Objective:To construct the sphingosine kinase 1(SPHK1)overexpression lentiviral vector,and to establish the SKOV3 lentiviral stable transfection cell line.Methods:According to the SPHK1 data information provided by the National Center for Biotechnology Information(NCBI)database,the primers were designed and synthesized,the target gene was amplified,and connected to the GV492 plasmid treated with Bam HⅠ and AgeⅠ restriction enzymes to construct the SPHK1 overexpression lentiviral vector;the positive clones were selected for PCR and sequencing identification;the lentiviral plasmid and the lentiviral packaging auxiliary plasmid were co-transfected into the HEK-293T cells for packaging and titer determination;according to the measured optimal multiplicity of infection(MOI)of 10,the corresponding lentiviral amounts in various groups were transfected into the SKOV3 cells,and the SKOV3 cells were divided into blank group(without treatment),GV492 control group(GV492 control lentivirus infected SKOV3 cells),and GV492-SPHK1 overexpression group(GV492-SPHK1 overexpression lentivirus infected SKOV3 cells,ov-SPHK1 group);the optimal concentration of 2 mg·L-1 puromycin was used to screen the stably transfected SKOV3 cell line;after 48 h,the medium was changed and replaced with 1 mg·L-1 puromycin for screening for 14 d;the morphology and fluorescence expression of the cells were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of SPHK1 mRNA in the SKOV3 cells in various groups;Western blotting method was used to detect the expression level of SPHK1 protein in the SKOV3 cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the SPHK1 overexpression lentiviral vector was completely consistent with the target sequence,and the SPHK1 overexpression lentiviral vector was successfully constructed;the titer determination results showed that the lentiviral titers in GV492 control group and ov-SPHK1 group were 5×1011 and 8×1011 TU·L?1,respectively;the SKOV3 cells in GV492 control group and ov-SPHK1 group were in good state and showed strong fluorescence expression,suggesting that the SKOV3 stable transfection cell line overexpressing SPHK1 was successfully established;the RT-qPCR results showed that compared with blank group and GV492 control group,the expression level of SPHK1 mRNA in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01);the Western blotting results showed that compared with blank group and GV492 control group,the expression level of SPHK1 protein in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01).Conclusion:The SPHK1 overexpression lentiviral vector is successfully constructed,and the SKOV3 stable transfection cell line is established.

Objective:To investigate the effect of polystyrene microplastics(PS-MPs)combined with high-fat treatment on vascular endothelial cells.Methods:Human umbilical vein endothelial cells(HUVECs)were cultured in the DMEM medium containing 5%fe-tal bovine serum.HUVECs were treated with conventional culture,high-fat treatment,and PS-MPs combined with high-fat treatment.The experiment was conducted in the three groups of control group,high-fat treatment group and PS-MPs+high-fat treatment group.CCK-8 assay was used to measure cell viability,F-actin staining was used to observe cell morphological changes,and flow cytometry,scratch assay,and tube formation assay were used to measure the apoptosis,migration,and tube-forming ability of cells.Results:After HUVECs were exposed to the high-fat environment,there was a significant reduction in cell viability,shrinkage of cells,a signifi-cant increase in cell apoptosis,and significant reductions in cell migration and tube-forming ability.Compared with the high-fat treat-ment group,there were no significant changes in cell viability,cell morphology,cell apoptosis,and cell migration ability after PS-MPs combined with high-fat treatment,but the tube-forming ability of cells was further impaired.Conclusion:High-fat treatment will affect cell viability,change cell morphology,and damage vascular endothelial cell function,and PS-MPs combined with high-fat treat-ment can aggravate the damage of vascular endothelial cell function.

Objective:To investigate the application value of fecal Syndecan-2 (SDC2) gene methylated SDC2 (m SDC2) detection in colorectal cancer (CRC) screening among urban residents in Guangzhou City. Methods:A cross-sectional study was conducted in Shitan Town, Zengcheng District, Guangzhou City from July to December 2022. A community-based screening program for CRC was conducted among residents aged 40-74 years old. m SDC2 detection was employed in the participants, and those with positive results should be recommended to receive colonoscopy examination. The positive rate of m SDC2 detection, colonoscopy compliance rate, detection rate of intestinal lesions and clinicopathological characteristics were observed. The relationship between cycle threshold (CT) value of m SDC2 and intestinal lesions was explored. Further, the cost-effectiveness of screening was evaluated. Results:A total of 8 189 fecal samples were collected from 8 877 participants with the recovery rate of 92.25%. 8 048 qualified samples were enrolled in this study, consisted of 3 182 males (39.54%) and 4 866 females (60.46%), with the average age of 56 years old (40-74 years). The positive rate of m SDC2 detection was 7.99% (643/8 048), and the compliance rate of colonoscopy was 73.10% (470/643). 20 cases (4.25%) of colorectal cancer, 109 cases (23.19%) of advanced adenoma, 145 cases (30.85%) of non-advanced adenoma, 79 cases (16.81%) of polyps were detected. The detection rate of intestinal lesions was 75.11% and indicated significant differences in gender and age. 20 CRCs included 15 of stage 0-I, 4 of stage Ⅱ-Ⅲ and 1 of unknown stage. The CT value of m SDC2 was negatively correlated with the proportion of advanced colorectal neoplasms ( χ2=16.063, P<0.001). The total cost of the screening was 4.339 5 million yuan, the screening benefit was 28.506 2 million yuan, and the benefit-cost ratio was 6.57. Conclusion:The CRC screening strategy of fecal m SDC2 detection combined with colonoscopy has high colonoscopy compliance and detection rate of intestinal lesions, which is conducive to the detection of early CRCs, and has good cost-effectiveness. This study suggests that this method may be applied to the general CRC screening in China and contribute to the prevention of CRC. The CT value of m SDC2 may have a certain suggestion on the malignant degree of intestinal tumors.

Objective:To investigate the effect of subacute exposure of Di(2-ethylhexyl)phthalate(DEHP)on endometrial decidualization and early pregnancy miscarriage in mice.Methods:CD1 mice were orally administrated with 300(low-dose group),1000(medium-dose group),or 3000 mg·kg-1·d-1 DEHP(1/10 LD50,high-dose group)for 28 days,respectively.An early natural pregnancy model and an artificially induced decidualization model were established.The uterine tissues were collected on D7 of natural pregnancy and D8 of artificially induced decidualization,respectively.The effects of a subacute exposure to DEHP on the decidualization of mice were detected by HE staining,Masson staining,TUNEL assay,and Western blotting.A model of spontaneous abortion was constructed in mice after subacute exposure to 300 mg·kg-1·d-1 DEHP,and the effect of impaired decidualization on pregnancy was investigated by observing the pregnancy outcome on the 10th day of gestation.Results:Compared with the control group,the conception rate was significantly decreased in the high-dose DEHP subacute exposure group(P＜0.05).HE staining showed that,compared with the control group,the decidual stromal cells in the low-and medium-dose exposure groups were disorganized,the nuclei of the cells were irregular,the cytoplasmic staining was uneven,and the number of polymorphonuclear cells was significantly reduced.Masson staining showed that compared with the control group,the collagen fibers in the decidua region of the DEHP low-dose group and the medium-dose group were more distributed,more abundant and more disorderly.TUNEL assay showed increased apoptosis in the decidua area compared to the control group.Western blotting showed that the expression of BMP2,a marker molecule for endometrial decidualization,was significantly reduced(P＜0.05 or P＜0.01).The abortion rate and embryo resorption rate were increased,and the number of embryos,uterine wet weight,uterine area and placenta wet weight were decreased in DEHP low-dose group compared to the control group stimulated by mifepristone,an abortifacient drug(P＜0.05 or P＜0.01).Conclusion:Subacute exposure to DEHP leads to impaired endometrial decidualization during early pregnancy and exacerbates the risk of adverse pregnancy outcomes in mice.

Objective:To investigate the application value of fecal Syndecan-2 (SDC2) gene methylated SDC2 (m SDC2) detection in colorectal cancer (CRC) screening among urban residents in Guangzhou City. Methods:A cross-sectional study was conducted in Shitan Town, Zengcheng District, Guangzhou City from July to December 2022. A community-based screening program for CRC was conducted among residents aged 40-74 years old. m SDC2 detection was employed in the participants, and those with positive results should be recommended to receive colonoscopy examination. The positive rate of m SDC2 detection, colonoscopy compliance rate, detection rate of intestinal lesions and clinicopathological characteristics were observed. The relationship between cycle threshold (CT) value of m SDC2 and intestinal lesions was explored. Further, the cost-effectiveness of screening was evaluated. Results:A total of 8 189 fecal samples were collected from 8 877 participants with the recovery rate of 92.25%. 8 048 qualified samples were enrolled in this study, consisted of 3 182 males (39.54%) and 4 866 females (60.46%), with the average age of 56 years old (40-74 years). The positive rate of m SDC2 detection was 7.99% (643/8 048), and the compliance rate of colonoscopy was 73.10% (470/643). 20 cases (4.25%) of colorectal cancer, 109 cases (23.19%) of advanced adenoma, 145 cases (30.85%) of non-advanced adenoma, 79 cases (16.81%) of polyps were detected. The detection rate of intestinal lesions was 75.11% and indicated significant differences in gender and age. 20 CRCs included 15 of stage 0-I, 4 of stage Ⅱ-Ⅲ and 1 of unknown stage. The CT value of m SDC2 was negatively correlated with the proportion of advanced colorectal neoplasms ( χ2=16.063, P<0.001). The total cost of the screening was 4.339 5 million yuan, the screening benefit was 28.506 2 million yuan, and the benefit-cost ratio was 6.57. Conclusion:The CRC screening strategy of fecal m SDC2 detection combined with colonoscopy has high colonoscopy compliance and detection rate of intestinal lesions, which is conducive to the detection of early CRCs, and has good cost-effectiveness. This study suggests that this method may be applied to the general CRC screening in China and contribute to the prevention of CRC. The CT value of m SDC2 may have a certain suggestion on the malignant degree of intestinal tumors.

AIM:To explore the roles and associations of hepatocyte nuclear factor 4 alpha(HNF4A)and mu-protocadherin(MUCDHL)in the kidney of diabetic mice.METHODS:(1)A cohort of six 12-week-old db/m mice and six db/db mice were selected and maintained on a standard diet until 16 weeks.The protein levels of fibronectin(FN),collagen type III(Col-III),E-cadherin,α-smooth muscle actin(α-SMA),HNF4A,Snail and MUCDHL in renal tissues were scrutinized using Western blot.Immunohistochemical staining was conducted to observe the distribution and expres-sion of FN,HNF4A and MUCDHL.(2)Mouse renal tubular epithelial cells(mRTEC)were cultured in vitro and catego-rized into groups:normal glucose(NG)group,high glucose(HG)group,overexpression control groups(NG+vector and HG+vector),overexpression groups(NG+OE-MUCDHL,HG+OE-MUCDHL,NG+OE-HNF4A and HG+OE-HNF4A),knockdown control groups(NG+control and HG+control),and knockdown groups(NG+si-MUCDHL,HG+si-MUCDHL,NG+si-HNF4A and HG+si-HNF4A).The relevant protein levels were also detected by Western blot.RESULTS:(1)In db/db group,elevated body weight,blood glucose and urine albumin-to-creatinine ratio(UACR)indicated significant re-nal injury.Compared with db/m group,the mice in db/db group exhibited increased expression of FN,Col-III,α-SMA and Snail,and decreased expression of E-cadherin,HNF4A and MUCDHL.MUCDHL was predominantly expressed in the apical membrane of renal tubular epithelial cells,FN in the tubular mesenchyme,and HNF4A in the plasma and nu-cleus of renal tubular cells.(2)In HG group,there was an up-regulation in the expression of fibrosis-related proteins and a down-regulation in the expression of E-cadherin,HNF4A and MUCDHL compared with NG group.Overexpression of MUCDHL led to a decrease in the expression of FN,Col-III,α-SMA and Snail proteins,an increase in the expression of E-cadherin and MUCDHL proteins,and unaltered expression of HNF4A.Knockdown of MUCDHL resulted in a reversal of the aforementioned effects,with HNF4A expression remaining unaltered.Overexpression of HNF4A led to an increased ex-pression of MUCDHL,and the expression changes of the remaining indicators were consistent with the overexpression of MUCDHL.Knockdown of HNF4A reversed the aforementioned effects.MUCDHL may represent a downstream target gene of HNF4A.CONCLUSION:The diminished expression of HNF4A and MUCDHL in the renal tubules of diabetic mice implies their involvement in the progression of renal fibrosis in diabetic kidney disease(DKD).HNF4A may potentially impede the progression of renal fibrosis in DKD by up-regulating the expression of MUCDHL.

Objective To investigate the serum levels of homocysteine(HCY)and cystatin C(Cys-C)in patients with antineutrophil cytoplasmic antibody(ANCA)-associated vasculitis(AAV)associated renal injury,and to explore the diagnostic value of combined detection of HCY and Cys-C in ANCA-associated renal injury.Methods We selected 70 ANCA-positive patients with renal injury admitted to the Department of Nephrology in our hospital from January 2016 to January 2019.Another 48 patients with ANCA-positive but not complicated with renal injury were in simple ANCA positive group while 50 patients with physical examination who came to the hospital during the same period were in control group.Healthy patients served as the control group.By comparing the differences in the test results of different groups,the area under the curve(AUC)of each test index for ANCA-positive renal injury was determined,and the correlation and diagnostic value of combined detection of HCY and Cys-C for ANCA-related renal injury were calculated.Results The levels of HCY and Cys-C in the ANCA positive combined with renal injury group,the pure ANCA positive group,and the control group differed significantly(P＜0.05).The positive rates of HCY and Cys-C in the two groups were significantly different(P＜0.05).The sensitivity and specificity of HCY and Cys-C in combination for the diagnosis of ANCA-positive related renal injury were significantly higher than those of the above indicators alone(P＜0.05).ROC curve results showed that HCY combined with Cys-C had a better diagnosis for patients with positive ANCA and associated renal injury and a higher correlation.Conclusion HCY combined with Cys-C is closely correlated to ANCA-related renal injury,and the combined detection has a positive effect on the diagnosis of patients with ANCA-positive related renal injury,and has guiding value for the treatment of patients.

Objective:To analyze assessment value of the hemodynamics of posterior cerebral artery(PCA)detected by transcranial color-coded Doppler(TCCD)ultrasound for occurring cerebral infarction in patients with fetal-type posterior cerebral artery(FTP).Methods:A total of 300 patients with suspected cerebrovascular disease admitted to the Department of Neurology of The Second Hospital of Harbin City from January,2020 to December 31,2023 were retrospectively selected.In these patients,7 cases with cerebral hemorrhage(without complication of cerebral infarction)did not be included.According to the clinical manifestations and the results of imaging examination,293 patients were divided into transient ischemic attack(TIA)group(176 cases)and acute ischemic stroke(AIS)group(117 cases).The differences in FTP detection between TCCD examination and magnetic resonance angiography(MRA)were compared.The differences in PCA hemodynamics among TIA,anterior circulation infarction,posterior circulation infarction and lacunar infarction were compared.The proportions of patients with TIA,anterior circulation infarction,posterior circulation infarction and lacunar infarction combined with FTP were also compared,and the PCA hemodynamics of patients with posterior circulation infarction of bilateral cFTP,unilateral cFTP,bilateral pFTP,unilateral pFTP and nFTP were compared.The proportions of patients with transient ischemic attack(TIA)who occurring AIS at bilateral cFTP,unilateral cFTP,bilateral pFTP,unilateral pFTP and nFTP in short term were compared.The receiver operating characteristic(ROC)curve and area under curve(AUC)were used to assess the predictive value of abnormal PCA hemodynamics of TIA patients,who combined with and/or without FTP,occurred AIS in short term.Results:MRA revealed that a total of 89 patients combined with FTP in 293 patients,of which 14 cases were bilateral cFTP(accounting for 15.73%),and 25 cases were unilateral cFTP(accounting for 28.09%),and 8 cases were bilateral pFTP(accounting for 8.99%),and 42 cases were unilateral pFTP(accounting for 4 7.19%).According to TCCD testing,it was found that a total of 89 cases combined with FTP in 293 patients,of which 16 cases were bilateral cFTP(accounting for 17.98%),and 23 cases were unilateral cFTP(accounting for 25.84%),and 8 cases were bilateral pFTP(accounting for 8.99%),and 42 cases were unilateral pFTP(accounting for 47.19%).There was favorable consistency between TCCD and MRA(Kappa=0.899).Under TCCD,the blood flow velocity(Vp)and resistance index(RI)of systolic stage of PCA in patients with posterior circulation infarction were significantly higher than those in patients with TIA,anterior circulation infarction,and lacunar cerebral infarction(F=15.392,9.032,P<0.05),respectively.Patients with posterior circulation infarction were more likely to occur bilateral cFTP.The Vp,Vm and Vd of patients with bilateral cFTP were significantly higher than those of patients with bilateral pFTP,unilateral pFTP and non-FTP(F=14.932,8.884,6.054,P<0.05),respectively.The proportion of occurring AIS in TIA patients with bilateral cFTP was significantly higher than that in TIA patients with unilateral cFTP,bilateral pFTP,unilateral pFTP and nFTP(Z=6.883,7.568,6.253,6.772,P<0.05),and the proportions of occurring AIS in TIA patients with unilateral cFTP,bilateral pFTP and unilateral pFTP were significantly higher than that in TIA patients with nFTP(Z=5.986,6.877,6.856,P<0.05),respectively.A total of 45 cases(accounting for 25.57%)of 176 TIA patients occurred AAIS within 3 months after they discharged.The AUC value of predictive value of Vp for occurring AIS in 45 TIA patients with FTP was 0.818,which was significantly higher than that(AUC=0.589)for occurring AIS in 131 TIA patients with non-FTP.Conclusion:Patients with posterior circulation cerebral infarction are prone to occur PCA hemodynamic abnormalities.In patients with posterior circulation infarction,the PCA blood flow velocity in patients with bilateral cFTP significantly accelerates.PCA hemodynamic ultrasound examination has a certain of predictive value for occurring AIS in short term in TIA patients who combine with FTP.

Objective To examine the relationship between the expressions of mismatch repair proteins,MutS homolog 2(MSH2)and MutS homolog 6(MSH6),and villin and the pathological features in patients with colon cancer.Methods A total of 310 cases of colon cancer patients who were treated at our hospital between January 2017 and September 2021 were selected.The diagnosis of colon cancer of all patients was verified by pathological evaluation.Immunohistochemistry was used to determine the protein expressions of MSH2,MSH6,and villin.The correlation between the expressions of MSH2,MSH6,and villin and the clinicopathological parameters in patients with colon cancer was analyzed accordingly.Multivariate logistic regression was used to analyze the correlation between the expressions of MSH2,MSH6,and villin and the clinicopathological parameters of colon cancer.Kaplan-Meier survival curve was used to compare the 2-year survival rates of colon cancer patients with different expression levels of the proteins.Results Among the 310 patients with colon cancer,the negative expression rates of MSH2,MSH6,and villin proteins in cancer tissues were 8.71％(27/310),9.35％(29/310),and 46.13％(143/310),respectively.The negative expression rates of the three proteins in tissues adjacent to cancer were 3.23％(10/310),4.19％(13/310),and 9.68％(30/310),respectively.The negative expression rates of the three proteins in cancer tissues were all higher than those in adjacent tissues(P＜0.05).Regression analysis showed that the expression of MSH2 and MSH6 in cancer tissues was correlated with the age,the location of tumor lesions,tumor differentiation degree,and lymph node metastasis in colon cancer patients(P＜0.05).The expression of villin in the cancer tissue is correlated with the depth of tumor infiltration,lymph node metastasis,distant metastasis,and clinical staging status in colon cancer patients(P＜0.05).The 2-year survival rates of patients with negative expressions of MSH2 and MSH6 were 51.85％and 44.83％,respectively,which were lower than those of patients with positive expression of MSH2 and MSH6(79.51％and 80.43％,P＜0.05).Thirteen patients(4.1％)had negative expression of MSH2,MSH6,and villin(referred to as"triple negative expressions")in the cancer tissues,and their 2-year survival rate was 30.77％,which was lower than that of colon cancer patients who did not meet the criteria for triple negative expressions(79.12％[235/297],P＜0.05).Conclusion The expressions of MSH2,MSH6,and villin are closely correlated with the pathological features of colon cancer patients.Evaluating the expression of the three proteins may assist in the clinical diagnosis,treatment,and prognosis evaluation of colon cancer.

Objective:To analyze the predictors of poor outcomes after emergency intracerebral thrombectomy based on the characteristics of cerebral angiography.Methods:A total of 146 patients with acute ischemic stroke (AIS) who received endovascular treatment in Loudi Central Hospital from March 2019 to February 2022 were included in the study, and digital subtraction angiography (DSA) was performed on the patients. The patients were divided into a good prognosis group (95 cases) and a poor prognosis group (51 cases) by the modified Rankin scale 3 months after operation. Gender, age, time from onset to visit, time from onset to puncture, proportion of intravenous thrombolysis, occlusion site, treatment strategy, National Institute of Health Stroke Scale (NIHSS) score, core infarct volume, ischemic hypoperfusion volume, collateral circulation classification, and venous drainage status were compared between the two groups score; Logistic regression was used to analyze the risk factors affecting the poor prognosis of patients; Receive Operating Characteristic (ROC) curve was used to analyze the predictive value of collateral circulation classification and venous drainage status score for poor prognosis of patients, and the differences in general data and imaging data were compared between groups with different collateral circulation grades and venous drainage status.Results:Compared with the good outcome group, the time from onset to visit, NIHSS score, core infarct volume, ischemic hypoperfusion volume, the proportion of thrombectomy alone, and collateral circulation classification in the poor outcome group [2 (2, 3) levels. 2 (1, 2) level] and venous drainage score [5 (4, 6) points vs. 6 (6, 8) points] increased ( P<0.05), and the proportion of recanalization grade 2b/3 decreased ( P<0.05); NIHSS score, collateral circulation grade and venous drainage status were predictors of poor outcome within 3 months after mechanical thrombectomy ( OR = 2.51, 1.93, 2.61, P<0.05); collateral circulation grade and venous drainage score predicted mechanical thrombectomy in patients with AIS, the area under curve (AUC) of poor outcome after thrombectomy were 0.714 and 0.829, respectively; the time from onset to visit between patients with poor collateral circulation, moderate and good AIS [(236.95 ± 21.03) min, (250.41 ± 21.32) min, (255.72 ± 20.98 min)], core infarct volume [52 (17, 80) ml, 25 (15.5, 30) ml, 15 (10, 25) ml] and venous drainage scores [5 (4, 6) points, 5 (5, 8) points, 5 (5, 8) points] were significantly different ( P<0.05); time from onset to visit in patients with poor venous drainage, moderate and good AIS (234.81 ± 21.22 min), (256.83 ± 20.88) min, (258.97 ± 21.35) min], core infarct volume [17(13, 45) ml, 26(25, 29) ml, 20 (11, 29) ml] and collateral circulation classification [2 (1, 2) level, 2 (1, 3) level, 2 (2, 3) level] were significantly different ( P<0.05). Conclusions:Collateral grading and venous drainage scores based on DSA imaging were predictors of poor outcomes within 3 months of mechanical arterial thrombectomy in patients with AIS.