Objective: To develop a RHCE genotyping assay based on kompetitive allele-specific PCR (KASP) and assess its clinical accuracy for RhCE blood group determination. Methods: KASP primers were designed to interrogate three RHCE loci: the 109 bp insertion/deletion in intron 2, c. 307T>C, and c. 676C>G. A total of 1 194 RhD-positive inpatients from Chengdu were typed by both KASP genotyping and manual tube serology. Discordant samples (n=10) were retested by both methods and further resolved by Sanger sequencing. An additional 377 cases were tested for the c. 48C>G locus to evaluate the predictive accuracy of individual loci and combined locus testing for RhC antigen. Results: Genotyping concordance with serology was 100.0% for both the c. 676C>G locus (RhE/Rhe) and the c. 307T>C locus (Rhc). For RhC prediction using the 109 bp insertion, overall accuracy was 99.7% (1 191/1 194); the 3 discordant cases were confirmed by Sanger sequencing to be false negatives attributable to 109 bp deletion in intron 2. Testing the c. 48C>G allele for RhC prediction yielded 7 false positives, with an accuracy of 98.1% (370/377). RhC antigen status was determined by combining the 109 bp insertion and the c. 48C allele. After excluding 10 samples with inconsistent results between the two loci, the accuracy reached 100% in the remaining 367 samples. When both loci were applied in combination, accuracy reached 100% in the 367 cases with concordant results. Among the 1 194 patients, CCee (45.8%) and CcEe (31.7%) were the most common RhCE phenotypes. The e antigen had the highest positivity rate (92.2%), and the Ce haplotype was the most frequent (66.9%). Conclusion: The KASP-based RHCE genotyping method achieves high accuracy for clinical RhCE typing. Combining the 109 bp insertion/deletion with the c. 48C allele significantly improves RhC antigen prediction compared with either locus alone. This method was applied to RhCE genotyping of 1 194 RhD-positive inpatients in Chengdu, providing local RhCE phenotype and haplotype distribution data to support RhCE-matched transfusion practice.

Objective:To analyze the clinical characteristics of mannose phosphate isomerase-congenital disorders of glycosylation (MPI-CDG) and evaluated the outcomes following D-mannose treatment.Methods:This case-series study analyzed clinical manifestations, laboratory findings, imaging results, genetic data, and outcomes after D-mannose therapy in 5 children with MPI-CDG diagnosed at the Children′s Hospital of Fudan University between December 2014 and December 2024.Results:The age of onset ranged from 0.3 to 0.4 years in all 5 children, who initially presented with diarrhea and hypoglycemia. Associated manifestations included short stature (3 cases), anemia (3 cases), splenomegaly (3 cases), hepatomegaly (4 cases), elevated transaminases (4 cases), and hypoalbuminemia (4 cases). Liver pathology revealed hepatic fibrosis in 3 cases. Genetic testing identified 8 variants in the MPI gene, including 2 novel variants. Following D-mannose treatment, diarrhea and hypoglycemia resolved within 1-2 weeks in all children, with concurrent improvement in anemia. Notably except for Patient 1, who developed progressive splenomegaly, worsening hepatic fibrosis, and portal hypertension despite persistently normal transaminase and albumin levels, the other 4 children showed improvement in transaminase levels, resolution of hypoalbuminemia and amelioration of imaging abnormalities.Conclusions:MPI-CDG typically manifests in infancy with diarrhea and hypoglycemia, often accompanied by multi-system involvement. D-mannose treatment significantly improves metabolic abnormalities and most organ damages. However, close surveillance of liver status is warranted due to the risk of hepatic fibrosis progression in some cases.

Objective:To analyze the clinical characteristics of mannose phosphate isomerase-congenital disorders of glycosylation (MPI-CDG) and evaluated the outcomes following D-mannose treatment.Methods:This case-series study analyzed clinical manifestations, laboratory findings, imaging results, genetic data, and outcomes after D-mannose therapy in 5 children with MPI-CDG diagnosed at the Children′s Hospital of Fudan University between December 2014 and December 2024.Results:The age of onset ranged from 0.3 to 0.4 years in all 5 children, who initially presented with diarrhea and hypoglycemia. Associated manifestations included short stature (3 cases), anemia (3 cases), splenomegaly (3 cases), hepatomegaly (4 cases), elevated transaminases (4 cases), and hypoalbuminemia (4 cases). Liver pathology revealed hepatic fibrosis in 3 cases. Genetic testing identified 8 variants in the MPI gene, including 2 novel variants. Following D-mannose treatment, diarrhea and hypoglycemia resolved within 1-2 weeks in all children, with concurrent improvement in anemia. Notably except for Patient 1, who developed progressive splenomegaly, worsening hepatic fibrosis, and portal hypertension despite persistently normal transaminase and albumin levels, the other 4 children showed improvement in transaminase levels, resolution of hypoalbuminemia and amelioration of imaging abnormalities.Conclusions:MPI-CDG typically manifests in infancy with diarrhea and hypoglycemia, often accompanied by multi-system involvement. D-mannose treatment significantly improves metabolic abnormalities and most organ damages. However, close surveillance of liver status is warranted due to the risk of hepatic fibrosis progression in some cases.

The wheeled mobile robot(WMR)was introduced in terms of concept,development route and application progress in medical service support in the world.The advantages and disadvantages of the WMRs from some countries were analyzed,and the key technologies of WMR were described.It's pointed out the WMR would be enhanced in obstacle-crossing ability,battlefield sensing and information interaction and endurance.[Chinese Medical Equipment Journal,2024,45(3):86-94]

In infants with severe bronchopulmonary dysplasia(sBPD),severe pulmonary lobar emphysema may occur as a complication,contributing to significant impairment in ventilation.Clinical management of these infants is extremely challenging and some may require lobectomy to improve ventilation.However,prior to the lobectomy,it is very difficult to assess whether the remaining lung parenchyma would be able to sustain adequate ventilation postoperatively.In addition,preoperative planning and perioperative management are also quite challenging in these patients.This paper reports the utility of selective bronchial occlusion in assessing the safety and efficacy of lobectomy in a case of sBPD complicated by severe right upper lobar emphysema.Since infants with sBPD already have poor lung development and significant lung injury,lobectomy should be viewed as a non-traditional therapy and be carried out with extreme caution.Selective bronchial occlusion test can be an effective tool in assessing the risks and benefits of lobectomy in cases with sBPD and lobar emphysema.However,given the technical difficulty,successful application of this technique requires close collaboration of an experienced interdisciplinary team.

Aim To explore the effect and mechanism of the artesunate(ART)on hepatocellular carcinoma(HCC).Methods The cell lines MHCC-97H and HCC-LM3 were used to be detected.MTT and clone formation were used to determine the cell proliferation;Wound healing was used to detect the cell migration;Transwell was used to test the cell invasion.Flow-cy-tometry was used to detect cell apoptosis and cell cy-cle.RNA-seq and qRT-PCR was used to detect the genes expression.Results The proliferation,migra-tion and invasion of treated cells were obviously inhibi-ted(P＜0.01).Moreover,the apoptosis rate in-creased significantly,so did the proportion of G2/M cells.Transcriptomic analysis identified GADD45A as a potential target of ART through RNA-sequencing da-ta,and suggested that ART might induce apoptosis and cell cycle arrest through regulating the expression of GADD45A.In addition,the results of mechanism studies and signaling analysis suggested that GADD45A had interaction with its upstream gene NACC1(nucle-us accumbens associated 1).Moreover,after ART treatment,the expressions of GADD45A and NACC1 were changed significantly.Conclusion ART may be a potential drug to resist HCC by affecting the expres-sion of GADD45A and its upstream gene NACC1,which provides a new drug,a new direction and a new method for the clinical treatment of HCC.

Objective To investigate the inhibitory effects of Wumeiwan on oxidative stress injury of ulcerative colitis mice induced by dextran sulfate sodium(DSS)by regulating Kelch-like ECH related protein 1(Keap-1)-nuclear factor E2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathwayand.Methods Forty C57BL/6 mice were randomly divided into five groups:normal group,model group,positive control group,experimental-L,-H groups.UC mice model were induced by free access to 2％DSS water.Mice in normal and model group were orally administered with 0.9％NaCl,mice in positive control group were orally treated with Mesalazine solution(0.005 g·10 g-1·d-1),while mice in experimental groups were orally administered with Wumeiwan decoction at the dose of 0.13 and 0.26 g·10 g-1·d-1,respectively.All the drugs were administered for consecutive 7 days,1 times a day.The levels of disease activity index(DAI)and the colon length were scored.The levels of superoxide dismutase(SOD),catalase(CAT),cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS)in colon tissue of mice were determined by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)method.The level of Keap-1,Nrf2,HO-1 proteins in colon tissue were determined by Western blot method.Results The levels of DAI of seventh day in normal group,positive control group,experimental-L,-H groups were 0、(2.62±0.33),(1.87±0.35),(1.87±0.35)and(1.58±0.35);the colon lengths were(8.16±0.47)、(5.98±0.24),(7.58±0.38),(7.33±0.24)and(7.48±0.51)cm;the SOD mRNA were 1.01±0.16、0.40±0.01,1.43±0.45,0.65±0.01 and 0.83±0.02;the CAT mRNA were 1.01±0.20、0.45±0.01,0.84±0.02,0.68±0.07 and 0.87±0.05;the COX-2 mRNA were 1.03±0.33、16.65±0.60,4.78±0.25,14.07±0.60 and 7.39±0.15;the iNOS mRNA were 1.04±0.40、20.71±0.66,8.09±0.93,15.44±0.68 and 11.66±0.06;the levels of Keap-1 were 1.22±0.16、1.10±0.05,1.18±0.05,1.94±0.08 and 1.17±0.08;the levels of Nrf2 were 1.12±0.16、0.76±0.15,0.65±0.13,0.70±0.16 and 0.82±0.18;the levels of HO-1 were 1.34±0.15、1.00±0.12,0.89±0.10,1.50±0.18 and 1.40±0.13,respectively.Significant difference was found between normal group and model group(P＜0.01,P＜0.05);significant difference was also found between the experimental-L,-H groups and model group(P＜0.01,P＜0.05).Conclusion Wumeiwan can inhibit oxidative stress in mice with UC,the mechanisms may be related to adjusted the expression of Keap-1-Nrf2/HO-1 signaling pathway protein in colon.

Objective: To establish an optimized method for isolation and purification of microglia from aged rat brain tissue, and the phenotype of microglia was detected by flow cytometry. Methods: With young rats(3 months old) as control, the brain tissues of aged rats were immediately processed into single cell suspensions by mechanical dissociation and enzymatic digestion using type IV collagenase. Microglia were isolated on Percoll gradients(30%-37%-70%). The cells were stained with fluorescence-labeled antibodies and the phenotype of microglia was detected by flow cytometry. Results: This study developed a method that enzymatic digestion and mechanical dissociation combined with density gradient centrifugation. More single cells could be obtained by using this method. And the survival rate of cells was more than 90%. The flow cytometric analysis showed that the expression of M1 microglia marker CD86 and MHC Ⅱ increased(P<0.01), and the expression of M2 microglia marker CD200R increased(P<0.01) in aged rats compared with that in young rats. Conclusion The use of type IV collagenase and mechanical digestion combined with density gradient centrifugation is good for isolating and purifying microglia from adult and aged rat brain tissue.

Objective:To study the mechanism of electroacupuncture (EA) on improving insulin resistance and in vitro fertilization and embryo transfer (IVF-ET) pregnancy outcome in polycystic ovary syndrome (PCOS) patients with phlegm-damp syndrome based on DNA methylation and insulin metabolism signaling pathway. Methods:In this prospective randomized controlled study, 100 PCOS patients with phlegm-damp syndrome who underwent IVF-ET in Department of Reproduction and Genetics in Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2020 to December 2022 were enrolled and divided into a treatment group (EA therapy) and control group (placebo needling) by random number table, 50 patients in each group. Fixed antagonist regimen was used to promote ovulation in the two groups. Patients received EA therapy and placebo needling respectively twice a week from the menstrual cycle before oocyte retrieval till human chorionic gonadotrophin injection day. The granulosa cells were collected. The improvement of phlegm and dampness syndrome, homeostasis model assessment insulin resistance (HOMA-IR), dosage and number of days of gonadotropins (Gn) used, number of oocytes retrieved and high-quality embryos, fertilization rate, clinical pregnancy rate, early miscarriage rate and live birth rate of patients in the two groups were compared. Bisulfite amplicon sequencing was used to evaluate the methylation levels of the INSR gene promoter region in ovarian granulosa cells of patients in the two groups. Real-time polymerase chain reaction and Western blotting technology were used to detect the expression of INSR, PI3K and GLUT4. Results:The phlegm-dampness score (15.23±1.57) and HOMA-IR (2.82±0.39) of the experimental group after treatment were significantly lower than those before treatment (21.65±3.61 and 3.34±0.56), and the differences were statistically significant ( P<0.001, P=0.014). The differences of the phlegm-dampness score (-5.76±2.86) and HOMA-IR (-2.67±0.06) before and after treatment in the experimental group were significantly greater than those in control group (-1.64±0.84, -0.11±0.04), and the differences were all statistically significant ( P<0.001, P=0.021). In the experimental group, the dosage of Gn used by patients [(2 119.53±338.28) U] and the duration of Gn used [(10.16±1.25) d] were significantly reduced compared with control group [(2 405.65±434.20) U, P=0.005; (10.94±1.46) d, P=0.026], and the number of high-quality embryos (3.54±1.04) was significantly increased compared with control group (2.66±1.87, P=0.014). Fertilization rate [66.91% (552/825)], clinical pregnancy rate [63.27% (31/49)] and live birth rate [51.02% (25/49)] were significantly higher in the experimental group than in control group [60.20% (475/789), 41.67% (20/48), 31.25% (15/48)], and the differences were all statistically significant ( P=0.005, P=0.033, P=0.048). There were no significant differences in the number of oocytes retrieved and early abortion rate between the two groups (all P>0.05). The methylation levels of INSR gene promoter sites 38, 47, 56, 59, 94 and 143 in the experimental group were significantly lower than those in control group (all P<0.05), but there were no significant differences in methylation levels of sites 71, 74, 154, 156 and 162 between the two groups (all P>0.05). Compared with control group, the mRNA and protein expression of INSR, PI3K and GLUT4 was up-regulated in the experimental group (all P<0.05). Conclusion:EA may improve IR by down-regulating INSR methylation levels and up-regulating INSR, PI3K and GLUT4 expression, thereby enhancing the quality of embryos and improving the pregnancy outcome in PCOS patients.

Objective:To study the mechanism of electroacupuncture (EA) on improving insulin resistance and in vitro fertilization and embryo transfer (IVF-ET) pregnancy outcome in polycystic ovary syndrome (PCOS) patients with phlegm-damp syndrome based on DNA methylation and insulin metabolism signaling pathway. Methods:In this prospective randomized controlled study, 100 PCOS patients with phlegm-damp syndrome who underwent IVF-ET in Department of Reproduction and Genetics in Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2020 to December 2022 were enrolled and divided into a treatment group (EA therapy) and control group (placebo needling) by random number table, 50 patients in each group. Fixed antagonist regimen was used to promote ovulation in the two groups. Patients received EA therapy and placebo needling respectively twice a week from the menstrual cycle before oocyte retrieval till human chorionic gonadotrophin injection day. The granulosa cells were collected. The improvement of phlegm and dampness syndrome, homeostasis model assessment insulin resistance (HOMA-IR), dosage and number of days of gonadotropins (Gn) used, number of oocytes retrieved and high-quality embryos, fertilization rate, clinical pregnancy rate, early miscarriage rate and live birth rate of patients in the two groups were compared. Bisulfite amplicon sequencing was used to evaluate the methylation levels of the INSR gene promoter region in ovarian granulosa cells of patients in the two groups. Real-time polymerase chain reaction and Western blotting technology were used to detect the expression of INSR, PI3K and GLUT4. Results:The phlegm-dampness score (15.23±1.57) and HOMA-IR (2.82±0.39) of the experimental group after treatment were significantly lower than those before treatment (21.65±3.61 and 3.34±0.56), and the differences were statistically significant ( P<0.001, P=0.014). The differences of the phlegm-dampness score (-5.76±2.86) and HOMA-IR (-2.67±0.06) before and after treatment in the experimental group were significantly greater than those in control group (-1.64±0.84, -0.11±0.04), and the differences were all statistically significant ( P<0.001, P=0.021). In the experimental group, the dosage of Gn used by patients [(2 119.53±338.28) U] and the duration of Gn used [(10.16±1.25) d] were significantly reduced compared with control group [(2 405.65±434.20) U, P=0.005; (10.94±1.46) d, P=0.026], and the number of high-quality embryos (3.54±1.04) was significantly increased compared with control group (2.66±1.87, P=0.014). Fertilization rate [66.91% (552/825)], clinical pregnancy rate [63.27% (31/49)] and live birth rate [51.02% (25/49)] were significantly higher in the experimental group than in control group [60.20% (475/789), 41.67% (20/48), 31.25% (15/48)], and the differences were all statistically significant ( P=0.005, P=0.033, P=0.048). There were no significant differences in the number of oocytes retrieved and early abortion rate between the two groups (all P>0.05). The methylation levels of INSR gene promoter sites 38, 47, 56, 59, 94 and 143 in the experimental group were significantly lower than those in control group (all P<0.05), but there were no significant differences in methylation levels of sites 71, 74, 154, 156 and 162 between the two groups (all P>0.05). Compared with control group, the mRNA and protein expression of INSR, PI3K and GLUT4 was up-regulated in the experimental group (all P<0.05). Conclusion:EA may improve IR by down-regulating INSR methylation levels and up-regulating INSR, PI3K and GLUT4 expression, thereby enhancing the quality of embryos and improving the pregnancy outcome in PCOS patients.