The effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation in acute leukemia / 中华血液学杂志

Objective: To study the effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia (AL) and its significance as molecular marker to dynamically monitor minimal residual disease (MRD) . Methods: Retrospectively analyzed those AL patients who underwent allo-HSCT in the First Hospital Affiliated to Zhejiang University School of Medicine during Jan 2016 to Dec 2017, a total number of 314 cases, 163 males and 151 females, median age was 30 (9-64) years old. Comparing the difference of WT1 expression at diagnosed, pre-HSCT and after HSCT. Using the receiver operating characteristic (ROC) curve to determine the WT1 threshold at different time so as to predict relapse. The threshold of WT1 expression before transplantation was 1.010%, within 3 months after HSCT was 0.079% and 6 months after HSCT was 0.375%. According to these thresholds, WT1 positive patients were divided into low expression groups and high expression groups. Analyzed the relationship between overall survival (OS) , disease-free survival (DFS) , cumulative incidence of relapse (CIR) and WT1 expression. Results: The OS and DFS of high expression group pre-HSCT were lower than low expression group [69.2% (9/13) vs 89.1% (57/64) , χ(2)=4.086, P=0.043; 53.8% (7/13) vs 87.5% (56/64) , χ(2)=9.766, P=0.002], CIR was higher than low expression group [30.8% (4/13) vs 7.8% (5/64) , P=0.017]. There was no significant difference of OS and DFS between high expression and low expression group of 3 months after HSCT (P=0.558, P=0.269) . The OS and DFS of high expression group of 6 months after transplantation were both lower than low expression group (P=0.049, P=0.035) . Multivariate analysis showed that WT1>0.375% when 6 months after transplantation was the only independent prognostic factor for shorter DFS (P=0.022) . There was no statistically significant difference in CIR between the high-expression group and the low-expression group 3 months after transplantation and 6 months after transplantation (P=0.114, P=0.306) . Conclusion: High expression of WT1 before and after HSCT was an adverse prognosis factor. It is of clinical practical value to use WT1 as a transplant recommendation index for patients with acute leukemia and as a marker to monitor MRD dynamically.

Effects of shangke jiegu tablet on the gene expressions of osteoprotegerin and osteoprotegerin ligand in the repairing process of mandibular defect rabbits / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the mechanism of Shangke Jiegu Tablet (SJT)in repairing the mandibular defect.</p><p><b>METHODS</b>Totally 72 healthy male New Zealand rabbits were randomly divided into the normal control group (n = 24), the model group (n = 24), and the SJT group (n = 24). Then the mandibular defect model was established. Animals in the normal control group and the model group were fed with normal forage, while those in the SJT group were fed with SJT forage. On the day 7, 14, 28, and 56 after model establishment, 6 rabbits were killed in each group. The bone was collected from the mandibular defect. The gene expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) were detected by means of RT-PCR. The positive dyeing strength and area of the bone tissue were detected by means of immunohistochemical technique.</p><p><b>RESULTS</b>Compared with the normal control group, the degree of OPGmRNA expression was remarkably up-regulated on day 7 after model establishment (P < 0.05) and the degree of OPGLmRNA expression was remarkably up-regulated on day 14 after model establishment (P < 0.05) in the model group. Compared with the model group, the degree of OPGmRNA expression was remarkably up-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were stronger and broader on day 14, 28, and 56 after model establishment in the SJT group. The degree of OPGLmRNA expression was remarkably down-regulated (P < 0.05), and the positive dyeing strength and area of bone tissue were weaker and smaller on day 14 after model establishment in the SJT group. The ratio of OPGmRNA/OPGLmRNA was remarkably up-regulated on day 14, 28, and 56 after model establishment (P < 0.05).</p><p><b>CONCLUSION</b>The effect mechanism of promoting mandibular defect repairing by SJT may be correlated to regulating the expressions of OPGmRNA and OPGLmRNA.</p>

Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of mycophenolic acid (MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>MSCs were treated with MPA at the concentration of 1 μ mol/L, 10 μ mol/L, 50 μ mol/L, and 100 μ mol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5'-monophosphate dehydrogenase (IMPDH) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining, Ca(2+) quantification and real-time PCR.</p><p><b>RESULTS</b>In the range of 1 μ mol/L to 100 μ mol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH I and IMPDH II. von Kossa staining and Ca(2+) quantification indicated that MPA inhibited osteogenic differentiation of MSCs, and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix.</p><p><b>CONCLUSION</b>MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration-and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.</p>

Effects of blocking inhibitory KIR receptors on cytotoxic activity of human NK cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody on cytotoxic activity of human NK cells.</p><p><b>METHODS</b>Human peripheral blood NK cells were isolated by Rosettesep NK sorting kit. The cytotoxic activity of NK cells against human leukemia NB4, K-562, Raji cells and allogeneic mature or dendritic cells (DCs) was detected before or after KIR2DL1 and KIR2DL2/2DL3 were blocked. The effect of NK cells on T lymphocyte proliferation was detected by mixed lymphocyte reaction and TGF-β1 concentration in culture supernatant was measured.</p><p><b>RESULTS</b>The cytotoxicity of NK cells to NB4 cells was augmented with increasing concentration of the antibody. Combination of both antibodies enhanced killing activity of NK cells. NK cells had strong cytotoxicity to K-562 cells, but were not enhanced by the blockade of inhibitory receptors. The cytotoxicity to Raji cells was not evidently augmented. The cytotoxicity of NK cells to mature DC was enhanced remarkably with the increase of concentration of the antibodies (2.20% ±1.10% compared with 37.59% ±5.06%, P<0.05). In mixed lymphocyte reaction, the blockade of two antibodies enhanced the inhibition effect of NK cells on T cell proliferation (77.85% ± 8.31% compared with 43.05% ± 5.95%, P<0.05) and the content of TGF-β1 in the supernatant was increased.</p><p><b>CONCLUSION</b>The cytotoxic effects of human NK cells against target cells were significantly enhanced with the blockade of inhibitory KIR receptor; and the cytokine TGF-β1 secreted by NK cells further inhibits T cells proliferation.</p>

Preparation of monoclonal antibodies against human mesenchymal stem cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (McAbs) against human mesenchymal stem cells (hMSCs) and to study their biological characteristics.</p><p><b>METHODS</b>BALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry.</p><p><b>RESULT</b>Five hybridoma cell lines were successfully established, which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells, the relative affinities of 5 McAbs were 1x10(6) (ZUB1), 1x10(5) (ZUB4), 1x10(6) (ZUC3), ZUE12 (1x10(5)) and 1x10(5) (ZUF10), respectively. Isotype analysis showed that ZUB1, ZUE12, ZUF10 against the same isotype, while ZUC3, ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87.39% (ZUB1), 88.07% (ZUB4), 88.12% (ZUC3), 69.89% (ZUE12) and 83.67% (ZUF10).</p><p><b>CONCLUSION</b>The prepared five McAbs can specifically react against hMSCs, which can be used for selection and study of hMCSs.</p>

Study on the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell lines / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>Detecting the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell line cells on the base of determining its genomic structure and its four pseudogenes to clarify if hTRF1 mutation is one of the factors of the activation of telomerase.</p><p><b>METHODS</b>hTRF1cDNA sequences were obtained from GenBank, its genome structure and pseudogenes were forecasted by BLAST and other biology information programs and then testified by sequencing. Real-time RT-PCR was used to detect the expression of hTRF1mRNA in 10 cell line cells, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji. Telomerase activities of cells were detected by using telomeric repeat amplification (TRAP)-ELISA protocol. PCR and sequencing were used to detect mutation of each exon of hTRF1 in 10 cell line cells.</p><p><b>RESULTS</b>hTRF1 gene, mapped to 8q13, was divided into 10 exons and spans 38.6 kb. Four processed pseudogenes of hTRF1 located on chromosome 13, 18, 21 and X respectively, was named as PsihTRF1-13, PsihTRF1-18, PsihTRF1-21 and PsihTRF1-X respectively. All cell line cells showed positive telomerase activity. The expression of hTRF1 was significantly lower in malignant hematopoietic cell lines cells (0.0338, 0.0108-0.0749) than in normal mononuclear cells (0.0493, 0.0369-0.128) (P=0.004). But no significant mutation was found in all exons of hTRF1 in 10 cell line cells. Four variants were found in part of intron 1, 2 and 8 of hTRF1. Their infection on gene function is unknown and needs further studies.</p><p><b>CONCLUSION</b>hTRF1 mutation is probably not one of the main factors for telomerase activation in malignant hematopoietic disease.</p>

Inhibition of acetamide-45 on airway smooth muscle contraction induced by electric field stimulation and methacholine in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of the new antiallergic agent N-(pyridin-4-yl)-(indol-3-yl) acetamide-45 (acetamide-45) on electric field stimulation (EFS)-and methacholine-induced contraction of airway smooth muscle in vitro.</p><p><b>METHODS</b>Contractions were induced by EFS in isolated trachea and bronchus of rats or by cumulative methacholine concentrations in isolated trachea of guinea pigs. Changes in isometric force of isolated airway smooth muscle were measured by force transducers and recorded on a multi-channel polygraph recorder.</p><p><b>RESULTS</b>Acetamide-45 inhibited the contraction induced by EFS in isolated rat airway. The IC50 was 10.74 (95% CI 8.87-13.00) micromol.L(-1) and 18.83 (95% CI 14.57-24.33) micromol.L(-1) in tracheae and bronchi, respectively. Acetamide-45 also inhibited methacholine-induced contractile response of isolated guinea pig trachea in a concentration-dependent manner. At concentrations of 3, 10, 30 micromol.L(-1) acetamide-45 significantly decreased maximal contractile response of methacholine by 24.6%-43.2% and increased EC50 of methacholine by 3.1-to 21.4-fold.</p><p><b>CONCLUSION</b>Acetamide-45 inhibits EFS-or methacholine-induced contraction of isolated airway smooth muscle, and these effects might be non-specific inhibition on cholinergic receptor.</p>

Isolation of Tara protein and its gene cloning / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene.</p><p><b>METHODS</b>The co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting.</p><p><b>RESULTS</b>Tara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase.</p><p><b>CONCLUSION</b>Tara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.</p>

Expression of human telomere repeat binding factor 1 (TRF1) in acute leukemia cells and its correlation with telomerase activities / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the expression of human telomere repeat binding factor 1 (TRF1) to investigate the correlation of telomerase activity with acute leukemia.</p><p><b>METHODS</b>Leukemic cells were collected from 30 cases of acute leukemia. Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of TRF1 and hTERT mRNA in leukemic cells.</p><p><b>RESULTS</b>TRF1 mRNA expression was 0.0126 (0.0127-0.0546) in acute non-lymphocytic leukemia (ANLL), which was lower than that in normal mononuclear cells [0.0457 (0.00839-0.262), P<0.001], but its expression in acute lymphoblastic leukemia (ALL) cells [0.0745 (1.92 x 10(-6)-0.193)] had no significant difference compared with that in normal mononuclear cells. TRF1 expression in ANLL cells was significantly lower than that in ALL cells (P=0.001). The expressions of TRF1 mRNA in AL cells and normal mononuclear cells had no significant correlation with expression of hTERT mRNA (r=-0.173, P=0.207).</p><p><b>CONCLUSION</b>The expression of TRF1 is lower in ANLL cells, which indicates TRF1 may have some effect on telomerase activity by regulating telomere length in ANLL cells.</p>

Expression of Pin1 in malignant hematopoietic cells and its relation with cell cycle / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the expression of peptidyl-prolyl cis/trans isomerase (PPIase or Pin1) in malignant hematopoietic cells and its relation with cell cycle.</p><p><b>METHODS</b>Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of Pin1 mRNA in malignant hematopoietic cell lines and normal mononuclear cells separated from bone marrow. HeLa cells were blocked with Thymidine and Nocodazole in different cell phases and then the expression of Pin1 mRNA and protein were detected by realtime-PCR and immunoblotting.</p><p><b>RESULTS</b>The expression of Pin1 in malignant hematopoietic cell lines was significantly higher than that in normal controls (0.339 +/-0.093 compared with 0.038 +/-0.005, P<0.01). Its expression in myeloid malignant hematopoietic cell lines was significantly higher than that in normal controls (0.388 +/-0.115 compared with 0.038 +/-0.005, P<0.01) and so was the malignant lymphocytic cell lines (0.226 +/-0.166 compared with 0.038 +/-0.005, P<0.01). The expression of Pin1 was closely correlated with cell cycle. It was the highest in G1 phase and the lowest in S phase (110.762 +/-16.737 compared with 4.080 +/-0.634, P<0.01).</p><p><b>CONCLUSION</b>Pin1 is overexpressed in malignant hematopoietic cell lines and its expression is different during cell cycle that is highest in G1 phase and lowest in S phase.</p>