The incorporation of site-specific recombination systems can help to overcome bottlenecks in livestock transgenic technology. For evaluating the efficiency of Cre/lox mediated DNA recombination in embryos and somatic cells, a working model was established using rat mammary carcinoma cell line SHZ-88, aimed at creation of and use repeatedly of selected "friendly loci" in transgenic livestock. An integration vector pTE-lox2272-DsRed-loxP-GFP-loxP, which red fluorescence gene DsRed served as the first target gene and green fluorescence gene GFP as marker gene, was constructed for introduction of acceptor loci in genome. At the same time a replacement vector pT-lox2272-neo-loxP in which Neo coding sequence served as the second target gene was also constructed for replacing DsRed gene. Transgenic cell clones were produced by electroporating SHZ-88 cell with the integration vector. Cells from three transgenic clones selected randomly were further amplified and were then co-electroporated with the replacement vector as well as cre gene. Analysis of the expression patterns of DsRed and GFP indicated that among the 1 070 cell colonies the efficiency on marker GFP deletion was 91.1% and the efficiency on gene replacement was 29.3%. Molecular analysis by PCR and Southern blotting confirmed that the color patterns as expressed by cell colonies could represent the actual molecular events. This working model mediated by Cre/lox system should be useful for the improvement of the present animal transgenic technology.

Objective To invastigate the effect of living donor liver transplantation(LDLT) with middle hepatic vein(MHV) and LDLT without MHV on the donor. Methods Between August 2007and August 2008, 62 LDLT were performed, 30 LDLT with MHV (group A), 32 LDLT without MHV (group B). Before operation, comprehensive assessment the graft size, remnant liver volume (RLV), fatty liver, MHV type, and the level of portal hypertension in recipient to determine whether harvested MHV. The graft was harvested depending on the port vein and hepatic artery ischemia-line,ultrasound was used to definite the branch of MHV. The donor operative time, intraoperative blood loss, postoperative hospital stay, bilirubin, INR, ALT, albumin, and complications were recorded in detail. Results Operative time, blood loss, hospital stay between group A and group B were not significant difference. But the bilirubin, INR, ALT in group A was higher than group B; Albumin recovered slower and with more ascites in group A. The early complications post-operation were similar in the two groups. There were 2 cases with wound infection in group A and 3 in group B. 3 cases complicated with cholestasis in group A and 1 case in group B, all of them gradually recovered to normal after 2 weeks. 2 cases occurred cross-section bile leakage in either group, the patients administrated with anti-infection, external drainage and recovered;4-7 days after operation, there were 6 cases in group A and 5 cases in group B occurred delayed gastric emptying, it was alleviated after adjust diet, encouraging activities and administrated with gastrointestinal motility drugs. Conclusion Right graft with the middle hepatic vein may lead donor recovery delayed, but with precise evaluation, especially MHV type, remnant liver volume (RLV%)＞30%, preserve the drainage of V4b and the artery segment Ⅳ,it's can ensure the donor's safety.

BACKGROUND:Because of its anti-inflammatory and antioxidant activities,Apelin-13 plays an effective role in the treatment of common clinical diseases such as neuroinflammation,cardiovascular injury and pneumonia.However,there is no relevant basic research on whether Apelin-13 also has a good effect in the treatment of inflammatory bone loss. OBJECTIVE:To explore the therapeutic effect and mechanism of Apelin-13 on inflammatory bone loss,in order to find potential drugs for the treatment of inflammatory bone loss. METHODS:(1)In vitro experiment:RAW264.7 cells were divided into three groups:control group,lipopolysaccharide group and treatment group.The control group was only added with DMEM complete medium;lipopolysaccharide group was added with lipopolysaccharide(100 ng/mL)induced inflammation DMEM medium;and the treatment group was added with 10 nmol/L Apelin-13+lipopolysaccharide induced inflammation DMEM medium.Then,24 hours after lipopolysaccharide induced inflammation,western blot was used to detect the marker proteins inducible nitric oxide synthase and CD86 of M1 macrophages,and cell immunofluorescence was extracted to detect the expression of inducible nitric oxide synthase.Finally,the same amount of receptor activator of nuclear factor-κB ligand(RANKL;50 ng/ml)was added to the control group,lipopolysaccharide group and treatment group to induce osteoclasts.The results of osteoclast induction were evaluated by tartrate-resistant acid phosphatase staining and F-actin staining after 6 days of induction.(2)In vivo experiment:Eighteen male C57bl/6 mice were randomly divided into three groups:sham group,lipopolysaccharide group and treatment group.The sham group received intraperitoneal injection of 0.1 mL of PBS;the lipopolysaccharide group was injected with 0.1 mL of PBS diluent containing lipopolysaccharide(5 mg/kg);and the treatment group was injected with 0.1 mL of PBS diluent containing lipopolysaccharide(5 mg/kg)+Apelin-13(100 μg/kg).After 7 days of continuous intraperitoneal injection,the mice in each group were killed on the 8th day,and two femurs of each mouse were collected.Half of them were scanned by micro-CT and analyzed by bone mineral density,and the other half were stained by hematoxylin-eosin staining RESULTS AND CONCLUSION:(1)In vitro experiment:Western blot results showed that the expressions of inducible nitric oxide synthase and CD86 in the lipopolysaccharide group were significantly higher than those in the control group,and Apelin-13 could significantly inhibit the M1 polarization of macrophages induced by lipopolysaccharide.Cell immunofluorescence results also showed that the expression of inducible nitric oxide synthase in the treatment group was lower than that in the lipopolysaccharide group.Besides,tartrate-resistant acid phosphatase staining and F-actin staining results showed that Apelin-13 inhibited the abnormal activation and bone resorption of lipopolysaccharide induced osteoclasts.(2)In vivo experiment:The results of micro-CT showed that systemic inflammation led to significant bone loss in the distal femur,while Apelin-13 could significantly inhibit bone loss in vivo.Hematoxylin-eosin staining results also showed that Apelin-13 could effectively alleviate inflammation induced bone loss in the distal femur of mice.To conclude,Apelin-13 can alleviate bone loss induced by systemic inflammation by inhibiting M1 polarization of macrophages,inhibiting abnormal activation of osteoclasts and bone resorption.

Objective: To investigate the relationship between psoas muscle index (PMI) and early postoperative survival rate and the incidence of complications after liver transplantation in adults. Methods: The clinical data of 225 patients (male, n=184; female, n=41) underwent liver transplantation at the Organ Transplantation Department of First Central Clinic Institute of Tianjin Medical University from January 2014 to December 2016 were analyzed, retrospectively.Original disease: hepatitis B liver cirrhosis(44 cases), hepatitis C cirrhosis(10 cases), autoimmune liver cirrhosis(29 cases), other benign liver diseases(24 cases), liver cirrhosis with liver cancer(116 cases), hilar cholangiocarcinoma(1 case) and hepatic vascular sarcoma(1 case). The area of bilateral psoas muscle on the lower edge level of the third lumbar vertebral body was measured through preoperative CT image.The PMI was calculated using this formula: bilateral psoas muscle area (mm²)/the square of the body height (m²). According to the receiver operating characteristic curve and cut-off values, the male and female patients were divided into low PMI group and high PMI group respectively.The χ² test, Fisher exact test and t-test was used to compare the differences in perioperative data, survival rate and postoperative complications between the two groups, respectively. Results: There were 44 patients in the low PMI group, and 181 patients in the high PMI group. ICU time was longer (82.5(62.0-128.0) hours vs.69.1(56.0-104.0) hours; P=0.006) and preoperative blood urea nitrogen level (5.86(4.35-15.52) mmol/L vs. 4.94(4.05-7.06) mmol/L; P=0.012) was higher in the low PMI group than those in the high PMI group. Incidence rates of grade 5 complication (18.2%) and grade 4a complication (18.2%) were higher in the low PMI group, and 120-day cumulative survival rate was lower than that in high PMI group(81.8% vs. 95.6%, P=0.001). On the other hand, there were no significant differences in preoperative white blood cell count level, serum creatinine level, operative time, anhepatic period time, intraoperative blood loss, and incidence of postoperative grade 3 complications between the two groups(all P>0.05). Conclusions There is a significant correlation between PMI and early postoperative survival rate and incidence of complications.Patients with lower PMI has poor prognosis after liver transplantation.

Objective: To report the operation methods and clinical effects of repairing finger tip defect with the free tibial dorsal nerve flap of the second toe. Methods: 13 patients with finger tip defects were repaired by the tibial dorsal nerve flap of the second toe. The area of finger tip defect was 2.5 cm×1.5 cm-1.3 cm×1.0 cm, and the area of cutting flap was 2.7 cm×1.7 cm-1.5 cm×1.1 cm. All donor site defects on the second toe were covered with full-thickness skin graft. Results: There were 13 cases in this group, and all the flaps and skin grafts were survived. Postoperative follow-up ranged from 6 to 18 months, with an average of 13 months. The appearance of the fingers was satisfied and the sensory recovery was good. Two-point discrimination of the flaps returned to 7-13 mm, with an average of 9 mm. According to the total active move(TAM)scale, results were excellent in 11 fingers, good in 1 finger, and fair in 1 finger. The donor site skin graft was well healed, the second toe pulp was full, and the two-point discrimination of the toe pulps were 6-10 mm, with an average of 8 mm. Conclusions Compared to the traditional method of repairing finger tip defect with the tibial inherent nerve flap of the second toe, our new method can reduce the damage to the donor site, and we can repair finger tip defect as well as the traditional one at the same time. So it was a better operative method to repair finger tip defect with the tibial dorsal nerve flap of the second toe.

Objective The critical genes associated with exhausted CD8+T cells were screened and validated by mapping the single-cell transcriptome profile of high-grade serous ovarian cancer(HGSOC).Methods The specific subtypes of T cells in the tumor microenvironment were analyzed using the single-cell sequencing data from the early stage of laboratory(SRA database:PRJNA756768)and integrating 5 HGSOC sequencing from the database,and the differentiation trajectory of T cell subsets was ex-plored through pseudotime analysis.Differential gene enrichment was used to determine immunosuppressed CD8+IL-2Low and CD8+IFN-γLow T cell subsets and differential genes,and candidate molecules closely related to exhausted CD8+T cells were screened based on patient prognosis.Flow cytometry was used to analyze the expression of PHLDA1 on CD8+T cells,CD4+T cells and Treg cells dur-ing the activation to exhaustion process of T cells in human PBMCs.ELISA was used to detect the levels of IFN-γ and IL-2 secreted by CD8+T cells in PHLDA1High and PHLDA1Low.Finally,flow cytometry was used to analyze the association between PHLDA1 and ex-hausted markers PD-1 and TIM-3.Results The results showed that T cells were grouped in three ways:(1)IL-2High and IL-2Low;(2)IFN-γHigh and IFN-γLow;and(3)exhausted and cytotoxic CD8+T cells.Subsequently,the intersection of its differentially expressed genes was taken,and the key gene PHLDA1 was ultimately screened.Flow cytometry analysis suggested that during the process of T cell activation to exhaustion,the expression of PHLDA1 continued to increase on CD8+T cells,CD4+T cells and Treg cells;The ELISA results showed that the levels of IFN-γ and IL-2 secreted by CD8+PHLDA1High T cells were significantly lower than those of CD8+PHLDA1Low T cells.Meanwhile,the CD8+PHLDA1High T cell subset could simultaneously cover the exhausted T cell types of CD8+TIM-3+and CD8+PD-1+.Conclusion Based on single-cell sequencing data,this study identified PHLDA1 as a key molecule responsi-ble for CD8+T cell exhaustion in OC,providing new insights for immunotherapy of OC.

Objective To explore risk factors of early lung infection after liver transplantation and to construct a prediction model of early lung infection after liver transplantation.Methods The clinical data of 269 patients who underwent orthotopic liver transplantation for the first time were retrospectively analyzed.Patients were divided into the infected group(n=97)and the non-infected group(n=172)according to whether pulmonary infection occurred within 30 days after operation.The preoperative general data,preoperative laboratory examination results,intraoperative and postoperative data of the patients were collected.Multivariate Logistic regression analysis were used to screen risk factors of pulmonary infection.Based on the results of multivariate analysis,the prediction model was constructed and the prediction efficiency of the model was evaluated.Results Univariate and multivariate Logistic regression analysis showed that preoperative PNI≤41.70(OR=1.972,95%CI:1.047-3.714,P=0.036),LMR≤1.52(OR=2.020,95%CI:1.102-3.705,P=0.023),MELD score＞10.72(OR=1.985,95%CI:1.103-3.573,P=0.022),operative time＞448.00 min(OR=2.676,95%CI:1.515-4.727,P=0.001)and intensive care unit(ICU)hospitalization time＞4.0 days(OR=2.623,95%CI:1.335-5.154,P=0.005)were independent risk factors for early pulmonary infection after liver transplantation.The ROC area under the curve(AUC)of the prediction model based on the results of multivariate Logistic regression analysis was 0.768,the sensitivity was 80.41%and the specificity was 60.47%.Conclusion The prediction model based on PNI,LMR,MELD score,operation time and ICU hospitalization time can effectively predict the occurrence of early pulmonary infection after liver transplantation.

Objective To synthesize a novel 18 F labeled probe targeting translocator protein ( TSPO) ligand 2-( 5, 7-diethyl-2-( 4-( 2-fluoroethoxy ) phenyl ) pyrazolo [ 1, 5-a ] pyrimidin-3-yl )-N, N-diethylacet-amide (VUIIS1008), and evaluate its biodistribution and imaging in rheumatoid arthritis (RA) model. Methods The tosylate substrate was labeled with 18 F using a tosyloxy for fluorine nucleophilic aliphatic substitution to obtain 18 F-VUIIS1008. The labeling efficiency, radiochemical purity, and stability in vitro were determined. In vitro cellular uptake and competitive binding assay were performed on RAW264.7 mac-rophage cells. Biodistribution and microPET/CT imaging were investigated on RA mice established by Com-plete Freund's Adjuvant. Two-sample t test was used to analyze the data. Results 18 F-VUIIS1008 was syn-thesized with the labeling yield up to (41.00±5.00)％, the radiochemical purity>98.00％, and the specific radioactivity >1. 52 × 108 MBq/mmol. 18 F-VUIIS1008 was highly stable with the radiochemical purity >98. 00％ at 4 h after incubation in mouse serum. In vitro, it also exhibited high specific TSPO binding in RAW264.7 macrophage cells. The uptake ratio was (14.00±0.30)％ at 1 h after incubation, and decreased significantly ((4.00±0.70)％;t=12.894, P<0.05) after adding excessive unlabeled VUIIS1008. The half maximal inhibitory concentration (IC50) of 18F-VUIIS1008 binding to TSPO was 0.05 nmol/L in RAW264.7 macrophage cells. In vivo distribution results showed that the uptake of 18 F-VUIIS1008 in the left arthritic ankles reached the peak value of (1.33±0.02) percentage activity of injection dose per gram of tissue (％ID/g) at 1 h after injection. The radioactivity ratio of left ankle arthritic tissue to blood ( A/B) and to normal muscle ( A/M) was 4.40±0.22 and 1.65±0.07 respectively. MicroPET/CT imaging demonstrated that 18F-VUIIS1008 could specifically target and retained in the inflammation site. Conclusion 18 F-VUIIS1008 can be easily synthe-sized with high radiochemical purity and can clearly visualized in RA imaging with low background, suggesting its potential as a novel promising molecular probe targeting TSPO for RA PET imaging.

Weightlessness in the space environment affects astronauts' learning memory and cognitive function. Repetitive transcranial magnetic stimulation has been shown to be effective in improving cognitive dysfunction. In this study, we investigated the effects of repetitive transcranial magnetic stimulation on neural excitability and ion channels in simulated weightlessness mice from a neurophysiological perspective. Young C57 mice were divided into control, hindlimb unloading and magnetic stimulation groups. The mice in the hindlimb unloading and magnetic stimulation groups were treated with hindlimb unloading for 14 days to establish a simulated weightlessness model, while the mice in the magnetic stimulation group were subjected to 14 days of repetitive transcranial magnetic stimulation. Using isolated brain slice patch clamp experiments, the relevant indexes of action potential and the kinetic property changes of voltage-gated sodium and potassium channels were detected to analyze the excitability of neurons and their ion channel mechanisms. The results showed that the behavioral cognitive ability and neuronal excitability of the mice decreased significantly with hindlimb unloading. Repetitive transcranial magnetic stimulation could significantly improve the cognitive impairment and neuroelectrophysiological indexes of the hindlimb unloading mice. Repetitive transcranial magnetic stimulation may change the activation, inactivation and reactivation process of sodium and potassium ion channels by promoting sodium ion outflow and inhibiting potassium ion, and affect the dynamic characteristics of ion channels, so as to enhance the excitability of single neurons and improve the cognitive damage and spatial memory ability of hindlimb unloading mice.
Animals ; Mice ; Transcranial Magnetic Stimulation ; Hindlimb Suspension ; Neurons ; Cognitive Dysfunction ; Brain

Transcranial magneto-acoustic electrical stimulation (TMAES) is a novel method of brain nerve regulation and research, which uses induction current generated by the coupling of ultrasound and magnetic field to regulate neural electrical activity in different brain regions. As the second special envoy of nerve signal, calcium plays a key role in nerve signal transmission. In order to investigate the effect of TMAES on prefrontal cortex electrical activity, 15 mice were divided into control group, ultrasound stimulation (TUS) group and TMAES group. The TMAES group received 2.6 W/cm ² and 0.3 T of magnetic induction intensity, the TUS group received only ultrasound stimulation, and the control group received no ultrasound and magnetic field for one week. The calcium ion concentration in the prefrontal cortex of mice was recorded in real time by optical fiber photometric detection technology. The new object recognition experiment was conducted to compare the behavioral differences and the time-frequency distribution of calcium signal in each group. The results showed that the mean value of calcium transient signal in the TMAES group was (4.84 ± 0.11)% within 10 s after the stimulation, which was higher than that in the TUS group (4.40 ± 0.10)% and the control group (4.22 ± 0.08)%, and the waveform of calcium transient signal was slower, suggesting that calcium metabolism was faster. The main energy band of the TMAES group was 0-20 Hz, that of the TUS group was 0-12 Hz and that of the control group was 0-8 Hz. The cognitive index was 0.71 in the TMAES group, 0.63 in the TUS group, and 0.58 in the control group, indicating that both ultrasonic and magneto-acoustic stimulation could improve the cognitive ability of mice, but the effect of the TMAES group was better than that of the TUS group. These results suggest that TMAES can change the calcium homeostasis of prefrontal cortex nerve clusters, regulate the discharge activity of prefrontal nerve clusters, and promote cognitive function. The results of this study provide data support and reference for further exploration of the deep neural mechanism of TMAES.
Acoustics ; Animals ; Brain ; Calcium ; Electric Stimulation ; Mice ; Prefrontal Cortex ; Transcranial Direct Current Stimulation ; Transcranial Magnetic Stimulation