Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.
Cartilage/*cytology ; Cell Proliferation ; Cell Transformation, Viral ; Cells, Cultured ; Fetus ; Simian virus 40/*genetics ; Stem Cells/*cytology ; Transfection

Abstract: Objective　To explore the correlation between monocyte to high-density lipoprotein cholesterol ratio (MHR) and insulin resistance (IR) in male patients with type 2 diabetes mellitus (T2DM) combined with metabolic-related fatty liver disease (MAFLD). Methods A total of 454 male patients with T2DM combined with MAFLD in National Metabolic Management Center (MMC) of the Affiliated Hospital of Jiangsu University from May 2018 to July 2020 were enrolled. The general clinical data of subjects were collected, blood routine and biochemical indexes were tested, homeostasis model insulin resistance index (HOMA-IR) was calculated, visceral fat area (VFA) and subcutaneous fat area (SFA) were measured. Accordingtothe MHR quartile, patients were divided into group Q1 (MHR≤0.38), group Q2 (0.380.64) to compare the differences in measured indicators above. In addition, patients were divided into two groups according to HOMA-IR, HOMA-IR<2.5 and HOMA-IR≥2.5, and the differences in MHR were compared. Results The patients were divided into four groups according to MHR:group Q1 (n=115), group Q2 (n=110), group Q3 (n=120) and group Q4 (n=109). Fasting insulin (FINS) were respectively 6.17(4.20,9.76), 7.73(4.94,10.66), 8.92(5.32,11.33) and 9.13(5.25,12.27) mU/L, 2-hour postprandial insulin were 22.75(12.87,39.59), 27.55(16.44,39.77), 30.98(17.46,43.11) and 31.28(18.54,45.92) U/L. HOMA-IR were 3.12(1.63,4.25), 3.72(2.26,4.66), 3.87(2.48,5.44) and 3.95(2.42,5.31). Neutrophil (Neu) were 3.10(2.60,3.70), 3.20(2.50,3.93), 3.60(2.80,4.28), 4.20(3.30,5.00)×109/L. Subcutaneous fat area (SFA) were (181.27±53.60), (192.64±62.41), (199.53±61.40) and (203.69±71.51) cm2. They all increased gradually. However, the levels of high-density lipoprotein cholesterol (HDL-c) [1.18(1.06,1.35), 1.02(0.86,1.17), 0.96(0.80,1.03) and 0.80(0.69,0.92) mmol/L] and low-density lipoprotein cholesterol (LDL-c) [(3.00±0.79), (2.76±0.83), (2.67±0.85) and (2.59±0.92) mmol/L] decreased gradually. Pearson's or Spearman's correlation analysis showed that MHR was positively correlated with FINS, 2-hour postprandial insulin (2hINS), HOMA-IR, VFA and SFA (r=0.190, 0.153, 0.184, 0.114, 0.127, P<0.05). The coronary heart disease history, systolic blood pressure,diastolic blood pressure,fasting plasmaglucose (FPG), FINS, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood uric acid (Ur), body mass index (BMI), VFA, SFA and MHR of patients in group HOMA-IR≥2.5 were higher than group HOMA-IR<2.5 (P<0.05). Conclusion　MHR is positively correlated with IR in male patients with T2DM combined with MAFLD, and as MHR increases, the degree of IR is higher.

Objective To study the biological effects of pulsed electromagnetic fields (PEMFs) on the pro-liferation of immunomagnetically separated human precartilaginous stem cells (PSCs) in vitro. Methods The cells from an aborted fetus's metaphysis were digested using collagenase. The PSCs were isolated by magnetic cell sorting (MACS), then subcultured and amplified. Flow cytometry, immunohistochemistry, immunofluorescence and RT-PCR analysis were performed to identify the purified PSCs. The PSCs were stimulated by PEMFs at 50 Hz frequency and 1 mT intensity. Cell proliferation was measured at different time points using methyl thiazolyl tetrazolium ( MTT), and the cell growth curve was plotted. Flow cytometry was applied to measure the cell cycle and apoptosis. Results The PSCs were successfully cultured. There was fibroblast growth factor receptor-3 (FGFR-3) on their sur-face. Cell proliferation was promoted after 4 and 6 days of PEMF stimulation. The percentage of cells at the S phase was higher than in a control group. Early, late and total rates of apoptosis in the experimental group decreased signifi-cantly. Conclusion PEMFs can enhance the proliferation and inhibit the apoptosis of human PSCs, and it is possi-ble to cultivate the high density human PSCs in vitro.

AIM:To investigate the effect of cervical lymphatic blockage(CLB)on blood pressure(BP)in conscious unrestrained rats.METHODS:Sprague-Dawley(SD)rats were adopted and randomly divided into two groups as Sham operated group and CLB group.By means of monitoring hemodynamic change in conscious unrestrained rats,twenty-four-hour blood pressure(SBP,DBP and MAP),blood pressure variability(BPV),heart rate(HR)and heart rate variability(HRV)were respectively measured before sham and cervical lymphatic blockage operation as their baseline and at 1st,3rd,7th,11th,15th days after operation.Meanwhile,arterial baroreflex sensitivity(BRS)was measured before and at 1st,7th,15th days after operation.RESULTS:SBP,DBP,MAP and HR significantly decreased at 1st day after CLB operation and their lowest values appeared at 7th day.The tendency of their alternation was descending early and then ascending whereas reverse alterations of BPV and HRV were observed.BRS reduced in CLB rats with no apparent recovery from 7th day.CONCLUSION:CLB results in reduction of blood pressure and dysfunction of nervous regulation on cardiovascular system in conscious unrestrained rats.

Objective To evaluate the clinical diagnostic value of 64-slice computed tomography coronary angiography (CTCA) for the detection of significant coronary artery stenosis ( ≥50% lumen reduction). Methods We enrolled 61 patients (47 male, 14 female; mean age 62.8 ± 9.7 years) with suspected coronary artery disease into the study and performed selective coronary angiography (SCA) within 2 weeks after CTCA examination. Results One patient was excluded from the analysis because of unsuccessful CTCA evaluation as a result of severe coronary artery calcification in 4 vessels. 240 vessels of remaining 60 patients were achieved good coronary artery imaging by CTCA. Sensitivity,specificity and positive and negative predictive value of CTCA for detecting significant coronary artery sternosis on a per-vessel basis were 90.0 % ,91.9% ,84. 7% and 94.8% ,respectively. Conclusions Our data indicated that 64-slice CTCA is a noninvasive method to reliably rule out significant coronary sternosis with high diagnostic value in negative prediction.

Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; DNA, Neoplasm ; genetics ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Infant ; Lymphoma, B-Cell ; diagnosis ; genetics ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; genetics ; Male ; Middle Aged ; Paraffin Embedding ; Polymerase Chain Reaction ; Young Adult

Objective To investigate the expression and significance of mRNAand exon mutationof NT5C2 gene in acute lymphoblastic leukemia (ALL) bone marrow.Methods Case control study design was used in this study.Bone marrow samples were collected from ALL patients in Anhui Provincial Cancer Hospital in recent 4 years.The patientswere divided into the initial diagnosis group , the complete remission group and the recurrence group.And they could specifically be divided into 36 patients initially diagnosed, 36 patients who achievedcomplete remission and 16 patients who relapsed with children B -ALL,15 patients initially diagnosed,15 patients who achievedcomplete remission and 9 patients who relapsed with children T -ALL, 18 patients initially diagnosed,18 patients who achievedcomplete remission and 12 patients who relapsed with adult B-ALL, and 11 patients initially diagnosed,11 patients who achievedcomplete remission and 6 patients who relapsed with adult B -ALL.The initial diagnosis,complete remission and recurrence samples were matched.8 children and 8 adults without hematologic malignanciewere used as controls .Real-time PCR was performed to detect the level of NT5C2 mRNAin ALL patients.The exons of NT5C2 gene were cloned and sequenced for the common mutations in all cases .The results of NT5C2 mRNA levels in different groups were performed using non -parametric test by SPSS16.0 analytics software, and then non-parametric test together with correlation analysis was analyzed between NT 5C2 mRNA levels of different initial diagnosis groups and gender, age, leukocyte level and risk classification .Results (1)The expression of NT5C2 mRNA levels of recurrence group were higher than that of initial diagnosis group ,complete remission group and controls in children and adult B -ALL respectively(P <0.01).(2)NT5C2 mRNA expression in children and adult T-ALL showed no difference in initial diagnosis ,complete remission, recurrence group and controls (P >0.05).(3)NT5C2 mRNA expression of initial diagnosis group in children and adult B -ALL and T-ALL was not correlated with risk classification (P >0.05).(4)A newheterozygousmutation p.P414A of NT5C2 was discovered in a recurrencesample.Conclusions (1) High expression ofNT5C2 mRNA is associated with recurrence inchildren and adult B-ALL, and it may be an indicator of monitoring recurrence .(2)The incidence of exons mutation of NT5C2 gene in ALL is low in China.

Objective To evaluate the safety and efficacy of ultrosonography-guided two-stage ERCP for management of emergent pancreatobiliary diseases during pregnancy.Methods The clinical data of 32 consecutive pregnant women with suspected emergent pancreatobiliary diseases,who were treated with two-stage ultrasonography-guided ERCP from January 2005 to May 2010,were analyzed retrospectively.All patients were treated with ERCP in two stages. In stage Ⅰ,all the patients underwent ultrasonographyguided biliary cannulation,sphincterotomy with small incision and plastic stent placement during pregnancy.In stage Ⅱ,routine ERCP was performed in two weeks after delivery.The success rate of first ERCP,relief of clinical manifestation,improvement of lab indices,maternal and fetal complications were evaluated.Results All patients presented with fever,biliary colic and jaundice.Abdominal ultrasonography or magnetic resonance cholagiopancreatography (MRCP) revealed dilated common bile duct (CBD) in all patients,with CBD stones in 24 and acute cholangitis in 8.Bile duct cannulation and stent placement were successful in all patients.All patients markedly improved after first ERCP,with laboratory indices significantly improved at 1week after the procedure ( P ＜ 0.01 ).In stage Ⅱ,CBD stones were retrieved in 24 patients under ERCP,including 2 cases of mechanical lithotripsy.Bile duct stenosis was found in 3 patients and plastic stents were replaced.Biliary bleeding occurred in 1 case,mild acute pancreatitis in another,with a complication rate at 6.25％.Patients and babies were followed up for 12 months and found to be healthy.Conclusion Twostage ultrosonography-guided ERCP is safe and effective for pregnant patients with emergent pancreatobiliary diseases,which might be regarded as the first choice for such patients to avoid radiation.

G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. Conclusion It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.