Objective: To identify an optimal back-flush solution for leukocyte-depleted filters that maximizes peripheral blood mononuclear cell (PBMC) recovery with high viability, long-term storage stability, and sterility of the harvested residues, thereby providing a clinically translatable strategy. Methods: Three sterile bag-packaged solutions—Saline, Solvent, and Hanks' balanced salt solution (HBSS)—were used to back-flush randomly assigned leukocyte-depleted filters. Nucleated cell recovery rate and viability of the harvested residues were compared. The optimal solution identified was applied to an expanded sample set. PBMC viability and yield were evaluated after 1h vs 48h storage of the residues. PBMCs isolated from the residues were cryopreserved in liquid nitrogen for 1 month, followed by post-thaw comparisons of viability and T-cell expansion capacity. Results: The Solvent group achieved the highest and most consistent nucleated cell recovery rate. Post-flush recovery rate from filters after 400 mL whole blood processing was (21.3±1.6)% for the Solvent group, significantly higher than Saline group (19.2±6.3)% and HBSS group (11.2±5.0)%, with residues from all groups maintaining viability >90%. No biologically significant difference in residue viability was observed between 48h vs 1h storage groups (93.3±2.3)% vs (95.7±1.8)%). PBMC recovery rates from residues showed no statistical difference between 48h vs 1h storage groups [(48.2%±9.5%)vs (40.41%±8.35%), P>0.05], with (17.7±2.6)×10 cells. After 1-month cryopreservation and 10-day expansion, PBMCs isolated from 48-hour-stored residues retained (91.2±3.2)% viability and achieved a (61.9±15.9)-fold expansion. Conclusion: The bag-packaged Solvent, as a back-flush solution, enables sterile acquisition of leukocyte-depleted filter residues through closed-system tubing connections. These residues maintained PBMC viability and recovery rates after 48h storage at 2℃-8℃, with post-cryopreservation (1-month liquid nitrogen) viability and expansion capacity remaining stable. This protocol complies with blood bank regulatory criteria, addresses the concerns about the infectious window period in cell therapy raw materials, and provides a clinically translatable strategy for PBMC-based applications.

OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

Objective:To compare the efficacy of body posture combined with pharmacotherapy and pharmacotherapy alone in the treatment of chronic subdural hematoma(CSDH).Methods:Firstly, retrospective case series study was conducted. Thirty cases of CSDH that had received body posture combined with pharmacotherapy at Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University from December 2016 to October 2020 were studied retrospectively. Twenty-seven patients were male, and 3 patients were female. The age of patients ( M(IQR)) was 66(16) years (range:28 to 84). Nineteen patients had unilateral hematoma, and 11 patients had bilateral hematoma. All patients received pharmacotherapy and body posture therapy that was to raise their lower limbs 20 to 30 cm with leg lift pad and get abdominal compressed with customized abdominal belt in supine position. Patients were required to maintain the body posture as much as possible, with the maximum to 16 to 18 hours per day. Patients with unilateral hematoma should tilt the head to the affected side and avoid tilting it to the opposite side. For patients with bilateral hematoma, there was no need for head lateralization. Patient were treated with oral dexamethasone and atorvastatin simultaneously. The preliminary efficacy of body posture combined with pharmacotherapy was determined by hematoma improvement rate which was analyzed by Clopper-Pearson method. Then, the multi-center, prospective, randomized controlled trial had carried out in 9 medical centers from August 2020 to November 2021. The stratified block randomization method was adopted. Patients were randomized in a ratio of 1∶1 to either receive pharmacotherapy alone(the control group) or body posture combined with pharmacotherapy(the experiment group) for 3 months and followed up for 6 months. Effective treatment was defined as complete absorption of hematoma, or the hematoma volume decreased by more than 10 ml and Markwalder grading scale score had improved by more than 1 point compared to the baseline. The efficacy rate and surgery conversion rate at 3 months and recurrence at 6 months were observed. Comparison between groups was performed with paired sample t test, Mann-Whitney U test, χ2 test, corrected χ2 test, or Fisher exact probability method. Logistic regression was used to compare the effective rate and operation rate between the two groups. Results:In the respective study, 30 patients completed follow-up 13 to 353 days after treatment. At the last follow-up, the incidence of almost complete absorption or significantly absorption of hematoma (hematoma volume was significantly reduced accompanied by symptom improvement) was 93.3%. The 95% CI for the incidence that analyzed by the Clopper-Pearson method was 77.9% to 99.2%. One hundred and six patients were enrolled in the multicenter study. Fifty-five patients underwent body posture combined with pharmacotherapy. The age was 74(17) years (range:26 to 92). Thirty-nine patients were males and 16 were females. Fifty-one patients underwent pharmacotherapy alone. The age was 69(12) years (range:48 to 84). Thirty-seven patients were males and 14 were females. The length of body posture recorded in diary card was (15.7±2.3) hours(range:7.6 to 19.3 hours). The efficacy rate in the body posture combined with pharmacotherapy group and pharmacotherapy alone group were 83.6% (46/55) and 56.9% (29/51), respectively at 3 months. The result of the logistic regression analysis showed that the efficacy of body posture combined with pharmacotherapy group was better than that of pharmacotherapy alone group ( OR=3.88,95% CI:1.57 to 9.58, P=0.003). Surgery rate in the body posture combined with pharmacotherapy group and pharmacotherapy alone group were 5.5% (3/55) and 21.6% (11/51) respectively. The result of Logistic regression showed that the pharmacotherapy alone group was more likely to be converted to surgery ( OR=0.21,95% CI:0.05 to 0.80, P=0.023). At the 6 months, no recurrence of cases was found in the body posture combined with pharmacotherapy group. However, the recurrence rate of pharmacotherapy alone group was 6.3% (3/48), there was no significant difference between the two groups ( P>0.05). Conclusion:The effect of body posture combined with pharmacotherapy for chronic subdural hematoma is better than that of pharmacotherapy alone.

Objective:To explore the clinical significance of skeletonized lymph node dissection of No.12 lymph nodes after neoadjuvant therapy in patients with advanced gastric cancer.Methods:For this retrospective case-cohort study we collected data from patients with advanced gastric cancer who underwent neoadjuvant chemotherapy and D2 or more extensive curative resection including No.12 lymph node dissection at the First Affiliated Hospital of Sun Yat-sen University from January, 2011 to December, 2022. Patients were divided into two groups based on whether they received skeletonized dissection of No.12 lymph nodes: 177 cases were in the skeletonized group, and 55 cases were in the nonskeletonized group. The differences of prognosis between the two groups were compared, and logistic regression models were used to analyze the factors affecting No.12 lymph node metastasis in the overall cohort and No.12b or No.12p lymph node metastasis in the skeletonized group.Results:A total of 232 patients were included, with 84 females (36.2%) and 148 males (63.8%), with an average age of 56.4±11.6 years. The proportion of female and ycT4 patients was significantly higher in the skeletonized group than in the nonskeletonized group (both P<0.05). Among all 232 patients, No. 12a metastasis occurred in 14 cases (6.0%). In the skeletonized group of 177 patients, No. 12b and No. 12p metastases were observed in 6 patients each (3.4%), and 4 patients had concurrent metastases in both No. 12b and No. 12a. The 5-year overall survival (OS) rates were 45.5% in the skeletonized group and 42.8% in the nonskeletonized group, with no statistical difference (HR=0.755, 95%CI: 0.488-1.168, P=0.580). The 5-year disease-free survival (DFS) rates were 39.8% and 41.0%, respectively, also with no statistical difference (HR=0.775, 95%CI: 0.513-1.172, P=0.584). 5-year OS for patients without No.12 lymph node metastasis was 48.8%, which was higher than the 15.9% for those with metastasis (HR=0.349, 95% CI: 0.209-0.584, P=0.003). Additionally, the 5-year DFS for those without metastasis was 44.3%, significantly higher than the 5.7% for those with metastasis (HR=0.444, 95%CI: 0.276-0.716, P<0.001). For patients without No. 12b or No. 12p lymph node metastasis, the 5-year OS was 47.6%, and the 5-year DFS was 42.3%, both of which were significantly higher than the 16.7% and 8.3% for those with No.12b or No. 12p lymph node metastasis, respectively (HR=0.353, 95%CI: 0.183-0.681, P=0.005; HR=0.457, 95%CI: 0.244-0.855, P=0.006). Multivariate analysis showed that more advanced ypN stage (OR=3.908, 95%CI:1.638-9.323, P=0.002) and tumor location in the lower stomach or whole stomach (OR=3.533, 95%CI: 1.312-9.511, P=0.012) were independent risk factors for No.12 lymph node metastasis and also for No.12b and No.12p lymph node metastasis (OR=2.426, 95%CI: 1.212-4.856, P=0.012 and OR=4.908, 95%CI:1.182-20.373, P=0.028, respectively). Conclusion:Patients with advanced gastric cancer who have more advanced ypN stage and tumor location in the lower stomach or whole stomach have a higher risk of No.12b and No.12p metastasis and thus require further skeletonized lymph node dissection of No.12.

Objective:To explore the clinical significance of skeletonized lymph node dissection of No.12 lymph nodes after neoadjuvant therapy in patients with advanced gastric cancer.Methods:For this retrospective case-cohort study we collected data from patients with advanced gastric cancer who underwent neoadjuvant chemotherapy and D2 or more extensive curative resection including No.12 lymph node dissection at the First Affiliated Hospital of Sun Yat-sen University from January, 2011 to December, 2022. Patients were divided into two groups based on whether they received skeletonized dissection of No.12 lymph nodes: 177 cases were in the skeletonized group, and 55 cases were in the nonskeletonized group. The differences of prognosis between the two groups were compared, and logistic regression models were used to analyze the factors affecting No.12 lymph node metastasis in the overall cohort and No.12b or No.12p lymph node metastasis in the skeletonized group.Results:A total of 232 patients were included, with 84 females (36.2%) and 148 males (63.8%), with an average age of 56.4±11.6 years. The proportion of female and ycT4 patients was significantly higher in the skeletonized group than in the nonskeletonized group (both P<0.05). Among all 232 patients, No. 12a metastasis occurred in 14 cases (6.0%). In the skeletonized group of 177 patients, No. 12b and No. 12p metastases were observed in 6 patients each (3.4%), and 4 patients had concurrent metastases in both No. 12b and No. 12a. The 5-year overall survival (OS) rates were 45.5% in the skeletonized group and 42.8% in the nonskeletonized group, with no statistical difference (HR=0.755, 95%CI: 0.488-1.168, P=0.580). The 5-year disease-free survival (DFS) rates were 39.8% and 41.0%, respectively, also with no statistical difference (HR=0.775, 95%CI: 0.513-1.172, P=0.584). 5-year OS for patients without No.12 lymph node metastasis was 48.8%, which was higher than the 15.9% for those with metastasis (HR=0.349, 95% CI: 0.209-0.584, P=0.003). Additionally, the 5-year DFS for those without metastasis was 44.3%, significantly higher than the 5.7% for those with metastasis (HR=0.444, 95%CI: 0.276-0.716, P<0.001). For patients without No. 12b or No. 12p lymph node metastasis, the 5-year OS was 47.6%, and the 5-year DFS was 42.3%, both of which were significantly higher than the 16.7% and 8.3% for those with No.12b or No. 12p lymph node metastasis, respectively (HR=0.353, 95%CI: 0.183-0.681, P=0.005; HR=0.457, 95%CI: 0.244-0.855, P=0.006). Multivariate analysis showed that more advanced ypN stage (OR=3.908, 95%CI:1.638-9.323, P=0.002) and tumor location in the lower stomach or whole stomach (OR=3.533, 95%CI: 1.312-9.511, P=0.012) were independent risk factors for No.12 lymph node metastasis and also for No.12b and No.12p lymph node metastasis (OR=2.426, 95%CI: 1.212-4.856, P=0.012 and OR=4.908, 95%CI:1.182-20.373, P=0.028, respectively). Conclusion:Patients with advanced gastric cancer who have more advanced ypN stage and tumor location in the lower stomach or whole stomach have a higher risk of No.12b and No.12p metastasis and thus require further skeletonized lymph node dissection of No.12.

Objective:To compare the efficacy of body posture combined with pharmacotherapy and pharmacotherapy alone in the treatment of chronic subdural hematoma(CSDH).Methods:Firstly, retrospective case series study was conducted. Thirty cases of CSDH that had received body posture combined with pharmacotherapy at Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University from December 2016 to October 2020 were studied retrospectively. Twenty-seven patients were male, and 3 patients were female. The age of patients ( M(IQR)) was 66(16) years (range:28 to 84). Nineteen patients had unilateral hematoma, and 11 patients had bilateral hematoma. All patients received pharmacotherapy and body posture therapy that was to raise their lower limbs 20 to 30 cm with leg lift pad and get abdominal compressed with customized abdominal belt in supine position. Patients were required to maintain the body posture as much as possible, with the maximum to 16 to 18 hours per day. Patients with unilateral hematoma should tilt the head to the affected side and avoid tilting it to the opposite side. For patients with bilateral hematoma, there was no need for head lateralization. Patient were treated with oral dexamethasone and atorvastatin simultaneously. The preliminary efficacy of body posture combined with pharmacotherapy was determined by hematoma improvement rate which was analyzed by Clopper-Pearson method. Then, the multi-center, prospective, randomized controlled trial had carried out in 9 medical centers from August 2020 to November 2021. The stratified block randomization method was adopted. Patients were randomized in a ratio of 1∶1 to either receive pharmacotherapy alone(the control group) or body posture combined with pharmacotherapy(the experiment group) for 3 months and followed up for 6 months. Effective treatment was defined as complete absorption of hematoma, or the hematoma volume decreased by more than 10 ml and Markwalder grading scale score had improved by more than 1 point compared to the baseline. The efficacy rate and surgery conversion rate at 3 months and recurrence at 6 months were observed. Comparison between groups was performed with paired sample t test, Mann-Whitney U test, χ2 test, corrected χ2 test, or Fisher exact probability method. Logistic regression was used to compare the effective rate and operation rate between the two groups. Results:In the respective study, 30 patients completed follow-up 13 to 353 days after treatment. At the last follow-up, the incidence of almost complete absorption or significantly absorption of hematoma (hematoma volume was significantly reduced accompanied by symptom improvement) was 93.3%. The 95% CI for the incidence that analyzed by the Clopper-Pearson method was 77.9% to 99.2%. One hundred and six patients were enrolled in the multicenter study. Fifty-five patients underwent body posture combined with pharmacotherapy. The age was 74(17) years (range:26 to 92). Thirty-nine patients were males and 16 were females. Fifty-one patients underwent pharmacotherapy alone. The age was 69(12) years (range:48 to 84). Thirty-seven patients were males and 14 were females. The length of body posture recorded in diary card was (15.7±2.3) hours(range:7.6 to 19.3 hours). The efficacy rate in the body posture combined with pharmacotherapy group and pharmacotherapy alone group were 83.6% (46/55) and 56.9% (29/51), respectively at 3 months. The result of the logistic regression analysis showed that the efficacy of body posture combined with pharmacotherapy group was better than that of pharmacotherapy alone group ( OR=3.88,95% CI:1.57 to 9.58, P=0.003). Surgery rate in the body posture combined with pharmacotherapy group and pharmacotherapy alone group were 5.5% (3/55) and 21.6% (11/51) respectively. The result of Logistic regression showed that the pharmacotherapy alone group was more likely to be converted to surgery ( OR=0.21,95% CI:0.05 to 0.80, P=0.023). At the 6 months, no recurrence of cases was found in the body posture combined with pharmacotherapy group. However, the recurrence rate of pharmacotherapy alone group was 6.3% (3/48), there was no significant difference between the two groups ( P>0.05). Conclusion:The effect of body posture combined with pharmacotherapy for chronic subdural hematoma is better than that of pharmacotherapy alone.

Objective:To investigate the application value of laparoscopic natural orifice specimen extraction surgery (NOSES) based on purse-string suture for sigmoid colon and upper rectal cancer.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 14 patients undergoing laparoscopic NOSES based on purse-string suture for sigmoid colon and upper rectal cancer in The First Affiliated Hospital of Sun Yat-sen University from October 2022 to June 2023 were collected. There were 8 males and 6 females, aged (56±10)years. Observation indicators: (1) surgical conditions; (2) postoperative conditions; (3) follow-up. Measurement data with normal distribution were represented as Mean± SD. Count data were described as absolute numbers. Results:(1) Surgical conditions. All patients underwent laparoscopic NOSES based on purse-string suture for sigmoid colon and upper rectal cancer successfully, without conversion to open surgery. The operation time of 14 patients was (162±32)minutes, and the volume of intraoperative blood loss was (22±12)mL. (2) Postoperative conditions. Time to postoperative first out-of-bed activity, time to postoperative first flatus, time to postoperative first drinking, time to postoperative initial liquid food intake, duration of postoperative hospital stay of 14 patients were (1.6±0.7)days, (2.1±0.6)days, (2.4±0.6)days, (3.8±1.0)days, (6.0±0.9)days, respectively. None of patient had perioperative complications such as postoperative anastomotic leakage and bleeding. (3) Follow-up. All 14 patients were followed up for (9.7±1.9)months. There was no postoperative recurrence, metastasis or death in 14 pati-ents.Conclusion:The laparoscopic NOSES based on purse-string suture can be used for sigmoid colon and upper rectal cancer, which is safe and feasible.

Objective This study aims to analyze the relationship between reproductive tract microecological changes,metabolic differences,and pregnancy outcomes at different time points in the frozen-thawed embryo transfer cycle while patients are undergoing hormone replacement therapy,which will be a breakthrough point for improving outcomes.Methods A total of 20 women undergoing frozen-thawed single blastocyst transfer for the first time at the Reproductive Medicine Center of Fujian Maternal and Child Health Hospital between July 2022 and January 2023 were recruited for this study.Their vaginal and cervical secretions were collected for 16S rRNA sequencing and non-targeted metabolomics analysis on days 2-5 of menstruation,day 7 after estrogen replacement therapy started,the day when progesterone was added,and the day of transplantation.The subjects were divided into different groups according to their clinical pregnancy status and the sequencing results were analyzed using bioinformatics methods.Results 1)The alpha-diversity index of the vaginal and cervical microbiota was higher on days 2-5 of menstruation(P<0.01),but did not differ significantly on day 7 after oral estrogen replacement therapy started,the day of progesterone administration,and the day of transplantation(P≥0.1).2)Both the pregnant group and the non-pregnant group showed a variety of microorganisms and metabolites with significant differences in the lower reproductive tract at different time points.3)Microbial analysis at different time points showed that there were significant differences in vaginal flora,including Peptoniphilus,Enterocloster,Finegoldia,Klebsiella,Anaerobutyricum,Agathobaculum,Sporanaerobacter,Bilophila,Prevotella,and Anaerococcus in the pregnant group(P<0.05).4)Metabolite analysis at different time points showed that there were significant differences in 3-hydroxybenzoic acid,linatine,(R)-amphetamine,hydroxychloroquine,and L-altarate in the vaginal secretions of the pregnant group(P<0.05),and that there were significant differences in isocitric acid,quassin,citrinin,and 12(R)-HETE in the cervical secretions(P<0.05).5)Metabolite analysis at different time points showed that,in the non-pregnant group,there were significant differences in linatine,decanoyl-L-carnitine,aspartame,sphingosine,and hydroxychloroquine in the vaginal secretions(P<0.05),and the isocitric acid,quassin,ctrinin,and 12(R)-HETE in the cervical secretions(P<0.05).6)Combined microbiome and metabolomics analysis showed that certain metabolites were significantly associated with microbial communities,especially Klebsiella.Conclusions Significant differences in the microbiota genera and metabolites at different time points were found during the frozen-embryo transfer cycle of hormone replacement therapy,which may be used as potential biomarkers to predict pregnancy outcomes of embryo transfer.

Objective: To investigate the design of free perforator flap, and the efficacy of utilizing perforator flaps for Ⅳ degree Ischia-sacral ulcer treatment. Methods: From January 2010 to October 2016, 18 patients with Ⅳ degree ischia-sacral ulcer were treated. The surface area of the sacral tail ranged from 4 cm×5 cm to 8 cm ×12 cm.Doppler sonography was used to detect potential perforator.All defects were repaired with free perforator flaps, designed based on the size and shape of the wound. The flap size ranged from 6 cm×11 cm to 9 cm×15 cm. Results: One perforator flap went dehiscence after surgery, repaired by V-Y flap. All the rest of perforator flaps survived well, after 3-24 months follow-up. Flap texture and appearance was good, no ulcer reoccurred. Conclusions The free perforator flap is a simple technique.It does not need to tracethe trunk of vessels, and it does not cause major morbidities to the buttocks. Therefore, it is one of the ideal ways to repair Ⅳ degree Ischia-sacral ulcer.

Objective: To explore the effects of combined transplantation of the rat Schwann cells and fibroblasts (Fbs) on the nerve regeneration of denervated perforator flaps in rats and the mechanism. Methods: (1) Fbs were isolated from the trunk of 2 Sprague-Dawley (SD) rats embryos of 14-16 days′ pregnancy and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The protein expressions of fibronectin and Ephrin-B2 were observed by immunohistochemical method. The mRNA expression of Ephrin-B2 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (n=3). (2) Schwann cells were isolated from the bilateral sciatic nerves and brachial plexus nerves of 45 SD rats born for 1-3 days and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The rate of S100 positive cells was detected by immunofluorescence method and flow cytometer, with sample numbers of 9 and 3 respectively. (3) In Dulbecco′s modified Eagle medium (DMEM) high glucose medium, 1 mL Fbs and 1 mL Schwann cells both in the concentration of 1×10⁵ cells/mL were co-cultured as Schwann cells+ Fbs co-culture group, and 2 mL Schwann cells in the concentration of 1×10⁵ cells/mL were cultured alone as Schwann cells alone culture group, with 5 wells in each group. The clusters of Schwann cells in the two groups were observed and counted under inverted phase contrast microscope at post culture hour (PCH) 6 and 24 respectively. The clusters of Schwann cells in Schwann cells+ Fbs co-culture group were observed by immunofluorescence method at PCH 24 too. The protein expressions of EphB2, Sox2, and N-cadherin in Schwann cells of two groups at PCH 24 were detected by Western blotting (n=20). (4) Totally 100 8-week-old male SD rats were selected, and an in situ replanted peritoneal denervated perforator flap was made in each rat. According to the random number table, the rats were divided into simple flap group, Fbs alone transplantation group, Schwann cells alone transplantation group, Schwann cells+ Fbs co-transplantation group, with 25 rats in each group. Flaps of rats in Fbs alone transplantation group and Schwann cells alone transplantation group were injected with 0.4 mL Fb and 0.4 mL Schwann cells respectively (2×10⁶ cells each). Flaps of rats in Schwann cells+ Fbs co-transplantation group were injected with 0.4 mL Fbs and Schwann cells mixed cells (totally 2×10⁶ cells, cell number ratio: 1∶1), and flaps of rats of simple flap group were injected with the same volume of DMEM high glucose medium. On post injection day (PID) 2, 5, 7, 9, and 14, 5 rats in each group were selected respectively according to the random number table. The flap tissue was collected, and the number, diameter, and arrangement of regenerated nerves were observed by immunofluorescence method. Data were processed with completely random designed t test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results: (1) The third passage of cells isolated and cultured from the rat embryo trunks were uniform in size and shape, long spindle-shaped, with a large proportion of nuclei. Strong positive expressions of fibronectin and Ephrin-B2 protein in cells were observed, and the mRNA expression of Ephrin-B2 was 0.004 1±0.000 8. The cells were identified as Fbs. (2) After 5 days of culture, the primary cells isolated from the sciatic nerves and brachial plexus nerves of neonatal rats were elongated in cell bodies and grew in nest, fence, or vortex-like shape. The third passage of cells were detected by immunofluorescence method and flow cytometer, and the corresponding S100 positive cell rates were (95.9±1.0)% and (95.8±1.1)% respectively. The cells were identified as Schwann cells. (3) At PCH 6 and 24, the cluster numbers of Schwann cells in Schwann cells+ Fbs co-culture group were significantly higher than those of Schwann cells alone culture group (t=6.500, 10.614, P<0.01). At PCH 24, the Schwann cells in Schwann cells+ Fbs co-culture group aggregated into clusters, Fbs dispersed around the Schwann cell clusters, and the protein expressions of EphB2, N-cadherin, and Sox2 in Schwann cells were significantly higher than those in Schwann cells alone culture group (t=2.975, 19.717, 11.159, P<0.05 or P<0.01). (4) On PID 2, a small number of scattered, disordered, short, and thin nerve fibers were observed in the flap tissue of rats in the four groups. From PID 5 to 14, the number of nerve fibers in the flap tissue of rats of Schwann cells+ Fbs co-transplantation group increased gradually, and the nerve fibers were with long diameter and arranged orderly. The number of nerve fibers in the flap tissue of rats of Schwann cells alone transplantation group increased, but the nerve fibers were with short diameter and arranged disorderly, and the number was smaller than that of Schwann cells+ Fbs co-transplantation group. In simple flap group and Fbs alone transplantation group, the nerve fibers in the flap tissue of rats gradually degenerated with gradually decreased number or even disappeared. Conclusions The combined transplantation of Fbs and Schwann cells in rats can regulate Schwann cells migration and clustering by activating Ephrin/Eph-Sox2-N-cadherin signaling pathway, thus promoting the orderly nerve regeneration of denervated perforator flaps in rats.