ObjectiveTo investigate the mechanism of action of modified Shaofu Zhuyutang in antagonizing cellular pyroptosis and fibrosis in ectopic endometrial tissues of endometriosis through the Toll-like receptor 4/nuclear factor-κB/NOD-like receptor protein 3 （TRL4/NF-κB/NLPR3） signaling pathway. MethodsSeventy-two SPF-grade female SD rats were randomly divided into a sham-operated group （n = 12） and a modeling group （n = 60）. The rats in the sham-operated group underwent a caesarean section， while the rats in the modeling group were used to establish an endometriosis model through the auto-transplantation method. After successful modeling， the animals were randomly divided into the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang low-， medium-， and high-dose groups （7.5， 15， 30 g·kg^-1）， with 12 animals in each group. After 4 weeks of drug administration， voluntary activity and heat pain latency were observed. The rats were sacrificed for tissue collection， and Masson staining were used to observe histopathological changes in the endometrial tissues. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum levels of interleukin-18 （IL-18）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and transforming growth factor-β （TGF-β）. Immunohistochemistry （IHC） was used to detect the protein expression area of tumor necrosis factor-related factor 6 （TRAF6） and NLPR3 in the endometrial tissues. Immunofluorescence （IF） was used to detect the relative fluorescence intensity of Caspase-1 and gasdermin D （GSDMD） in the endometrial tissues. Western blot was employed to measure the relative expression of TRL4， myeloid differentiation factor 88 （MyD88）， TRAF6， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， and NLPR3 proteins in endometrial tissues. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 in the endometrial tissues. ResultsCompared with the sham-operated group， rats in the model group showed reduced voluntary activity and shorter heat pain latency. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were elevated. The relative expression areas of TRAF6 and NLPR3 proteins were increased， and the relative fluorescence intensity of Caspase-1 and GSDMD was enhanced. The relative expression of TRL4， MyD88， TRAF6， NF-κB p65， p-NF-κB p65， and NLPR3 proteins， along with the expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA， were significantly increased （P<0.01）. Compared with the model group， rats in the progesterone group and the modified Shaofu Zhuyutang medium- and high-dose groups exhibited improved voluntary activity， longer heat pain latency， the fibrosis of endometrial tissue is alleviated. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were decreased. The relative expression areas of TRAF6 and NLPR3 proteins decreased， and the relative fluorescence intensity of Caspase-1 and GSDMD weakened. The relative expression of TRL4， MyD88， TRAF6， p-NF-κB p65， NLPR3 proteins， and TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA expression were reduced （P<0.05， P<0.01）. ConclusionModified Shaofu Zhuyutang may play a therapeutic role in endometriosis by interfering with key proteins in the TRL4/NF-κB/NLPR3 signaling pathway， reducing NLRP3 inflammasome-induced cellular pyroptosis， antagonizing the fibrosis process in ectopic endometrial tissues， improving the inflammatory microenvironment in the pelvic cavity， and alleviating pain.

ObjectiveTo observe the effects of modified Shaofu Zhuyutang on key proteins of the epidermal growth factor receptor （EGFR）/phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway in SD rats with endometriosis. MethodsAfter successful establishment of an endometriosis model in 60 female SD rats of SPF grade via the auto-transplantation method， the rats were randomly divided into a model group， modified Shaofu Zhuyutang high-， medium-， and low-dose groups， and a gestrinone group， with another 12 rats serving as a blank group. The blank and model groups were administered 10 mL·kg^-1 normal saline， while the high-， medium-， and low-dose groups received 30， 15， and 7.5 g·kg^-1 modified Shaofu Zhuyutang， respectively. The gestrinone group was administered 0.25 mg·kg^-1 gestrinone suspension. After four weeks of treatment， uterine contractions were induced with 2 U of oxytocin， and the writhing response of rats was observed. After 24 h， the rats were euthanized， and the weight and volume of ectopic endometrial tissue were recorded. Hematoxylin-eosin （HE） staining was used to observe pathological changes in endometrial tissues， while the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay was used to evaluate the apoptosis rate of endometrial tissues. Immunofluorescence was used to detect the relative expression areas of the B-cell lymphoma-2 gene-associated promoter （Bad） and B-cell lymphoma-2 （Bcl-2） proteins in endometrial tissues. Serum levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， epidermal growth factor （EGF）， and EGFR were measured by enzyme-linked immunosorbent assay （ELISA）. The relative protein expression levels of EGFR， PI3K， phosphorylated PI3K （p-PI3K）， Akt， and phosphorylated Akt （p-Akt） in endometrial tissues were analyzed by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of EGFR， PI3K， and Akt. ResultsCompared with the blank group， the model group showed endometrial thickening， glandular and mesenchymal hyperplasia， a significant decrease in the relative expression area of Bad in ectopic endometrial tissues， a significant increase in the relative expression area of Bcl-2， and a significant reduction in the apoptosis rate as indicated by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly elevated （P<0.01）. The relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were also significantly increased （P<0.01）. Compared with the model group， the high- and medium-dose groups of modified Shaofu Zhuyutang and the gestrinone group exhibited reduced glandular and mesenchymal hyperplasia to varying degrees， with dilated glandular lumens. The number of writhing responses was significantly reduced， the latency to writhing response was significantly prolonged， and the weight and volume of ectopic endometrial tissue were significantly decreased. The relative expression area of Bad in ectopic endometrial tissue was significantly increased， the relative expression area of Bcl-2 was significantly decreased， and the apoptosis rate was significantly elevated as shown by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly reduced， and the relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were significantly decreased （P<0.05，P<0.01）. ConclusionModified Shaofu Zhuyutang may exert therapeutic effects on endometriosis by interfering with key proteins of the EGFR/PI3K/Akt signaling pathway and inducing apoptosis in ectopic endometrial tissue.

ObjectiveTo investigate the mechanism of modified Shaofu Zhuyutang in inducing ferroptosis in ectopic endometrial tissues of rats with endometriosis through the murine double minute 4 （MDM4）/tumor suppressor p53/glutathione peroxidase 4 （GPX4） signaling pathway. MethodsSeventy SPF-grade female SD rats were randomly divided into a blank group （n = 10）， a sham-operated group （n = 10）， and a modeling group （n = 50）. The sham-operated group underwent laparotomy， while the modeling group was subjected to the autotransplantation method to establish an endometriosis model. After successful modeling， the animals were randomly assigned to the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang high-， medium-， and low-dose groups （30， 15， and 7.5 g·kg^-1， respectively）， with 10 rats per group. After four weeks of drug administration， the rats were euthanized for sample collection. The weight and volume of ectopic endometrial tissues were recorded for each group. Transmission electron microscopy （TEM） was employed to observe ultrastructural changes in endometrial tissues， while Prussian blue staining was used to assess iron ion deposition. Serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The relative levels of Fe²⁺， malondialdehyde （MDA）， and glutathione （GSH） in endometrial tissues were determined by colorimetric assay. Immunofluorescence （IF） was used to detect the relative fluorescence intensities of GSH and GPX4 in endometrial tissues. The relative expression levels of phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， MDM4， p53， and GPX4 proteins were detected by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to assess the mRNA expression of PI3K， Akt， MDM4， p53， and GPX4. ResultsCompared with the blank and sham-operated groups， the model group exhibited reduced ferroptotic damage in ultrastructural observations， decreased ferroptotic aggregates and positive iron ion expression area on Prussian blue staining， elevated serum IL-6， IL-1β， and TNF-α levels， reduced Fe²⁺ and MDA content， increased GSH content in endometrial tissues， and enhanced GSH and GPX4 fluorescence intensities （P<0.01）. The protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 were elevated， while those of p53 were decreased （P<0.01）. Compared with the model group， in the progesterone group and the modified Shaofu Zhuyutang high- and medium-dose groups， ferroptotic damage in ultrastructural observations was exacerbated to varying degrees by TEM， and ferroptotic aggregates and positive iron ion expression areas were increased on Prussian blue staining. Serum IL-6， IL-1β， and TNF-α levels decreased， Fe²⁺ and MDA content increased， and GSH content decreased in endometrial tissues. GSH and GPX4 fluorescence intensities weakened， while the protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 decreased， and those of p53 increased （P<0.05， P<0.01）. Conclusionmodified Shaofu Zhuyutang may exert therapeutic effects in endometriosis by inducing ferroptosis in ectopic endometrial tissues， alleviating inflammatory responses， and modulating key proteins in the MDM4/p53/GPX4 signaling pathway.

This paper summarized Professor ZHANG Zhiwen's clinical experience in treating pulmonary nodules. Professor ZHANG proposed that the pathogenesis of pulmonary nodules is impairment of qi and body fluid distribution， sweating pores constraint and block， binding of phlegm and stasis， and long-term accumulation transforming into toxin， with abnormal circulation of qi， blood and body fluid as the root， and phlegm， stasis and toxin as the branch. According to the evolution of pathogenesis， a dynamic diagnosis and treatment idea of "regulating qi and body fluid， opening sweating pores， penetrating kenang （窠囊）， and preventing cancer toxins" is proposed， including the methods of regulating qi and body fluid， and calming the five zang （脏） organs with the modified self-prescribed Tiaoqi Huatan Decoction （调气化痰汤）， opening sweating pores and unblocking qi and blood with the modified self-prescribed Kaixuan Tongyu Decoction （开玄通郁汤）， penetrating kenang and dissipating masses and stagnation with the modified self-prescribed Tounang Sanjie Decoction （透囊散结汤）， rectifying the healthy qi and preventing cancer toxins with the modified self-prescribed Shenqi Fuzheng Jiedu Decoction （参芪扶正解毒汤）. These four methods can be used alone， in combination， or in sequence， and can provide ideas for the diagnosis and treatment of pulmonary nodules with traditional Chinese medicine.

Objective To investigate the potential molecular mechanism of modified Shaofu Zhuyu Decoction in treating endometriosis from the perspective of autophagy in ectopic endometrial tissue cells mediated by the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Methods Seventy-two female SD rats were divided into the blank group,the model group,the gestrinone group and the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups according to the random number table method,12 rats in each group. Construction of an endometriosis model used auto-transplantation method. The rats in the gestrinone group were gavaged with 0.25mg/(kg·d) of gestrinone suspension,the rats in the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups were gavaged with 30,15,and 7.5g/(kg·d) of modified Shaofu Zhuyu Decoction,respectively,and the rats in the blank and model groups were gavaged with an equal amount of physiological saline,respectively. 4 weeks of continuous treatment. At the end of drug administration,2 U of oxytocin was given to each rat to induce contractions,and the pain of the rats was observed;the weight and volume of ectopic endometrial tissue of the rats were recorded. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of rat endometrial tissue in each group;transmission electron microscope was used to observe the ultrastructure of rat endometrial tissue. Enzyme-linked immunosorbent assay was used to detect the serum levels of oestradiol (E2),progesterone (P),interleukin-1β (IL-β),interleukin-6 (IL-6),tumour necrosis factor-α (TNF-α),transforming growth factor-β (TGF-β),epidermal growth factor (EGF),and EGFR in the rat serum of each group. Mean fluorescence intensity of autophagy-associated protein yeast Atg6 homologue (Beclin-1) and microtubule-associated protein 1 light chain 3 (LC3B) in endometrial tissues of rat endometrium in each group by immunofluorescence assay. Protein expression levels of EGFR,PI3K,phosphorylated phosphatidylinositol 3-kinase (p-PI3K),AKT,and phosphorylated protein kinase B (p-AKT) in endometrial tissues of rats in each group were examined by protein blotting. Real-time fluorescence PCR was used to detect EGFR,PI3K,and AKT mRNA expression levels.Results Compared with the blank group,the rats in the model group showed typical torsion reaction,ectopic endometrial tissue was visible in the abdominal wall,and the HE result showed thickening of the ectopic endometrial tissue,mesenchymal hyperplasia,and glandular dilatation;only autophagic vesicles were seen in the ultrastructure,and no obvious autophagic structures were seen in some of the fields of view;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,and EGFR levels were elevated and P levels were decreased in rats;autophagy-related proteins Beclin-1 and LC3B decreased in average fluorescence intensity;protein expressions of EGFR,PI3K,AKT,p-PI3K,p-AKT was elevated;and mRNA expressions of EGFR,PI3K,AKT was elevated (P<0.05). Compared with the model group,the gestrinone group and modified Shaofu Zhuyu Decoction high-,medium-dose groups showed a decrease in the number of torsion and a prolongation of the latency time of the torsion response,a decrease in the volume and mass of ectopic endometrial tissues,a decrease in the degree of pathological damage as shown by HE,and the appearance of lysosomes,autophagolysosomes and autophagic vesicles in different degrees in the ultrastructure;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,EGFR levels were decreased,and P levels were increased;the mean fluorescence intensity of autophagy-related proteins Beclin-1 and LC3B was increased;the protein expression of EGFR,PI3K,AKT,p-PI3K,and p-AKT was decreased;and the mRNA expression of EGFR,PI3K,and AKT was decreased (P<0.05). Conclusion The mechanism of modified modified Shaofu Zhuyu Decoction for the treatment of endometriosis may be related to the induction of autophagy of ectopic endometrial tissues mediated by the EGFR/PI3K/AKT signaling pathway and the attenuation of inflammatory responses.

Objective:To evaluate the clinical practice of intensive care unit (ICU) physicians at Hebei General Hospital in identifying patients meeting the diagnostic criteria for acute respiratory distress syndrome (ARDS) and the current status of invasive mechanical ventilation management and adjunctive therapy in these patients, and to analyze the incidence and clinical outcomes of ARDS.Methods:A retrospective cohort study was conducted. The patients who were hospitalized in the ICU of Hebei General Hospital from April 10, 2017 to June 30, 2022 and met the Berlin definition diagnostic criteria for ARDS were enrolled as study subjects. Artificial intelligence (AI) technology was applied to search the basic information (age, gender, height, body weight, etc.), auxiliary examination, electronic medical record, non-drug doctor's advice, drug doctor's advice, critical report, scoring system, monitoring master table and other data of the above medical records in the electronic medical record system of the hospital. The first set of laboratory indicators sequentially retrieved from the system daily from 05: 00 to 10: 00 and vital signs and mechanical ventilation-related parameters recorded in the "critical care report" at 06: 00 daily were extracted, and outcome indicators of the patients were collected.Results:After screening and analysis, a total of 255 patients who met the ARDS diagnostic criteria were finally enrolled. The overall incidence of ARDS in the ICU accounted for 3.4% (255/7?434) of the total number of ICU patients, of which mild, moderate and severe ARDS accounted for 22.4% (57/255), 49.0% (125/255), and 28.6% (73/255), respectively, while the recognition rates of clinical doctors were 71.9% (41/57), 58.4% (73/125) and 71.2% (52/73), respectively. During the ICU stay, 250 patients (98.0%) received only invasive mechanical ventilation, while 5 patients (2.0%) received both non-invasive and invasive mechanical ventilation. The tidal volume/ideal body weight of ARDS patients was 7.64 (6.49, 9.01) mL/kg, and the positive end-expiratory pressure (PEEP) was 8.0 (5.0, 10.0) cmH 2O (1 cmH 2O≈0.098 kPa). In addition, during the diagnosis and detection of ARDS, only 7 patients were recorded the platform pressure and 6 patients were recorded the drive pressure. Regarding adjunctive therapies, 137 patients (53.7%) received deep sedation, 26 patients (10.2%) underwent lung recruitment, 55 patients (21.6%) received prone ventilation, 42 patients (16.5%) were treated with high-dose steroids, 19 patients (7.5%) were treated with neuromuscular blockade, and 8 patients (3.1%) were treated with extracorporeal membrane oxygenation (ECMO). Finally, 70 patients (27.5%) were discharged automatically, while 50 patients (19.6%) died in the ICU, of which the ICU mortality of mild, moderate, and severe ARDS patients were 15.8% (9/57), 22.4% (28/125), and 17.8% (13/73), respectively. After follow-up, it was found that all 70 patients discharged automatically died within 28 days after discharge, and the overall ICU mortality adjusted accordingly was 47.1% (120/255). Conclusions:The overall incidence of ARDS in ICU patients at Hebei General Hospital is relatively low, with a high recognition rate by clinical physicians. Despite the high level of compliance and implementation of lung protective ventilation strategies and auxiliary treatment measures, it is still necessary to further improve the level of standardization in the implementation of small tidal volume and respiratory mechanics monitoring. For the implementation of auxiliary measures such as prone ventilation, it is necessary to further improve the enthusiasm of medical staff. The mortality in ICU is relatively low in ARDS patients, while the rate of spontaneous discharge is relatively high.

Objective:To observe the expression of deleted in malignant brain tumor protein 1 (DMBT1) in rat acute respiratory distress syndrome (ARDS) model induced by sepsis and its relationship with ARDS related biomarkers.Methods:Forty-eight healthy male rats were randomly divided into sham operation group (Sham group) and ARDS model group, and the rats in each group were further divided into three subgroups at 6, 12 and 24 hours after operation, with 8 rats in each subgroup. The rats in the Sham group were exposed to the cecum only, and sepsis induced ARDS model was reproduced by cecal ligation and puncture (CLP) in the ARDS model group. The general performance was observed at 6, 12, 24 hours after operation. Abdominal aortic blood of rats was collected, and the levels of DMBT1, surfactant-associated protein D (SP-D), vascular endothelial growth factor (VEGF), interleukins (IL-6, IL-10) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the lung wet/dry weight (W/D) ratio was determined. The lung tissue pathological changes were observed under light microscope after hematoxylin-eosin (HE) staining, and the lung tissue injury score was evaluated. The expression of DMBT1 protein in lung tissue was determined by Western blotting. The relationship between the serum DMBT1 and SP-D, VEGF, IL-6, IL-10, lung tissue injury score were analyzed by Pearson correlation analysis.Results:Rats in the ARDS model group showed obvious pathological manifestations after operation. The alveolar structure destruction, inflammatory cell infiltration, and alveolar hemorrhage were observed under microscope. Compared with the Sham group, the lung tissue injury score and the lung W/D ratio at 12 hours after operation in the ARDS model group were significantly increased (lung tissue injury score: 3.35±0.13 vs. 1.16±0.07, lung W/D ratio: 5.36±0.44 vs. 4.38±0.35, both P < 0.05), and pulmonary edema was present, which suggested that the ARDS model caused by CLP was successfully reproduced. The results of ELISA and Western blotting showed that the levels of serum DMBT1, SP-D, VEGF and IL-6 in the ARDS model group increased gradually with time, while the level of IL-10 increased first and then decreased. Compared with the Sham group, the levels of DMBT1 in serum and the expressions of DMBT1 protein in lung tissue in the ARDS model group were significantly increased from 6 hours after operation [serum (ng/L) : 231.96±19.17 vs. 187.44±10.19, lung tissue (DMBT1/β-actin): 2.05±0.19 vs. 0.93±0.25, both P < 0.05], and the levels of SP-D, VEGF, IL-6 and IL-10 in serum were significantly increased from 12 hours after operation [SP-D (ng/L): 73.35±8.05 vs. 43.28±5.77, VEGF (ng/L): 89.85±8.47 vs. 43.19±5.11, IL-6 (ng/L): 36.01±2.48 vs. 17.49±1.77, IL-10 (ng/L): 84.55±8.41 vs. 39.83±5.02, all P < 0.05]. Pearson correlation analysis showed that serum DMBT1 was positively correlated with serum SP-D, VEGF, IL-6, IL-10 and lung injury score at 12 hours and 24 hours in the ARDS model group (12 hours: r values were 0.946, 0.942, 0.931, 0.936, 0.748, respectively; 24 hours: r values were 0.892, 0.945, 0.951, 0.918, 0.973, respectively; all P < 0.05). Conclusion:DMBT1 is a novel early biomarker of ARDS by affecting alveolar epithelial cell, alveolar capillary permeability and inflammatory response.

Objective To investigate the clinical efficacy of fluoroscopy-guided intestinal adhesion lysis, as a new non-surgical method, in treating incomplete adhesive small intestinal obstruction in order to improve the therapeutic results of adhesive intestinal obstruction. Methods A total of 93 patients with incomplete adhesive small intestinal obstruction were enrolled in this study. The patients were divided into study group (n=49) and control group (n=44). Fluoroscopy-guided intestinal adhesion lysis together with restoration of inter-intestinal loop enterocele was carried out for the patients of the study group , while traditional conservative surgical therapy was employed for the patients of the control group. The study group was comparable with the control group in patients’ age, gender, medical history, disease course, X-ray findings, etc. Results Of the 49 cases in the study group, complete cure was obtained in 40 with a cure rate of 81.6%. The mean hospitalization day was 0.3 day, and the average operation time was 3.25 hours. Among the 44 patients in the control group, complete cure was obtained in 37 with a cure rate of 84.1%. The mean hospitalization day was 7.6 days, and the average therapeutic time was 183.26 hours. Conclusion For the treatment of incomplete adhesive small intestinal obstruction , the therapeutic efficacy of fluoroscopy-guided intestinal adhesion lysis together with restoration of inter-intestinal loop enterocele is better than that of traditional conservative surgical therapy.

ObjectiveTo explore the application and significance of assisted laparoscopic hepatectomy (ALH) in living-donor-hepatectomy.MethodsWe successfully performed 7 cases of ALH of right hepatectomy on living donor from 30/5/2011 to 1/9/2011.ResultsThe donors recovered well with ratio of remnant lver:32.10％ ～38.31 ％,good liver fuction,little pain and no surgical complications.All the wound sutured intracuteneously was taken out stitches 7 days after operation and healed perfectly.Liver function got normal 2 weeks after operation.Conclusions ALH,which gives the consideration to both the minimal invasion of laparoscopic surgery and safe of open surgery,can be applied safely in hepatectomy of living donor and highly acceptible for donor and receptor.

We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Fusion ; genetics ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection