ObjectiveTo study the expression of transforming growth factor beta-1 (TGF-?1),platelet endothelial cell adhesion molecule-1 (PECAM-1,CD31) in prostate cancer tissues and their correlation with prostate specific antigen (PSA) values and TNM staging.MethodsUsing immunohistochemical method 36 prostate cancer specimens were tested for TGF-?1 expression, and using CD31 for marking vascular endothelial cells,the tumor microvascular density (MVD) was counted. Twelve normal tissue specimens were taken from the non-tumorous tissues adjacent to the prostate cancer as controls. The correlation of TGF-?1 expression and MVD with PSA values,TNM staging,pathologic grading and bone metastasis was analyzed in combination with the clinical data.ResultsThe positive expression rate of TGF-?1 in prostate cancer was 88.89%(32/36),while it was 16.67%(2/12)in controls, showing statistically significant difference between them ( P 20 ng/ml, MVD was (81.5?12.2) mm 2 ( P 20 ng/ml,23 cases had the TGF-?1 expression rate of 100% ( P

Objective To discuss the diagnostic value of ultrasonography in piriformis syndrome . Methods Ultrasonography was performed in thirty‐eight patients with unilateral piriformis syndrome and forty healthy volunteers . The morphological structures and the internal echoes of their bilateral piriformises and sciatic nerves were observed and their thicknesses were measured . These parameters of the patients and voluteers were recorded and compared . Results The ultrasonographic images of piriformis and sciatic nerve of the healthy voluteers showed no abnormal change . The thickness difference of their bilateral piriformises and sciatic nerves had no statistical significance ( P > 0 .05 ) . The ultrasonography image of the morphological structure and the internal echo of the sick side piriformis and sciatic nerve of the patients with piriformis syndrome showed a change ,that the sick side piriformis was significantly thicker than the healthy side piriformis [(25 .74 ± 3 .12) mm vs (22 .48 ± 2 .60) mm , P < 0 .05] . The area under the operator characteristic curve ( AUC ) for the thickness difference of bilateral piriformises in diagnosing piriformis syndrome was 0 .896 ,with the optimal cut‐off value of 2 .15 mm . However ,the thickness difference of their bilateral sciatic nerves had no statistical significance ( P >0 .05) . Conclusions Ultrasonography can show piriformis and sciatic nerve clearly . The ultrasonographic images and the thickness difference of the bilateral piriformises is helpful to diagnose piriformis syndrome ,and can provide more informations for clinic .

Objective Set up virtual experimental teaching platform of medical molecular biology, and explore effective operating system of virtual experiment teaching. Methods 400 students of majored in clinical medicine in Kunming Medical University in Grade 2011 were randomly divided into the virtual experiment teaching group (n = 195 ) and the traditional experiment teaching group (n = 205 ). We realized the teaching effect by questionnaire survey, and analyzed the final exam results of two groups statistically. Results The experiment teaching way of virtual experiment has been widely accepted by students, and it could help students to understand and master experiment operations and theory knowledges.No statistical difference was found between two groups on the final exam. Conclusion Virtual experiment technology as a new teaching method has a lot of advantages, but it can't completely replace traditional experiments. We should use both the two kinds of teaching methods reasonally in the medical molecular biology experiment teaching.

Objective To explore the labeling method of rat adipose-derived stromal cells,and observe the stem cell characteristics and the activities of EGFP-positive adipose-derived stromal cells (EGFP-ADSCs) in vitro and in vivo.Methods ADSCs were transfected for 12 h with enhanced green fluorescent protein gene (EGFP) carried by lentivirus(Lv-EGFP) vector at different value of MOI (0,5,10,25,50,100,respectively).The rate of EGFP expression and fluorescence intensity were evaluated by flow cytometric analysis and fluorescence microscopy,and cell viability was detected by MTT-test after transfection.Secondly,cells were exposed either to adipogenic medium or osteogenic medium,then stained with Oil Red O and Alizarin Red S.Cell growth was investigated on frozen longitudinal sections when EGFP-ADSCs were injected into acellular nerves to build tissue-engineered peripheral nerves repairing sciatic nerve defects in rats for 1 week in vivo.Results EGFP-positive rate and fluorescence intensity peak at 4 days after transfection.The rate of EGFP expression was 0.13％,31.09％,75.33％,92.66％,96.70％,98.38％ for MOI =0,1,5,25,50,100,respectively.The positive rate between the experimental group and control (MOI =0) existed significantly difference (P ＜ 0.05) ; the difference between MOI =1,5 groups and MOI =25,50,100 groups were also observed (P ＜ 0.05).There was no statistical difference in EGFP-positive rate and cell proliferation activity among MOI =25,50,100 groups (P ＞ 0.05).MOI =25 was chosen as best scheme to transfect ADSCs for subsequent experiments.Osteogenic and adipogenic differentiation for 20 days,orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red O staining.At 1 week in vivo,EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections.Conclusion Lv-EGFP transfection does not affect the ADSCs activity and their osteogenic and adipogenic differentiation,so could be as a tracing method for ADSCs-tissue-engineered peripheral nerves repairing nerve defects.

Objective To investigate the diagnostic value of multiparametric MRI in early prostate cancer(PCa) based on PI-RADS version 2.Methods 27 surgically-proved early PCa patients were collected in this retrospective study.T2WI,DWI and DCE were evaluated by two blinded radiologists.By 12 sub-region classification method the possibility of the presence of cancer at each sub-region was scored according to the PI-RADS V2.The receiver operating characteristic (ROC) curve was used to analyze the diagnosic efficacy of the following 4 protocols:T2WI alone(protocol 1),T2WI+DWI(protocol 2),T2WI+DCE(protocol 3),T2WI+DWI+DCE(protocol 4).The sensitivity,specificity and accuracy for each protocol were calculated.The average scores of cancerous sub-regions and non-cancerous sub-regions were calculated and the independent sample t test was used to compare the four protocols.Results 324 sub-regions were analyzed in 27 early PCa patients and then divided into 119 cancerous sub-regions and 205 non-cancerous sub-regions,including 64 peripheral zone cancerous sub-regions and transition zone cancerous sub-regions.In protocol 1-4, the average scores of cancerous sub-regions in orderwere 3.13±1.19,3.27±1.15,3.28±1.23, 3.33±1.16,respectively.Non-cancerous sub-regions's scores in order were 1.98±0.90,1.91±0.91, 2.03±0.99,1.94±0.96 respectively and there were significant differences among each protocol (P<0.05).The area under the ROC curve of the 4 protocols for region-based analysis were displayed in descending order: protocol 4 (0.819), protocol 2 (0.810), protocol 3 (0.772), protocol 1 (0.765) and there were no significant differences between any two protocols (P>0.05).In four protocols, the sensitivity in order were 45.40%, 56.30%, 59.70%, 61.34%, while the specificity in order were 95.10%, 96.10%, 89.80%, 96.60%, and the accuracy in order were 76.85%, 81.48%, 78.70%, 83.65%.Conclusion Multiparametric MRI can improve the diagnostic accuracy for the detection of early PCa, and T2WI+DWI+DCE is with the highest value.The PI-RADS V2 system is a better semi quantitative method for evaluation of early PCa.

Objective:To investigate the effects of fluoride exposure on proliferation, apoptosis and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in mice.Methods:BMSCs were isolated and cultured from femur bone marrow of C57BL/6 mice (6 - 8 weeks). The cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. The cells were cultured in media with a final fluoride concentration of 0.0, 0.1, 1.0, 5.0, 10.0, 15.0, 20.0 and 40.0 mg/L, respectively. The effects of different fluoride concentrations on BMSCs cell proliferation (CCK8 method), apoptosis (flow cytometry analysis), osteogenic differentiation ability [alizarin red and alkaline phosphatase (ALP) staining] were detected. Western blot was applied to detect the levels of apoptosis-related proteins [poly ADP-ribose polymerase (PARP)], mitogen-activated protein kinase (MAPK) pathway member proteins [extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase (JNK), p38 and phosphorylated ERK, JNK, p38 (p-ERK, p-JNK, p-p38)], osteogenic differentiation-related protein [Runt-related transcription factor 2 (Runx2), ALP] and Wnt/β-catenin pathway member proteins [glycogen synthase kinase-3β (GSK3β), phosphorylated GSK3β (p-GSK3β) and β-catenin]. Immunocytofluorescense staining was applied to evaluate the expression levels of p-GSK3β and β-catenin. The two pathways (MAPK and Wnt/β-catenin) were blocked by SP600125 and DKK-1, respectively, to testify their involvement in mechanisms of apoptosis and osteogenic differentiation.Results:The mouse BMSCs were successfully isolated and cultured. Flow cytometry analysis showed that the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. The comparison of cell proliferation at three time points (24, 48 and 72 h) in each concentration group was statistically significant ( F = 65.36, 160.04 and 365.32, P < 0.001), and the comparison of early apoptosis (24 h) in each concentration group was statistically significant ( F = 214.04, P < 0.001); compared with the 0.0 mg/L group, the cell proliferation in 15.0, 20.0 and 40.0 mg/L groups decreased, and the early apoptosis rate in 10.0, 15.0 and 20.0 mg/L groups increased ( P < 0.05). When cells were treated with 15.0 mg/L fluoride for 0 - 24 h, the p-JNK/JNK ratio was higher at 2, 4, 8, 12, 18 and 24 h compared with that at 0 min ( P < 0.05); compared with the fluoride group (15.0 mg/L), the early apoptosis rate of cells after SP600125 block decreased ( P < 0.05), and the protein expression levels of PARP and p-JNK decreased ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, in 0.1 and 1.0 mg/L groups ALP staining was enhanced and the number of calcified nodules increased, and the protein expression levels of Runx2 and ALP in the 0.1 and 1.0 mg/L groups were higher ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, the p-GSK3β/GSK3β ratio and β-catenin protein level were significantly higher in the 0.1 and 1.0 mg/L groups ( P < 0.05); and compared with the fluoride group (1.0 mg/L), addition of DKK-1 significantly decreased the protein expression levels of p-GSK3β and β-catenin and reduced the nuclear entry of β-catenin, and ALP staining decreased and the number of calcified nodules decreased. Conclusions:High concentration of fluoride (> 10.0 mg/L) inhibits the proliferation and promotes apoptosis of BMSCs, while low concentration of fluoride (0.1, 1.0 mg/L) promotes osteogenic differentiation. The MAPK/JNK pathway and the classical Wnt pathway are involved in the above cellular processes, respectively.

Objective ToexplorethevalueofMRIindifferentiatingbetweenmass-formingchronicpancreatitis(MFCP)andpancreatic carcinoma(PC).Methods MRIdataof19caseswith MFCP,36caseswithPCand30normalcontrolcasesconfirmedbypathology orfollow-upwereanalyzedretrospectively.AllofthesubjectsunderwentroutineMRIandDWIscan.MRIcharacteristicsofdiseases andnormalpancreaswereanalyzed,andADCvalueswerecomparedamongthethreegroups.Results TheaverageADCvalueofthe MFCPgroupwas(1.41±0.25)×10-3 mm2/s,higherthanthatofthePCgroup (1.13±0.11)×10-3 mm2/s(P<0.05),andlower thanthatofthenormalcontrolgroup(1.50±0.27)×10-3 mm2/s(P<0.05).IntheT2WI,enhancedscanningarterialphase,andenhanced scanningportalphase,thesignalcharacteristicsofthelesionswerestatisticallydifferentbetweentheMFCPandPCgroup (P<0.05).The sensitivityandspecificityofthecombinationT2WI,enhancedarterialimagingandADCvaluewere86.9%and88.9%indifferentiatingMFCPand PC,whichwasbetterthananysinglemethod.Conclusion MRImulti-sequencecombinationisoneoftheeffectivemethodsforidentifyingPCand MFCP,andhasreferencevalueforclinicaldiagnosis.

OBJECTIVETo establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs.

METHODSA total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs.

RESULTSAfter separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%).

CONCLUSIONManual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

Objective:To explore whether left ventricular interstitial fibrosis is associated with left atrial enlargement and left atrial dysfunction in patients of hypertrophic cardiomyopathy(HCM) with preserved ejection fraction.Methods:From October 2018 to September 2021, 59 HCM including 30 with enlarged maximal left artrial volume index (LAVI max), 29 with normal LAVI max and 28 age-and gender-matched controls were retrospectively enrolled. Imaging protocol included cine sequence, late gadolinium enhancement and T 1 mapping.The relationships between left ventricular mass index (LVMI), quantitative myocardial fibrosis and left atrial-related indexes were analyzed. One-way analysis of variance with Bonferroni post hoc correction or Kruskal-Wallis was performed for continuous variables. Categorical variables were assessed using the Chi-square test or Fisher′s exact test. Pearson or Spearman analysis was used for linear or monotonic nonlinear correlations. Results:The left ventricular end-diastolic volume index, left ventricular end-systolic volume index, left ventricular cardiac output and LVMI of HCM with enlarged LAVI max group were higher than HCM with normal LAVI max group and control group( P<0.05).Correlation analysis showed that LVMI correlated positively with LAVI max( r=0.780, P<0.001) and minimal left artrial volume index (LAVI min) ( r=0.816, P<0.001), extracellular volume correlated positively with LAVI max( r=0.462, P<0.001) and LAVI min( r=0.483, P<0.001),%LGE was correlated positively with LAVI max( r=0.311, P<0.05) and LAVI min( r=0.327, P<0.05),left ventricular index interstitial volume was correlated negatively with left atrial ejection fraction of reservoir ( r=-0.669, P<0.001),left atrial ejection fraction of conduit ( r=-0.472, P<0.001),left atrial ejection fraction of pump ( r=-0.518, P<0.001)and left atrial expansion index( r=-0.626, P<0.001). Conclusion:There is association between LVMI and fibrosis and left atrial enlargement and phases dysfunction in HCM with preserved ejection fraction.

OBJECTIVE：To establish a method for the determination of aloesin in plasma of rats ，and to investigate pharmacokinetic characteristics of aloesin. METHODS ：The plasma samples were precipitated with methanol. Using aloeresin D as internal standard ，the plasma concentration of aloesin was determined by LC-MS/MS. The determination was performed on Synergi Hydro-RP column with mobile phase consisted of 0.1‰ formic acid-methanol （gradient elution ）at the flow rate of 0.50 mL/min. The column temperature was 30 ℃，and sample size was 5 µL. The electrospray ionization source was applied to carry out negative ion detection with multiple reaction monitoring mode . The ion transitions for quantitative analysis were m/z 393.1→272.9（aloesin） and m/z 555.3→144.9（internal standard ），respectively. The concentration of aloesin in venous blood was determined by above method at 0.083，0.167，0.333，0.667，1，1.5，2.5，4，6，8，10 h after intravenous injection （3.35 mg/kg）and intragastric administration（16.75 mg/kg）of aloesin. DAS 3.0 software was used to calculate pharmacokinetic parameters. RESULTS ：The linear range of aloesin were 1-600 ng/mL（r＝0.994 5）. The lower limit of quantification was 1 ng/mL，and RSDs of within and between batches were less than 15％；accuracies within and between batches were within ±15％. The matrix factors were （92.74± 4.33）％-（94.84±2.57）％，and extraction recoveries were （69.04±2.13）％-（75.03±2.84）％；the deviation between the measured results of the stability test and the theoretical values were within ±15％. After intravenous injection and intragastric administration of aloesin ，main pharmacokinetic parameters were as follows ：cmax were（10 693.3±2 745.3）and（223.3±36.2）ng/mL；t1/2 were （2.45±1.45）and（3.33±1.91）h；AUC0－24h were（4 190.6±883.6）and（1 210.1±93.9）ng·h/mL（n＝3）. Absolute bioavailabi- lity was 11.13％. CONCLUSIONS ：The established method is rapid and sensitive for plasma determination of aloesin ，and suitable for its pharmacokinetic study.