Objective:To investigate the possible mechanism of mesenchymal stem cells (MSC) secreting hepatocyte growth factor (HGF).Methods:① C57BL/6 mouse mesenchymal stem cells (mMSC) were cultured in vitro, and mMSC with high expression of chemokine receptor 7 (CXCR7) were transduced by lentivirus plasmid. Blank control group and empty carrier control group were set at the same time. After 20 generations of cell culture, the transfection efficiency was identified by fluorescence microscopy and flow cytometry. The mRNA expression levels of CXCR7 in mMSC were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). ② mMSC with passage number 4-6 were divided into MSC control group [MSC-blank group, 100 μg/L lipopolysaccharide (LPS) was added to wild-type MSC], highly expressed CXCR7 group (MSC-OE-CXCR7 group, 100 μg/L LPS was added to mMSC transduced by lentivirus plasmid with high expression of CXCR7), highly expressed CXCR7 control group (MSC-OENC-CXCR7 group, 100 μg/L LPS was added to mMSC transduced by no load lentivirus plasmid), CXCR4 inhibitor group (MSC-IE-CXCR4 group, 100 μg/L LPS was added to mMSC after 0.1 mg/L CXCR4 inhibitor TC14012 pretreatment for 24 hours), and CXCR4 inhibitor control group (MSC-IENC-CXCR4 group, 100 μg/L LPS was added to mMSC after DMEM culture medium with equal amount of TC14012 pretreatment for 24 hours). Cells in each group were collected after treatment with LPS, and mRNA expression of inhibitor of differentiation-1 (ID-1) was detected by RT-PCR. The cell supernatant was collected, and the levels of HGF were detected by enzyme linked immunosorbent assay (ELISA). Results:① The high expression of CXCR7 for mMSC which were transduced through lentivirus plasmid were successfully constructed detected by fluorescence microscope and flow cytometry. Compared with the blank control group, the expression of CXCR7 mRNA in the lentivirus with high expression of CXCR7 group was significantly increased (2 -ΔΔCt: 5.81±0.97 vs. 1.02±0.12, P < 0.05). There was no significant difference in CXCR7 mRNA expression between the empty carrier control group and the blank control group (2 -ΔΔCt: 0.95±0.22 vs. 1.02±0.12, P > 0.05). ②Compared with the MSC-blank group, high expression of CXCR7 in MSC-OE-CXCR7 group or inhibition of CXCR4 in MSC-IE-CXCR4 group could induce high expression of ID-1 mRNA in mMSC (2 -ΔΔCt: 5.56±0.66, 2.47±0.58 vs. 1.00±0.10, both P < 0.05) and increase HGF exocrine level (ng/L: 632.02±149.98, 217.21±40.53 vs. 108.53±24.62, both P < 0.05). However, there were no significant differences in ID-1 mRNA expression and HGF exocrine level of mMSC among MSC-OENC-CXCR7 group, MSC-IENC-CXCR4 group and MSC-blank group [ID-1 mRNA (2 -ΔΔCt): 1.01±0.27, 1.21±0.32 vs. 1.00±0.10, HGF (ng/L): 133.56±25.19, 107.11±25.30 vs. 108.53±24.62, both P > 0.05]. Conclusion:High expression of CXCR7 or inhibition of CXCR4 in MSC can increase the expression of ID-1 and promote the secretion of HGF, thus promoting pulmonary microvascular endothelial repair.

Objective:To discuss the effect of parthenolide(PTL)on the apoptosis of the chondrocytes under mechanical stretch stress by regulating the expression of piezo type mechanosensitive ion channel component 1(Piezo1),and to clarify the related mechanism.Methods:The chondrocytes were divided into 0%,5%,10%,15%,and 20%stretch groups according to the stretch variable.Additionally,the chondrocytes were divided into control group,20%stretch group,20%stretch+5 μmol·L-1 PTL group,20%stretch+10 μmol·L-1 PTL group,and 20%stretch+20 μmol·L-1 PTL group.The Piezo1 short hairpin RNA(shRNA)interference lentivirus(sh-Piezo1)or shRNA-NC lentivirus were used to infect the chondrocytes,and the chondrocytes were divided into sh-Piezo1 group and sh-NC group,and also set up blank control group.The chondrocytes were also devided into 20%stretch group,20%stretch+PTL group,20%stretch+sh-Piezo1 group,and 20%stretch+sh-Piezo1+PTL group.Hoechst 33258 fluorescence staining was used to observe the morphology of the nuclear in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups;spectrophotometry was used to detect the cysteinyl aspartate specific proteinase(Caspase)-3 activities in the cells in various groups;CCK-8 method was used to detect the proliferation rates of the cells in various groups;Fluo-4/AM fluorescent probe method was used to detect the calicium ion(Ca2+)levels in the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Piezo1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of Piezo1 protein in the cells in various groups.Results:The Hoechst 33258 fluorescence staining resuts showed that as the increasing of stretch,the number of the chondrocytes with fragmented and densely stained nuclei in 0%,5%,10%,15%,and 20%stretch groups were gradually increased.The flow cytometry results showed that compared with 0%stretch group,the apoptotic rates of the chondrocytes in 5%,10%,15%,and 20%stretch groups were significantly increased(P<0.01);compared with control group,the apoptotic rate of the chondrocytes in 20%stretch group was significantly increased(P<0.05);compared with 20%stretch group,the apoptotic rates of the chondrocytes in 20%stretch+5 μmol·L-1 PTL group,20%stretch+10 μmol·L-1 PTL group,and 20%stretch+20 μmol·L-1 PTL group were significantly decreased(P<0.05);compared with 20%stretch group,the apoptotic rates of chondrocytes in 20%stretch+PTL group and 20%stretch+sh-Piezo1 group were significantly decreased(P<0.05).The spectrophotometry results showed that compared with 0%stretch group,the Caspase-3 activities in the chondrocytes in 5%,10%,15%,and 20%stretch groups were significantly increased(P<0.01);compared with control group,the Caspase-3 activity in the chondrocytes in 20%stretch group was significantly increased(P<0.05);compared with 20%stretch group,the Caspase-3 activities in the chondrocytes in 20%stretch+5 μmol·L-1 PTL group,20%stretch+10 μmol·L-1 PTL group,and 20%stretch+20 μmol·L-1 PTL group were significantly decreased(P<0.05).Compared with 20%stretch group,the Caspase-3 activities in the chondrocytes in 20%stretch+PTL group and 20%stretch+sh-Piezo1 group were significantly decreased(P<0.05).The CCK-8 method results showed that compared with 0 μmol·L-1 PTL group,the proliferation rates of the chondrocytes in 40.00,80.00,and 160.00 μmol·L-1 PTL groups were significantly decreased(P<0.05),indicating that 20.00 μmol·L-1 PTL was the maximum non-toxic concentration.The Fluo-4/AM fluorescent probe method results showed that compared with control group,the Ca2+level in the chondrocytes in 20%stretch group was significantly increased(P<0.05);compared with 20%stretch group,the Ca2+levels in the chondrocytes in 20%stretch+5 μmol·L-1 PTL group,20%stretch+10 μmol·L-1 PTL group,and 20%stretch+20 μmol·L-1 PTL group were significantly decreased(P<0.05);compared with 20%stretch group,the Ca2+levels in the chondrocytes in 20%stretch+PTL group and 20%stretch+sh-Piezo1 group were significantly decreased(P<0.05).The RT-qPCR results showed that compared with blank control group and sh-NC group,the expression level of Piezo1 mRNA in the chondrocytes in sh-Piezo1 group was significantly decreased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of Piezo1 protein in the chondrocytes in 20%stretch group was significantly increased(P<0.05);compared with 20%stretch group,the expression levels of Piezo1 protein in the chondrocytes in 20%stretch+5 μmol·L-1 PTL group,20%stretch+10 μmol·L-1 PTL group,and 20%stretch+20 μmol·L-1 PTL group were significantly decreased(P<0.05);compared with blank control group and sh-NC group,the expression level of Piezo1 protein in the chondrocytes in sh-Piezo1 group was significantly decreased(P<0.05).Conclusion:PTL can inhibit the apoptosis of the chondrocyte induced by high-intensity cyclic mechanical stretch stress,and its mechanism may be related to inhibiting the Piezo1-mediated Ca2+influx-induced apoptosis.

Objective:To establish a HPLC method for determinating 9 components simultaneously in Swertia chirayita. Methods:By useing water Sunfire C18 column (4.6 mm× 250 mm,5 μm); Gradient elution was carried out with methanol-0.05% phosphoric acid solution as mobile phase. Setting the column temperature at 30 ℃, the flow rate at 1.0 ml/min, and the detection wavelength at 254 nm.Results:9 components showed good linear relationship within the injection quality range. The recovery rates of wertiamarin, Gentiopicroside, Angelica glycosides,Mangiferin, Isolysine, Gentianoside, Diol glycoside, 8-hydroxy-1,3,5 trimethoxyketone, and Daisy leaf gentinone were 95.38%, 92.41%, 95.14%, 91.87%, 92.24%, 92.51%, 95.08%, 91.72%, 95.74% ( n=6). Conclusion:The method is simple, efficient, sensitive, accurate, economical and practical, with repeatability and stability. It could provide reference for the quality control and comprehensive utilization of Swertia chirayita.

Objective:To investigate methods of molecular diagnosis and clinical features of 46, XY disorders of sexual development(DSD).Methods:A total of 206 cases of 46, XY DSD patients, who visited the Shanghai Ninth People′s Hospital, Shanghai Jiaotong University School of Medicine, from July 2009 to June 2021, underwent AA chip based on multiplex PCR and probe-capture-targeted next-generation sequencing. Clinical features of patients with genetic diagnosis were analyzed.Results:Among 206 patients, the diagnostic rate of patients with micropenis, hypospadias and cryptorchidism was the highest, up to 75.28%. Almost all patients had different degrees of undermasculinized external genitalia. The most frequent phenotype was micropenis with hypospadias(87.25%). Only one gene variant was detected in 81 patients(39.32%), multiple genetic variants were detected in 104 patients(50.49%), and no gene variant was identified in 21 patients(10.19%). 107 patients had definite genetic diagnosis, with a diagnostic rate of 51.94% by adding the pathogenic and likely pathogenic ratios following the American College of Medical Genetics and Genomics(ACMG) guidelines, including 40 patients of steroid 5α-reductase type 2(SRD5A2) variants(37.38%), 36 patients of androgen receptor(AR) variants(33.64%), 13 patients of steroidogenic factor 1(NR5A1) variants(16.82%), 6 patients of 17β-hydroxysteroid dehydrogenases 3(HSD17B3) variants(5.61%), 2 patients of 17α-hydroxylase/17, 20-lyase enzyme(CYP17A1), Wilms′ tumor 1(WT1) and GATA binding protein 4(GATA4) variants(1.87%), and one patient of luteinizing hormone receptor(LHCGR) variant(0.93%). Gynecomastia was found in 29 of 81 postpubertal patients, of which 25(86.21%) had AR variants.Conclusions:46, XY DSD presents complex clinical manifestations and molecular etiologies. Targeted nextgeneration sequencing has the advantages of high throughput, high efficiency and low cost, which has a high value especially in etiological diagnosis of 46, XY DSD with large genetic heterogeneity.

Background/Aims: Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provide an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Methods: Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). Results: The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and highrisk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed an HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC=0.8333, 0.8145 and 0.7958 for validation cohort, respectively). Conclusions We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.

Background/Aims: Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provide an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Methods: Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). Results: The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and highrisk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed an HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC=0.8333, 0.8145 and 0.7958 for validation cohort, respectively). Conclusions We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.

Background/Aims: Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provide an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Methods: Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). Results: The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and highrisk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed an HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC=0.8333, 0.8145 and 0.7958 for validation cohort, respectively). Conclusions We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

ObjectiveTo explore the effect of repetitive peripheral magnetic stimulation on upper limb motor function rehabilitation of stroke patients after contralateral seventh cervical nerve transfer (CC7). MethodsFrom May, 2020, to May, 2022, 34 stroke patients with hemiplegia underwent CC7 in Jing'an District Centre Hospital of Shanghai were randomly divided into control group (n = 17) and observation group (n = 17). Both groups received conventional rehabilitation. The observation group accepted repetitive peripheral magnetic stimulation, and the control group received sham stimulation, for eight weeks. They were assessed with Fugl-Meyer Assessment-Upper Extremities (FMA-UE) and Hua-Shan Grading of Upper Extremity (H-S grading) before and after treatment. ResultsTwo cases dropped down in each group. There was difference in gender between two groups (χ² = 6.136, P < 0.05). After treatment, the scores of FMA-UE and H-S grading significantly improved in both groups (t > 4.000, P < 0.01), and the improvement was better in the observation group than in the control group (t > 2.362, P < 0.05). ConclusionRepetitive peripheral magnetic stimulation could improve the motor function of upper limb and hand of stroke patients with hemiplegia after CC7.

【Objective】 To clarify the role and molecular mechanism of Tanshinone ⅡA (TanⅡA) in the pathological integration of granule cells in the dentate gyrus (DG) by using the mouse model of temporal lobe epilepsy (TLE). 【Methods】 Status epilepticus (SE) was induced in the mice with pilocarpine and treated with TanⅡA 5 mg/kg. After two months, Morris water maze was used to examine the spatial learning and memory ability and video surveillance was used to monitor spontaneous seizures. The DG was removed for staining of Timm, Prox-1, DCX and SynⅠ. PTEN, p-AKT, and p-S6 expressions were observed by Western blotting. 【Results】 TanⅡA decreased Timm score, SynⅠ, PSD-95 and pS6 levels, and increased the level of PTEN in the DG, and attenuated the formation of mossy fiber sproutings and basal dendrites of the granule cells. Video surveillance showed that TanⅡA reduced the frequency of Racine’ grade 5 seizures. 【Conclusion】 TanⅡA can effectively attenuate the abnormal integration of the granule cells in the DG by regulating PTEN/AKT/mTOR pathway and thus plays an anti-epileptic role.