Effects of VEGF-Notch Signaling Pathway on Proliferation and Apoptosis of Bone Marrow MSC in Patients with Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To evaluate the regulation of VEGF-Notch signaling pathway on proliferation and apoptosis of mesenchymal stem cells (MSC) in the patients with aplastic anemia (AA).@*METHODS@#The bone marrow specimens of AA patients were collected for isolation and identification of BM MSC. Westen blot was used to detect the expression of VEGF-Notch signaling pathway-related proteins (VEGF, VEGFR, Notch 1, Jagged 1, Delta-like 1 and Hes1). The VEGF (100 ng/ml) and DAPT (γ-secretase inhibitor, 10 μmol/L) were respectively added into MSC culture system in oder to activate and inhibit the signaling transduction of VEGF-Notch in BM MSC. The proliferation, apoptosis and cell cycle of MSC in AA patients were detected by CCK8 assay and flow cyfometry. The adipogenic differentiation of BM MSC was detected by oil red O staining.@*RESULTS@#The VEGF-Notch signaling pathway was significantly inhibited in AA BM tissues and AA MSC (P＜0.05) detected by Western blot. The intervention of VEGF and DAPT significantly activated and inhibited VEGF-Notch signaling in AA MSC, respectively. CCK8 assay showed that VEGF intervention significantly promoted the proliferation of MSC in AA patients (P＜0.05). Flow cytometry showed that VEGF significantly inhibited apoptosis of MSCs by blocking S phase cells (P＜0.05).@*CONCLUSION@#The activation of VEGF-Notch can restore the proliferation function of MSC in AA patients.

Pure total flavonoids from Citrus paradisi Macfad induce leukemia cell apoptosis in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the potential effect of pure total flavonoids from Citrus paradisi Macfad peel (PTFC) on the proliferation of human myeloid leukemia cells Kasumi-1, HL-60 and K562, and the underlying mechanisms.</p><p><b>METHODS</b>PTFC was extracted from Citrus paradisi Macfad peel and was identified by high performance liquid chromatography. The effect of PTFC on the proliferation and apoptosis of leukemia cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, fluorescent microscopy and flow cytometry, respectively. The effect of PTFC on the expression levels of apoptosis-related regulators was determined by Western blot assay.</p><p><b>RESULTS</b>Treatment with PTFC inhibited leukemia cell proliferation in a dose- and time-dependent manner and triggered Kasumi-1 cell apoptosis. Treatment with PTFC significantly increased the levels of activated poly adenosine diphosphate-ribosepolymerase and caspase-3/-9, but reduced the levels of Mcl-1 expression in Kasumi-1 cells. However, PTFC did not obviously induce HL-60 cell apoptosis.</p><p><b>CONCLUSION</b>PTFC inhibited leukemia cell proliferation and induced their apoptosis by modulating apoptosisrelated regulator expression in leukemia cells in vitro.</p>

Analysis of clinical features of traditional Chinese medicine symptoms and syndromes of 220 patients with chronic aplastic anemia / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study Chinese medicine (CM) syndrome types of chronic aplastic anemia (CAA) patients and the distribution laws of typical CM symptoms in different genders.</p><p><b>METHODS</b>From June 2002 to June 2012, 220 CAA outpatients/inpatients at Department of Hematology, Zhejiang Chinese Medical Hospital were recruited. Patients' symptoms and signs, as well as four diagnostic information at the first onset were collected. CM syndrome differentiation was performed. The syndrome types and typical symptoms were analyzed.</p><p><b>RESULTS</b>(1) In the 220 CAA patients, there were 121 cases of Shen yang deficiency syndrome (55.0%), 18 of Shen yin deficiency syndrome type (8.18%), 81 cases of Shen yin-yang deficiency syndrome (36.82%). (2) The distribution of typical symptoms: fatigue and shortness of breath (77.12% males and 73.53% females), pale complexion (64.41% males and 57.84% females), low temperature of four limbs (12.71% males and 26.47% females), spontaneous perspiration and night sweating (32.20% males and 26.47% females), dry mouth and throat (6.78% males and 6.86% females), feverish feelings in palms and soles (14.41% males and 20.59% females), loose stool (6.78% males and 2.94% females), petechiae and ecchymosis (42.37% males and 43.14% females).</p><p><b>CONCLUSIONS</b>Shen yang deficiency syndrome was most often seen in CAA patients at the initial diagnosis, followed by Shen yin-yang deficiency syndrome. Shen yin deficiency syndrome was the least seen. In CM symptoms, fatigue and shortness of breath were most common seen, followed by pale complexion, skin petechia and ecchymosis.</p>

Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: correlation with the clinico-pathological features of aplastic anemia / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology.</p><p><b>METHODS</b>Thirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α.</p><p><b>RESULTS</b>Counts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively).</p><p><b>CONCLUSION</b>AA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.</p>

Apoptosis of multiple myeloid cells induced by polysaccharides extracts from Hedyotis diffusa and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the proliferation inhibition and apoptosis effects of polysaccharides extracts from Hedyotis diffusa (PEHD) on multiple myeloma (MM) cell line RPMI 8226 cells in vitro, so as to provide experimental theory for the clinical application in the treatment of MM.</p><p><b>METHODS</b>MTT assay was used to examine the effects of PEHD on cell growth. The apoptotic cells were analyzed by flow cytometry with AnnexinⅤ/PI staining. Hoechst staining was used to observe the morphological changes of RPMI 8226 cell apoptosis. The expression levels of caspase-3,-8,-9, PARP, nucleoprotein NF-κB protein and other channel protein were assayed by Western blotting method.</p><p><b>RESULTS</b>The growth of RPMI 8226 cells were suppressed after treatment with PEHD, the highest inhibition rate reached to 92.3%, the results in the doses from 1 to 4 mg/ml showed a dose-and-time-dependent manner. The proportion of apoptotic cells in 1, 2 and 3 mg/ml PEHD treatment groups for 24 h were 22.52%, 62.31% and 69.94%, respectively, and significantly higher than that of control 8.93%. After treated with PEHD, apoptotic body appeared in RPMI 8226 cells nucleus and the number of apoptotic body increased in a dose-dependent manner. With the increasing of PEHD concentration, the expression of caspase-8,-9,-3 and PARP protein increased. The expression of Mcl-1, Bcl-xl, Bid and Bim protein decreased gradually, but the expression of Bax, Bak and Bad protein increased, and the expression of p-AKT protein (60 kDa) and NF-κB obviously decreased.</p><p><b>CONCLUSION</b>PEHD could inhibited the growth of RPMI 8226 cells and displayed a dose-and-time-dependent manner, its mechanism may involve cell apoptosis induction, which was associated with the activation of caspase-8, caspase-9, and caspase-3 protein and the down-regulation of p-AKT and NF-κB protein expression.</p>

Effects of Hedyotis diffusa Willd injection on the proliferation of RPMI 8226 cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the inhibition of Hedyotis diffusa Willd Injection (HDI) on the proliferation of RPMI 8226 cells and its mechanisms.</p><p><b>METHODS</b>The inhibition of HDI on the proliferation of RPMI 8226 cells was detected by MTT and the drug concentrations for further researches were screened out. The apoptosis rate was detected using Annexin V-PI of flow cytometry. The cell cycle distribution was detected by PI. The expressions of adhesion molecule FITC-CD44 and PE-CD49d were detected. The IL-6 and VEGF concentrations of cell supernatants were tested by ELISA. The mRNA expressions of Bax, Bcl-2, Caspase-3, Survivin, IL-6, and VEGF were detected by RT-PCR.</p><p><b>RESULTS</b>HDI could inhibit the proliferation of RPMI 8226 cells. Meanwhile, it induced their early apoptosis, arresting them at G1 phase in a concentration-dependent manner. The VEGF concentrations were down-regulated after acted by 0, 20, 40, and 60 microL/mL HDI in a dose-dependent manner (P< 0.01). The IL-6 content increased (P<0.01). The expressions of CD44 and CD49d were up-regulated in a concentration-dependent manner. After acted by 40 microL/mL HDI, the Survivin mRNA level was significantly downregulated (P<0.01), the mRNA levels of Bcl-2, IL-6, and VEGF were significantly up-regulated (P<0.01), but the up-regulation of Bax and Caspase-3 mRNA levels were not so obvious (P>0.05).</p><p><b>CONCLUSIONS</b>HDI could inhibit the proliferation of RPMI 8226 cells. Its mechanisms might be correlated with early apoptosis induction, G1 phase arresting, VEGF secretion lowering, and Survivin mRNA transcription level down-regulating.</p>

Treatment of severe aplastic anemia by immunosuppressor anti-lymphocyte globulin/anti-thymus globulin as the chief medicine in combination with Chinese drugs / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the therapeutic effect of combined therapy with Chinese drugs and immuno-suppressors, mainly anti-lymphocyte globulin/anti-thymus globulin (ALG/ATG), for the treatment of severe aplastic anemia (SAA), the efficacy associated factors and adverse effects as well.</p><p><b>METHODS</b>A retrospective analysis was conducted on 65 patients with SAA treated by combined therapy which was supplemented with cyclosporin A, androgen, hematopoietic growth factor, etc.</p><p><b>RESULTS</b>Of the 57 patients followed-up, 26 (45.6%) were basically cured, 15 (26.3%) remitted, and 8 (14.0%) improved markedly, the total effective rate being 85.9%. By separately comparing with a single item of clinical data, it was shown that the therapeutic effectiveness was correlated, to a certain extent, with age, illness duration, neutrophil count, and bone marrow proliferation in patients before treatment, as well as with infection that occurred in the follow-up period. It was obviously higher in patients with peripheral neutrophil count > 0.5 x 10 10(9)/L (P<0.05). Various degrees of serum sickness-like reactions occurred in the treatment of 36 patients, including fever in 36 (63.2%), skin rash in 8 (14.0%), and musculoskeletal pain in 5 (8.8%).</p><p><b>CONCLUSIONS</b>The therapeutic effect of combined therapy with Chinese drugs and ALG/ATG in treating SAA could be affirmed, showing some superiority as compared with Western medicine alone. The patients' age, duration of illness, neutrophil count, and bone marrow proliferation before treatment, and degree of infection that occurred could affect the therapeutic efficacy to a certain extent. Adverse reactions resulting from the combined therapy are less, showing the toxicity reducing and effect enhancing action of Chinese drugs.</p>

Effects of combined therapy of human leucocyte antigen haploidentical related bone marrow transplantation and Chinese medicine in treating leukaemia / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the feasibility of human leucocyte antigen (HLA) haploidentical related T-cell undepleted allogeneic bone marrow transplantation (Allo-BMT) combined with Chinese medicine for the treatment of leukaemia.</p><p><b>METHODS</b>Four patients with chronic myeloblastic leukemia (CML) and 4 with acute myeloid leukemia (AML) received allo-BMT with graft from 1 - 3 HLA-mismatched related donors. All patients were pre-treated with standardized conditioning regimen consisting of high dose Ara-c, cyclophosphamide (CY) and total body irradiation (TBI) or busulfan. Donors were given G-CSF 250 microg/d for 7 days prior to marrow harvest. To prevent GVHD, besides application of CSA and MTX, ATG 2.5 mg/kg was given everyday for 4 days before transplantation, and MMF 1.0 g per day starting from the 7th day after transplantation. Chinese medicine for replenishing qi and yin and strengthening Pi and Wei was administrated orally after transplantation.</p><p><b>RESULTS</b>Successful haematopoietic reconstruction was seen in all patients. The median days for reaching of granulocyte >0.5 X 10(9)/L and platelet > 20 x 10(9)/L were 12 (range 10-14) and 20 (range 18-25) days respectively. GVHD of skin in various grade was seen in 7 patients, among whom only one advanced to grade IV; besides, 2 accompanied with GVHD of gut, one with hemorrhagic cystitis, one died for concurrent infection on the 81st day, and one left hospital of his own accord on the 46th day. The median follow-up duration was 18 (range 2-32) months, during this period six patients were alive in a disease-free situation.</p><p><b>CONCLUSION</b>Combined therapy of HLA haploidentical related T-cell undepleted Allo-BMT and Chinese medicine plus immunosuppressants with pre-harvest G-CSF application in doner could effectively reduce the incidence of acute severe GVHD and raise the disease-free survival rate in treating leukaemia.</p>