Objective To verify the passage stability of recombinant human interferon(rhIFN) α2b working seed strain at key generations through the construction of a 15 L scale-down model, in order to ensure the stability and effectiveness of rhIFNα2b during the production process.Methods The rhIFNα2b lyophilized strain was revived and subcultured, and glycerol stocks of the 0, 10 th, 20 th, 30 th, 40 th and 50 th generations of the culture were preserved respectively. The glycerol stocks of each generation preserved in the subculture stability study were selected and the fermentation-expression experiments were conducted in a 15 L reduced-scale model fermentation system under simulated production conditions. By real-time monitoring of the growth curve of the cells and combining with endpoint detection of product expression level,biological activity, plasmid stability, genetic integrity and microbial purity, a comprehensive comparison was made to determine whether the limited subculture times could cover the existing culture process.Results The rhIFNα2b working seed strains from passages 0, 10, 20, 30, 40, and 50 exhibited good stability and consistency after continuous subculturing.All strains showed typical E. coli colony morphology on LB agar plates with no contamination by foreign microorganisms.Antibiotic resistance profiles remained unchanged, Gram staining confirmed Gram-negative bacterial characteristics, and biochemical profiles were consistent with typical E.coli traits. The target gene expression levels consistently exceeded 20%without a declining trend, and the fermentation activity remained above 1. 0 × 108 IU/L. Plasmid restriction analysis produced bands consistent with theoretical expectations, with no observed sequence mutations, and the plasmid loss rates remained below 30%, indicating robust genetic stability.Conclusion The rhIFNα2b working seed strains demonstrated good fermen-tation stability under scaling-down simulated production conditions in the 15L bioreactor. The current passage limitations of the strain effectively meet the requirements of the updated production process, providing crucial data to support process improvement and optimization.

Objective:To investigate the safety and therapeutic effect of split liver transplantation (SLT) in clinical application.Methods:This is a retrospective case-series study. The clinical data of 203 consecutive SLT, 79 living donor liver transplantation (LDLT) and 1 298 whole liver transplantation (WLT) performed at the Third Affiliated Hospital of Sun Yat-sen University from July 2014 to July 2023 were retrospectively analyzed. Two hundred and three SLT liver grafts were obtained from 109 donors. One hundred and twenty-seven grafts were generated by in vitro splitting and 76 grafts were generated by in vivo splitting. There were 90 adult recipients and 113 pediatric recipients. According to time, SLT patients were divided into two groups: the early SLT group (40 cases, from July 2014 to December 2017) and the mature SLT technology group (163 cases, from January 2018 to July 2023). The survival of each group was analyzed and the main factors affecting the survival rate of SLT were analyzed. The Kaplan-Meier method and Log-rank test were used for survival analysis.Results:The cumulative survival rates at 1-, 3-, and 5-year were 74.58%, 71.47%, and 71.47% in the early SLT group, and 88.03%, 87.23%, and 87.23% in the mature SLT group, respectively. Survival rates in the mature SLT group were significantly higher than those in the early SLT group ( χ2=5.560, P=0.018). The cumulative survival rates at 1-, 3- and 5-year were 93.41%, 93.41%, 89.95% in the LDLT group and 87.38%, 81.98%, 77.04% in the WLT group, respectively. There was no significant difference among the mature SLT group, the LDLT group and the WLT group ( χ2=4.016, P=0.134). Abdominal hemorrhage, infection, primary liver graft nonfunction,and portal vein thrombosis were the main causes of early postoperative death. Conclusion:SLT can achieve results comparable to those of WLT and LDLT in mature technology liver transplant centers, but it needs to go through a certain time learning curve.

Objective:To investigate the safety and therapeutic effect of split liver transplantation (SLT) in clinical application.Methods:This is a retrospective case-series study. The clinical data of 203 consecutive SLT, 79 living donor liver transplantation (LDLT) and 1 298 whole liver transplantation (WLT) performed at the Third Affiliated Hospital of Sun Yat-sen University from July 2014 to July 2023 were retrospectively analyzed. Two hundred and three SLT liver grafts were obtained from 109 donors. One hundred and twenty-seven grafts were generated by in vitro splitting and 76 grafts were generated by in vivo splitting. There were 90 adult recipients and 113 pediatric recipients. According to time, SLT patients were divided into two groups: the early SLT group (40 cases, from July 2014 to December 2017) and the mature SLT technology group (163 cases, from January 2018 to July 2023). The survival of each group was analyzed and the main factors affecting the survival rate of SLT were analyzed. The Kaplan-Meier method and Log-rank test were used for survival analysis.Results:The cumulative survival rates at 1-, 3-, and 5-year were 74.58%, 71.47%, and 71.47% in the early SLT group, and 88.03%, 87.23%, and 87.23% in the mature SLT group, respectively. Survival rates in the mature SLT group were significantly higher than those in the early SLT group ( χ2=5.560, P=0.018). The cumulative survival rates at 1-, 3- and 5-year were 93.41%, 93.41%, 89.95% in the LDLT group and 87.38%, 81.98%, 77.04% in the WLT group, respectively. There was no significant difference among the mature SLT group, the LDLT group and the WLT group ( χ2=4.016, P=0.134). Abdominal hemorrhage, infection, primary liver graft nonfunction,and portal vein thrombosis were the main causes of early postoperative death. Conclusion:SLT can achieve results comparable to those of WLT and LDLT in mature technology liver transplant centers, but it needs to go through a certain time learning curve.

Objective:To investigate the anatomic classification and reconstruction of right intrahepatic bile duct in the donor liver of split liver transplantation (SLT).Methods:The retrospective and descriptive study was constructed. The clinical data of 85 patients who underwent SLT in the Third Affiliated Hospital of Sun Yat-sen University from July 2014 to January 2022 were collected. There were 65 males and 20 females, aged 45(range, 1-82)years. Observation indicators: (1) surgical conditions; (2) anatomy of right intrahepatic bile duct; (3) bile duct reconstruction; (4) postoperative biliary complications; (5) follow-up. Measurement data with normal distribution were represented as Mean± SD, and measurement data with skewed distribution were represented as M(range) or M( Q1, Q3).Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Results:(1) Surgical conditions. Of the 85 donor livers, 11 donor livers were split between the left and right hemilivers, and 74 donor livers were split between the classic right trilobe and left lateral lobe. The cold ischemia time of 85 donor livers was 291(273, 354)minutes, and the operation time, anhepatic phase time and volume of intraoperative blood transfusion of 85 recipients were (497±97)minutes, 51(40, 80)minutes and 8(7, 12)U. (2) Anatomy of right intrahepatic bile duct. Of the 85 donor livers, there were 47 donor livers with classic bile duct anatomical model (type 1), of the ratio as 55.3%(47/85), and 38 donor livers with anatomical variants, of the ratio as 44.7%(38/85). Of the 38 donor livers with anatomical variants, 7 donor livers were type 2, 16 donor livers were type 3a, 2 donor livers were type 3b, 2 donor livers were type 3c, 1 donor liver was type 4, 3 donor livers were type 5a, 4 donor livers were type 5b, 3 donor livers were type 6. For bile duct splitting patterns of the 85 donor livers, 84 donor livers were split with the main trunk of common hepatic duct preserving in the right hemiliver or right trilobe, and 1 donor liver were treated with complete left and right hemiliver splitting to preserve the main trunk of the common hepatic duct in the left hemiliver and the right hemiliver in the right hepatic duct (type 1 bile duct anatomical model). There were 84 donor livers with only one bile duct opening, and 1 donor liver with two bile duct openings (type 3c bile duct anatomical model). (3) Bile duct reconstruction. Of the 85 recipients, there were 69 recipients with common bile duct end-to-end anastomosis to common bile duct of donor liver (38 donor livers with type 1 bile duct anatomical model, 5 donor livers with type 2 bile duct anatomical model, 14 donor livers with type 3a bile duct anatomical model, 2 donor livers with type 3b bile duct anatomical model, 1 donor liver with type 4 bile duct anatomical model, 3 donor livers with type 5a bile duct anatomical model, 4 donor livers with type 5b bile duct anatomical model, 2 donor livers with type 6 bile duct anatomical model), 11 recipients with jejunum anastomosis to common bile duct of donor liver (7 donor livers with type 1 bile duct anatomical model, 2 donor livers with type 2 bile duct anatomical model, 1 donor liver with type 3c bile duct anatomical model, 1 donor liver with type 6 bile duct anatomical model), 3 recipients with jejunum anastomosis to common hepatic duct of donor liver (1 donor liver with type 1 bile duct anatomical model, 2 donor livers with type 3a bile duct anatomical model), 1 recipient with jejunum anastomosis to right hepatic duct of donor liver (type 1 bile duct anatomical model), 1 recipient with common hepatic duct end-to-end anastomosis to right posterior branch of donor liver combined with jejunum of the recipient Roux-en-y anastomosis to common hepatic duct of donor liver (type 3c bile duct anatomical model). (4) Postoperative biliary complications. Of the 85 recipients, 6 cases had postoperative biliary complications, with an incidence of 7.1% (6/85). Of the 6 recipients with postoperative biliary complications, there were 5 recipients with donor liver with type 1 bile duct anatomical model, including 3 cases undergoing postoperative biliary stricture with biliary leakage and 2 cases undergoing postoperative biliary anastomotic stricture, 1 recipient with donor liver with type 3b bile duct anatomical model and undergoing postoperative biliary anastomotic stricture and bile leakage in the liver section. Cases with biliary complications were 5 in the 47 recipients with donor liver with classic bile duct anatomical model and 1 in the 38 recipients with donor liver with anato-mical variants, showing no significant difference between them ( P>0.05). (5) Follow-up. There were 83 recipients receiving followed up for 52(12,96)months. During the follow-up period, 2 recipients died due to non-biliary complication factors (1 donor liver with type 1 bile duct anatomical model and 1 donor liver with 3a bile duct anatomical model). Conclusion:The anatomical classification of right intrahepatic bile duct of donor liver in SLT is mainly classical bile duct anatomical model, and the bile duct reconstruction scheme is mainly common bile duct of donor liver end-to-end anasto-mosis to common bile duct of recipient.

【Objective】 To construct the secretory expression system of insect cells to express the secretory TSHR A subunit protein in the ovarian cells of Spotoma oryzae (sf9). 【Methods】 A recombinant plasmid containing the target protein was constructed, and then the positive bacmid was screened out by the blue and white spots experiment. The verified bacmid was transfected into SF9 insect cells to obtain recombinant baculovirus. The virus was amplified, and the titer level was detected by virus plaque assay. Finally, Western blotting was used to identify the expression of the recombinant protein and optimize the expression conditions. 【Results】 During the construction of the protein expression system, PCR identification and sequencing results confirmed the correctness of the sequences of the recombinant plasmid and the recombinant bacmid. After the transfection of the bacmid, the signs of virus budding were observed in sf9 cells. The virus was collected and amplified. The titer of P1 generation virus was 2×10⁷ pfu/m according to the plaque assay. The recombinant protein was identified by Western blotting and confirmed to be exogenous into the culture medium. The optimal condition for virus infection and protein expression was 72 h after the infection when the multiplicity of infection (MOI) was 1. 【Conclusion】 We constructed an insect cell expression system secreting TSHR 22-289 (55 ku), and the protein could be successfully glycolyzed. This system provides a preliminary basis for the construction and production of its industrial platform and also provides a useful tool for studies on TSHR protein and prevention of GO in the future.

Objective:To analyze the molecular mechanism of Zhenqi Fuzheng Capsules in the adjuvant treatment of AIDS by network pharmacology method and molecular docking technology.Methods:The active components and targets of Zhenqi Fuzheng Capsules were obtained through TCMSP, and the AIDS-related targets were obtained through GeneCards, OMIM and DrugBank databases. The intersection target PPI network was constructed through STRING 11.5 database, and Cytoscape 3.9.1 software was used for network topology analysis; Metascape database was used for GO function and KEGG pathway enrichment analysis of core targets; Cytoscape 3.9.1 was used to construct Zhenqi Fuzheng Capsules component-target-pathway network; Autodock Tools software was used to carry out molecular docking of core targets and active components.Results:Totally 31 active components and 180 targets of Zhenqi Fuzheng Capsules were screened out. TNF, IL6, AKT1, IL1B, TP53, VEGFA, RELA, EGFR and CASP3 were identified as the core targets. GO functional enrichment analysis obtained 1 436 biological processes, 53 cellular components, and 117 molecular functions. KEGG pathway enrichment analysis obtained 167 pathways, which were related to pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, and IL-17 signaling pathway was closely related. Molecular docking results showed that core targets such as AKT1 and TNF had good binding activity to quercetin, kaempferol, and luteolin.Conclusion:The main active components of Zhenqi Fuzheng Capsules in the adjuvant treatment of AIDS are quercetin, kaempferol and luteolin, which may treat AIDS through the IL-17 signaling pathway.

Objective To introduce the simplified "All in one" hepatic vein reconstruction in right split liver transplantation, and to investigate the clinical indications, surgical procedures and clinical prognosis of this technique. Methods Clinical data of 2 recipients undergoing right split liver transplantation were retrospectively analyzed, and the simplified "All in one" hepatic vein reconstruction of right liver lobe was summarized and analyzed. In 2 cases, the right liver lobe was split in vivo. In case 1, the liver parenchyma was split until the first and second porta hepatis, and then the liver was obtained by whole-liver perfusion after cutting off the right hepatic duct, and the hepatic blood vessels were isolated ex vivo. In case 2, the left liver lobe was obtained during splitting in vivo and the right liver lobe was obtained after perfusion in vivo. During donor liver splitting in two cases, the common trunk of the middle hepatic vein was maintained in the left liver lobe, the S5 and S8 hepatic veins of the right liver lobe were reconstructed by the same donor iliac artery, and directly anastomosed with the gap between the left and middle hepatic veins of the inferior vena cava, thus reconstructing the integrity of the posterior inferior vena cava. Results The simplified "All in one" right hepatic vein reconstruction was adopted. The anhepatic phase of two recipients undergoing liver transplantation was 41 and 36 min. After the liver was incised open, the blood flow of the donor liver was normal, the iliac artery bypass was fully filled, liver congestion or swelling was not observed, and liver function was properly recovered after surgery. Two recipients were subject to postoperative follow-up for 23 and 10 months, respectively, No complications related to hepatic venous outflow tracts were noted, such as hepatic vein and inferior vena cava. Conclusions "All in one" hepatic vein reconstruction may simplify the procedures of hepatic venous outflow tract reconstruction, shorten the anhepatic phase and reduce the incidence of postoperative hepatic vein complications in complete right liver split transplantation.

Objective To investigate the anatomical classification of left intrahepatic bile duct (LHD) and the pattern of bile duct reconstruction during pediatric split liver transplantation and their relationship with postoperative biliary complications. Methods Clinical data of 75 pediatric recipients undergoing split liver transplantation were analyzed retrospectively. Before splitting the donor liver, iopromide injection was used for retrograde cholangiography through the common bile duct. According to the patterns of intrahepatic bile ducts in the second, third and fourth segments, the anatomical classification of LHD of the donor liver was determined. The biliary reconstruction regimens for different classification types of LHD were summarized. The incidence and treatment of biliary complications after pediatric split liver transplantation were analyzed. Results Among 75 donor livers, the anatomical classification of LHD included 57 cases (76%) of type Ⅰ, 9 cases (12%) of type Ⅱ, 4 cases (5%) of type Ⅲ and 5 cases (7%) of type Ⅳ LHD, respectively. Among 75 pediatric recipients, 69 cases (53 cases of type Ⅰ, 8 type Ⅱ, 4 type Ⅲ and 4 type Ⅳ) underwent the left hepatic duct-jejunum Roux-en-Y anastomosis, 1 case received common bile duct-jejunum Roux-en-Y anastomosis (type Ⅳ), and 5 cases underwent the left hepatic duct-common bile duct end-to-end anastomosis (4 cases of type Ⅰ and 1 type Ⅱ). Postoperative biliary complications occurred in 6 cases (8%), including 3 cases of biliary anastomotic stenosis, 2 cases of biliary anastomotic leakage and 1 case of bile leakage on the hepatic resection surface. Among 6 recipients, 4 cases were classified as type Ⅰ and 2 cases of type Ⅲ LHD. No significant difference was observed in the incidence of biliary complications between typical type and anatomical variant type of LHD (all P > 0.05). Among 3 recipients with biliary anastomotic stenosis, 2 cases underwent percutaneous transhepatic cholangial and drainage (PTCD) and 1 case repeatedly received biliary-intestinal anastomosis. Two cases of biliary anastomotic leakage underwent PTCD and 1 case of bile leakage on the hepatic resection surface received local drainage. All 6 children survived after treatment. Conclusions Anatomical variation of LHD can be observed in 24% of donor livers, and type Ⅱ accounts for the highest proportion of 12%. Prior to donor liver splitting, routine cholangiography and fine biliary anastomosis may effectively lower the incidence of biliary complications. The incidence of postoperative biliary complications is not significantly associated with the anatomical classification of LHD.

Objective:To analyze the clinical features and SCN4A gentic background of a family with hypokalemic periodic paralysis.Methods:Peripheral blood samples and clinical data were collected from the proband, his brother and parents, and genomic DNA was extracted from these blood samples. Genome-wide exome sequencing was conducted to determine the mutation site in the proband and then allele-specific oligonucleotide primers were designed based on the mutation site. Polymerase chain reaction (PCR) was performed to detect the mutation site to further identify the causative gene in the family.Results:The patient was a 19-years-old male, Han nationality. The patient presented with periodic paralysis while hypokalemia at the same time. His father and grandpa have a similar medical history in the family. A hybrid missense variation (p.R672H) was identified in exon 12 of SCN4A gene in the proband. The same mutation was also detect in the proband's father.Conclusions:The heterozygous missense variation of SCN4A gene (p.R672H) found in this study resulted in familial hypokalemic periodic paralysis. Our research provided reference for the future genetic counseling of this patient and enriched the research data on the relationship between genotype and clinical manifestations.