Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To evaluate the role of the CC chemokine ligand 2/CC chemokine receptor 2 (CCL2/CCR2) signaling pathway in electroacupuncture (EA)-induced reduction of spinal cord injury (SCI) in rats.Methods:Sixty clean-grade healthy adult female Sprague-Dawley rats, weighing 210-250 g, were divided into 5 groups ( n=12 each) using a random number table method: sham operation group (group S), group SCI, SCI+ Anti-CCL2 group (group SCI+ A), SCI+ EA group (group SCI+ EA), and spinal cord injury+ EA+ rCCL2 group (group SCI+ EA+ R). The SCI model was established using the Allen method in anesthetized animals. Group S only underwent spinous process and laminectomy without damaging the spinal cord. In SCI+ A group, CCL2 neutralizing antibody 50 μg/kg was intrathecally injected at 0, 3 and 6 days after successful development of the SCI model. On the 7th day after the successful development of the SCI model, Jiaji, Dazhui and Mingmen acupoints were stimulated with a depth of 2 mm, voltage of 12-15 mV and frequency of 2 Hz for 30 min once a day for 7 consecutive days in SCI+ EA group. In SCI+ EA+ R group, recombinant rat CCL2 2.5 μg/kg was intrathecally injected at the site of injury at 0, 3 and 6 days after successful development of the SCI model, and the remaining treatments were similar to those in SCI+ EA group. At 1 day before developing the model, 0, 3, 7, 14, 21 and 28 days after developing the model, the mechanical paw withdraw threshold (MWT) and thermal paw withdrawal latency (TWL) were measured, and the motor function was assessed by BBB score. The rats were sacrificed after the final behavioral testing, and their spinal cord tissues were obtained for determination of the expression of CCL2 and CCR2 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction), the expression of GFAP (by immunofluorescence), contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay) and for examination of the pathological changes (using HE staining). Results:Compared with S group, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI group ( P<0.05). Compared with SCI group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 7 days after developing the model in SCI+ A group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 14 days after developing the model in SCI+ EA group, and the expression of CCL2 and CCR2 protein and mRNA and GFAP was significantly down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in SCI+ A and SCI+ EA groups ( P<0.05). Compared with SCI+ EA group, the MWT and BBB scores were significantly decreased at 14 days after developing the model, the TWL was shortened, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI+ EA+ R group ( P<0.05). Compared with SCI+ A and SCI+ EA groups, the histopathological injury were significantly attenuated in SCI group, and the histopathological injury was aggravated in SCI+ EA+ R group. Conclusions:The CCL2/CCR2 signaling pathway is involved in the process by which EA reduces SCI, and the mechanism is related to the inhibition of astrocyte activation, thereby reducing the inflammatory response.

Objective To evaluate the practical application of the Media Fill Test(MFT)in assessing aseptic compounding competency of personnel in Pharmacy Intravenous Admixture Services(PIVAS).Methods A multicenter controlled study was conducted across six tertiary hospitals(center ①-⑥)in China.Participants were divided into an inexperienced group(Group A,n=118)and an experienced group(Group B,n=118),each performing five MFT operations.Positive controls validated medium efficacy.Contamination rates and pass rates were analyzed using chi-square and Fisher's exact tests.Results Valid samples included 584 for Group A and 588 for Group B.The sample pass rate was 66.78%(390/584)in Group A and 91.67%(539/588)in Group B,while personnel pass rates were 46.15%(54/117)and 80.51%(95/118),respectively,with significant intergroup differences(both P<0.01).All centers except Center ⑥ showed significantly higher pass rates in Group B(all P<0.05).Conclusion MFT effectively differentiates technical proficiency levels and is suitable for training evaluation of novice PIVAS staff.

Sacroiliac complex injuries are commonly seen in high-energy pelvic fractures. The injuries make a big difference in treatment patterns due to the diverse injury types, posing considerable challenges in formulating optimal treatment strategies, and hence are persistent clinical difficulties in orthopedic trauma. The clinical management of sacroiliac complex injuries presents several key challenges such as a non-negligible rate of missed diagnoses in associated vascular and visceral injuries, absence of standardized protocols for surgical approaches and reduction-fixation strategies across different injury patterns, and ongoing controversies regarding surgical indications and optimal timing for patients combined with concomitant lumbosacral plexus injuries. Currently, no systematic clinical guidelines are available for the diagnosis and treatment of sacroiliac complex injuries both domestically and internationally. To this end, the Pelvic and Acetabular Surgery Group, Orthopedic Branch, China International Exchange and Promotive Association for Medical and Health Care and Orthopedic Physician Branch, Chinese Medical Doctor Association organized a panel of domestic experts in the field to develop the Clinical guideline for the diagnosis and treatment of sacroiliac complex injuries ( version 2025), based on evidence-based medicine and adhering to the principles of scientific rigor, clinical applicability, and innovation. These guidelines provided 11 recommendations covering diagnosis, therapeutic principles and techniques, management protocols for lumbosacral plexus injuries, outcome evaluation, and postoperative rehabilitation pathways, etc., aiming to standardize the clinical management of sacroiliac complex injuries.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective To evaluate the practical application of the Media Fill Test(MFT)in assessing aseptic compounding competency of personnel in Pharmacy Intravenous Admixture Services(PIVAS).Methods A multicenter controlled study was conducted across six tertiary hospitals(center ①-⑥)in China.Participants were divided into an inexperienced group(Group A,n=118)and an experienced group(Group B,n=118),each performing five MFT operations.Positive controls validated medium efficacy.Contamination rates and pass rates were analyzed using chi-square and Fisher's exact tests.Results Valid samples included 584 for Group A and 588 for Group B.The sample pass rate was 66.78%(390/584)in Group A and 91.67%(539/588)in Group B,while personnel pass rates were 46.15%(54/117)and 80.51%(95/118),respectively,with significant intergroup differences(both P<0.01).All centers except Center ⑥ showed significantly higher pass rates in Group B(all P<0.05).Conclusion MFT effectively differentiates technical proficiency levels and is suitable for training evaluation of novice PIVAS staff.

Objective:To evaluate the role of the CC chemokine ligand 2/CC chemokine receptor 2 (CCL2/CCR2) signaling pathway in electroacupuncture (EA)-induced reduction of spinal cord injury (SCI) in rats.Methods:Sixty clean-grade healthy adult female Sprague-Dawley rats, weighing 210-250 g, were divided into 5 groups ( n=12 each) using a random number table method: sham operation group (group S), group SCI, SCI+ Anti-CCL2 group (group SCI+ A), SCI+ EA group (group SCI+ EA), and spinal cord injury+ EA+ rCCL2 group (group SCI+ EA+ R). The SCI model was established using the Allen method in anesthetized animals. Group S only underwent spinous process and laminectomy without damaging the spinal cord. In SCI+ A group, CCL2 neutralizing antibody 50 μg/kg was intrathecally injected at 0, 3 and 6 days after successful development of the SCI model. On the 7th day after the successful development of the SCI model, Jiaji, Dazhui and Mingmen acupoints were stimulated with a depth of 2 mm, voltage of 12-15 mV and frequency of 2 Hz for 30 min once a day for 7 consecutive days in SCI+ EA group. In SCI+ EA+ R group, recombinant rat CCL2 2.5 μg/kg was intrathecally injected at the site of injury at 0, 3 and 6 days after successful development of the SCI model, and the remaining treatments were similar to those in SCI+ EA group. At 1 day before developing the model, 0, 3, 7, 14, 21 and 28 days after developing the model, the mechanical paw withdraw threshold (MWT) and thermal paw withdrawal latency (TWL) were measured, and the motor function was assessed by BBB score. The rats were sacrificed after the final behavioral testing, and their spinal cord tissues were obtained for determination of the expression of CCL2 and CCR2 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction), the expression of GFAP (by immunofluorescence), contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay) and for examination of the pathological changes (using HE staining). Results:Compared with S group, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI group ( P<0.05). Compared with SCI group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 7 days after developing the model in SCI+ A group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 14 days after developing the model in SCI+ EA group, and the expression of CCL2 and CCR2 protein and mRNA and GFAP was significantly down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in SCI+ A and SCI+ EA groups ( P<0.05). Compared with SCI+ EA group, the MWT and BBB scores were significantly decreased at 14 days after developing the model, the TWL was shortened, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI+ EA+ R group ( P<0.05). Compared with SCI+ A and SCI+ EA groups, the histopathological injury were significantly attenuated in SCI group, and the histopathological injury was aggravated in SCI+ EA+ R group. Conclusions:The CCL2/CCR2 signaling pathway is involved in the process by which EA reduces SCI, and the mechanism is related to the inhibition of astrocyte activation, thereby reducing the inflammatory response.

Sacroiliac complex injuries are commonly seen in high-energy pelvic fractures. The injuries make a big difference in treatment patterns due to the diverse injury types, posing considerable challenges in formulating optimal treatment strategies, and hence are persistent clinical difficulties in orthopedic trauma. The clinical management of sacroiliac complex injuries presents several key challenges such as a non-negligible rate of missed diagnoses in associated vascular and visceral injuries, absence of standardized protocols for surgical approaches and reduction-fixation strategies across different injury patterns, and ongoing controversies regarding surgical indications and optimal timing for patients combined with concomitant lumbosacral plexus injuries. Currently, no systematic clinical guidelines are available for the diagnosis and treatment of sacroiliac complex injuries both domestically and internationally. To this end, the Pelvic and Acetabular Surgery Group, Orthopedic Branch, China International Exchange and Promotive Association for Medical and Health Care and Orthopedic Physician Branch, Chinese Medical Doctor Association organized a panel of domestic experts in the field to develop the Clinical guideline for the diagnosis and treatment of sacroiliac complex injuries ( version 2025), based on evidence-based medicine and adhering to the principles of scientific rigor, clinical applicability, and innovation. These guidelines provided 11 recommendations covering diagnosis, therapeutic principles and techniques, management protocols for lumbosacral plexus injuries, outcome evaluation, and postoperative rehabilitation pathways, etc., aiming to standardize the clinical management of sacroiliac complex injuries.

Objective:To investigate the efficacy and safety of platelet-rich plasma (PRP) in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS).Methods:This prospective study included patients diagnosed with IC/BPS who received multiple PRP injections at our hospital from January 2023, to April 2024. Inclusion criteria included patients over 18 years old with symptoms of urinary frequency, urgency, pain, and pelvic pain, patients who were required to have confirmed non-ulcerative IC/BPS via cystoscopic hydrodistension under anesthesia and exclusion of other diseases through urodynamic and imaging tests, patients who failed to improve after more than three treatments including lifestyle intervention, intravesical hyaluronic acid instillation, botulinum toxin injection, or cystoscopic hydrodistension, and they all signed sign informed consent for the procedures. Exclusion criteria included active urinary tract infection, use of anticoagulants or antiplatelet drugs, T-cell lymphoma causing systemic autoimmune disease, platelet count below 100×10 9/L, hemoglobin level below 100 g/L, severe cardiovascular disease, or other conditions that preclude platelet apheresis. Approximately 100 ml of PRP was collected from each patient using a blood cell separator, with 1 ml reserved for platelet detection and the remaining PRP divided into six bags, each containing about 15 ml. Patients received one PRP injection per month, for a total of six injections. Each injection involved 30 evenly distributed sites on the bladder wall under cystoscopic visualization, with 0.5 ml injected per site. The primary endpoint was global response assessment (GRA), conducted approximately one month after each procedure, with GRA≥5 indicating improvement and GRA≥6 indicating good efficacy. Secondary endpoints included assessments of 24-hour urinary frequency, nocturia, functional bladder capacity, O'Leary-Sant scores [including the IC Symptom Index (IC-SI) and IC Problem Index (IC-PI)], pelvic pain and urgency/frequency (PUF) scores, visual analogue scale (VAS) for pain, and quality of life (QOL) scores, evaluated preoperatively and one month after each treatment. Results:A total of 21 patients were enrolled in the study, including 2 males and 19 females, with an average age of (37.10±14.75) years and an average preoperative disease duration of (4.05±3.25) years. The average bladder capacity under general anesthesia before the procedure was (502.86±150.84) ml. Among these patients, 11 completed six injections, 6 completed five injections, and 4 completed four injections. After four treatments, 17 patients had a GRA≥5, and 11 patients had a GRA≥6, with an improvement rate (GRA≥5) of 80.95%. Following the most recent treatment, 18 patients had a GRA≥5, and 12 had a GRA≥6, with an overall improvement rate (GRA≥5) of 85.71%. Comparison of clinical data before surgery and one month after the fourth treatment showed significant differences in functional bladder capacity [(203.10±77.74) ml vs. (254.52±115.95 ml)], O'Leary-Sant scores [(20.90±7.94) vs. (15.81±8.47)], VAS scores [(3.55±3.00) vs. (2.14±2.22)], and QOL scores [(4.24±1.00) vs. (3.43±1.29)] ( P<0.05). In 17 patients who completed five injections, comparison of data one month after the fourth and fifth treatments showed significant differences in O'Leary-Sant scores [(15.41±8.64) vs. (13.65±8.13)] ( P<0.05). In 11 patients who completed six injections, there were no statistically significant differences in any indicators between one month after the fifth and sixth treatments. As for surgical complications, only one patient experienced gross hematuria after the fourth treatment, and no other significant postoperative complications were observed. Conclusions:Intravesical PRP injections for the treatment of IC/BPS are effective and safe.

Objective:To investigate the efficacy and safety of platelet-rich plasma (PRP) in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS).Methods:This prospective study included patients diagnosed with IC/BPS who received multiple PRP injections at our hospital from January 2023, to April 2024. Inclusion criteria included patients over 18 years old with symptoms of urinary frequency, urgency, pain, and pelvic pain, patients who were required to have confirmed non-ulcerative IC/BPS via cystoscopic hydrodistension under anesthesia and exclusion of other diseases through urodynamic and imaging tests, patients who failed to improve after more than three treatments including lifestyle intervention, intravesical hyaluronic acid instillation, botulinum toxin injection, or cystoscopic hydrodistension, and they all signed sign informed consent for the procedures. Exclusion criteria included active urinary tract infection, use of anticoagulants or antiplatelet drugs, T-cell lymphoma causing systemic autoimmune disease, platelet count below 100×10 9/L, hemoglobin level below 100 g/L, severe cardiovascular disease, or other conditions that preclude platelet apheresis. Approximately 100 ml of PRP was collected from each patient using a blood cell separator, with 1 ml reserved for platelet detection and the remaining PRP divided into six bags, each containing about 15 ml. Patients received one PRP injection per month, for a total of six injections. Each injection involved 30 evenly distributed sites on the bladder wall under cystoscopic visualization, with 0.5 ml injected per site. The primary endpoint was global response assessment (GRA), conducted approximately one month after each procedure, with GRA≥5 indicating improvement and GRA≥6 indicating good efficacy. Secondary endpoints included assessments of 24-hour urinary frequency, nocturia, functional bladder capacity, O'Leary-Sant scores [including the IC Symptom Index (IC-SI) and IC Problem Index (IC-PI)], pelvic pain and urgency/frequency (PUF) scores, visual analogue scale (VAS) for pain, and quality of life (QOL) scores, evaluated preoperatively and one month after each treatment. Results:A total of 21 patients were enrolled in the study, including 2 males and 19 females, with an average age of (37.10±14.75) years and an average preoperative disease duration of (4.05±3.25) years. The average bladder capacity under general anesthesia before the procedure was (502.86±150.84) ml. Among these patients, 11 completed six injections, 6 completed five injections, and 4 completed four injections. After four treatments, 17 patients had a GRA≥5, and 11 patients had a GRA≥6, with an improvement rate (GRA≥5) of 80.95%. Following the most recent treatment, 18 patients had a GRA≥5, and 12 had a GRA≥6, with an overall improvement rate (GRA≥5) of 85.71%. Comparison of clinical data before surgery and one month after the fourth treatment showed significant differences in functional bladder capacity [(203.10±77.74) ml vs. (254.52±115.95 ml)], O'Leary-Sant scores [(20.90±7.94) vs. (15.81±8.47)], VAS scores [(3.55±3.00) vs. (2.14±2.22)], and QOL scores [(4.24±1.00) vs. (3.43±1.29)] ( P<0.05). In 17 patients who completed five injections, comparison of data one month after the fourth and fifth treatments showed significant differences in O'Leary-Sant scores [(15.41±8.64) vs. (13.65±8.13)] ( P<0.05). In 11 patients who completed six injections, there were no statistically significant differences in any indicators between one month after the fifth and sixth treatments. As for surgical complications, only one patient experienced gross hematuria after the fourth treatment, and no other significant postoperative complications were observed. Conclusions:Intravesical PRP injections for the treatment of IC/BPS are effective and safe.