Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the safety and efficacy of umbilical cord-derived mesenchymal stem cells (MSCs) infusion in patients with steroid-resistant severe acute graft-versus-host disease (aGVHD).</p><p><b>METHODS</b>A total of 19 patients with steroid-resistant severe aGVHD received MSCs infusion treatment. The treatment response, transplantation-related mortality, events associated with infusion and relapse rate were analyzed.</p><p><b>RESULTS</b>Two patients with grade II, 5 patients with grade III and 12 patients with grade IV aGVHD received a total of 58 infusions of MSCs. The mean total dose of MSCs was 2.13 (range 0.60 - 7.20)×10(6) cells per kg bodyweight. Seven patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. Eleven patients had a complete response and 4 had a partial response and 4 had no response. No patients had side-effects during or immediately after infusions, and no MSCs related tumorigenesis was detected to date. Eleven patients survived and 8 died, 4 for aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1 for underlying leukemia relapse. The cell viability of freshly prepared MSCs is 93% (92% - 95%) by trypan blue staining. The cell viability of programmatically frozen and thawed MSCs is 72% (70% - 74%).</p><p><b>CONCLUSION</b>Infusion of umbilical cord-derived MSCs expanded in vitro is an effective therapy for patients with steroid-resistant severe aGVHD without negative impact on relapse. Freshly prepared MSCs are superior to frozen and thawed cells in terms of cell viability.</p>

Homing characteristics of graft cells in leukemia mice after allogenic bone marrow transplantation with different conditioning regimens / 中国实验血液学杂志

The aim of this study was to investigate the homing characteristics of bone marrow cells in leukemia mice after allogenic bone marrow transplantation with different conditioning regimens on the basis of a leukemia mouse model. Allogenic bone marrow transplantation was performed after three different kinds of conditioning regimen, including nonmyeloablative conditioning regimen (5 Gy (60)Co γ ray total body irradiation, A group), radiotherapeutic myeloablative conditioning regimen (9 Gy (60)Co γ ray total body irradiation, B group) and chemotherapeutic myeloablative conditioning regimen (large dose chemotherapy, C group). In the recipient mice, the nucleated cell number in peripheral blood, bone marrow and spleen was counted, the percentage of positive cells capable of connecting with FITC labeled anti-mouse H-2K(b) antibody was detected by flow cytometry and the homing ratio in bone marrow and spleen was calculated at 24, 48, 72, 96 h after bone marrow transplantation. The results showed that donor myeloid cells displayed homing and then mobilization (going out of home) in group A; homing, mobilization, and rehoming in group B and C, and there was a little delay of homing in the spleen in group C. In bone marrow, the homing efficiency of A group was the highest in early period and the lowest [(0.90 ± 0.09)%] in the fourth day with the mobilization of myeloid cells (P < 0.05), and the homing efficiency of B and C groups was lower in the early period and the highest [(2.17 ± 0.26)%, B group] in the fourth day with the rehoming of myeloid cells (P < 0.05). In spleen, the homing efficiency was similar to that in bone marrow and there still was a little delay in C group. It is concluded that the homing ratio is high in the early period and decrease obviously in 72 h after bone marrow of leukemia mice treated with nonmyeloablative conditioning regimen. The homing ratio is low in the early period and increases obviously in 72 h after bone marrow of leukemia mice treated with radio-or chemotherapeutic myeloablative conditioning regimens. The homing ratio does not obviously change between the early period and 72 h after bone marrow of leukemia mice treated with chemotherapeutic myeloablative conditioning regimen, and lies between group A and B.

A case of Richter syndrome transformed from chronic lymphocytic leukemia with karyotype aberration of trisomy 12 / 中国实验血液学杂志

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.

Comparison of the biological characteristics of serum-free and fetal bovine serum-contained medium cultured umbilical cord-derived mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the differences of biological characteristics between human umbilical cord-derived mesenchymal stem cells (UC-MSCs) cultured by serum-free medium or fetal bovine serum-contained complete medium to establish a xenogeneic protein-free UC-MSCs culture system.</p><p><b>METHODS</b>Healthy human umbilical cord segments were digested with collagenase. UC-MSCs were cultured by serum-free MesenCult-XF medium and FBS-based αMEM complete medium, then analyzed the morphology, immunophenotype, expansion potential, lineage differentiation potential, karyotype and immunosuppression of early passages.</p><p><b>RESULTS</b>The average cell diameters of UC-MSCs in suspension cultured by serum-free medium and FBS-based medium were 26 (18 - 39) µm and 35 (20 - 61) µm, respectively. Cell expansion folds with serum free medium and FBS-based medium were (5.2 ± 0.2) and (3.5 ± 0.1) respectively, in the first five passages. The expansion potential of serum-free medium cultured UC-MSCs was significantly higher than FBS-based medium cultured ones (P < 0.05). A panel of markers CD29, CD44, CD90, CD73, CD105 and HLA-ABC expressed on human UC-MSCs. Hematopoietic lineage markers CD34, CD45 and HLA-DR were not detectable on UC-MSCs. The cpm were (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured MSCs were added to the cultures at MSCs/T cell ratios of 1:100, 1:10 and 1:5. While the cpm was (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured UC-MSCs were added to the cultures. The immunosuppressive potential of serum-free medium-cultured UC-MSCs was higher than serum-contained medium cultured ones at three different MSC/T cell ratios (P < 0.05).</p><p><b>CONCLUSION</b>Compare with serum-contained medium cultured early passages of UC-MSCs, the cell diameter of serum-free medium cultured UC-MSCs was smaller with higher expansion potential. No xenogeneic proteins were presented in UC-MSCs preparations when cultured with serum-free medium. Human UC-MSCs suppressed T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of serum-free medium cultured UC-MSCs was higher than FBS-based medium cultured ones.</p>

Clinical analysis on 40 patients with chronic lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum β2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum β2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

Apoptosis-inducing effect of C-MYC siRNA on acute lymphoblastic leukemia Jurkat cell line / 中国实验血液学杂志

The study was purposed to investigate the effects of C-MYC siRNA on the proliferation and apoptosis of acute lymphoblastic Jurkat cell line. siRNA targeting the site 1545-1565 of C-MYC mRNA was designed and chemically synthesized, then C-MYC siRNA was transfected into Jurkat cells by the transfer agent (HiPerFect Transfection Reagent), the morphological changes were observed under inverted microscope; the tetrazole compound (MTS) was applied to draw the cell growth curve; the cell colony test was used to detect the effect of C-MYC siRNA on the proliferation of Jurkat cells; the flow cytometry and TUNEL method were used to analyze the apoptosis of Jurkat cells. The results showed that after Jurkat cells were treated with different concentrations of C-MYC siRNA, the growth of Jurkat cells was inhibited to various degrees, inhibitory rate was enhanced as C-MYC siRNA concentration increased. C-MYC siRNA also could obviously inhibit the cell clony formation. The apoptosis of cells could be detected by flow cytometry and TUNEL method, the apoptosis rate of cells increased along with prolonging of treatment with C-MYC siRNA. It is concluded that the chemically synthesized C-MYC siRNA can inhibit significantly the proliferation and induce the apoptosis of Jurkat cells.

Effects of small interference RNA against c-myc on expression of c-myc and h-tert genes in acute lymphoblastic leukemia cell line Jurkat / 中国实验血液学杂志

This study was purposed to investigate the effect of small interfering RNA against c-myc on c-myc, h-tert gene and protein expressions in acute lymphoblastic leukemia cell line (Jurkat cells), so as to provide new methods and targets for gene therapy of leukemia. The siRNA against target sites 1545-1565 of c-myc mRNA was chemically synthesized and was transfected into Jurkat cells by transfectant. The c-myc, h-tert mRNA and protein expression levels before and after treatment with c-myc siRNA were detected by RT-PCR and Western blot, respectively. The results showed that c-myc siRNA obviously inhibited the proliferation of Jurkat cells, the half inhibitory concentration (IC50) for 48 hours was about 75 nmol/L. c-myc siRNA could decrease c-myc, h-tert mRNA and C-MYC, h-TERT protein expression levels of Jurkat cells. It is concluded that c-myc siRNA significantly reduce c-myc, h-tert mRNA and protein expression levels through inhibiting c-myc mRNA expression and decreasing intracellular level of C-MYC protein.

Calcium ionophore induces the differentiation of chronic myeloid leukemia cells into dendritic cells / 中国实验血液学杂志

The aim of this study was to investigate the ability of calcium ionophore (CI ) to induce the differentiation of CML cells into dendritic cells (DC), to analyze the P210 expression in DCs and to evaluate the stimulatory effect of CML-DC on production of cytotoxic activity against CML cells via activating the autologous T cells. The mononuclear cells were isolated from bone marrow of CML patients whose WBC counts were more than 30x10(9)/L when samples were collected, then the lymphocytes and monocytes were discarded by pouring out supernatant twice at different culture time point. Slightly adherent cells were cultured in RPMI 1640 containing 10% FCS, with or without CI (375 ng/ml) and GM-CSF (200 ng/ml) at 37 degrees C, 5% CO2, fully humidified atmosphere for 96 hours. The cell morphology was observed under the inverted microscope and electron microscope; the expression of CD antigens was analyzed with flow cytometry; the P210 expression was measured with Western blot. LDH assay was used to evaluate the effect of cultured CML cells (CML-DC) generating cytotoxic T lymphocyte (CTL) activity against CML cells. The results indicated that after treatment with calcium ionophore and GM-CSF for 96 hours, CML cells showed DC morphological characteristics under inverted microscope and electron microscope. The expression of CD83, CD86, CD40, CD80 and HLA-DR increased remarkably. P210 was expressed in the CML-DC, but the expression level was lower than that in CML cells without CI and GM-CSF treatment. LDH assay showed that the CTL activity against CML was found greater in autologous T cells activated by CML-DC than that by CML cells. It is concluded that the CML cells can be induced to quickly differentiate into DC when cultured with CI and GM-CSF. CML-DC expresses P210, but the expression level is lower than that in CML cells. CML-DC can stimulate autologous T cells to produce CTL against CML.

Apoptosis of human myeloid leukemia cell line HL-60 cells induced by siRNA targeting gene c-myc / 中国实验血液学杂志

This study was purposed to investigate the effects of siRNA targeting c-myc on apoptosis induction, proliferation in inhibition as well as c-myc protein and mRNA expression in human myeloid leukemia cell line HL-60 cells. C-myc siRNA synthesized in vitro was transfected into HL-60 cells by liposome. Changes of cell morphology were observed. Growth inhibition was detected by MTT assay and colony formation assay, and cell apoptosis was determined by DNA ladder. The expressions of c-myc mRNA and protein were detected by RT-PCR and Western-blot respectively. The results indicated that c-myc siRNA remarkably inhibited the cell proliferation, with an IC(50) value of 150 nmol/L. Data of DNA ladder showed that HL-60 cells apoptosis could be efficiently induced by c-myc siRNA, the apoptosis rate positively correlated with the time duration of treatment with drugs. The c-myc mRNA and protein expressions on HL-60 cells decreased after treatment with c-myc siRNA, which negatively correlated with time duration of treatment. It is concluded that c-myc siRNA can efficiently induce growth inhibition, decrease the expressions of c-myc mRNA and protein, and induce apoptosis in HL-60 cells.

In vitro inducing effect of dendritic cells cotransfected with survivin and granulocyte-macrophage colony-stimulating factor on cytotoxic T cell to kill leukemic cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Survivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells.</p><p><b>METHODS</b>After derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL.</p><p><b>RESULTS</b>Expression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group.</p><p><b>CONCLUSION</b>DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.</p>

Demethylation and transcription of p16 gene in malignant lymphoma cell line CA46 induced by EGCG / 中国实验血液学杂志

The study was purposed to investigate the possible mechanism of epigallocatechin-3-gallate (EGCG) induced p16 gene demethylation and transcription regulation in the malignant lymphoma cell line-CA46. The induced growth inhibition of CA46 cells was assayed by growth curve and MTT; the DNA content of CA46 cells was analyzed by flow cytometry after being exposed to EGCG; the methylation status of the p16 gene in CA46 cell line before and after treatment with EGCG was detected by the nested-methylation specific PCR and DNA sequencing; the mRNA of p16 and DNA methyltransferases (DNMT3A and DNMT3B) gene were determined by RT-PCR. The results showed that in comparison with the control, all the 3 different concentration of EGCG were able to inhibit the growth of malignancy cell lines and increase the cell number in G(0)/G(1) phase. After treatment with EGCG for 48 hours, the methylation level was apparently attenuated in a concentration-dependent manner. Expression of p16 gene in untreated group was mild while in the treated groups it had been greatly strengthened, as compared with untreated group, the gray scale ratio of p16 to beta-actin 1 treated with EGCG (6, 12, 24) microg/ml was increased from (0.05 +/- 0. 01) to (0.19 +/- 0.03), (0.39 +/- 0.10), (0.85 +/- 0.09) respectively, exhibiting a significant difference (p < 0.05); as compared with the untreated group, after treatment with EGCG for 48 hours, the expressions of DNMT3A and DNMT3B were obviously down-regulated. It is concluded that EGCG can activate and up-regulate the expression of p16 gene mRNA which inhibits the proliferation of CA46 cell through inducing the G(0)/G(1) arrest by demethylation and/or by inhibiting DNMT3A and DNMT3B gene.

Induction of anti-lymphoma cytotoxic T cell effect by dendritic cells transfected with recombinant adenovirus vectors carrying survivin gene / 中国实验血液学杂志

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.

Effect of reduced glutathione as anti-leukemic immune adjuvant / 中国实验血液学杂志

To investigate the reversal effect of reduced glutathione (GSH) on suppression of NK cells by reactive oxygen metabolites (ROM) in K562 cells, interleukin-2 (IL-2) or mononuclear cell (Mo) was added in cultured cell line of K562 cells and NK cells, the yield of ROM and K562 cell suppression rate were observed. Then the histamine dihydrochloride (DHT) or GSH was added in the mixed cultured cell lines, the ROM production and K562 cell suppression rate were observed. The results showed that the ROM yield increased from 33.17 +/- 5.08 U/L to 223.59 +/- 9.41 U/L by IL-2, and K562 cell suppression rate increased from 65.56% to 85.89% by IL-2 (P < 0.01). The ROM yields were 389.79 +/- 43.83 U/ml, 456.74 +/- 42.77 U/ml and 601.42 +/- 21.92 U/ml respectively, and K562 cell suppression rates were 82.36%, 81.36% and 48.09% respectively, when Mo was added in the mixed cultured cell lines under ratios of E/Mo being 10/2, 10/5 and 10/10. When E/Mo was 10/2, DHT or GSH was added in the mixed cultured cell line ROM yield decreased from 389.79 +/- 3.83 U/L to 50.21 +/- 2.4 U/L or -3.58 +/- 9.49 U/L (P < 0.05) respectively. With increase of concentration of DHT or GSH, the ROM yield in the mixed cultured cell line decreased (P < 0.05), the K562 cell suppression rate increased from 82.53% to 94.64% or 96.39% (P < 0.05), the more ROM yield, the less K562 suppression rate (P < 0.05). When E/Mo is 10/5 or 10/10, the ROM yield decreased by the high concentration of DHT or GSH (P < 0.05), but the K562 cell suppression rate not increased by every concentration of DHT or GSH. GSH was as effective as DHT in the reversing ROM and increasing K562 cell suppression rate. It is concluded that GSH may reverse ROM and increase K562 cell suppression rate, and GSH is as effective as DHT, but GSH has less side-effect than DHT. Therefore, GSH would be better antileukemia immune adjuvant.

Construction of recombinant adenovirus vector containing human survivin gene and its expression in dendritic cells / 中国实验血液学杂志

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.

Experimental investigation of non-heart-beating donor in the rat lung transplantation / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the impact of different warm ischemia time on structure and function of the non-heart-beating donor lung and to find out the feasibility of non-heart-beating donor in rat lung transplantation.</p><p><b>METHODS</b>Sixty Sprague-Dawley rats were randomly divided into 3 groups: heart-beating donor (HBD) group, non-heart-beating donor (NHBD) with 30 minutes of warm ischemia time (WIT) group and NHBD with 60 minutes of WIT group. Each group has 10 pairs (the donors and the recipients). The donor lungs of group HBD were flushed with low potassium dextran (LPD) solution at 4 degrees C after asystolia while the lungs of group NHBD-30 and group NHBD-60 remained ventilated at the room temperature for 30 and 60 minutes after asystolia and then were flushed with LPD solution. All the donor lungs remained inflated when they were stored in LPD solution at 4 degrees C for 4 hours. The recipient rat underwent left thoracotomy, and then orthotopic left lung transplantation. Followed by a right thoracotomy, the right pulmonary hilum were ligated with one-hour reperfusion and ventilation.</p><p><b>RESULTS</b>All the recipients in group HBD and group NHBD-30 survived the observation period of one hour with excellent gas exchange, whereas 4 of recipients in group NHBD-60 survived for 10 minutes after the ligation of right pulmonary hilum and 3 for 20 minutes. The pulmonary compliance, ultrastructure, energy metabolite and other markers revealed no significant differences between group HBD and group NHBD-30 (P > 0.05). But the differences between group NHBD-60 and other two groups were significant (P < 0.05).</p><p><b>CONCLUSIONS</b>The adoption of non-heart-beating donor could be a safe and effective method to expand the lung donor pool. The NHBD lung with 30 minutes of WIT may be suitable for lung transplantation in rat.</p>

Bcl-2 antisense oligonucleotides suppress HL-60 cell growth in a SCID mouse mode / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effects of bcl-2 antisense phosphorothioate oligonucleotides (ASPO) on suppression of HL-60 cell growth in SCID mice and to investigate the feasibility of purging leukemia cells plus bcl-2 ASPO used in vitro.</p><p><b>METHODS</b>1 x 10(7) viable HL-60 cells were treated with 10 micro mol/L bcl-2 ASPO seven days before the intraperitoneal (IP) inoculation to the SCID mice, Treatment with sense oligonucleotides (SPO) was similar as for the controls. 35 days after the inoculation, all the SCID mice of both groups were sacrificed and their peripheral blood, bone marrow, liver and spleen were examined using half nested RT-PCR and histopathology for detecting the appearance and distribution of the HL-60 cells treated beforehand with antisense or sense oligonucleotides respectively.</p><p><b>RESULTS</b>ASPO could down regulate the expression of bcl-2 resulting in both inhibition of growth and induction of apoptosis in treated HL-60 cells, which failed to develop leukemia in SCID mice at all. However, SPO treated HL-60 cells still behaved their own ways and proliferated agressively, and developed leukemia at last.</p><p><b>CONCLUSION</b>The bcl-2 ASPO enables to suppress HL-60 cell growth and prevent the development of leukemia in the SCID mice. The purging leukemia cells used are seemed liable in inhibiting the development of leukemia in SCID mouse model.</p>

Oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation / 药学学报

<p><b>AIM</b>To explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.</p><p><b>METHODS</b>Intracellular mean fluorescence intensity was measured by flow cytometry.</p><p><b>RESULTS</b>After treatment with FITC-labeled G3139 at the concentration of 0.60 mumol.L-1 for 4 h, the G3139 uptake into peripheral blood mononuclear cell and bone marrow mononuclear cell in hematological tumor patients was significantly higher than that in normal control. There was different uptake of G3139 among the malignant hematological tumor cell strains, and the uptake in cells derived from monocyte, B lymphocyte and myeloid cell was much higher than that in cells derived from T lymphocyte. After treatment with all-trans retinoic acid (ATRA), HL60 cell proliferation was markedly inhibited and the uptake of G3139 decreased significantly.</p><p><b>CONCLUSION</b>Hematological tumor cells were capable of taking up oligonucleotide, and the oligonucleotide uptake in hematological tumor cells is related to its cellular species and its activation.</p>

Bone Marrow Cells Activated by Autologous Dendritic Cells Purges Bone Marrow from Patients with Chronic Myelogenous Leukemia / 中国实验血液学杂志

In order to investigate the effect of autologous dendritic cells (DCs) activating bone marrow cells and purging bone marrow from chronic myelogenous leukemia (CML) patients, DCs were separated by negative selection system of human cells from bone marrow mononuclear cells (BMMNCs) of 2 CML patients in hematological remission and harvested after 3 days of culture in IMDM containing autologous plasma, rhGM-CSF and rhTNFalpha at 37 degrees C, 5% CO(2) humidified atmosphere. BMMNCs from the patients were also used to set up long-term culture (LTC) system in T-25 plastic flasks. The LTCs included three groups, i.e., control, addition of rhIL-2, and co-culture with autologous DCs. Half of non-adherent cells were collected, counted and assayed for CFU-GM weekly. Then, equivalent volume of fresh medium was replaced to maintain the culture. The culture was discontinued if the non-adherent cells count was less than 2 x 10(5). Adherent cells were collected for CFU-GM assay and flow cytometry for CD34 and P210. The colonies originating from the adherent cells were picked up under the inverted microscope. RNA was extracted, and BCR/ABL measured by nested reverse transcription polymerase chain reaction (RT-PCR). The results showed that the CFU-GM yields of non-adherent cells declined after 1 to 2 weeks co-cultured with autologous DCs, and it paralleled with group with rhIL-2. P210(+) cell percentage was also decreased. From the third week on, however, the decrease of CFU-GM yields slowed down, while CFU-GM in the system with rhIL-2 continued to fall. In system co-cultured with autologous DCs, the adherent cells contained the least percentagcs of CD34(+) cells and P210(+) cells percentage. However, the expression of BCR/ABL in CFU-GM colonies derieved from the adherent cells of DCs co-cultured had no significant difference with those from the culture without DCs. Our results suggest that co-culture of marrow cells with autologous DCs could significantly diminish the leukemic progenitors cells including both mature and primitive progenitor cells. Autologous dendritic cells might be used for ex vivo purging of CML marrow.