OBJECTIVETo explore the effects and possible mechanisms of decitabine on Molt4 in vitro.

METHODSEffects of decitabine on cells proliferation were detected by using CCK-8, the apoptosis by Annexin V-FITC, cell cycles by propidium iodide-FACS. Discrepancy genes were screened by RNA-seq technique. The CpG methylation of lactoferrin (LTF) gene in Molt4 cells were identified by Bisulfite sequencing PCR (BSP). The expression of LTF mRNA in Molt4 by RT-PCR and LTF protein expression were analyzed by Western blot.

RESULTSDecitabine effectively inhibited proliferation and induced apoptosis for Molt4 cells by an time- and dose-dependent manners. Cell cycles were arrested at the G₀/G₁ phase. The promoter methylation degree of LTF gene in Molt4 cells was 72.3% before decitabine treatment and decreased to 45.0% after treatment with 0.50 μmol/L decitabine for 72 h. After the reduction of methylation, expression of its mRNA and protein increased, meanwhile caspase 3 and caspase 9 protein expression levels increased.

CONCLUSIONThe demethylating drug decitabine can induce apoptosis, detain cell cycle at phase G₀/G₁, inhibit proliferation and up-regulate LTF gene expression in Molt4 cells. LTF may become a new target for acute T lymphoblastic leukemia.

Antimetabolites, Antineoplastic ; Apoptosis ; Azacitidine ; analogs & derivatives ; Caspase 3 ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Methylation ; Humans ; Lactoferrin ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Promoter Regions, Genetic

Objective To analyze the stability and accuracy of the equipment for three-dimensional ultrasound-based image-guided radiation therapy (3DUS-IGRT) in daily practice, and to provide a basis for clinical application of radiotherapy for soft tissue tumors.Methods A specific calibration phantom was used for continuous calibration and quality control of the 3DUS-IGRT equipment in a year.The method for daily quality control of ultrasound-guided equipment was explored, and its stability and accuracy were monitored.Results The phantom position errors in both Sim and Guide stations of the 3DUS-IGRT equipment were within 1 mm.Conclusions The 3DUS-IGRT equipment has a stable performance with the support of a complete set of stringent and accurate calibration and quality control, which provides a new image-guided method for precise radiotherapy for soft tissue tumors.

Objective To explore the feasibility and effectiveness of external ureteral clipping and insertion of double J tube in laparoscopic ureterobladder reimplantation. Methods The clinical data of 21 patients with lower ureteral stricture undergoing laparoscopic ureterobladder reimplantation from Mar. 2014 to Mar. 2016 were retrospectively analyzed. Twelve patients receiving conventional laparoscopic ureterobladder reimplantation were assigned to control group, and 9 patients receiving laparoscopy-assisted external ureterobladder reimplantation were assigned to improvement group. The ureter of the patients in the improvement group was separated under laparoscopy, the narrow ureter was severed, and the proximal ureter was taken out from the 12-mm Trocar. Then, the broken end of ureter was clipped under euthyphoria, and the double J tubes were inserted and fixed on the ureteral mucosa with 4-0 absorbable sutures and then sent back to the pelvis. The bladder was mobilized under laparoscopy, and the bladder and ureter were anastomosed. Results The surgery was successfully completed in all the 21 patients without transferring to open surgery or urine leakage after operation. The operation time was significantly shorter in the improvement group versus the control group ([77±17] min vs [104±20] min, P<0.05). All patients were followed up for at least 6 months after operation. Both ultrasonography and intravenous pyelography showed no ureteral anastomotic stricture. The hydronephrosis was alleviated to some extent. Cystography showed that 2 patients had reflux, with 1 case in each group. Conclusion In laparoscopic ureteral reimplantation, external ureteral clipping and insertion of double J tube are minimally invasive, and can shorten operation time and make the surgery less complex.

[Abstract] Objective To evaluate the clinical value of Sysmex XN-9000 hematology analyzer for detecting peripheral blood nucleated red blood cells (NRBC). Methods A total of 16 273 peripheral blood samples were collected and examined by Sysmex XN-9000 hematology analyzer and microscopic manual detection. The sensitivity, specificity and positive prediction value and negative predictive value of NRBC were measured by manual measurement under microscope. The 248 specimens positive by both methods were used as subjects, and the correlation between the two methods for detecting NRBC was analyzed. The disease types of 277 patients positive of NRBC were analyzed by microscopic examination. Results The sensitivity of NRBC detected by Sysmex XN-9000 hematology analyzer was 89.5%, specificity was 99.6%, positive predictive value was 80.5%, and negative predictive value was 99.8%. There was a positive correlation between the percentages of NRBC detected by the two methods (rs＝0.813, P0.001). Among the 277 NRBC-positive patients, 173 had hematological diseases and 104 had no hematologic diseases, and there were significant differences in NRBC counts between the two groups (median: 0.38×109/L vs 0.16×109/L, P0.05), and the percentages of NRBC were not significantly different (median: 2.95% vs 3.60%, P＝0.835 1). Among patients with hematological diseases, NRBC was mainly present in patients with acute lymphoblastic leukemia (31 cases), acute myeloid leukemia (55 cases), malignant lymphoma (39 cases) and multiple myeloma (18 cases). Among patients without hematologic diseases, NRBC was mainly present in those with solid cancer (24 cases) and cirrhosis hemorrhage (36 cases). Conclusion The Sysmex XN-9000 hematology analyzer can detect NRBC with high accuracy, and it thus has a promising clinical application value.

[Abstract] Objective To analyze the difference of the carbohydrate antigen 242 (CA242) results measured by Luminex flow cytometry fluorescence immunoassay with different calibrations and different ambient temperatures, so as to evaluate the effect of ambient temperature on the measurement results. Methods Two CA242 samples with different concentrations (median- and high-value) were selected and the determination was repeated at 11 temperature points (18.3, 20.1, 21.5, 22.2, 24.1, 25.0, 25.4, 26.5, 27.4, 28.5, and 30.0 ℃) for 5 times by Luminex flow cytometry fluorescence immunoassay. The average value of the detection result at the calibration temperature (25 ℃) was taken as the reference value, and the biases of the detection results at different ambient temperatures were calculated. Forty-nine CA242 specimens covering high-, median- and low-values were examined at 20, 25 and 30 ℃ by Luminex flow cytometry fluorescence immunoassay, and the results were compared and correlation analysis was carried out. Results The biases of the median- and high-value samples at 18.3 ℃ were －35.6% and －29.4%, respectively. The difference between the measurement results at 20 ℃ and 25 ℃ was significant for the 49 specimens (P0.001), and there was a linear correlation between them (Y＝0.676 9X＋0.374 7, R2＝0.990 5). The difference of measurement results was significant between 30 ℃ and 25 ℃ (P0.001), and there was a linear correlation between them (Y＝0.896 6X＋0.227 0, R2＝0.999 4). Conclusion Too low or too high ambient temperature has a negative effect on the determination of CA242 by Luminex flow cytometry fluorescence immunoassay. The ambient temperature should be kept relatively constant and controlled within the range of calibration temperature ±4 ℃.

Objective To investigate the preliminary experience of laparoscopic partial nephrectomy in patients with adherent perinephric fat. Methods We retrospectively analyzed the clinical data of 16 patients with adherent perinephric fat undergoing laparoscopic partial nephrectomy by the single surgeon in the Department of Urology of Changhai Hospital, Second Military Medical University between Jan. 2016 and Dec. 2016. For the patients with serious adherent perinephric fat, the tumor could be located by the intra-operative B-mode ultrasound after exposing the general boundaries of tumor and normal renal tissues, and the boundaries were marked with electric knife. The adherent adipose tissue and tumor were both resected with scissors along the marking line after blocking the blood flow. Results The operations were successfully completed in all the 16 patients, and the intra-operative B-mode ultrasound positioning was used in 5 cases. The maximal diameter of tumor was 2.1-3.5 (2.8±0.4) cm, the Mayo adhesive probability (MAP) score was 3-5 (3.8±0.7), the renal artery occlusion time was 20-31 (25.6±3.1) min, and the operation time was 90-133 (112.0±10.5) min. During a follow-up of 6-20 months, no patients had recurrence or metastasis. Conclusion It is important to locate the tumor boundaries during laparoscopic partial nephrectomy in patients with adherent perinephric fat, and the intra-operative B-mode ultrasound can be used if necessary. The tumor and adherent adipose tissue must be both resected to avoid forced separation of perirenal fat from renal capsule.

Objective: To observe the expression of SRYCsex determining region Y)-box 9 (SOX9) and collagen type E alpha 1 (COL2A1) in human cervical intervertebral disc nucleus pulposus (NP) tissues of various degeneration degrees, so as to discuss the role of their expression during cervical intervertebral disc degeneration(CIDD). Methods: Totally 36 human cervical intervertebral disc specimens and their pre-operation MRI findings were obtained from surgical patients or patients with cervical trauma, and they were classified into five grades according to the Miyazaki grading system. The expression of SOX9 and COL2A1 was examined by fluorescence quantitative RT-PCR, Western blotting analysis and immunohistochemistry staining. The relationship between SOX9, COL2A1 expression and the degrees of CIDD was analyzed, and the relationship between SOX9 and COL2A1 was also discussed. Results: Both SOX9 and COL2A1 were expressed in NP tissues, with SOX9 seen in the nucleus and COL2A1 in the extracellular matrix. The expression of SOX9 and COL2A1 mRNA and protein gradually decreased as Miyazaki I progressed to Miyazaki V. We also found a linear relationship between COL2A1 (Y) and SOX9 (X) expression: Y=0. 923X-0. 059 (P<0. 001). Conclusion: The expression of SOX9 and COL2A1 mRNA decrease with the increase of CIDD severity, with SOX9 and COL2A1 mRNA having a similar decreasing tendency. SOX9 positively regulate COL2A1 expression, indicating that changes of SOX9 and COL2A1 gene might be one of the molecular mechanisms of CIDD.

Objective To prepare an absorbable crosslinked micro orous hemostatic starch (ACMHS), to observe its hemostasis effect in the liver, ki dney and femoral artery, and to discuss the hemostasis mechanism. Methods Thirty adult healthy New Zealand rabbits of either sex, weighing 2,000-2,250 g, were equally randomized into 3 groups: liver bleeding group (group A), kidney bleeding group (group B) and femoral artery bleeding group (group C). Each group was further divided into a test group and a control group randomly (n=5) according to different hemostatic powders, ACMHS was used in the test group(group Al, Bl, CI), and Arista™ was used in the control group (group A2, B2, C2). The bleeding amount and bleeding time were accurately recorded. The pathological changes at incision and in the surrounding tissues of the liver, kidney and femoral artery were observed by hematoxylin-eosin staining after test. Results All the bleeding amounts of group Al and A2 were (1. 02 ± 0. 10) g and (1. 03 ± 0. 09) g, and the bleeding time periods of group Al and A2 were (92. 6 ± 5. 16) s and (95. 6 ± 5. 31) s, respectively (P>0. 05). All the bleeding amounts of group Bl and B2 were (0. 92 ± 0. 09) g and (0. 94 ± 0. 08) g, and the bleeding time periods of group Bl and B2 were (87. 5 ± 7. 48) s and (88. 8 ± 6. 54) s, respectively (P>0. 05). All the hemostatic efforts failed in group C. Hematoxylin-eosin staining showed accumulation of red blood cells at the incision and in surrounding tissues of the liver and kidney in group A and group B, without evidence of burn. Conclusion ACMHS has the same hemostatic function as Arista™ in parenchymatous organ injury, but it is not suitable for bleeding of femoral artery injury with high pressure.

Genetic susceptibility exists in intervertebral disc degeneration (IDD) diseases, which may be associated with gene polymorphisms. Recently, much progress has been made in the relationship between IDD susceptibliity and gene single nucleotide polymorphism(SNP). The genes associated with IDD manly include 3 types: genes associated with intervertebral disc stability (about structure and metabolism), genes associated with inflammation, and genes associated with pain signal channel. Studies of SNP in genes associated with IDD can Hp to identify its molecular mechanism, paving a way for individualized prevention and treatment of IDD.

Objective To investigate the correlation between the quantitative results,the semi-quantitative scores from ultrasound elastography and the distribution of myofibroblasts (MFS)in breast tumor,and to analyze the value of quantitative results and the semi-quantitative score from ultrasound elastography in the diagnosis of breast tumor.Methods Thirty eight patients with breast lesions underwent ultrasound elasticity examinations,tissue dispersion quantitative analysis technique was used to assess the 11 characteristic quantities and the corresponding strain ratios in all lesions,and the score of ultrasonic elastography was evaluated.38 cases were divided into benign and malignant group according to pathological diagnosis results.The expression levels of CD34 andα-SMA protein in breast tissue were examined by immunohistochemistry.The distribution patterns of MFS in breast tumor were analyzed. The correlation between the quantitative results, the semi-quantitative scores from ultrasound elastography and the distribution of MFS in breast tumor was studied.Results The expression level of CD34 in malignant group was significantly higher than that in benign group, while the expression level of α-SMA was significantly higher than that in benign group;the differences in the expression levels of CD34 andα-SMA between benign and malignant breast tumor patients were statistically significant (P < 0.01 ).The masses observed in malignant group by elastography were shown in blue,while most of the masses in benign group were shown in green.There were statistically significant differences between two groups in elastography scores (P < 0.05 ). There was a anegative correlation between the CD34 expression in tumor tissue and the score of ultrasound elastography (r=-0.423 7,P =0.027 3).There was a negative correlation between the CD34 expression and the score of ultrasound elastography (r=-0.423 7,P =0.027 3),while the positive correlation was found between theα-SMA expression and the score of ultrasound elastography (r=0.397 0,P =0.014 2).The CD34 expression was significantly correlated with the average relative strain value,entropy,area ratio of low-strain region,kurtosis, skewness and inverse difference moment (P <0.05).Among the 11 characteristics,CD34 was positively correlated with the average relative strain value (r=0.385 6,P =0.016 8)and entropy (r=0.380 5,P =0.018 5);CD34 was negatively correlated with area ratio of low-strain region (r = - 0.351 7,P = 0.030 4),kurtosis (r =-0.427 7,P =0.007 4),skewness (r=-0.394 6,P =0.014 2),inverse difference moment (r = -0.344 3, P =0.034 3),angular second moment (r = - 0.484 9,P = 0.002 0)and strain ration (r = - 0.379 0,P =0.047 5);CD34 was not correlated with standard deviation,complexity and contrast (P > 0.05).The α-SMA espression was positively correlated with kurtosis (r = 0.356 9,P = 0.027 8),skewness (r = 0.323 0,P =0.047 9),area ratio of low-strain region (r=0.382 0,P =0.021 6)and strain ratio (r=0.403 3,P =0.012 0). Conclusion The features and its scores of ultrasound elastography are correlated with the distribution of MFS in breast tumor,suggesting that the ultrasound elastography is very informative and helpful in the diagnosis of breast tumor.