Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

Exosomes derived from M1 macrophages enhance viability of theca cells

AIMTo investigate the effects of exosomes secreted by M1 macrophages on the viability of ovarian follicular theca cells and the possible mechanism. METHODSRaw264.7 mouse macrophages were treated with lipopolysaccharide (LPS) to establish a model of M1 macrophages. Exosomes secreted by the macrophages were isolated by ultracentrifugation and identified by electron microscopy, Western blot, and flow nanoanalyzer. Primary mouse ovarian follicular theca cells were co-incubated with PKH67 fluorescence-labeled exosomes to detect whether exosomes secreted by the macrophages were uptaken by the theca cells. The cell viability was measured by CCK-8 assay. Flow cytometric analysis was used to evaluate the cell cycle distribution. The expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) was determined by qPCR and Western blot. RESULTSThe results of electron microscopy, Western blot, and flow nanoanalyzer verified that the macrophages secreted exosomes. The co-incubation of PKH67 fluorescence-labeled exosomes and theca cells confirmed that the theca cells were able to ingest large numbers of exosomes secreted by the macrophages. M1 macrophage-derived exosomes significantly enhanced the viability of theca cells by shifting the cells from G₁ phase to S phase. The expression of CDKN1B was up-regulated in theca cells treated with M1 macrophage-derived exosomes as compared with the controls. CONCLUSION M1 macrophages-derived exosomes enhance the viability of theca cells in association with down-regulated expression of CDKN1B.     

Effect of inhibiting release of exosomes on biological characteristics of bone marrow mesenchymal stem cells

AIM:To investigate the effect of inhibiting the release of exosomes on the biological characteristics of bone marrow mesenchymal stem cells (BM-MSCs) and the underlying mechanisms. METHODS:The exosome releasing-deficient mouse model was constructed by knockout of Rab27a using TALEN technique. The BM-MSCs were isolated and cultured. The exosomes were extracted from the culture medium using total exosome isolation kit and quantified by nanoparticle tracking analysis (NTA). The size and morphology of the exosomes were observed under transmission electron microscope. To evaluate the proliferation ability of BM-MSCs, the BM-MSCs were labeled with 5-ethynyl-2'-deoxyuridine (EdU) and the expression level of proliferating cell nuclear antigen (PCNA) was determined by Western blot. Moreover, hypoxia tolerance of BM-MSCs in vitro was evaluated via TUNEL staining and MTS assay. RESULTS:The count of exosomes released by BM-MSCs isolated from Rab27a knockout mice was significantly reduced. Inhibition of exosome release resulted in decreases in the viability of the BM-MSCs and their resistance to hypoxia. CONCLUSION:Inhibition of exosome release from the BM-MSCs results in significantly decreased proliferation ability and resistance to hypoxia.     

Decidual macrophages modulate trophoblast cells by transferring exosomes in unexplained recurrent spontaneous abortion

AIM: To investigate whether decidual macrophages have ability to regulate trophoblast cells by secreting exosomes in unexplained recurrent spontaneous abortion (URSA). METHODS: The decidual macrophages were isolated from deciduas of URSA and normal early pregnant induced abortion women. Transmission electron microscopy revealed the morphology and size of the macrophages-derived exosomes. Besides, the characterization of the exosomes was confirmed by the presence of known exosomal marker CD63 by Western blot. The HTR8/Snveo cells were treated with exosomes extracted from the decidual macrophages of URSA or normal early pregnanty induced abortion women. Confocal microscopy, MTS assay and Transwell assay were used to explore the effect of URSA macrophages-derived exosomes on viabi-lity and migration ability of trophoblasts cells. RESULTS: Electron microscopy showed exosomes secreted by macrophages displayed a round-shaped appearance, ranging 40~80 nm in diameter. The results of Western blot indicated that exosomes exacted from the macrophages expressed CD63, a member of tetraspanin family protein. Confocal microscopy revealed the exosomes were taken up by the HTR8/Snveo cells. MTS assay indicated the viability of HTR8/Snveo cells was declined when incubated with the URSA macrophages-derived exosomes (P<0.05). In URSA macrophages-derived exosomes group, the migration ability of the HTR8/Snveo cells was decreased significantly as compared with normal early pregnant induced abortion macrophages-derived exosome (P<0.05). CONCLUSION: Decidual macrophages of URSA inhibits the viability and migratory properties of trophoblast cells by exosomes, which participates in regulating maternal-fetal immune in URSA.     

Isolation of exosomes and effect of stellate ganglion block on the number of exosomes in post-hemorrhagic shock mesenteric lymph of rats

AIM To isolate the exosomes in mesenteric lymph, verify the source of exosomes, and observe the effect of stellate ganglion block （SGB） on the number of exosomes in post-hemorrhagic shock mesenteric lymph （PHSML） of rats. METHODS Twenty-four male rats were randomly divided into sham, sham+SGB, shock, and shock+SGB groups. SGB was performed before the establishment of hemorrhagic shock model using the routine methods in our lab. The PHSML was drained for exosomes isolation. The exosomes were identified through particle size analysis and CD63 protein expression. The expression of epithelial cell adhesion molecule （EpCAM） was detected to identify whether the exosomes were derived from epithelial cell. The number of exosomes in various mesenteric lymphs was measured using the flow cytometry. RESULTS The diameter of granular material extracted from mesenteric lymph was about 100 nm. The positive expression of exosomes pecific protein CD63 indicated the successful isolation of exosomes, and the EpCAM expression verified the exosomes were derived from intestinal epithelial cells. The number of exosomes in mesenteric lymph isolated from the rats of Shock group was obviously increased compared to that from the Sham group （P<0.05）, while the exosomes from the Shock+SGB group was markedly decreased when compared to Shock group （P<0.05）. CONCLUSION The current study establishes the isolation technique of exosomes in mesenteric lymph, and proved the exosomes were derived from the intestinal epithelial cells. SGB treatment reduces the number of exosomes in PHSML.     

Preliminary study of bone marrow mesenchymal stem cell-derived exosomes in repair of spinal cord injury in rats

AIM:To study the influence of bone marrow mesenchymol stem cell-drived exosomes (BMSC-exosomes) on hindlimb activity, and the numbers of reactive astrocytes and residual neurons in spinal cord injury (SCI) rats. METHODS:BMSCs were cultured using the whole bone marrow adherent culture method and surface markers CD90 and CD34 were verified by flow cytometry. Exosomes were isolated by ultracentrifugation and the morphology of exosomes was observed under transmission electron microscope. The protein markers CD63 and CD9 were verified by Western blot. After exosomes were applied to SCI rats, the Basso, Beattie and Bresnahan locomotor rating scale score, the Nissl staining of the lesion site, and the numbers of reactive astrocytes and residual neurons were assessed at various time points. RESULTS:Transmission electron microscopic observation revealed the presence of saucer-shaped vesicles. BMSC-exosomes were found to express high levels of CD63 and CD9. Compared with injury group, significant improvement of hindlimb activity scores from day 14 after injury in treatment group was observed (P<0.05), and less reactive astrocytes and more residual neurons from day 7 after injury were also observed (P<0.05). CONCLUSION:BMSC-exosomes inhibit reactive astrocytes and death of neurons, and improve hindlimb activity in the rats after SCI.     

Pancreatic cancer cell-secreted exosomes promote apoptosis of β cells via activation of mitochondrial apoptotic pathways

AIM: To investigate the effects of exosomes secreted by pancreatic cancer cells on the viability and function of β cells and the possible mechanism. METHODS: ExoQuick-TC kit was used to extract exosomes in the supernatants of mouse pancreatic cancer Pan02 and MPC-83 cells, and the extracted exosomes were identified by transmission electron microscopy. Fluorescence-labeled exosomes were incubated with mouse insulinoma MIN6 cells for 48 h to detect whether exosomes secreted by pancreatic cancer cells were uptaken by MIN6 cells. MTT and glucose-stimulated insulin secretion (GSIS) assays were conducted to examine cell viability and insulin secretion of MIN6 cells after incubating with exosomes. The expression of miR-204 and Bcl-2 mRNA in MIN6 cells was detected by qPCR. The protein expression of Bcl-2, Bax, caspase-3 and cytochrome C (Cyt-C) in MIN6 cells was determined by Western blot. RESULTS: The results of transmission electron microscopy showed that both Pan02 cells and MPC-83 cells secreted exosomes, and Pan02 cells secreted more. The co-incubation results of fluorescence-labeled exosomes and MIN6 cells confirmed that MIN6 cells were able to ingest large amounts of exosomes secreted by pancreatic cancer cells. The results of MTT and GSIS assays showed that the viability and the level of high glucose-stimulated insulin secretion of MIN6 cells in exosome treatment group significantly decreased compared with nontreatment group (P<0.01). The results of qPCR showed that the exosomes secreted by pancreatic cancer cells were rich in miR-204, and the mRNA expression of Bcl-2 in MIN6 cells was significantly down-regulated by exosome incubation (P<0.01). The results of Western blot showed that the protein expression of Bcl-2 in the MIN6 cells treated with exosomes was significantly down-regulated (P<0.05), and the protein levels of Bax, cleaved caspase-3 and Cyt-C in exosomes treatment group were significantly up-regulated (P<0.01). CONCLUSION: Pancreatic cancer cells secrete exosomes. The exosomes secreted by pancreatic cancer cells are ingested by β cells, and reduce the viability and insulin secretion of β cells. The mechanism may be related to the increase in exosomal miR-204 in the β cells. Increasing miR-204 may inhibit the expression of Bcl-2 and promote the activation of mitochondrial apoptosis in β cells.     

Progress in the study on establishment and characteristics of viral L6565 cell clone

The biological characteristics of viral L6565 leukemia cell clone were as follows: (1) The chromosome counts varied 38～114 , and stem cells were 42; (2) Virus particles type A and type C found in the cytoplasm of clone cells; (3) X-C assays were positive, c-myc and c-fos gene overexpressed in clone cells; (4) Differential markers CD4, CD8, CD45R were negative, CD45RO λ were positive; (5) The supernatant of clone cells could induce T or B lymphocytic leukemia/lymphoma and granulocytic leukemia in SSB strain mice. The leukemogenic effect of concentrate supernatant was stronger than non-concentrate supernatant (P<0.05); (6) The antisense of the c-myc gene induced low expression of c-myc protein, and inhibited the growth of viral L6565 clone cells.     

Differentiation of mesenchymal stem cells derived from human umbilical cord

AIM: To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchymal stem cells(hUCMSCs) into neuron-like cells and to provide some evidence for the transplantation of hUCMSCs for spinal cord injury. METHODS: The hUCMSCs were isolated from human umbilical cord digested with collagenase Ⅱ. The hUCMSCs was verified by flow cytometry analysis. The passage 5 cells were randomly divided into 4 groups. The differentiation of hUCMSCs was induced by bFGF in group A, bFGF and BDNF in group B, or BHA, bFGF and BDNF in group C, while the cells in group D served as a control group cultured with DMEM-F12 and 10% FBS. Two weeks later, the expression of nestin, neurofilament protein H(NEFH) and glial fibrillary acidic protein(GFAP) was detected by real-time PCR and immunocytochemistry. The morphological changes of cells were observed under an atomic force microscope. RESULTS: Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion. hUCMSCs expressed CD29, CD44 and CD105, but no CD34, CD45 or HLA-DR. After cultured with inducing medium for 2 weeks, the cells were successfully induced into neuron-like cells. The appearance of the cells had great change. The induced hUCMSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope. The result of real-time PCR showed that nestin was positive in A, B and C groups, and NEFH was positive in A and B groups, but GFAP was negative in 4 groups. The difference of nestin and NEFH expression among the induced groups was significant(P<0.05). CONCLUSION: Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion in vitro, and all the hUCMACs presented stable biological properties. Moreover, hUCMSCs were induced to differentiate into neuron-like cells in vitro via bFGF combined with BDNF.     

植物果实发育调控的分子机理研究进展

果实的发育与成熟是一个复杂的过程,果实大小、形状、颜色、品质、风味等都随着果实发
育和成熟而变化,并受一系列果实发育相关基因的影响和调控。研究植物果实发育调控的分子机理对于今
后提高果实品质具有重要的意义。因此,本文主要综述了拟南芥和番茄中果实发育与成熟相关基因的发掘
与相互作用,以及果实发育调控的分子机理研究进展,为今后的果实发育研究和育种工作的开展提供一定
的理论指导。     

Effect of Radix Tetrastigma hemsleyani flavone on apoptosis and MAPK signaling pathway in myeloid leukemia NB-4 cells

AIM:To study the effects of Radix Tetrastigma hemsleyani flavone (RTHF) on the viability, apoptosis and MAPK signaling pathway in human acute promyelocytic leukemia NB-4 cells. METHODS:The inhibitory effects of RTHF on the viability and proliferation of NB-4 cells were measured by CCK-8 assay and BrdU test. Flow cytometry was used to analyze the apoptosis of NB-4 cells induced by RTHF, and the cell cycle distribution after RTHF treatment. The levels of apoptosis- and MAPK pathway-related proteins in the NB4 cells were determined by Western blot. RESULTS:RTHF inhibited the viability and proliferation of NB-4 cells in a time- and dose-dependent manner, and the IC₅₀ at 48 h was 2.26 g/L. RTHF blocked NB-4 cells into the cell proliferation cycle, with stagnation in the G₂ phase. Meanwhile, RTHF induced apoptosis of the cells, down-regulated the expression of anti-apoptotic protein Bcl-2, and up-regulated the expression of pro-apoptotic proteins Bax, caspase-3 and Cyt-C, in a dose-dependent manner (P<0.05). The expression of ERK5 was decreased, and p38 was increased induced after RTHF treatment. However, no obvious change of ERK1/2 and JNK after RTHF treatment was observed. CONCLUSION:RTHF effectively inhibits the viability and proliferation, and induces apoptosis of leukemic NB-4 cells in vitro. Its mechanism may be related to signaling pathways of p38 MAPK and apoptosis proteins.     

Study on Cx43 communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells in vitro

AIM: To approach the gap junction distribution and communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.METHODS: MSCs were isolated by extraction of bone marrow specimens,gradient centrifugation and the adherence of culture plates.MSCs were culture in vitro,treated with 5-azacytidine and incubated for 24 h.The induced MSCs,which had been incubated for 2,3 and 4 weeks,were divided into group Ⅰ,Ⅱ and Ⅲ.In addition,the normal cardiomyocyte cells were used as control group.The distribution of connexin 43(Cx43) and the mean fluorescence redistribution rate were detected in every group with the laser scanning confocal microscope.RESULTS: Cx43 protein grain density in induced MSCs was increased with the lasting of incubation by quantitive analysis of Cx43 distribution.After 4 weeks,the Cx43 protein density in induced MSCs was nonsignificant deviation with control group (63.87±12.43,64.87±12.15,P>0.05).The diversify tendency of the mean fluorescence redistribution rate was approximation with the result of Cx43 in every group.The results showed that groupⅠ was 19.59%±6.08%,groupⅡ was 37.17%±3.84%,groupⅢ was 46.82%±2.69%,and control group was 49.71%±5.53%.CONCLUSION: MSCs can be differentiated to cardiomyocyte-like cells,which have been induced and incubated for 4 weeks in vitro.The communicational function of those MSCs is similar to the normal cardiomyocyte cells.     

Effect of interleukin 18 gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes

AIM: To study the effect of interleukin 18( IL-8 ) gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes.METHODS: Exosomes isolated from the supernatants of IL-18 gene-modified NCI-H460 lung cancer cells (IL-18/H460), pcDNA3.1⁺ vector-modified cancer cells (DNA3.1/H460) and non-modified NCI-H460 lung cancer cells (NCI-H460) were observed under transmission electron microscope.The expression of heat-shock protein 70(HSP70),human leukocyte antigen(HLA) and IL-18 were determined by Western blotting.T lymphocytes were activated by exosomes or exosome-pulsed dendritic cells(DCs).The activity of T cells for killing lung cancer cells were detected by lactate dehydrogenase (LDH) method.The killing rates were calculated and compared.RESULTS: Exosomes showed typical morphous under transmission electron microscope.The protein levels of HSP70 and HLA were detected in the exosomes of all 3 groups, and IL-18 protein was only observed in IL-18/H460 group.The killing rates of exosome-activated T cells in IL-18/H460 group with the ratio of effector cell to target cell at 25∶ 1, 10∶ 1 and 5∶ 1 were (38.45±5.42)%, (25.17±3.94)% and (11.75±3.22)%, respectively.The killing rates of exosome-pulsed DC-activated T cells in this group were (89.05±4.06)%, (64.97±6.02)% and (40.16±4.98)%, respectively.The killing rates in IL-18/H460 group were higher than those in DNA3.1/H460 group and NCI-H460 group.The anti-tumor efficacy of exosome-pulsed DC－activated T cells was stronger than that of exosome-activated T cells.CONCLUSION: IL-18 gene modification enhances the anti-tumor activity induced by NCI-H460 lung cancer cell-derived exosomes.     

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34⁺ cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34⁺ cells simultaneously expressed mRNA for IL-1(α,β),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGFβ₁ ,TNFα and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGFβ 1 , TNFα and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.     

Electrophysiological characteristics of the membrane current of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells

AIM: To study the electrophysiological characteristics of the membrane currents of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells (MSCs).METHODS: MSCs were induced,cultured and identified according to the reference.At the fourth week after treatment with 5-azacytidine(5-aza),cardiomyocytes-like cells were detected for the membrane current with the whole-cell patch-clamp technique and compared with the undifferentiated MSCs.RESULTS: The undifferentiated MSCs only expressed potassium currents.MSCs were stained positive for troponin T after treatment with 5-aza,and two kinds of inward currents and three kinds of outward currents were expressed.They respectively were the fast inward sodium current (I_Na),the L-type calcium current (I_Ca),the transient outward potassium current (I_to),the ultra-rapid delayed rectifier potassium current (I_kur) and the slow delayed rectifier potassium current (I_ks).Compared with the undifferentiated MSCs,the potassium currents of cardiomyocytes-like cells derived from MSCs were mainly made up of I_kur and I_ks.CONCLUSION: After treatment with 5-azacytidine,MSCs are differentiated into cardiomyocytes-like cells,which express the current of I_Na,I_Ca,I_to,I_kur and I_ks.     

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages, macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

Ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells during their differentiation into neurons and astrocytes

AIM: To observe the ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells (ADSC) differentiating into neurons and astrocytes. METHODS: ADSC were cultured, amplified and induced with β-mercaptoethanol or 3-isobutyl-1-methylxanthine (IBMX). The expression of nestin in the induced cells was determined by the method of immunofluorescence. The ultrastructure of the induced neural stem cells was observed under transmission electron microscope.RESULTS: The expression of nestin reached the peak at 3 h in β-mercaptoethanol group and at 14 d in IBMX group, and the positive rate of the former (86%) was significantly higher than that of the latter (23%). However, the duration of the induction in IBMX group was obviously longer than that in β-mercaptoethanol group. The ultrastructure of the induced neural stem cells in the two groups was similar under the transmission electron microscope, only with some differences in organelles.CONCLUSION: In the process of ADSC differentiating into astrocytes or neurons with the induction of β-mercaptoethanol or IBMX, the different changes of ultrastructure in ADSC-derived neural stem cells occur in some organelles in cytoplasm, not in nucleus.     

The feature of clonal expansion of TCR Vβ T cells from peripheral blood in patients with acute monoblastic leukemia

AIM: To investigate the distribution and clonal expansion of TCR Vβ subfamily T cells in patients with acute monoblastic leukemia (M5). METHODS: The CDR3 of TCR Vβ 24 subfamily genes were amplified in peripheral blood mononuclear cells from 9 cases with M5 using RT-PCR and the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. RESULTS: Only 1-10 Vβ subfamily T cells were identified in M5 cases. Genescan analysis showed that oligoclonal (clonal expansion) T cells were found in some Vβ subfamilies from 8 cases with M5, Vβ 2 oligoclonal T cells were identified in six cases, whereas Vβ 7- or Vβ 9 clonal expansion T cells were detected in the other two cases, respectively. In addition, except Vβ 2, Vβ 7 or Vβ 21 oligoclonal T cells could be detected in two cases, respectively. CONCLUSION: The skew distribution and clonal expansion of TCR Vβ subfamily T cells could be found in patients with M5.It may be a specific ant i-leukemia immune response with which the host T cells were activated by the leukemia-associated-antigen.The clonal expansion T cells were tendentious in Vβ 2,which may be related to the M5 leukemia cells as sociated antigen.     

Inhibitory effect of propolis on proliferation of K562 cells in vitro

AIM: To study the effect of propolis on the proliferation of K562 cells.METHODS: K562 cells were cultured in vitro. Cell proliferation was measured by MTT method. The apoptotic rate was determined by flow cytometry. RT-PCR was applied to detect mRNA expression of Nup98. The protein level of Nup98 was determined by Western blotting. RESULTS: The inhibitory rates of proliferation induced by propolis at the concentrations of 2 mg/L, 20 mg/L and 200 mg/L were obviously higher than that in control cells in a time-and dose-dependent manner. The apoptotic rate was increased in a dose-dependent manner. High concentration of propolis down-regulated the expression of Nup98 at mRNA and protein levels. CONCLUSION: Propolis inhibits the proliferation and induces apoptosis in K562 cells. The mechanism may be related with down-regulation of Nup98.     

Expression of telomerase inhibitor Pinx1 in leukemia cells and its correlation with telomerase activity

AIM:To study the expression of telomerase inhibitor Pinx1 in acute leukemia cells and during the differentiation of acute promyelocytic leukemia cells,and to realize its effect on telomerase activity.METHODS:Realtime quantitative PCR with fluorescence probe hybridization was used to measure the expression of Pinx1 and hTERT mRNA in acute leukemia cells and during differentiation of NB4 cells induced by ATRA.The correlations between Pinx1 and hTERT expression were also analyzed.RESULTS:Pinx1 mRNA expression in acute leukemia samples (0.00312,5.42×10-4-0.024) was significantly higher than that in normal bone marrow mononuclear cells (7.89×10-4,0-0.00863,P<0.01).The expression of Pinx1 mRNA had significant positive correlation with hTERT mRNA expression (r=0.296,P<0.05).Pinx1 mRNA expression decreased during differentiation,its expression was positive correlated with hTERT mRNA expression (r=0.900,P<0.05).CONCLUSIONS:As an inhibitor of telomerase,however,Pinx1 also had the same direction of regulation with telomerase activity in acute leukemia cells,suggesting its expression variation may be a subsequent reaction induced by that of hTERT to stabilize telomerase activity.The exact mechanisms remained to be verified.