BACKGROUND:Heterotopic ossification of skeletal muscle is a clinically serious complication.For heterotopic ossification of skeletal muscles,the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE:To investigate the involvement of myocytes,fascia cells,and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4. METHODS:Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured,and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively,and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope.Myogenic cells(L6,derived from rats)and fibroblast-derived cells(derived from human)were co-cultured.After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β,osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining.Using transgenic animal FVB/N-TgN(TIE2-LacZ)182Sato mice,15 μL of adeno-associated virus-bone morphogenetic protein 4(5×1010 PFU/mL)were implanted in the thigh muscle space of genetic mice for 10 and 14 days.X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone. RESULTS AND CONCLUSION:(1)Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation.Compared with other groups,the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining(P<0.05)and a lower area of alkaline phosphatase staining(P<0.05),while the pure L6 group had a bigger area of alkaline phosphatase staining(P<0.05)but a smaller area of positive alcian blue and safarin O staining(P<0.05).(2)Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN(TIE2-LacZ)182Sato mice resulted in heterotopic ossification.(3)X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone.(4)These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle,but myogenic cells are the main source of osteoblasts.Tie2+ endothelial cells might not be the cell source for cartilage and bone.

SUMMARY The mesothelium,which consists of a monolayer of mesothelial cells,extends over the sur-face of the serosal cavities(pleura,pericardium,peritoneum and tunica vaginalis).Mesothelial tumours of the tunica vaginalis is rare compared with those arise from pleura or peritoneum.According to World Health Organization 2022 Classification of Urinary and Male Genital Tumours(5th edition),mesothelial tumours of the tunica vaginalis were categorized into adenomatoid tumour,well-differentiated papillary mesothelial tumour(WDPMT)and mesothelioma.Since WDPMT of tunica vaginalis was rare,there was no consensus concerning the treatment of it.In this case report,a 29-year-old man who had endured intermittent right scrotal pain for 8 months,aggravating scrotal pain for 2 weeks was admitted.No symptoms,such as frequent,urgent,or painful urination were shown.Physical examination revealed the enlargement and tenderness of right scrotum,with no signs of lifting pain.The most recent scrotal ultrasonography be-fore surgery revealed right hydrocele with maximum depth of 4 centimeters and poor blood flow of right testis.Under the circumstance of patient's chronic history of testicular hydrocele,he underwent an emer-gency operation of right scrotal exploration and hydrocelectomy under epidural anesthesia.After opening the vagina tunic cavity,spot-like bleeding was observed on the right testicle,epididymis and vaginalis surface.The vaginalis was obviously thickened and the inner and outer walls were smooth.The post-operative histopathology revealed a grayish-brown tissue with a thickness of 0.3-0.5 cm,smooth inner and outer walls,and a suspected WDPMT with a diameter of 1.5 cm.Immunohistochemical staining showed positive for Calretinin,BAP1,WT-1,CK5/6,D2-40 and P16,which confirmed the diagnosis of WDPMT.To sum up,the purpose of this case report was to raise awareness of a rare disease WDPMT,which was usually asymptomatic and could be diagnosed by pathology and immunohistochemistry.The disease should be differentiated from testicular torsion,epididymitis,orchitis and oblique inguinal hernia in symptoms,and from malignant mesothelioma and adenomatoid tumour in pathology.Because of the rarity of the cases,there was no unified standard for the treatment of WDPMT at present.The common treatment methods reported in literature included orchidectomy and vaginectomy.Due to the lack of un-derstanding of this disease,postoperative follow-up was still recommended for at least 5 years.

Objective:To investigate the correlation between serum hypoxia inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), and inflammatory factors and incision infection after anal fistula surgery.Methods:A retrospective analysis was conducted on the clinical data of 120 patients who underwent anal fistula thread hanging surgery at the First People′s Hospital of Shuangliu District, Chengdu from June 2022 to April 2023. The patients were divided into an infected group (36 cases) and a non infected group (84 cases) based on their postoperative incision infection status. The differences in serum HIF-1 α, VEGF, sTREM-1 and inflammatory factors [C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α)] between the infected and non infected groups were compared, and the correlation between CRP, IL-6, TNF-α, HIF-1 α, VEGF, sTREM-1 and postoperative infection of anal fistula was analyzed by Pearson correlation coefficient; The correlation between CRP, IL-6, TNF-α and HIF-1 α, VEGF, sTREM-1. And the efficacy of serum HIF-1 α, VEGF, sTREM-1, inflammatory factors and their individual and combined diagnosis of incision infection after anal fistula surgery was analyzed by receiver operating characteristic (ROC) curve analysis.Results:The levels of serum HIF-1 α, sTREM-1, CRP, IL-6, and TNF-α in the infected group were higher than those before surgery and higher than those in the uninfected group 3 days after surgery; VEGF levels were lower than preoperative levels and lower than those in the non infected group (all P<0.05). Pearson correlation analysis showed that HIF-1α, sTREM-1, CRP, IL-6, TNF-α were positively correlated with postoperative infection in anal fistula ( r=0.456, 0.494, 0.455, 0.510, 0.363, all P<0.05), while VEGF was negatively correlated with postoperative infection in anal fistula ( r=-0.462, P<0.05). CRP, IL-6, TNF-α were positively correlated with HIF-1 α and sTREM-1 ( r=0.574/0.611/0.653, 0.661/0.608/0.610, all P<0.05), while CRP, IL-6, TNF-α were negatively correlated with VEGF ( r=-0.200, -0.207, -0.254, all P<0.05). The area under the curve (AUC) of HIF-1 α, VEGF, sTREM-1, CRP, IL-6, and TNF-α for diagnosing incision infection after anal fistula surgery were 0.716, 0.787, 0.741, 0.678, 0.792, and 0.688, respectively. The AUC of the combined diagnosis of inflammatory factors and 6 data points for postoperative incision infection in anal fistula surgery were 0.836 and 0.921, respectively. Conclusions:Serum levels of HIF-1 α, sTREM-1, CRP, IL-6, and TNF-α are abnormally high in patients with incision infection after anal fistula surgery, while VEGF is abnormally low in expression. HIF-1 α, VEGF, sTREM-1, and inflammatory factors can be used as effective indicators for clinical diagnosis of incision infection after anal fistula surgery, and their combined diagnostic value is better. HIF-1 α and sTREM-1 are positively correlated with inflammatory factors, while VEGF is negatively correlated with inflammatory factors.

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.
Humans ; Apoptosis/genetics* ; bcl-2-Associated X Protein ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Leukemia, Myeloid, Acute/pathology* ; Luciferases ; MicroRNAs/metabolism* ; PAX9 Transcription Factor/genetics*

Based on single-cell sequencing of the hippocampi of 5x familiar Alzheimer's disease(5x FAD)and wild type mice at 2-,12-,and 24-month of age,we found an increased percentage of microglia in aging and Alzheimer's disease(AD)mice.Blood brain barrier injury may also have contributed to this increase.Immune regulation by microglia plays a major role in the progression of aging and AD,according to the functions of 41 intersecting differentially expressed genes in microglia.Signaling crosstalk between C-C motif chemokine ligand(CCL)and major histocompatibility complex-1 bridges intercellular communi-cation in the hippocampus during aging and AD.The amyloid precursor protein(APP)and colony stimulating factor(CSF)signals drive 5x FAD to deviate from aging track to AD occurrence among intercellular communication in hippocampus.Microglia are involved in the progression of aging and AD can be divided into 10 functional types.The strength of the interaction among microglial subtypes weakened with aging,and the CCL and CSF signaling pathways were the fundamental bridge of communication among microglial subtypes.

With the extensive application of highly sensitive genetic techniques in the field of prenatal diagnosis, prenatal chromosomal mosaicisms including true fetal mosaicisms and confined placental mosaicisms are frequently identified in clinical settings, and the diagnostic criteria and principle of genetic counseling and clinical management for such cases may vary significantly among healthcare centers across the country. This not only has brought challenges to laboratory technician, genetic counselor and fetal medicine doctor, but can also cause confusion and anxiety of the pregnant woman and their family members. In this regard, we have formulated a consensus over the prenatal diagnosis and genetic counseling for chromosomal mosaicisms with the aim to promote more accurate and rational evaluation for fetal chromosomal mosaicisms in prenatal clinics.
Consensus ; Female ; Genetic Counseling ; Humans ; Mosaicism ; Placenta ; Pregnancy ; Prenatal Diagnosis/methods*

Objective:To explore the correlation between SAM domain and HD domain-containing protein 1 (SAMHD1) and programmed death-ligand 1 (PD-L1) expression in lung adenocarcinoma.Methods:The expression of SAMHD1 in lung adenocarcinoma and its effect on prognosis were analyzed by online database GEPIA and Kaplan-Meier Plotter. The expression of SAMHD1 in lung adenocarcinoma cell lines was detected by quantitative real-time PCR (qPCR) and Western blotting. SAMHD1 gene was silenced in H1975, H1299 and LLC cells by small interfering RNA transfection and lentivirus infection, respectively. The mRNA and protein expression levels of PD-L1 in lung adenocarcinoma cells of control group, siSAMHD1-1 group and siSAMHD1-2 group were detected by qPCR and Western blotting. The membrane PD-L1 level was detected by flow cytometry. A mouse lung adenocarcinoma xenograft model was constructed. The PD-L1 levels in the tumor tissues of control group and shSAMHD1 group were detected by immunohistochemistry. Cell proliferation activities of the control, siSAMHD1-1 and siSAMHD1-2 groups were detected by CCK-8 assays.Results:The GEPIA database results showed that the mRNA expression of SAMHD1 in lung adenocarcinoma was lower than that in normal lung tissue (4.81±0.90 vs. 5.99±0.76, t=20.67, P<0.001) . The median overall survival time of patients with high SAMHD1 expression was significantly longer than that of patients with low SAMHD1 expression (109.0 months vs. 87.7 months, χ2=26.83, P=0.002) . The relative mRNA expression levels of SAMHD1 in A549, PC9, H1299 and H1975 cells were 1.00±0.02, 0.75±0.05, 3.49±0.19 and 7.25±0.38 ( F=589.00, P<0.001) , and the relative protein expression levels were 1.00±0.06, 0.34±0.07, 1.67±0.22 and 2.11±0.63 ( F=15.79, P=0.001) . In H1975 cells, the relative mRNA levels of PD-L1 in the control, siSAMHD1-1 and siSAMHD1-2 groups were 1.00±0.00, 1.54±0.26 and 2.89±0.13 ( F=102.30, P<0.001) , and the relative protein expression levels were 1.00±0.01, 1.50±0.10 and 1.52±0.33 ( F=6.65, P=0.030) . In H1299 cells, the relative mRNA levels of PD-L1 in the three groups were 1.00±0.08, 1.63±0.03 and 2.14±0.03 ( F=368.80, P<0.001) , and the relative protein levels of PD-L1 were 1.00±0.07, 1.88±0.35 and 2.05±0.38 ( F=10.66, P=0.011) . The expression level of PD-L1 in the siSAMHD1-1 and siSAMHD1-2 groups was higher than that in the control group (all P<0.05) . Flow cytometry results showed that in H1975 cells, the fluorescence intensity of membrane PD-L1 in the control, siSAMHD1-1 and siSAMHD1-2 groups were 246.83±27.59, 325.60±8.00 and 308.93±7.60 ( F=17.56, P=0.003) , and in H1299 cells, the fluorescence intensity of membrane PD-L1 in the three groups were 959.00±6.25, 1 084.33±7.64 and 1 085.33±21.22 ( F=86.74, P<0.001) . The fluorescence intensity of PD-L1 in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group (all P<0.05) . In xenograft mouse model, the H-SCORE of PD-L1 in the shSAMHD1 group was higher than that in the control group (7.99±1.10 vs. 4.49±0.43, t=5.13, P=0.007) . The proliferative activities of H1975 cells in the control group, siSAMHD1-1 group and siSAMHD1-2 group at 72 h were 0.50±0.02, 0.75±0.05 and 0.73±0.06 ( F=25.01, P=0.001) . The proliferative activities of H1299 cells in the three groups at 72 h were 0.80±0.01, 1.00±0.04 and 0.93±0.07 ( F=13.90, P=0.006) . The cell proliferation activity in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group (all P<0.05) . Conclusion:SAMHD1 silencing induces PD-L1 expression in lung adenocarcinoma.

Objective To investigate the efficacy and safety of two antiangiogenic drugs (recombinant human endostatin endostar and thalidomide ) combined with capecitabine and oxaliplatin (XELOX) regimens in the treatment of advanced colorectal cancer.Methods From January 2015 to May 2018,40 patients of advanced metastatic colorectal cancer with organ metastasis and non?resectable were selected from the first people′s hospital of Hefei and Anhui Provincial Hospital, and they were randomly divided into treatment and control group, with 20 cases in each group.The treatment group received intravenous infusion (IV) of endostar for continuous 7 days (day 1～7) combined with oral administration of thalidomide for continuous 14 days (day 1～14) plus XELOX regimens after the fifth dose of endostar (day 6～19),and the control group was treated with XELOX regimen (day6～19).Results The objective response rate (ORR) was 50%(10／20) and 20%(4／20) respectively (χ2＝3.956,P＜0.05),the disease control rate (DCR) was 85%( 17／20) and 70%(14／20) respectively ( χ2＝1.290,P＞0.05),and the median progression free survival (mPFS) was 6.8 in both groups.There was no significant difference in Karnofsky performance score ( KPS) and incidence of adverse reactions between the two groups before and after treatment (P＞0.05).Conclusion Combination of endostar and thalidamide plus XELOX regimen as first?line treatment have better antitumor activity and are well?tolerated in patients with advanced colorectal cancer.

ObjectiveTo investigate the changes in positive staining of CD34, CK7, and CK19 and amount of fibrous collagen deposition in patients with chronic hepatitis B (CHB) and the pathological basis affecting FibroTouch measurements. MethodsA retrospective analysis was performed for the clinical data of 72 CHB patients who visited Department of Liver Cirrhosis in Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from January 2015 to December 2017. The amount of positive immunohistochemical staining of CD34, CK7, and CK19 was calculated, as well as the amount of fibrous collagen deposition in Masson trichrome staining and liver stiffness measurement (LSM) by FibroTouch. The t-test was used for comparison of normally distributed continuous data between two groups. The Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data or continuous data with heterogeneity of variance between two groups. The chi-square test was used for comparison of categorical data between groups, and the Kruskal-Wallis H test was used for comparison of ranked data between multiple groups. The receiver operating characteristic (ROC) curve was used to analyze the value of LSM in the diagnosis of hepatitis B cirrhosis, and the logistic regression model was used for multivariate analysis. ResultsWith the increase in inflammation degree, there was no significant change in the amount of positive staining of CD34 (P＞0.05), while there were significant increases in the amount of positive staining of CK19 and the amount of fibrous collagen deposition (H=9.02 and 14.12, P=0011 and 0.001). With the progression of liver fibrosis, there were significant increases in the amount of positive staining of CD34 and CK7 and the amount of fibrous collagen deposition (H=10.26, 16.29, and 22.97, P=0.016, 0.001, and ＜0.001). The logistic regression analysis showed that the amount of positive staining of CK7 (Wald=4.756, P=0.029) and the amount of fibrous collagen deposition (Wald=4.757, P=0.029) were independent influencing factors for FibroTouch measurements. ConclusionIncreases in the amount of fibrous collagen deposition and the amount of positive staining of CK7 may lead to increased FibroTouch measurements.

Objective: To evaluate the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in prophylaxis neutropenia after chemotherapy in patients with lymphoma. Methods: This was a multicenter, single arm, open, phase Ⅳ clinical trial. Included 410 patients with lymphoma received multiple cycles of chemotherapy and PEG-rhG-CSF was administrated as prophylactic. The primary endpoint was the incidence of Ⅲ/Ⅳ grade neutropenia and febrile neutropenia (FN) after each chemotherapy cycle. Meanwhile the rate of antibiotics application during the whole period of chemotherapy was observed. Results: ①Among the 410 patients, 8 cases (1.95%) were contrary to the selected criteria, 35 cases (8.54%) lost, 19 cases (4.63%) experienced adverse events, 12 cases (2.93%) were eligible for the termination criteria, 15 cases (3.66%) develpoed disease progression or recurrence, thus the rest 321 cases (78.29%) were into the Per Protocol Set. ②During the first to fourth treatment cycles, the incidences of grade Ⅳ neutropenia after prophylactic use of PEG-rhG-CSF were 19.14% (49/256) , 12.5% (32/256) , 12.18% (24/197) , 13.61% (20/147) , respectively. The incidences of FN were 3.52% (9/256) , 0.39% (1/256) , 2.54% (5/197) , 2.04% (3/147) , respectively. After secondary prophylactic use of PEG-rhG-CSF, the incidences of Ⅳ grade neutropenia decreased from 61.54% (40/65) in the screening cycle to 16.92% (11/65) , 18.46% (12/65) and 20.75% (11/53) in 1-3 cycles, respectively. The incidences of FN decreased from 16.92% (11/65) in the screening cycle to 1.54% (1/65) , 4.62% (3/65) , 3.77% (2/53) in 1-3 cycles, respectively. ③The proportion of patients who received antibiotic therapy during the whole period of chemotherapy was 34.39% (141/410) . ④The incidence of adverse events associated with PEG-rhG-CSF was 4.63% (19/410) . The most common adverse events were bone pain[3.90% (16/410) ], fatigue (0.49%) and fever (0.24%) . Conclusion During the chemotherapy in patients with lymphoma, the prophylactic use of PEG-rhG-CSF could effectively reduce the incidences of grade Ⅲ/Ⅳ neutropenia and FN, which ensures that patients with lymphoma receive standard-dose chemotherapy to improve its cure rate.