OBJECTIVETo study the effect of homoharringtonine (HHT) alone or combined with 3-methyladenine (3-MA) , an autophagy inhibitor, on the apoptosis and autophagy of K562 cells.

METHODSK562 cells were treated with HHT(10 ng/ml) or HHT(10 ng/ml) combined with 3-MA (1.5 mmol/L) for 1 to 8 days. The apoptosis of treated cells was tested by means of flow cytometry(FCM), and the autophagy levels were tested with RT-PCR, Western blot and electron microscopy.

RESULTSIn the early stage of HHT-treated group, the apoptosis rate increased and decreased later. Beclin1 mRNA expression level and the LC3II/I ratio were declined firstly and increased later in HHT group. While combining with autophagy inhibitor 3-MA, both the Beclin1 mRNA expression level and the LC3II/I ratio were declined continually during the treated period. The activated caspase-3 protein expression level was also raised sustainability during both HHT and 3-MA cultured period.

CONCLUSIONSHHT can induce apoptosis of K562 cells, but the sustaining effect of HHT can induc autophagy of K562 cells, the combination of HHT with 3-MA may enhance the cytotoxicitic effect of HHT on K562 cells.

OBJECTIVETo explore the optimal surgical approach for Siewert II and III adenocarcinoma of esophagogastric junction (AEG).

METHODSClinical data of 135 patients with Siewert II and III AEG treated in our hospital from August 2007 to August 2012 were analyzed retrospectively. Of 135 patients, 57 received transthoracic path for the stomach and proximal gastrectomy, second station lymph node resection (transthoracic group), and 78 cases received transabdominal hiatal approach (transabdominal group). The intraoperative lymph node harvested, postoperative complications and 5-year survival rate were compared between the two groups.

RESULTSThere were no significant differences in the residual tumor positive margin and anastomotic leakage rate between the two groups (both P>0.05). Compared with transthoracic group, transabdominal group had more lymph node dissected (11.1±0.2 vs. 10.4±0.3, P=0.033], less postoperative cardiac [2.6% (2/78) vs. 19.3% (11/57), P<0.01] and pulmonary [7.7% (6/78) vs. 21.1% (12/57), P<0.05] morbidity, and short postoperative hospital stay [(13.4±0.1) d vs. (16.4±0.3) d, P<0.01]. A total of 128 cases were followed up for median 38 months (6 to 72 months). The 5-year survival rate in transthoracic group and transabdominal group was 29.8% and 34.6% respectively, without significant difference (P>0.05).

CONCLUSIONFor the treatment of patients with Siewert II and III AEG, transabdominal hiatal approach can remove more lymph nodes, reduce postoperative cardiopulmonary morbidity and shorten hospital stay.

Adenocarcinoma ; surgery ; Esophageal Neoplasms ; surgery ; Esophagogastric Junction ; pathology ; Gastrectomy ; Humans ; Lymph Node Excision ; Lymph Nodes ; pathology ; Postoperative Complications ; Retrospective Studies ; Stomach Neoplasms ; surgery ; Survival Rate

Objective To prepare serum calibrators for CRP measurement.Methods Fresh serum without infectious diseases , hemolysis, lipemia and choloplania were collected and divided into 3 groups, low, medium, and high, according to the CRP concentration.Each serum pool was mixed , filtered, sterilized and aliquoted.The materials were tested for homogeneity and stability.The values of the CRP was assigned by particle enhanced immunonephelpmetry , and calibrated with international reference materials.The expanded uncertainty was evaluated.Results The materials were tested to be homogeneous (Ubb﹤Ur, P>0.05) with Ubb values being 0, 0.125, 0, Ur values being 0.046, 0.213, 0.785, and F values being 0.803, 1.686, 0.966 in CL, CM, CH groups respectively.Stability study, where F values are 0.609, 0.259, and 1.557 at 22-25℃, 1.217, 4.583, and 0.893 at 2-8℃(P>0.05), showed that the materials were stable for at least 3 days at 22-25 ℃or 30 days at 2-8 ℃, respectively.The certified values of the 3 levels materials for CRP were ( 2.64 ±0.14 ) , ( 31.17 ±0.63 ) , ( 73.85 ±1.74 ) mg/L, respectively.Conclusion The calibrators prepared for serum CRP measurement were homogeneous , stable and accurately assigned and can be used to calibrate the CRP measure system.

OBJECTIVETo explore the mechanism of immunomodulatory activity of triptolide on primary immune thrombocytopenia (ITP)patients-derived plasmacytoid dendritic cells (pDCs).

METHODSpDCs in peripheral blood of ITP patients before therapy (group 1), ITP patients in complete response (ITP-CR, group 2) and healthy donors (group 3) were sorted by flow cytometry, then incubated with triptolide at 0, 5, 10 or 30 µg/L. After 24 hours, we collected the supernatants and then detected the concentrations of IFN-α, IL-6 and TNF-α using ELISA. After 5 days, the cultured cells were collected and CD11c, CD80 and CD86 expressions of myeloid dendritic cells (mDCs) were analyzed by flow cytometry, the morphology of mDC was observed by light microscope and electron microscope.

RESULTSAfter incubation with triptolide at 10 µg/L, the levels of IFN-α, IL-6 and TNF-α in group 1 \[(451.32 ± 85.77) ng/L, (105.68 ± 23.85) ng/L and (135.78 ± 30.62) ng/L\] and group 2 \[(391.71 ± 72.49) ng/L, (84.73 ± 17.77) ng/L and (108.16 ± 23.21) ng/L\] were significantly higher than those in group 3 \[(335.51 ± 67.54) ng/L, (73.62 ± 21.82) ng/L and (95.58 ± 32.85) ng/L\] (all P < 0.05); the levels of IFN-α, IL-6 and TNF-α in group 1 were significantly higher than those in group 2 (all P < 0.05) in a dose-dependent manner (P < 0.05). CD11c, CD80 and CD86 expressions of mDC in group1 and group 2 were significantly higher than those in group 3 (all P < 0.05); CD11c, CD80 and CD86 expressions of mDC in group 1 were significantly higher than those in group 2 (all P < 0.05) also in a dose-dependent manner (all P < 0.05). Triptolide could inhibit pDCs from differentiation into mDCs, the latter displayed more immature morphology than untreated-pDCs.

CONCLUSIONTriptolide could decrease the immune function of pDCs from ITP, inhibit pDCs from differentiation and maturation.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Phenanthrenes ; pharmacology ; Thrombocytopenia ; etiology ; immunology ; Young Adult

OBJECTIVETo investigate the proportion of Th22 cells in peripheral blood of patients with acute lymphoblastic leukemia (ALL) and evaluate its significance.

METHODSThe proportions of Th22 cells in peripheral blood of B-ALL and T-ALL patients before therapy (group 1), B-ALL and T-ALL patients in complete remission (ALL-CR, group 2) and healthy donors (group 3) were evaluated by flow cytometry. The cytokines IL-22, TGF-β, TNF-α and IL-6 in peripheral blood of each group were measured by enzyme-linked immunosorbent assay (ELISA). The levels of IL-22 mRNA in peripheral blood mononuclear cells of each group were examined by reverse transcription-PCR (RT-PCR).

RESULTSThe percentages of Th22 cells and the levels of IL-22, TNF-α, IL-6 and IL-22 mRNA in B-ALL and T-ALL patients before therapy were (0.44 ± 0.10)%, (10.9 ± 3.4) ng/L, (110.7 ± 26.5) ng/L, (60.2 ± 13.8) ng/L, 0.17 ± 0.04 and (0.46 ± 0.11)%, (11.2 ± 3.5) ng/L, (114.6 ± 27.0) ng/L, (58.7 ± 12.4) ng/L, 0.19 ± 0.04, respectively; Which in B-ALL and T-ALL patients in complete remission were(0.59 ± 0.15)%, (14.3 ± 4.1) ng/L, (142.5 ± 32.7) ng/L, (83.7 ± 18.9) ng/L, 0.25 ± 0.06 and(0.60 ± 0.15)%, (14.6 ± 4.3) ng/L, (140.4 ± 31.4) ng/L, (81.4 ± 18.2) ng/L, 0.26 ± 0.06, significantly lower than those in healthy donors \[(1.24 ± 0.31)%, (19.7 ± 6.6) ng/L, (238.3 ± 50.4) ng/L, (138.0 ± 27.1) ng/L, 0.49 ± 0.09\] (P < 0.01). The percentages of Th22 cells and the levels of IL-22, TNF-α, IL-6 and IL-22 mRNA in group l were lower than those in group 2 (P < 0.05), there was not significant difference between B-ALL and T-ALL (P > 0.05). But the levels of TGF-β in B-ALL and T-ALL patients before therapy \[(30.6 ± 8.2) ng/L, (31.4 ± 8.8) ng/L\] and in complete remission \[(24.2 ± 5.8) ng/L, (25.1 ± 6.1) ng/L\] were significantly higher than those in group 3\[(9.6 ± 2.8) ng/L\] (P < 0.01). However, the level of TGF-β in group 1 was higher than that of group 2 (P < 0.05), there was not significant difference between B-ALL and T-ALL (P > 0.05).

CONCLUSIONBoth the number and function of Th22 cells reduced in ALL patients. Th22 cells might be negatively correlated with ALL progression. The lower levels of TNF-α and IL-6, and overexpression of TGF-β in ALL patients might suppress the differentiation of Th22 cells.

Adolescent ; Adult ; Case-Control Studies ; Humans ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Interleukins ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Middle Aged ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; blood ; RNA, Messenger ; genetics ; T-Lymphocytes, Helper-Inducer ; metabolism ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Young Adult

This study was aimed to explore the change of K562 cell apoptosis at different time point after homoharringtonine (HHT) treatment and its mechanism. After treatment of K562 cells with 10 ng/ml HHT, the cell viability was tested with MTT assay; the expression of caspase-3 was detected with Western blot; the BCL-2 expression was analyzed with flow cytometry; the autophagosome was observed by electron microscopy. The results showed that the viability of K562 cells reduced gradually from day 1 to day 5 and ascended from day 6 to day 8 after HHT treatment. At the same time, the cleaved caspase-3 expression level of K562 cells increased gradually from day 1 to day 7, but reduced at the day 8 (P < 0.05). From day 1 to day 8 after HHT treatment, the BCL-2 expression level declined firstly and then went up (P < 0.05). Autophagosome was also seen remarkably at day 8 after HHT treatment. It is concluded that the apoptosis level of K562 cells after being treated with HHT enhances firstly and then declines , which may be associated with higher autophagy level in the late stage of HHT treatment.
Apoptosis ; drug effects ; Autophagy ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Flow Cytometry ; Harringtonines ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

The study was aimed to investigate the synergistically effect of interferon-α (IFN-α) and homoharringtonine (HHT) on the proliferation, apoptosis, cell cycle of K562 cells and the expression of β-catenin. The proliferation, apoptosis, cell cycle and β-catenin mRNA expression of K562 cells treated with IFN-α and/or HHT were assayed with MTT, flow cytometry or RT-PCR respectively. The results showed that HHT alone, but not IFN-α alone, displayed a proliferation inhibition, apoptosis induction, G(0)/G(1) phase block and down-regulation of β-catenin expression in K562 cells with concentration- and time-dependent manners. The expression level of β-catenin mRNA after being treated with HHT was 0.5576 ± 0.0373, which were lower than that in control group (0.9369 ± 0.0142). The down-regulation of β-catenin expression in group of IFN-α combined with HHT was higher significantly than that in HHT group (0.3737 ± 0.0529 vs 0.5576 ± 0.0373, P < 0.05). Otherwise, HHT combined with IFN-α did not demonstrate obvious toxicologic effect on bone marrow mononuclear cells. It is concluded that IFN-α combined with HHT can enhance the cytotoxic effect of HHT on K562 cells, which may be associated with down-regulation of β-catenin expression.
Cell Proliferation ; drug effects ; Harringtonines ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; K562 Cells ; beta Catenin ; genetics ; metabolism

Adult ; Bone Plates ; utilization ; Clavicle ; surgery ; Female ; Humans ; Internal Fixators ; trends ; Male ; Middle Aged ; Young Adult

This study was aimed to investigate the effect of homoharringtonine (HHT) on K562 cell proliferation, apoptosis and expression of BCL-2 and NF-κB proteins. The cells proliferation was assayed with MTT method, the cell apoptosis, cell cycle and BCL-2 expression were analyzed with flow cytometry, NF-κB protein expression was detected with Western blot. The results showed that HHT concentration-dependently inhibited proliferation of K562 cells, the IC50 at 48 h was 43.89 ng/ml. Treated with HHT 10 ng/ml for 48 h, K562 cell apoptosis significantly increased, cell cycle was blocked at G0/G1, the expression level of BCL-2 and NF-κB proteins was lower than that in control group (P < 0.05). It is concluded that HHT may inhibit the proliferation of K562 cells, and down-regulating expression levels of BCL-2 and NF-κB may be one of its anti-CML mechanisms.
Flow Cytometry ; Harringtonines ; pharmacology ; Humans ; K562 Cells ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

Objective:To evaluate the morbidity and causes of long-term abnormal sensation of breasts after typy Ⅰ dual-plane breast implant augmentation via endoscopic assisted axillary approach.Methods:A retrospective analysis was conducted on 53 patients who underwent endoscopic assisted transaxillary type Ⅰ dual plane breast implant augmentation by a same surgeon from July 2014 to December 2016. All patients were followed up for more than 18 months. Hyperesthesia, hypoesthesia and numbness of nipple-areola complex and imframammary fold and breast pain were accessed.Results:According to the palpation and consultation of the surgeon, a total of 10 patients presented with post-operative long-term abnormal sensation of breasts, of which 6(11.3% of the total number) patients presented with hypoesthesia of nipple-areola complex, 1 patient (1.9% of the total number)presented with hyperesthesia of nipple-areola complex, and 2 patients (3.8% of the total number)presented with hypoesthesia of inframammary fold region. One patient (1.9% of the total number) had long-term postoperative breast pain. According to the questionnaire designed to assess breast sensation, a total of 33 patients (64.7%) presented with long-term postoperative breast pain, a total of 12 patients (23.5%) presented with post-operative long-term hyperesthesia of nipple-areola complex, another 5 patients (9.8%) presented with transient hyperesthesia of nipple-areola complex.Conclusions:It is clear that the incidence of post-operative long-term abnormal breast sensation following endoscopic assisted transaxillary type Ⅰ dual-plane breast implant augmentation is comparatively low and controllable.And the main reason for this complication is the peripheral nerve innervation of the nipple areolar complex.