( 10/20 ) , and the boundaries between sludge and bile duct walls were vague in 15 cases (15/20).Obvious incrassation of bile duct walls in hepatic hilar region was observed in 16 cases (16/20), and dilation of intrahepatic bile ducts was observed in 19 cases (19/20).There were significant differences in the above 4 ultrasound features between two groups (all in P<0.05).Conclusions There are differences in the ultrasonograms of biliary sludge between different prognosis groups.Biliary sludge combined with ischemic-type biliary lesions suggests a poor prognosis , which may need more active interventional treatments.

Objective: To investigate the efficacy and adverse effects of sustained lung inflation (SLI) combined with pulmonary surfactant (PS) in the treatment of neonatal respiratory distress syndrome (NRDS). Methods: This prospective randomized controlled trial included 124 premature infants (gestational age <34 weeks and birth weight <2 000 g) diagnosed with NRDS and in need of PS treatment in Shenzhen Maternity & Child Healthcare Hospital affiliated to Southern Medical University from July 1, 2016 to October 31, 2018. They were randomly divided into experimental or control group, with 62 cases in each. Infants in the experimental group were treated with SLI using T-piece and intratracheal PS, while those in the control group were given PS only. Blood gas analysis and measurement of fraction of inspiration O₂ (FiO₂) and ratio of partial pressure of oxygen (PO₂) over FiO₂ were performed before and 1 h after PS injection. Results of the treatments and incidence of complications were compared. Paired samples t-test, two independent samples t-test, rank-sum test and Chi-square test were used for statistical analysis. Results: There were 56 participants in the experimental group and 54 in the control group who were eventually analyzed. In the experimental group, the pH value, partial pressure of carbon dioxide (PCO₂), FiO₂ and PO₂/FiO₂ at 1 h after PS injection were all improved compared with those before treatment [pH value: 7.26±0.09 vs 7.19±0.09, t=3.814; PCO₂: (51.5±12.6) vs (59.8±16.3) mmHg (1 mmHg=0.133 kPa), t=2.610; FiO₂: 26.0 (21.0-31.5)% vs 40.5 (38.5-51.5)%, U=392.000; PO₂/FiO₂: (284.6±117.9) vs (173.4±59.7) mmHg, t=6.427; all P<0.05]. The overall decrement of FiO₂ after PS injection in the experimental group was more significant than that in the control group [-10.0 (-15.0 to -5.0)% vs -5.0 (-8.0 to 0.0)%, U=706.500, P<0.001]. The experimental group had a higher rate of extubation within 24 h than the control group [80% (45/56) vs 71% (32/54), χ²=5.830, P=0.016]. However, no significant differences were shown in total mechanical ventilation time, non-invasive/high-flow nasal cannula ventilation time, the ratio of re-intubation within 72 h, or the incidence of air leak, bronchopulmonary dysplasia, periventricular-intraventricular hemorrhage, necrotizing enterocolitis or patent ductus arteriosus between the two groups (all P>0.05). Conclusions SLI combined with PS for NRDS babies can increase the rate of extubation within 24 h and promote the down-regulation of FiO₂ without causing significant complications.

Objective To investigate the value of three-dimensional contrast-enhanced ultrasound (3D-CEUS) in diagnosing the hepatic artery complications after liver transplantation.Methods A total of 26 liver transplant donors or recipients suspected with hepatic artery complications were examined by color Doppler ultrasound, two-dimensional contrast-enhanced ultrasound ( 2D-CEUS ) and 3D-CEUS.The successful reconstruction rates of 3D-CEUS and the three-dimensional image quality of reconstructed hepatic arteries were evaluated.Results In the 26 patients, 21 hepatic arteries could be visualized with a successful reconstruction rate 81%.The three-dimensional images of hepatic arteries by 3D-CEUS were continuous.The coronal ( Z-axle) , fine or circuitous branches or tributaries could be visualized well.The segmental hepatic arteries origin could be determined and the spatial relationship of vessels could be visualized definitely.Conclusions The 3D-CEUS images are intuitive , three-dimensional and clear , and have clinical application value in diagnosing of hepatic artery complications after liver transplantation.