Objective To construct a novel bioartificial liver (BAL) system and evaluate its functions in vitro. Methods Chinese experimental minipig hepatocytes were isolated by in situ recirculating collagenase perfusion method and 1.0?10~(10) hepatocytes were cultured in serum-free medium with restriction of(attachment), and using spinner method to form hepatocyte(spheroids).cted by inoculating the hepatocyte(spheroids) into cell circuit of a hollow fiber bioreactor from BIOLIV A3A. Observing the number and viability of the(hepatocytes), the changes of alanine aminotransferase (ALT), total bilirubin (TBI), albumin (ALB) in(circulating) hepatocyte suspension and(RPMI1640) medium; in addition, lidocaine metabolism test was(determined),(during) 6h circulation of the system. (Results) There were no significant differences in number and viability of the hepatocytes before and after 6h(circulation). The BAL system has relatively strong albumin synthesis and lidocaine(metabolism) functions. (Conclusions) The BAL system that we developed had ability to support liver functions and could be used in the treatment of liver failure, or to provide temporary liver support for candidates of liver(transplantation).

Objective To investigate the clinical value of Color Doppler Ultrasound in diagnosis of lymph node diseases. Methods We observed the ultrasound features of the 93 cases of swelled lymph nodes with Color Doppler ultrasound. The ultrasound features included pseudokidney sign, assessment on blood flow distribution, the Doppler resistive Index (RI), maximal flow rate (Vmax),the longitudinal axis compared to the diameter of a node (L/D ratio) . Results Out of 93 cases of clinically confirmed swelled lymph nodes,the concordance rate of Color Doppler Ultrasound was 86%and 88%in diagnosis of malignant lymph node disease and benign lymph node disease, respectively. 4 cases of lymph tuberculosis were misdiagnosed as lymphoma due to the similar ultrasound characteristics found in malignant lymph group,the rate of misdiagnosis was 8%. In the cases of proliferative lymph node diseases with Pseudokidney sign, 93% of the blood flow distribution was classified as grade 0-I. 90% of lymphadenitis were found with Pseudokidney sign,and 95%of those cases with blood flow distribution was classified as grade II-III. Malignant lymph diseases had no Pseudokidney sign, and 86%of blood flow distribution grade was as III. There was statistically significant difference in the L/D ratio and RI between benign lymph group and malignant lymph group (P<0.01) . There was statistically significant difference in Vmax between lymphoproliferative group and other groups (P<0.01) . Conclusions Pathological characteristics on different lymph node disease determin the ultrasound characteristics. Combined clinical data based on Pseudokidney sign, Blood Flow Distribution, RI value,Vmax value and L/D ratio can enhance the accuracy of various lymph node disease diagnosis.

Aim To investigate the effect of Rhein on the movement and invasiveness of human ovarian carci-noma cells with directional high lymphatic metastasis SKOV3-PM4 cells and explore the role of Rac1/LIMK1/cofilin signaling pathway. Methods Migration assay and invasion assay were used to observe the effect of Rhein on the metastatic and invasive ability of SK-OV3-PM4 cells in vitro. The effect of Rhein on the morphology and cytoskeleton ultrastructure of ovarian cancer cells was observed by laser scanning confocal microscope and scanning electron microscope. The protein expression level of Rac1,LIMK1,PAK1 and co-filin were detected by Western blot, respectively. Re-sults Rhein inhibited the abilities of cell invasion and migration of SKOV3-PM4 cells,and the inhibitory rate increased along with the increase of the concentration and treatment duration. After treated with 8. 80 μmol· L-1 ,17. 60 μmol · L-1 , 26. 40 μmol · L-1 of Rhein for 24 h,the abilities of migration and invasion of SK-OV3-PM4 cells were inhibited ( P <0. 05 ); the mor-phology and cytoskeleton ultrastructure of SKOV3-PM4 cells were changed, cellular pseudopod reduced, cell microfilament fractured and its distribution disordered, plasma membrane was uneven and cell gap widened . After treatment of Rhein and Rac1 inhibitor , Rac1 protein expression and the expression of P-LIMK1 , P-PAK1 and P-cofilin notably decreased in a dose-de-pendent manner compared with the control group ( P<0. 05 ) . After Rhein and Rhein plus Rac1 inhibitor treatment ,P-LIMK1, P-cofilin, P-PAK1 protein levels of SKOV3-PM4 cells significantly decreased compared with the control group , and the group of Rac1 inhibi-tor plus Rhein treatment, the phosphorylated protein decreased more significantly ( P <0. 05 ) . After Rac1 activator plus Rhein treatment, phosphorylated protein expression of P-LIMK1 ,P-PAK1 and P-cofilin upregu-lated significantly ( P <0. 05 ) . Conclusions Rhein may be a potential inhibitor of Rac1 and can inhibit the migrating and invasive capabilities of directional high lymphatic metastasis SKOV3-PM4 cells through down-regulating the phosphorylation of Rac1/LIMK1 /cofilin pathway associated protein.

Objective:This study aimed to investigate the effect of human lymphatic endothelial cells (HLECs) on proteins secreted by epithelial ovarian cancer (EOC) cells SKOV3-pm4 with highly directional lymphatic metastasis. Methods:The supernatants of the four groups of cultured cells (A, SKOV3;B, SKOV3+HLEC;C, SKOV3-PM4;and D, SKOV3-PM4+HLEC) were collected. The proteins of these cells were detected by antibody arrays and iTRAQ-2D-LC-MALDI-TOF/TOF/MS. The screened significantly differential proteins were further analyzed by bioinformatics and validated in the human serum and cell culture medium by ELISA. Results:Progranulin (GRN) and vascular endothelial growth factor A (VEGF-A) were upregulated between groups C and A. In addition, insulin-like growth factor binding protein-7 (IGFBP-7) and secreted protein acid rich in cysteine (SPARC) were downregulated between groups D and C. Comprehensive bioinformatics analysis revealed that IGFBP7 interacted with VEGFA. VEGF exhibited the highest expression in ovarian cancer and IGFBP7 exhibited the lowest expression compared with the serum of the normal control group. Statistically significant differences were observed between the two substances. Conclusion:The HLEC microenvironment is closely associated with directional metastasis in lymph nodes with differential proteins, including matricellular proteins and adhesion factors. In particular, the upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 were closely associated with the directional metastasis of EOC cells in lymph nodes.

Objective To establish the condition cultrue cell system and co-culture cell system with SKOV3/PM4,HUVEC and to study the changes of their biological characteristics. Methods The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium(e.g:the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium)and to establish the condition cultrue cell system and the co-culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy(TEM). The growth curve of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry was used to analyzed the cell cycles.In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope(LSCM). The expression of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) were detected by gelatin zymography. Results Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division,the mitotic index respectively were [(4.8 ± 0.8)%,(11.2 ± 0.3)%;P<0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively[(69.4±3.6)%, (48.4±4.6)%;P<0.05] and the raised cell ratio of G2/M phase, respectively [(5.2±1.6)%, (24.9±2.2)%;P<0.05]. Compared with the single culture HUVEC,the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [(2.7±0.5)%, (5.7±0.6)%;P<0.05]. The growth rate was slightly declined. In cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%,(79.0 ± 4.1)%;P<0.05] and the declined cell ratio in G2/M phase, respectively [(19.1±1.2)%, (3.3±0.5)%;P<0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P<0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co-culture SKOV3/PM4+HUVEC. Conclusion The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.

Objective To explore the clinical effect of complete transposition of arteriovenous in free flap artery crisis. Methods From October, 2009 to April, 2017, 13 cases of extremities tissue defect were repaired with free flaps.The intractable arterial crisis appeared after transplantation.Repeated anastomosis vessels were adapted but it was not relieved. Then the complete transposition of arteriovenous was adapted in the flaps. Namely the vein of the flap was anastomosed with the arterial in the recipient site to reconstruct the blood supply, and the arterial of the flap was anastomosed with the vein in the recipient site to reconstruct recirculation.Ten cases of hand defect and 3 cases of crus defect were repaired by 5 low abdominal flaps and 8 anterolateral thigh flaps. The tissue defect area was 16 cm× 7 cm-6 cm×4 cm and the flap area was 18 cm×8 cm-7 cm×4 cm. Results Ten flaps survived completely, the other 3 flaps almost survived that scab healed in 1 case and skin grafted in 2 cases. The flap for skin color was from purple red to dark red, and finally close to normal, and skin flap edge would have different degrees of ecchymosis; the bleed-ing from the incision of the skin flap was from dark red to bright red; the swelling of the flap was obvious in the early stage and the later swelling subsided. All cases were followed-up from 6 months to 32 months with an average of 16 months.The wounds healed well.The flaps had a clear boundary and soft texture. Conclusion Complete transposi-tion of the arteriovenous system can be used as an alternative in the presence of intractable arterial crisis after free flap transplantation, to save the flap and to reduce the trauma to the patient.

Objective:To discuss the ultrasonographic features of ganglioneuroma(GN), ganglioneuroblastoma-intermixed(GNBi), ganglioneuroblastoma-nodular(GNBn), neuroblastoma(NB) in children and to improve the ultrasound diagnostic accuracy of four kinds of neuroblastic tumors.Methods:Two hundred and seventy-nine patients with neuroblastic tumors(23GN, 44GNBi, 86GNBn, 126NB) confirmed by surgery and pathological diagnosis in Children′s Hospital Affiliated to Capital Institute of Pediatrics from June 2014 to February 2019 were retrospectively analysed. The ultrasonographic data were summarized and compared with pathological results.Results:The median age were 6.9 years in GN group, 3.9 years in GNBi group, 3.0 years in GNBn group and 2.0 years in NB group. The median values of serum neuron-specific enolase(NSE) were 6.3 μg/L in GN group, 9.6 μg/L in GNBi group, 22.6 μg/L in GNBn group and 40.7 μg/L in NB group respectively. There was no distant metastasis of GN, while 9% GNBi, 26% GNBn and 36% NB had distant metastasis; GN and GNBi group had no invasion to adjacent tissues and organs, while 9% GNBn and 15% NB lesions had invasion to adjacent tissues and organs; 91% GN, 70% GNBi, 58% GNBn lesions had complete capsule, while 44% NB lesions had incomplete capsule and 28% NB had no capsule; 96%GN, 57%GNBi lesions were round or oval, while 57%GNBn, 60%NB lesions showed irregular shape; 96%GN showed homogeneous hypoechoic, 75% GNBi had "cloudy" inhomogeneous echoe, while hyperechoic nodules were seen in 59% GNBn and 75% NB lesions; 65%GN lesions contained discrete and punctate calcifications, while 27%GNBi, 29%GNBn, 25%NB lesions contained coarse calcifications; 100%GN, 91%GNBi, 91%GNBn lesions had little to moderate blood flow, while 33%NB lesions had abundance blood flow; 4%GN, 41%GNBi, 51%GNBn, 49%NB lesions surrounded blood vessels; 13%GN, 5%GNBi, 6%GNBn, 8%NB lesions had spinal canal invasion; GN had no lymph node metastasis, but 48%GNBi, 59%GNBn, 56%NB lesions had lymph node metastasis.Conclusions:The ultrasonographic characteristics of GN are largely different from GNBi, GNBn and NB; There are some differences in ultrasonographic features of GNBi, GNBn and NB, but some of them are difficult to identify.

Objective To investigate the impact of cardiomyocyte ferroptosis on acute myocardial infarction(AMI)and its relationship with the expression of long non-coding RNA(lncRNA)GAS5.Methods An AMI model was established in eighteen male SD rats(6-8 weeks old),and then the subjected rats were randomly divided into blank control group,sham operation group,and model group,6 animals in each group.In 2 weeks after surgery,echocardiography was used to evaluate heart function,and TTC staining,HE staining and TUNEL staining were employed re-spectively to observe myocardial infarct area,histopathological changes of myocardial tissues and apoptosis in cardiomyocytes.Serum Fe2+concentration was measured with an iron ion detection kit.The expression levels of lncRNA GAS5,nuclear factor erythroid-derived 2-like 2(Nrf2),and glutathione peroxidase 4(GPX4)in myocardial tissues were detected with QT-PCR.Linear corre-lation analysis was performed to investigate the relationship between the expression of lncRNA GAS5,Nrf2 and GPX4 with Fe2+concentration.Western blotting was conducted for the protein levels of ferroptosis-related proteins,glutathione peroxidase 4(GPX4),nuclear factor erythroid-derived 2-like 2(Nrf2),glucose-regulated protein 78(GRP78),and cysteinyl aspartate specific proteinase 12(Caspase-12)in myocardial tissues.Results Western blotting revealed obvious in-creases in the protein levels of ferroptosis related proteins GRP78(1.11±0.13 vs 0.51±0.08,P＜0.01)and Caspase-12(1.23±0.05 vs 0.92±0.07,P＜0.05)and a decrease in those of ferroptosis inhibitor GPX4(0.27±0.11 vs 0.68±0.10,P＜0.01)and Nrf2(0.30±0.12 vs 0.58±0.04,P＜0.05)in the model group than the sham operation group.Additionally,QT-PCR showed that the mRNA levels of GPX4 and Nrf2 were notably lower,indicating cardiomyocyte ferroptosis,while that of lncRNA GAS5 was remarkably higher in the myocardial tissues of the model group than the sham operation group(P＜0.01).What's more,the expression of lncRNA GAS5 had a positive correlation with serum Fe2+concentration(P＜0.05).Conclusion AMI is closely associated with cardiomyocyte ferroptosis.lncRNA GAS5 is involved in the regulation of ferroptosis by Nrf2/GPX4 signaling pathway,which thus mediating the occurrence of heart failure after AMI.

Objective:To explore the application value and treatment effects of multiple free anterolateral perforator flaps of calf for reconstruction of multiple digital wounds in single hand.Methods:From August 2020 to March 2022, 12 patients with soft tissue defects in 35 digits were treated in Department of Hand Surgery, Suzhou Ruihua Orthopaedic Hospital. Ten patients were male and 2 were female, aged 25 to 58 years old. Of the patients, 1 had soft tissue defects in 5 digits, 3 in 4 digits, 2 in 3 digits and 6 in 2 digits. The size of defects was from 1.2 cm ×1.2 cm to 7.0 cm×3.5 cm after debridement. The vascular perforators discovered from intraoperative explorations were found originating from the superficial peroneal artery in 24 flaps, from the peroneal artery in 7 flaps and from the anterior tibial artery in 4 flaps. During surgery, the perforator artery and accompanying veins of the flaps were anastomosed with the proper digital artery and palmar or dorsal subcutaneous veins in the recipient site, respectively. The size of the flaps was from 1.5 cm×1.5 cm to 7.5 cm×4.0 cm. No nerve was affected in the surgery. The wound at donor sites in the calf was sutured directly. Regular postoperative follow-ups were conducted at outpatient clinics. The comprehensive evaluation scale of flap was used to assess the conditions of the donor and recipient sites.Results:In this study, all 35 soft tissue defects of digits in 12 patients were reconstructed by the anterolateral perforator flaps of calf. All the 35 flaps survived after surgery, with a 100% of survival rate. The patients were instructed to carry out early functional training after surgery. Follow-up lasted 6 to 24 months, with an average of 11 months. Twenty-five flaps were found in slightly swollen, and further flap thinning surgery were carried out 3 months after the primary surgery, while the rest of the flaps had good appearance and texture. At 6 months after surgery, all flaps recovered a partial deep and shallow sensory and sense of touch. All wound at donor sites in calf had one-stage healing without dysfunction. The comprehensive evaluation scale was excellent in 28 flaps and good in 7 flaps. The excellent and good rate was 100%.Conclusion:It is an effective method to use multiple free anterolateral perforator flaps of calf to reconstruct multiple digit defects in single hand. The flaps can be conveniently harvested and the multiple digital defects in single hand can be reconstructed in primary surgery with small damages to the donor sites and together with satisfactory clinical outcomes.

Objectives To evaluate the effectiveness and safety of peramivir trihydrate in patients with influenza.Methods This was a randomized,double-blind,double-dummy,placebo and positive control,multicenter clinical trial,comparing peramivir trihydrate with oseltamivir and placebo.The inclusive criteria were 15-70 years old,onset within 48 h,positive rapid influenza antigen test,and febrile(＞38℃) accompanied with at least two associated symptoms.The severe cases complicated with chronic pulmonary and cardiac diseases,malignancies,organ transplantation,hemodialysis,uncontrolled diabetes,immunocompromised status,pregnancy and coexistence of bacterium infections were excluded.All patients were randomized 2:2:1 to receive peramivir,oseltamivir and placebo respectively.The primary endpoint was the disease duration,the secondary endpoints included time to normal axillary temperature and normal living activities,viral response,and adverse effects.Results Following informed consent,133 patients were included in this study.Four patients were exclude due to missing medical records,not fitting inclusion or exclusion criteria and poor compliance.A total of 129 patients were finally analyzed,including 49 cases,54 cases and 26 cases in peramivir group,oseltamivir group and placebo group.The median disease duration were 96 (76,120)hours,105(90,124) hours,and 124 (104,172)hours in three groups respectively(P＞0.05).The time to normal axillary temperature,normal living activities and viral response were not significantly different in three groups(P＞0.05).Conclusion The value of antiviral therapy in patients with mild influenza needs to be further determined.