[Percentages and functions of natural killer cell subsets in peripheral blood of patients with severe aplastic anemia].

OBJECTIVE: To analyze the percentage and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cells in the pathogenesis of SAA. METHODS: By flow cytometry, the percentages of NK cells (CD3(-)CD56(+)CD16(+)) and its subsets [CD3(-)CD56(bright)CD16(-)(CD56(bright)), CD3(-)CD56(dim) CD16(+)(CD56(dim)), CD3(-)CD56(-)CD16(+)] in peripheral blood lymphocytes were detected in 12 untreated patients, 30 recovered patients and 13 normal controls respectively from April 2010 to December 2010 in our hospital. NK cells activating receptors (NKG2D and NKp46), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte and reticulocyte absolute value in peripheral blood, and hyperplasia degree, percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes absolute value in bone marrow were evaluated. RESULTS: (1) The percentages of NK cells (10.30% ± 6.08%) and CD56 bright cells (0.11%) in untreated patients were significantly lower than those of recovered patients (16.47% ± 8.29%, 0.68%, both P < 0.05) or normal controls (19.45% ± 6.88%, 0.53%, both P < 0.05). The percentage of CD56(dim) cells in untreated patients was significantly lower than that of normal controls (9.62% ± 6.04% vs 18.21% ± 7.16%, P < 0.05). The percentage of CD3(-)CD56(-)CD16(+) cells was significantly higher in recovered patients than that of untreated patients or normal controls (0.79% vs 0.37%, 0.41%, both P < 0.05). (2) The expression of NKp46 and perforin of NK cells in untreated (88.23%, 64.97% ± 21.61%) and recovered patients (82.97%, 66.14% ± 20.73%) were significantly higher than those of healthy controls (40.99%, 42.11% ± 27.25%, all P < 0.05). (3) The percentage of NK CD56(bright) and CD3(-)CD56(-)CD16(+) cells of patients was positively correlated with ANC% (r = 0.423, 0.609, 0.468 respectively, all P < 0.05) and the percentage of granulocytes in bone marrow (r = 0.357, 0.517, 0.434 respectively, all P < 0.05). The percentages of NK, CD56(bright), CD56(dim) and CD3(-)CD56(-)CD16(+) cells were positively correlated with the hyperplasic degree of bone marrow (r = 0.455, 0.412, 0.404, 0.451 respectively, all P < 0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow (r = -0.522, -0.435, -0.411, -0.547 respectively, all P < 0.05). The expression of NKG2D, NKp46, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage of NK CD56(bright), CD56(dim) cells and a higher expression of perforin may cause the over-function of T lymphocytes and thus lead to hematopoietic failure in SAA.

[Expression Level and Clinical Significance of OTC4 Gene in Patients with Myelodysplastic Syndromes].

OBJECTIVE: To investigate the expression characteristics and clinical value of OTC4 gene in patients with myelodysplastic syndrome (MDS). METHODS: Sixty-five patients with MDS were selected from June 2017 to April 2018, and 39 healthy subjects were selected as control group. Mononuclear cells were isolated from bone marrow collected by aseptic puncture. The OTC4 gene level of MDS patients was detected by RT-PCR, and the OTC4 protein of MDS patients was detected by Western blot. The survival curve of MDS patients was drawn by Kaplan-Meier. Cox multivariate analysis was used to analyze the independent prognostic factors. RESULTS: The relative expression level of OTC4 gene in MDS patients was significantly higher than that in the control group (P＜0.05). Western blot showed that the expression level of OTC4 protein in MDS patients was higher than that in the control group (P＜0.05). OTC4 gene expression level closely related with the leukocyte count, and the level of hemoglobin, and lactate dehydrogenase and platelet count in MDS patients (P＜0.05). CR rate of MDS patients with low OTC4 gene expression was 54.8%, which was higher than that of high OTC4 gene expression group (P＜0.05), while HI, SD and PD rates of MDS patients with low OTC4 gene expression were 9.7%, 12.9% and 6.5% respectively, which were lower than those of high OTC4 gene expression group (P＜0.05). Kaplan-Meier survival analysis showed that OS and DFS in patients with low OTC4 gene expression were superior to those with high OTC4 gene expression (P＜0.05). Multivariate Cox regression analysis showed that leukocyte count and OTC4 gene were independent influencing factors for OS (P＜0.05), platelet level and OTC4 gene expression were independent influencing factors for DFS (P＜0.05). CONCLUSION: OTC4 gene closely relates with the severity of MDS. The patients with lower expression of OTC4 gene have better prognosis, the detection of OTC4 gene has higher clinical value for evaluating the prognosis of MDS patients.

[Expression of SHP-1 mRNA in Patients with Myelogenous Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the expression and clinical significance of SHP-1 mRNA in patients with myelogenous leukemia. METHODS: The SYBR Green-based qRT-PCR was used to assess SHP-1 mRNA levels in 54 patients with chronic myelogenous leukemia (CML), 30 cases of de novo acute myelogenous leukemia (AML) and 10 persons without malignancy as controls. RESULTS: The relative expression levels of SHP-1 mRNA in control group (CG), chronic phase CML (CP-CML) group, advanced phase of CML (including accelerated phase CML and blastic phase CML) group and AML group were 1.15±0.62, 4.96±1.76, 2.60±0.90 and 0.45±0.20, respectively. The expression of SHP-1 mRNA in patients with CML significantly increased in comparison with that in CG(P<0.05). Meanwhile, the expression of SHP-1 mRNA in CP-CML group very significantly increased as compared with that in advanced stage of CML group(P<0.0001). The expression of SHP-1 mRNA in AML group significantly decreased as compared with that in CG group(P=0.0442). In CP-CML group, statistical analysis showed that SHP-1 mRNA expression at baseline in optimal responders (5.712±0.4476) was significantly higher than that in the suboptimal or failed responders (4.044±0.3701)(P=0.0090). Meanwhile, the SHP-1 mRNA expression in AML patients was higher than that in CR group (0.4984±0.05164) and non-CR group (0.3537±0.02388)(P=0.0017). CONCLUSION: The SHP-1 mRNA levels in CML patients are higher than that in AML patients, and probably correlats with disease progression of CML. The mRNA expression level of SHP-1 may be a molecular marker to predict early response to inatinib treatment in CP-CML and AML.

[Effects of Scutellaria Barbata on VEGF Expression in K562 Cells].

OBJECTIVE: To investigate the anti-angiogenesis effect of Scutellaria barbata extract(SBE) on chronic myeloid leukemia(CML) K562 cell line in vitro. METHODS: The proliferating activity after treating K562 cells with 0.5,1.0,2.0 and 4.0 g/ml SB for 24, 36, 48 hours were assessed by MTT assay. The level of vascular endothelial growth factor(VEGF) in the culture supematant of K562 cells was determined by ELISA; and the expression of VEGF mRNA was detected by RT-PCR. RESULTS: MTT assay showed that SBE could inhibit the proliferation of K562 cells in a dose-dependent manner (r=0.56); ELISA displayed that the concentration of VEGF in K562 cells in blank-control group was most high; after intervention of K562 cells by SBE (0.5,1.0,2.0 and 4.0 g/ml) for 48 h, the concentration of VEGF decreased, the comparison between different groups showed significant differences (P<0.05); after treatment with SBE for 48 h, the expression of VEGF mRNA in K562 cells decreased, the gray scale ratio of target gene/ß-actin declined, and the difference between various groups was statistically significant (P<0.05). Conclution: SBE can inhibit K562 cell proliferation, its action mechanism may related with the VEGF level concentration in K562 cells and down-regulation of VEGF mRNA expression.

[Overexpression of SHP-1 Enhances the Sensitivity of K562 Cells to Imatinib].

OBJECTIVE: To explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism. METHODS: K562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2. RESULTS: After exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect. CONCLUSION: SHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

[Clinicopathological and immunohistochemical study of extra-gastrointestinal stromal tumors arising from the omentum and mesentery].

OBJECTIVE: To explore the clinicopathological and immunohistochemical features of extra-gastrointestinal stromal tumors (EGIST) arising from the omentum and mesentery and to investigate the cellular origin of these tumors, prognostic factors, and the relationships with gastrointestinal stromal tumors. METHODS: Nineteen cases of mesenchymal neoplasms arising from the omentum and mesentery (previously diagnosed as smooth-muscle tumors or schwannomas) were studied morphological with a panel of immunohistochemistry including CD117 and CD34. RESULTS: Among the 19 cases, 14 tumors were confirmed to be EGIST, of which 6 tumors arose from the omentum and 8 cases located at the mesentery. The size of tumors ranged from 3.5cm to 29.0 cm (mean 12.4cm) in diameter. Histologically, there were 9 cases of mainly spindle cell type, 2 cases of mainly epithelioid cell type and 3 cases of mixed cell type. all EGIST expressed CD117 (14/14) and a percentage of them expressed also CD34 (8/14) and/or SMA (6/14), anyhow, all EGIST were negative for desmin and S-100 protein. Six patients with tumors arising from the omentum were all alive without evidence of disease (tumor-free). Among 7 cases with tumors of the mesentery, three patients died of the disease, 1 alive with the disease and 3 patients alive without evidence of the disease. CONCLUSIONS: EGIST were identical by their histological and immunohistochemical features with gastrointestinal stromal tumors (GIST). This tumor may arise from the multipotential mesenchymal stem cells. EGIST have various clinical behavior, and the parameters used for predicting the prognosis of GIST may not be completely suitable for EGIST evaluation.

[Expression and Clinical Significance of DNMT in Patients with Chronic Myeloid Leukemia].

OBJECTIVE: To investigate the expression and clinical significance of DNA methyltransferases (DNMT) mRNA in patients with chronic myeloid leukemia (CML). METHODS: The expression levels of DNMT mRNA in mononucllear cells (MNC) of bone marrow or in peripheral blood of 93 CML patients in 3 different phases and 10 normal controls (NC) were detected by SYBR Green flurescent quatitative PCR. RESULTS: The relative expression levels of DNMT1 mRNA in NC, chronic phase CML (CML-CP), accelerated phase (CML-AP) and blastic phase (CML-BP) were 1.45 ± 0.22, 1.83 ± 0.63, 2.95 ± 0.87 and 3.24 ± 1.39 resectively. The expression of DNMT1 mRNA showed no statistically significant difference between CML-CP and NC (P = 0.28). The expression of DNMT1 mRNA in advanced stages (including CML-AP and CML-BP) of CML obviously increased in comparison with CML-CP and NC (P < 0.05). The expression of DNMT1 mRNA in CML-AP was not significantly different from that in CML-BP (P = 0.336). The relative expression levels of DNMT3a mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.29 ± 0.34, 1.34 ± 0.46, 2.33 ± 1.05 and 3.18 ± 1.23 resectively. And the expression levels of DNMT3a mRNA were not statistically significantly different between CML-CP and NC (P = 0.844). The results showed that the expression of DNMT3a mRNA in the advanced phase of CML significantly increased in comparison with that in CML-CP and NC (P < 0.05). Meanwhile, the expression of DNMT3a mRNA in CML-AP was not different from that in CML-BP (P = 0.304). The relative expression levels of DNMT3b mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.37 ± 0.31, 16.41 ± 22.50, 9.36 ± 5.50 and 12.17 ± 13.44 resectively. It was also found that the level of DNMT3b mRNA in CML significantly increased in comparison with NC (P < 0.05), and that the between the 3 different phase of CML was not statistically significantly different (P >0.05). CONCLUSION: The expression of DNMT mRNA increases in advanced CML as compared with normal controls and CML-CP, and the increased levels of DNMT mRNA probably correlate with disease progression in CML.

Effects of Tongguan Capsule on post-myocardial infarction ventricular remodeling and cardiac function in rats.

OBJECTIVE: To observe the effects of Tongguan Capsule (TGC) on post-myocardial infarction ventricular remodeling and heart function in rats. METHODS: A rat model of acute myocardial infarction (AMI) was established by coronary ligation. Experimental rats were randomized to 4 groups including three model groups (Group A: captopril 5 mg/kg * day, n=7; Group B: TGC 10 g/kg * day, n=7; and Group C: placebo, n=8), and a sham-control group (Group D: blank control, n=6). Animals were treated for 4 weeks. The cardiac function of rats was assessed at the end of the experiment based on left ventricular ejection fraction (LVEF) and left ventricular short axis fractional shortening (LVFS) detected by colored echocardiography; meanwhile, the condition of ventricular remodeling was observed through the levels of left ventricular mass (LVM), plasma aldosterone (ALD), myocardial angiotensin II (Ang II) and myocardial collagen measurements. RESULTS: At the end of the experiment, LVEF and LVFS in Group A and B were improved significantly, while those in Group C were unchanged, the LVEF in Group A, B, C, and D was 0.57+/-0.46, 0.61+/-0.08, 0.36+/-0.55 and 0.76+/-0.02, respectively; and their LVFS was 0.31+/-0.52, 0.34+/-0.04, 0.23+/-0.57 and 0.45+/-0.03, respectively. The difference was statistically significant when comparing the two indexes in Group A and B with those in Group C and D (P<0.05). LVM, levels of plasma ALD and myocardial Ang II were lower in Group A and B than in Group C, but a comparison between Group A and B showed an insignificant difference in lowering LVM and ALD, while the lowering of Ang II was more significant in Group B than in Group A (754.7 +/- 18.7 pg/mL vs 952.6+/-17.6 pg/mL, P<0.05). Morphological examination showed that in Group A and B the swollen myocardial cells had shrunk, with regularly arranged myocardial fibers and decreased collagen proliferation, but the improvements in Group B were more significant. CONCLUSION: TGC could markedly improve the post-infarction ventricular remodeling and cardiac function in rats, showing that the efficacy was better than or equal to that of captopril.