Effect of spinal cord hemisection on bone fracture healing in rats / 中国组织工程研究

BACKGROUND: The nervous system has been proved to play an important role in fracture healing, and different nervous components exert different effects on bony callus.OBJECTIVE: To observe the callus volume and morphological changes in the fracture healing progress of the rats with spinal cord hemisection and fracture.METHODS: Forty-eight male Sprague-Dawley rats were divided into two groups, and the models of simple fracture and spinal cord hemisection by cutting the right spinal cord combined with fracture were established. The tibia was removed at 14 and 28 days after surgery, and the wet weight was measured, the fracture healing was observed by X-ray examination, morphological changes of the callus were observed through hematoxylin-eosin staining, and the expression level of calcitonin gene-related peptide mRNA in the broken bones was detected by SYBR Green qPCR.RESULTS AND CONCLUSION: In the combination group, the wet weight of the contralateral tibia was more than that of the affected side (P < 0.05), which both were more than those in the simple group (P < 0.05). X-ray findings showed that the combination group had the largest lateral callus, followed by the affected side, and the smallest in the simple group.Hematoxylin-eosin staining showed that in the combination group, the contralateral cells arranged in disorder, while the fibrous callus and cells in regular arrangement were found in the affected side; there were bony callus and cells in regular arrangement in the simple group at 28 days after surgery. SYBR Green qPCR results revealed that the expression level of calcitonin gene-related peptide mRNA in the simple group was significantly higher than that in the combination group (P < 0.05). To conclude, complete innervation is a necessary factor for fracture healing.

Resveratrol-downregulated phosphorylated liver kinase B1 is involved in senescence of acute myeloid leukemia stem cells / 华中科技大学学报（医学）（英德文版）

Senescence is an important obstacle to cancer development. Engaging a senescent response may be an effective way to cure acute myeloid leukemia (AML). The aim of this study was to examine the effect of resveratrol-downregulated phosphorylated liver kinase B1 (pLKB1) on the senescence of acute myeloid leukemia (AML) stem cells. The protein expressions of pLKB1 and Sirtuin 1 (SIRT1), a regulator of pLKB1, were measured in CD34(+)CD38(-) KG1a cells treated with resveratrol (40 μmol/L) or not by Western blotting. Senescence-related factors were examined, including p21 mRNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase (SA-β-gal) staining, cell proliferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that pLKB1 was highly expressed in CD34(+)CD38(-) KG1a cells, and resveratrol, which could downregulate pLKB1 through activation of SIRT1, induced senescence and apoptosis of CD34(+)CD38(-) KG1a cells. It was concluded that resveratrol-downregulated pLKB1 is involved in the senescence of AML stem cells.

Mechanism involving blm gene underlies repair of DNA damage of Jurkat cells induced by mitomycin C / 中国实验血液学杂志

The defect or block of apoptosis is an important factor involved in the drug resistance of tumor cells. Blm gene plays a great role in DNA damage and repair. This study was aimed to explore the relationship of blm gene expression with cell cycle and apoptosis after Jurkat DNA damage. The apoptosis rate and change of cell cycle were detected by flow cytometry, the expression level of blm mRNA in Jurkat cells was determined by semi-quantitative RT-PCR. The results indicated that after induction with 0.4 g/L of mitomycin C (MMC) for 24 hours the apoptosis rate of Jurkat cells were (11.42±0.013)%, and (66.08±1.60)% Jurkat cells were arrested in G2/M phase. After induction for 48 hours, the apoptosis rate of Jurkat cells declined from (11.42±0.013)% to (8.08±0.27)%, and cell count of Jurkat cells arrested in G2/M phase decreased from (66.08±1.60)% to (33.96±1.05)%. When induced with 0.4 g/L of MMC for 24 hours, the apoptosis rate of fibroblasts and the percentage of fibroblasts in G2/M, G0-G1 and S phase all showed no significant change until 48 hours. The range of apoptosis rate and the change of cell percentage in three phases were significantly different between Jurkat cells and fibroblasts (p<0.01). Expression level of blm mRNA in Jurkat cells was remarkably higher than that in normal fibroblasts (p<0.01), at 48 hours expression level of blm mRNA was remarkably higher than that at 24 hours. The 2 groups showed clear difference of blm mRNA expression after treated by MMC (p<0.01). It is concluded that the blm gene may play a significant role in repair of DNA damage of Jurkat cells after MMC induction. Abnormal expression of blm is correlated to the drug resistance of leukemia cells.

Effect of MLL-AF9 fusion gene silence of acute monocytic leukemia cell line THP-1 on cyclin-dependent kinase inhibitor p27 expression / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of MLL-AF9 fusion gene silence on p27 expression and transcription regulation in THP-1 cells.</p><p><b>METHODS</b>Small interference RNA (siRNA) fragments targeting THP-1 cells specific MLL-AF9 fusion gene were designed and constructed, and transfected into THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was examined by RT-PCR and the expression of MLL-AF9 and p27 protein was detected by Western blot. Chromatin immunoprecipitation (ChIP) assay was used to confirm whether MLL-AF9 binds to the p27 promoter in THP-1 cell.</p><p><b>RESULTS</b>SiRNA transfection efficiency was (69.1 +/- 1.8)%. The level of p27 expression was up-regulated at both mRNA [(0.84 +/- 0.12) vs (0.35 +/- 0.03) of control group] and protein levels after MLL-AF9 expression was significantly inhibited in siRNA-transfected cells (0.31 +/- 0.07) compared with that in the controls (1.25 +/- 0.13) (P<0.01). MLL-AF9 fusion protein bond to DNA fragment of p27 gene promoter region in THP-1 cell.</p><p><b>CONCLUSION</b>MLL-AF9 fusion gene silence up-regulates p27 gene expression, and the mechanism maybe the recovery of p27 gene expression due to MLL-AF9 fusion protein binding to p27 promoter.</p>

Effect of down-regulating mll-af9 gene expression on proliferation of acute monocytic leukemia cell line THP-1 / 中国实验血液学杂志

This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1.

Enhancement of Fas-mediated apoptosis in leukemic cell line HL-60 by Bay 11 - 7082 / 中国实验血液学杂志

The aim of study was to explore the effects of NF-kappaB inhibitor Bay 11 - 7082 on Fas/FasL system and Fas-mediated apoptosis in HL-60 cells. The mRNA and protein expression levels of Fas, FasL and XIAP after treatment with Bay 11 - 7082 were detected by RT-PCR and FCM respectively. The level of sFasL was detected by ELISA before and after treatment with Bay 11 - 7082; apoptosis was detected by FCM before and after treatment with Bay 11 - 7082. The results showed that after treating HL-60 cells with Bay 11 - 7082, the mRNA and protein levels of FasL and XIAP were lower than that of controls, the difference was significant by statistic analysis (p < 0.05). Neither the mRNA and protein levels of Fas, nor the level of sFasL changed significantly (p > 0.05). Apoptotic rate of HL-60 cells treated with Bay 11 - 7082 was significantly higher as compared with controls (p < 0.05). It is concluded that Bay 11 - 7082 can enhance Fas-mediated apoptosis in HL-60 cells by down-regulation of FasL and XIAP levels.

hMRE11 plays an important role in U937 cellular response to DNA double-strand breaks following etoposide / 中国实验血液学杂志

MRE11 plays an important role in the signal transduction of DNA damage response, therefore this study was purposed to explore the relationship between hMRE11 focus formation and DNA double-strand breaks (DSBs) caused by etoposide (VP-16) in human promonocytic cells U937. After U937 cells were treated with VP-16, the drug-induced DSBs were assessed by pulsed-field gel electrophoresis (PFGE), the gene transcription levels of hMRE11 were evaluated by RT-PCR, the nuclear focus formation of hMRE11 protein was examined using immunofluorescence technique, the cell cycle in parallel was analyzed by flow cytometry. The results showed that the percentage of U937 cells with DSBs induced by VP-16 raised from 13.0 +/- 2.3% in VP-16 2 microg/ml to 32.0 +/- 4.3% in VP-16 20 microg/ml (P < 0.01) along with increase of VP-16 dose. No difference of the hMRE11 mRNA level in U937 cells following the treatment with 100 microg/ml VP-16 at different times was discovered (P > 0.05). The hMRE11 protein was abundantly and uniformly distributed in the nuclei of untreated U937 cells outside of nucleoli, however, it formed discrete nuclear foci following VP-16 treatment. The mean value of nuclear foci increased by 5 to 20 times following the drug dosing (P < 0.01). An average of 5 nuclear foci per positive nucleus were observed at a dose of 2 microg/ml, and it was increased to an average of over 14 nuclear foci per positive nucleus after treating with VP-16 20 microg/ml. The percentage of nuclei containing hMRE11 nuclear foci also increased from less than 10% after treatment wiht VP-16 2 microg/ml to over 50% after VP-16 20 microg/ml (P < 0.01) following treatment with VP-16. After U937 cells were treated with 100 microg/ml VP-16 for 2 hours and fixed at 4, 8, 12 and 24 hours, the percentage of nuclei with hMRE11 nuclear foci increased to 61.54 +/- 3.6% (the control U937 cells: 0.47 +/- 1.17%, P < 0.01) at 8 hours, with a subsequent decrease in the percentage of nuclear foci-positive cells by 24 hours. The ratio of S-phase U937 cells at 8 hours after being treated with 100 microg/ml VP-16 for 2 hours was 47.55 +/- 2.35%, and that without 100 microg/ml VP-16 was 21.95 +/- 2.91% (P < 0.05). It is concluded that the nuclear focus formation of hMRE11 protein may be a response to DNA damage induced by topoisomerase II inhibitor VP-16 in human promonocytic cell line U937.

Comparison of immunophenotype and clinical manifestations between patients with M5a and M5b of acute monocytic leukemia / 中国实验血液学杂志

Acute monocytic leukemia is a distinct subtype of acute myeloid leukemia (AML) with characteristic biology and clinical features. This study was designed to compare the immunophenotypical features and clinical manifestations of the patients with AML-M(5a) to that of patients with AML-M(5b), and to identify differences between M(5a) and M(5b) and to explore their relations. A total of 58 cases of de novo adult patients with AML M(5) were investigated. Immunofluorescence analysis by flow cytometry was performed to determine the immunophenotype of the leukemic cells in all cases. Meanwhile, clinical data of these cases were studied retrospectively. The results showed that the immunophenotypes of monocytic leukemic cells in patients with AML M(5) were heterogeneous, and CD68 and CD11b were expressed higher in patients with AML M(5a), compared with that in patients with AML M(5b) (P < 0.01). The significant differences in sex, extramedullary infiltration, WBC counts of peripheral blood, complete remission rate and disease-free survival (DFS > 300 days) between the patients with AML M(5a) and M(5b) did not exist (P > 0.05). It is concluded that the special individual immunophenotype features can be detected in patients with either of AML M(5a) or M(5b), and that expressions of CD68 and CD11b were much higher in M(5a). It seems that the complete remission rate and disease-free survival of patients with M(5a) and M(5b) are not different from that of currently available therapy.

Depressing the immune escape of acute myelomonocytic leukemia via an anti-Fas ribozyme / 中国实验血液学杂志

In order to investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in CTLL-2 cells (mouse CTL cell line), and to explore a new way for enhancing the ability of T cells against Leukemia in donor lymphocytes infusion, CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 (mock-transfected) via electroporation. Fas expression on CTLL-2 cells was detected by RT-PCR and Western blot. The killing effect of CTL against WEHI-3 (mouse acute myelomonocytic leukemia cell line) highly expressing FasL in vitro was detected by MTT assay. The caspase-3 proteolytic activity and the apoptosis rate of CTLL-2 cells were detected by means of BD AproAlert Caspase-3 Colorimetric kit and FITC labeled Annexin-V apoptosis detecting kit respectively. The results showed that the anti-Fas ribozyme could be successfully introduced into mouse CTLL-2 cells; Fas expression on the surface of cells transfected with the ribozyme was obviously decreased, in comparison with control and mock-transfected cells; after cocultured with WEHI-3 cells, the viability of CTLL-2 cells transfeced with the ribozyme was significantly increased, as compared with other two groups; caspase-3 activity and apoptosis rate of the ribozyme-transfeced cells were significantly decreased, the killing effect of CTLL-2 transfected with the ribozyme was stronger than that of other groups. It is concluded that anti-Fas ribozyme can remarkably decrease Fas expression on CTLL-2 cells, so as to avoid Fas-mediated apoptosis by Fas ligand on WEHI-3 cells, and to enhance their killing activity against WEHI-3 cells, as a result, the immune escape of acute myelomonocytic leukemia was depressed.

Comparison of cytogenetics and clinical manifestations between M5a and M5b of acute monocytic leukemia / 中国实验血液学杂志

To compare the cytogenetic difference between M5a and M5b of acute monocytic leukemia and to study the correlation between karyotypes and clinical manifestations, a total of 58 cases of de novo adult AML M5 have been investigated. Chromosome metaphases of bone marrow cells were prepared by using direct method and 24 hours short-term culture. The karyotypes were analyzed by G-banding. Meanwhile, clinical information of these cases were studied retrospectively. The results showed that there were 28 with normal karyotype and 30 with aberrant karyotype in 58 cases. The frequency of normal karyotype in patients with M5b was significantly higher than that in patients with M5a (P = 0.0001). The 11q23 aberrations and trisomy 8 were more common in patients with M5a in comparison with patients with M5b (P < 0.01). The patients with AML M5 with aberrant karyotype had a higher incidence of hyperleukocytosis, extramedullary central nerve system infiltration, lower complete remission (CR) rate and shorter overall survival. It is concluded that acute monocytic leukemia is a series of heterogeneous diseases, a distinctive cytogenetic features can be observed between patients with AML M5a and M5b, these results will provide insights into the classification and pathogenesis mechanism of AML M5 at molecular level.

Expression of immune response molecules and function of fas ligand on surface of AML WEHI-3 cells / 中国实验血液学杂志

The purpose of this study was to investigate the expression of Fas, Fas ligand (FasL) and CD80 and function of FasL on the surface of acute myelomonocytic leukemia cells from WEHI-3 line. The expression of Fas, FasL and CD80 on the surface of WEHI-3 were detected by flow cytometry, the apoptosis of YAC-1 cell induced by FasL on the surface of WEHI-3 were detected by (3)H-TdR incorporation. The results showed that the expression rate of Fas, FasL and CD80 on the surface of WEHI-3 cells were (6.75 +/- 2.31)% (n = 5), (63.73 +/- 5.23)% (n = 5) and (5.06 +/- 0.41)% (n = 5) respectively. The apoptosis rate of YAC-1 cells (target cells) co-cultured with WEHI-3 cells (Effector cells) at the rate of 1:3, 1:10 and 1:30 were (26 +/- 4.5)%, (35 +/- 3.2)% and (43 +/- 2.7)% (n = 5) respectively. It is concluded that WEHI-3 cells have high expression of FasL and low expression of Fas and CD80 on their cell membrane, and can induce the apoptosis of Fas(+) YAC-1 cells.

Cytobiological and clinicobiological features of AML with 11q23 chromosome abnormalities / 中国实验血液学杂志

To investigate the interrelationship among morphology, immunology and clinical features in adult acute myeloid leukemia cases with 11q23 chromosome abnormalities, 210 newly diagnosed AML patients were retrospectively analyzed by cell morphology, immunophenotyping, G-banding or R-bamding analysis and clinical features. The results showed that 13 cases were found with 11q23 rearrangements or deletion (the incidence rate was 6.19%.), totally 84.6% showed the involvement with the monocytic lineage. Immunophenotyping tests indicated that AML cases with 11q23 abnormalities usually expressed the marker molecules of hematopoietic stem or progenitor cells, monocytic lineage cells, such as CD34, CD117, CD14, CD15 and CD11b. The complete remission rate of the cases with 11q23 abnormalities was comparable to that of the cases with normal karyotype (P = 0.075), but the median disease-free survival in the former was significantly lower than that in the latter (P < 0.001). It is concluded that the category AML with 11q23 abnormalities accounts for 6.19% of all the newly diagnosed AML cases, that seems to be closely associated with monocytic differentiation blocking with a dismal prognosis.

Expression of BLM mRNA in six tumor cell strains / 中国实验血液学杂志

The mutations of BLM gene may result in Bloom's syndrome which includes immunodeficiency, predisposition to malignant tumors and so on, and enhances sister chromati exchange (SCE), DNA replication failure, genome instability, and increases cancer susceptibility. This study was aimed to investigate the variability of mRNA expression level and cDNA structure of BLM gene in tumor cell strains so as to look for a new cancerogenic mechanism and to find a new therapeutic target. The expression level of mRNA and the structure of cDNA of BLM gene in six tumor cell strains and the normal human bone marrow mononuclear cells were detected with RT-PCR and DNA sequencing was performed. The results indicated that these tumors cells expressed BLM mRNA higher than the normal human bone marrow mononuclear cells (P < 0.01), but no cDNA sequence abnormality of BLM gene in these tumors cells was observed. It is concluded that the increase of expressing level of BLM mRNA may play an important role in the development of these tumors.

Recombinant adenovirus mediated hVEGF165 gene transfer promotes recovery of hematopoiesis in post-BMT mice / 中国实验血液学杂志

To investigate the effects of vascular endothelial growth factor (VEGF) on the recovery of hematopoiesis in post-BMT mice, the recombinant adenovirus Ad-EGFP/hVEGF(165) was injected into syngeneic BMT BALB/c mice via the tail vein. At day 10, 20, 30 after BMT, the in vivo expression of hVEGF(165) was measured. At the same different time points, the numbers of WBC, Plt, RBC in peripheral blood and MNC in bone marrow were counted. By the way, the bone marrow MNCs at day 30 post-BMT were used for further CFU-S assay. The results indicated that a long-term expression of hVEGF(165) in plasma and different organs was successfully mediated by recombinant adenovirus. At each time point of post-BMT, the numbers of WBC, Plt, RBC as well as bone marrow MNC observed in the group treated with recombinant adenovirus Ad-EGFP/hVEGF(165) were lower than those of the normal control group, but were higher than those in other testing groups (P < 0.05). The number of CFU-S (21.4 +/- 2.67) formed by bone marrow MNC at day 30 after BMT reached to the normal level (19.50 +/- 2.46) (P > 0.05), which was much higher than that in other groups (P < 0.05). It is concluded that hVEGF(165) gene transfer mediated by recombinant adenovirus plays a role of promoting the recovery of hematopoiesis in post-BMT mice.

Impact of trisomy 8 on cytobiological and clinical features of acute myelomonocytic and monocytic leukemia / 中国实验血液学杂志

To evaluate the impact of trisomy 8 on cytobiological and clinical features of acute myelomonocytic and monocytic leukemia (M(4), M(5)), a total of 56 cases of acute myelomonocytic and monocytic leukemia were investigated. Karyotypes were analyzed by G-banding or R-banding. The immunotypes in all cases were detected by flow cytometry. And the clinical characteristics at the first visit were analyzed retrospectively. The results showed that thirty-four of 56 (60.7%) patients had normal cytogenetics; 10 (17.9%) patients had trisomy 8 in their karyotypes, including 3 (5.4%) patients with trisomy 8 as the sole aberration; and 12 (21.4%) patents had other cytogenetic abnormalities (except trisomy 8). All trisomy 8 cases demonstrated a increased expression frequency of surface markers of myeloid progenitor cells CD34 (P < 0.01) and CD117 (P < 0.05) and a decreased expression frequency of surface markers of mature monocytes CD11c (P < 0.01) and CD14 (P < 0.05), compared with normal cytogenetics cases. Patients with trisomy 8 were slightly older (P < 0.05), which had lower percentages of peripheral blasts (P < 0.05) and lower WBC (P < 0.05) than the patients without trisomy 8. Patients with trisomy 8 had a shorter disease-free survival time than that of patients with normal cytogenetics (P < 0.05). It is concluded that trisomy 8 may play an important role in the pathogenesis and progression of acute myelomonocytic/monocytic leukemia (M(4)/M(5)), whic seems to be related with a block in differentiation of monocytes. Therefore, trisomy 8 may be an adverse prognostic factor for patients with M(4) or M(5).

Role of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells and its mechanism / 中国实验血液学杂志

To investigate the role and mechanism of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells, apoptosis of CD34(+) cells, mitochondrial membrane potential (MMP) and Fas antigen expression were detected by flow cytometry (FCM), the Bcl-2 protein expression was detected by immunohistochemistry, caspase-3 expression was determined by Western blot and caspase-3 activity analysis, colony-forming units (CFU) was performed by semi-solid methylcellulose culture. The results showed that when cells were store at -196 degrees C for 2 weeks or 4 weeks, apoptotic cells increased, gel electrophoresis displayed typical DNA ladder, and CFU decreased by 25.2% and 30.1%. The value of MMP reduced and expression of Bcl-2 protein was down-regulated during the freeze-thaw process, but the Fas antigen expression was not effected. However, only the 32 kD inactive caspase-3 proenzyme was detected in freshly isolated CD34(+) cells. After freeze-thaw, caspase-3 was activated and a cleavage of 20 kD protein was detected. Cryopreserved cells showed a 1.2-fold and 1.5-fold increase in caspase-3 activity, respectively. It is concluded that apoptosis plays an important role in cryoinjury of cord blood hematopoietic stem/progenitor cells, which triggers a mitochondrial apoptotic pathway that is caspase-dependent but does not require death receptors, where caspase-3 is the key effector.

Experimental study of inhibiting CTLL-2 cell apoptosis and enhancing cytotoxicity to Yac-1 cell by hammerhead ribozyme / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in mouse cytotoxic T lymphocyte (CTL) cell line--CTLL-2 cells, and explore a novel approach to enhance the ability of T cells against leukemia in donor lymphocytes infusion (DLI).</p><p><b>METHODS</b>A hammerhead ribozyme targeting the Fas mRNA was synthesized and transfected into CTLL-2 cells by electroporation. Fas expression in CTLL-2 cells was detected by using RT-PCR, Western blot and flow cytometry, CTLL-2 cells viability was measured by MTT assay, caspase-3 proteolytic activity by caspase-3 detection kit, and cell apoptosis by flow cytometry. Killing activity of CTLL-2 was detected by LDH releasing assay in vitro.</p><p><b>RESULTS</b>Expression of Fas mRNA and protein in CTLL-2 cells was reduced to 50% after transfection with anti-Fas ribozyme. Being treated with anti-Fas antibody (JO(2)), compared with control and mock-transfected cells, viability of CTLL-2 cells transfected with anti-Fas ribozyme increased by 1-fold, caspase-3 activity and apoptosis rate of ribozyme-transfected cells decreased to 50% and 37%, respectively, and cell killing activity was enhanced by 2-fold.</p><p><b>CONCLUSION</b>Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.</p>

Study on blocking the leukemia immune escape after BMT by Fas-Fas ligand pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.</p><p><b>METHODS</b>The recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT.</p><p><b>RESULTS</b>The adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5 x 10(11) pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P > 0.05), but indices in Group A were significantly lower than those in the latter three groups (P < 0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P < 0.01).</p><p><b>CONCLUSIONS</b>The transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.</p>

Study on the expression of hematopoietic growth factor gene in human umbilical vein endothelial cells using gene chip / 中国实验血液学杂志

In order to detect the hematopoietic growth factor gene expressed in human umbilical vein endothelial cells using gene chip, human umbilical vein endothelial cells (ECV304) were cultured in vitro and divided into VEGF group and control group in same medium. 50 ng/ml hVEGF165 was added in the VEGF group. After culture for 24 hours all cells were collected for total RNA extraction. Then, cDNAs were marked with Cy3 and Cy5 for control group and VEGF group, respectively, and hybridized with gene chip. Hybridization signals were collected and analyzed following scanning by laser co-focal microscopy. The results showed that a large number of hematopoietic growth factor and receptor genes (Epo/R, GM-CSF/R, G-CSF/R, LIF, IL-3, TPO, Flt-3, SCF) were expressed in both groups, while many other growth factors (VEGF, IGF2, PDGFA, PDGFB, TGFbeta1) and receptors (neuropilin-1, neuropilin-2, TGFbeta-R1)were expressed. The differentially expressed genes amounted to 24. It is concluded that many hematopoietic growth factors and receptors expressed by hUVECs could be analyzed in a short period by using gene chip, which provides a powerful method for further studies on characteristics of vascular endothelial cells.

Study on blocking the tumor immune escape by Fas ligand pathway / 中国实验血液学杂志

The expression of Fas ligand (FasL) on the membrane of many kinds of leukemia or solid tumor cells played an important role in the immune escape of tumor cells. This study was aimed to know if the soluble Fas (sFas), expressed by adenovirus, could block the immune escape of tumor cells by FasL pathway. The two recombinant adenoviral vectors, AdsFas with murine soluble Fas gene and AdEGFP with enhanced GFP protein gene, were constructed by homologous recombination between two plasmids in Escherichia coli with the AdEasy adenovirus vector system. The viruses were propagated and purified by two times ultracentrifugation. Their titres were detected by plaque assays. The expressed protein was evaluated by Western blot analysis. Then the tumor EL4 cells were infected with AdsFas and AdEGFP respectively. The apoptosis ratio of the target cells-YAC-1 cells induced by EL4 cells was respectively detected by (3)H-thymidine ((3)H-TdR) labeling. The results showed that the recombinant adenoviral vectors AdsFas and AdEGFP were successfully obtained. The titres of viruses purified by two times ultracentrifugation were up to 10(11) pfu/ml by plaque assays. The sFas protein was highly expressed in the target cells by Western blot analysis. After the EL4 cells were transfected with the adenoviruses AdsFas, the apoptosis rate of YAC-1 cells in the sFas transfection group (respectively 6%, 7% and 9% when the effector:target (E:T) was 3:1, 10:1 and 30:1) was obviously lower than that in the control group (respectively 28%, 37% and 45%), P < 0.01. But when the EL4 cells were transfected with AdEGFP, the apoptosis rate of YAC-1 cells (respectively 30%, 36% and 48%) was similar to the control group, P > 0.05. In conclusion, the transfer of sFas by adenovirus could inhibit the apoptosis of Fas(+) cells-YAC-1 cells induced by tumor EL4 cells. It showed that the transduction of sFas could block the effect of the immune escape of EL4 cells through FasL in vitro. These results thus provide a new direction to find a way to treat tumors.