Role of allograft inflammatory factor-1 in regulating the proliferation, migration and apoptosis of colorectal cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism.</p><p><b>METHODS</b>The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting.</p><p><b>RESULTS</b>AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression.</p><p><b>CONCLUSION</b>AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.</p>

Clinical Efficacy of Sorafenib Combined with Low Dose Cytarabine for Treating Patients with FLT3+ Relapsed and Refractory Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the efficacy and safety of sorafenib combined with low dose cytarabine for treating patients with FLT3(+) relapsed and refractory acute myeloid leukemia (FLT3(+) RR-AML).</p><p><b>METHODS</b>Seven patients with FLT3(+) RR-AML were treated with sorafenib and low dose cytarabine. The curative rate and adverse effects were observed in these patients.</p><p><b>RESULTS</b>Out of 7 RR-AML patients after treatment, 5 patients achieved complete remission (CR), 2 patients achieved partial remission (PR), and the overall response rate (ORR) after one course of therapy was 100%. No severe bleeding, nausea, vomiting and other side effects were found in these patients.</p><p><b>CONCLUSION</b>Sorafenib combined with low dose cytarabine can effectively induce the remission of FLT3(+) RR-AML patients, and is worth for further clinical trails to verify its safty and efficiency.</p>

Clinical Summarization of Allogeneic Hematopoietic Stem Cell Transplantation for Leukemia: A Report of 100 Cases / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the treatment outcome of a consecutive series of 100 leukemia patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The clinical data of leukemia patients received allo-HSCT were analyzed retrospectively, the therapeutic efficacy was summarized. 100 evaluable cases of leukemia included 47 cases of AML, 33 cases of ALL, 2 cases of AL (biphenotypic), 16 CML and 2 CMML. Before transplantation, 76 cases were in first complete remission, 9 cases in second or greater complete remission and 15 cases in non-remission or relapse. All the patients received peripheral blood hematopoietic stem cell transplantation (PBHSCT). The conditioning regimen of human leukocyte antigen (HLA)-matched allo-HSCT group was modified BuCy, but in HLA-mismatched group Fludarabine and anti-human thymocyte globulin (ATG) was added. CsA+MTX regimen was used for prophylaxis of graft-versus-host disease (GVHD) in HLA-identical allo-HSCT, while additional MMF was added in HLA-mismatched group. The average time of follow-up was 13 months.</p><p><b>RESULTS</b>At the last follow-up, 66.0% (66/100) patients survived, 53.0% (53/100) patients survived without leukemia, 28.0% (28/100) patients relapsed and 34.0% (34/100) patients died, 44.1% patients of them died from infectious pulmonary complications. During transplantation, 65.0% of the patients were suffered from lung infection. The overall survival (OS) and disease-free survival (DFS) of all cases was 60.9% and 48.8%, respectively. The recurrence rate was significantly higher in non-remission (66.7%) than in CR (21.2%) patients (P < 0.05). The cumulative incidence of GVHD in HLA-mismatched transplantation was 60.8%, which was significantly higher than that of HLA-matched transplantation (38.8%) (P < 0.05).</p><p><b>CONCLUSION</b>Allo-HSCT can cure a significant proportion of leukemia patients, especially for those in CR status. Since the incidence of infectious pulmonary complications after allo-HSCT is still high, much more attention should be paid to it. The comprehensive prognosis of HLA-matched transplantation is better than the HLA-mis-matched transplantation.</p>

Effect of quercetin on neural stem cell proliferation in the subventricular zone of rats after focal cerebral ischemia-reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of quercetin on the proliferation of neural stem cells in the subventricular zone (SVZ) of rats after focal cerebral ischemia.</p><p><b>METHODS</b>An adult rat model of middle cerebral artery occlusion (MCAO) model was established by placement of an intraluminal filament at the origin of the MCA. Quercetin was administered intraperitoneally in the rats at a dose of 50 mg/kg every 3 days starting at 6 h after MCAO, and BrdU (50 mg/kg daily) was also injected intraperitoneally starting at 4 h after MCAO. BrdU-positive cells in the SVZ were counted at 7, 14 and 21 days after MCAO.</p><p><b>RESULTS</b>Compared with the sham-operated group, the rats in the ischemic model group showed significantly increased BrdU-positive cells in the ipsilateral SVZ 7 days after MCAO, reaching the peak level on day 14 and beginning to decrease on day 21 (P<0.05). The number of ipsilateral BrdU-positive cells in quercetin group was significantly greater than that in the model group on days 7, 14 and 21 (P<0.05), and maintained the high level on day 21.</p><p><b>CONCLUSION</b>Quercetin can maintain a high level of neural stem cell proliferation in the SVZ after focal cerebral ischemia in adult rats.</p>

Modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui / 药学学报

To investigate the modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui with ussing chamber and rt-pcr, Rhodamine 123 (R123), a P-gp substrate and fluorescein sodium (CF), a model drug of non-P-gp substrate transported by a passive diffusion were taken as investigational drugs. Because these two drugs can be easily assayed and widely used in various research fields. The permeability of R123 or CF via Wistar rat jejunum membranes was evaluated by in vitro ussing chamber after oral administration of four different decoctions of Glycyrrhiza inflata and Kansui for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry in the receiving solution. Meanwhile the expression of mdr1a in P-glycoprotein was detected by real-time fluorescent quantitative PCR. After oral administration of combined decoction of the single drug, the absorptive directed permeability of R123 increased significantly (P < 0.01). On the other hand, Kansui and combine decoction of the two drugs also decrease the permeability of secretory directed transport (P < 0.05). No action of Glycyrrhiza inflata was found on the secretory transport of R123 [Papp = (2.56 +/- 0.38) x 10(-5), cm x s(-1)] across the jejunum tissues, while Papp of control group was found [Papp = (2.35 +/- 0.27) x 10(-5), cm x s(-1)]. After oral administration of Kansui decoction for 1 week and 2 weeks, the levels of mdr1a expression in Wistar rats were lower than that of the control group, but there were no significant difference in the results. Meanwhile, Glycyrrhiza inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. Kansui may slightly inhibit P-glycoprotein function in the intestinal membrane. For another, some compositions in Kansui inhibit P-glycoprotein function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-glycoprotein was enhanced by combination of Glycyrrhiza inflata and Kansui, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of Glycyrrhiza inflata and Kansui.

Protective effect of mild hypothermia on astrocytes with traumatic or ischemic injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the protective effect of mild hypothermia on rat astrocytes with traumatic or ischemic injury.</p><p><b>METHODS</b>Rat astrocytes in primary culture were subjected to scratching or hypoxic injury and exposed to normothermia (37 degrees celsius;) or hypothermia (34 or 32 degrees celsius;) for 24 h. The morphology of the astrocytes was evaluated by live/dead staining, and the cell injury was measured by lactate dehydrogenase (LDH) release assay.</p><p><b>RESULTS</b>As the temperature reduced the LDH release rate from the cells in hypoxic group decreased significantly, to (11.48 - or + 1.53)% at 34 degrees celsius; and (3.79 - or + 0.45)% at 32 degrees celsius; as compared to that in normothermia [(33.02 - or + 3.58)%] in the absence of rat white blood cells (WBC) (P<0.001). LDH release rate of the hypoxic cells further decreased in the presence of rat WBC to (51.14 - or + 2.17 )% at 37 degrees celsius;, (19.53 - or + 4.37)% at 34 degrees celsius; and (16.68 - or + 1.47)% at 32 degrees celsius; (P<0.001). In the scratched cells, with or without WBC, LDH release rate showed no significant variation between the 3 temperatures (P>0.05).</p><p><b>CONCLUSION</b>Mild hypothermia offers obvious protective effects on rat astrocytes against ischemic damage but not against mechanical injury.</p>

An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an enzyme-linked immunosorbent assay (ELISA) for determining anti-themocyte globulin (ATG) levels in serum samples.</p><p><b>METHODS</b>The microplate was coated with mouse anti-rabbit IgG monoclonal antibody, and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation.</p><p><b>RESULTS</b>The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 microg/ml, 1:40 and 1:2500, respectively. The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection. A linear relationship was established within the concentration range from 40 to 1000 ng/ml, with the coefficients of variation of 7.91 within assay and 5.22 between assays, respectively. Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG, and after 90 days ATG could still be detected.</p><p><b>CONCLUSION</b>The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting. In patients undergoing stem cell transplantation with ATG pretreatment, the ATG concentration gradually decreases but remains detectable 90 days after the administration.</p>

Construction of delta-pIRES2-EGFP plasmid and its expression in HEK293 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the delta-pIRES2-EGFP plasmid and investigate its expression in HEK293 cells.</p><p><b>METHODS</b>Full length cDNA of rat delta opioid receptor gene amplified from rat brain tissues using reverse transcription and nested PCR was cloned into pMD20 T vector. The delta cDNA was inserted into pIRES2-EGFP plasmid to construct the recombinant eukaryotic plasmid delta-pIRES2-EGFP, which was transfected into HEK293 cells via Lipofectamine2000. The expression of delta was examined under fluorescence microscope.</p><p><b>RESULTS</b>The recombinant delta-pIRES2-EGFP plasmid was successfully constructed, and high expression of delta was detected in HEK293 cells transfected by the plasmid.</p><p><b>CONCLUSION</b>delta-pIRES2-EGFP has been successfully cloned, which shows high expression of delta in HEK293 cells.</p>

Effect of small interfering RNA targeting multidrug resistance-related protein and bcl-2 on drug resistance and apoptosis of K562 and K562/ADM cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of small interfering RNA (siRNA) targeting multidrug resistance-related protein (MRP) and bcl-2 genes in modulating drug resistance and apoptosis of K562 and K562/ADM cells.</p><p><b>METHODS</b>Two siRNA constructs targeting respectively bcl-2 and MRP genes, were synthesized and transfected either alone or in combination into K562 and K562/ADM cells via lipofectamine2000. MTT assay was used to evaluate the viability of the transfected cells at 24, 48 and 72 h Post-fransfection, and RT-PCR was performed to determine the mRNA levels of bcl-2 and MRP. The effects of MRP siRNA and bcl2 siRNA on the apoptosis and the protein expression of Bcl-2 and MRP were evaluated with flow cytometry.</p><p><b>RESULTS</b>In K562/ADM cells, the IC (50) decreased from 12.81 microg/ml (ADM group) to 3.74 microg/ml (ADM+MRP siRNA group), 6.82 microg/ml (ADM+bcl2 siRNA group) and 2.51 microg/ml (ADM+MRP siRNA+bcl2 siRNA). Similarly, in K562 cells, the IC50 decreased significantly from 6.75 microg/ml (ADM) to 3.22 microg/ml (ADM+MRP siRNA), 3.56 microg/ml (ADM+bcl2 siRNA) and 1.84 microg/ml (ADM+MRP siRNA+bcl2 siRNA) (P<0.05). Flow cytometry demonstrated significantly increased apoptosis of the cells following MRP siRNA and bcl2 siRNA transfection, which also resulted in significantly decreased expressions of MRP and bcl-2 proteins (P<0.05).</p><p><b>CONCLUSION</b>Treatment with both MRP and bcl-2 siRNAs inhibits the target gene expression, and increases the drug sensitivity and apoptosis of K562 and K562/ADM cells.</p>

Thermotherapy enhances the sensitivity of lymphoma cell line Raji to chemotherapy in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of thermotherapy on the intracellular adriamycin concentration and apoptosis of Raji cells in vitro.</p><p><b>METHODS</b>The working concentration of adriamycin against Raji cells was determined with MTT assay. Raji cells were subjected to thermotherapy (at 40 degrees Celsius;, 41 degrees Celsius; or 42 degrees Celsius;) and chemotherapy with adriamycin alone or in conjunction, and the cell survival rates were determined 48 h after the treatment. The cell growth inhibition effect of the treatment was evaluated with MTT assay, and the apoptotic rates of Raji cells and alteration of intracellular adriamycin concentration were determined by flow cytometry.</p><p><b>RESULTS</b>The IC(50) of adriamycin was defined as the working concentration in the experiment. Thermotherapy at 40, 41 and 42 degrees Celsius; for 60 min in conjuction with chemotherapy significantly inhibited the growth of Raji cells (P<0.01). The results of flow cytometry showed that thermotherapy and adriamycin chemotherapy, used either alone or in combination, significantly increased the apoptotic rates of Raji cells (P<0.01), and thermotherapy remarkably increased the intracellular concentration of adriamycin.</p><p><b>CONCLUSION</b>Adriamycin chemotherapy combined thermotherapy for 60 min can increase the intracellular concentration of adriamycin and the apoptosis rates of Raji cells.</p>

ITS sequence of 9 Bupleurum species and its application in identification of Chaihu (Radix Buplurei) / 南方医科大学学报

<p><b>OBJECTIVE</b>The study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs.</p><p><b>METHOD</b>The ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2.</p><p><b>RESULTS</b>Homologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%.</p><p><b>CONCLUSION</b>The ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.</p>

Nuclear ribosomal DNA internal transcribed spacer 1 sequences of 4 Leonurus species / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the nuclear ribosome DNA (nrDNA) internal transcribed spacer (ITS) sequences of 4 Leonurus species, and the possibility of using them for molecular authentication of the crude drugs from the genus.</p><p><b>METHODS</b>The nrDNA ITS sequence (including ITS1, 5.8S rDNA, ITS2, and partial 18S rDNA and 26S rDNA) of L. japonicus and its 3 adulterant species were amplified and sequenced, and CLUSTRAL X and MEGA software was employed for analysis.</p><p><b>RESULTS</b>The variation of ITS1 and ITS2 between L. japonicus and its adulterant species ranged between 7.2% and 18.8% and between 14.2% and 27%, respectively. The phylogenic tree derived from the dendrograms based on the ITS sequence data contained some discrepancy from the traditional classification.</p><p><b>CONCLUSION</b>The nrDNA ITS sequences can be used potentially as efficient markers for identification of L. japonicus and its adulterants, and further study is needed for studying the phylogeny of Leonurus.</p>

Decrease of chronic graft-versus-host disease by adding anti-human thymocyte globulin to the conditioning regimen / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the influence of adding anti-human thymocyte globulin (ATG) into conditioning regimen on graft-versus-host disease (GVHD) and life quality of the patients of allo-peripheral blood stem cell transplantation (PBSCT).</p><p><b>METHODS</b>Patients were distributed into study (19 cases) and control (24 cases) groups at random. Median dose of rabbit ATG was added to the conditioning regimen based on the fludara, busufan and cyclophosphamide (FBC) in study group, and no ATG in the control group. Acute and chronic GVHD disease and Karnofsky scores were compared between two groups after allo-PBSCT.</p><p><b>RESULTS</b>The patients in the study group received a mean of 6.0 (3 - 9) x 10(8)/kg mononucleated cells and 5.5 (4.5 - 7.5) x 10(6)/kg in the control group. The mean CD(34)(+) cells number was 5.5 (3.0 - 6.5) x 10(5)/kg in the study and 5.0 (3.0 - 7.0) x 10(6)/kg in the control group respectively. Eighteen patients in the study group and in the control group were successfully engrafted. The mean time of absolute neutrophil count recovered more than 500/ micro l was 13 days and 12 days respectively. Acute GVHD occurred in 6 patients of the study group, and 15 of the control group. Seven patients suffered from chronic GVHD and 14 got 90 Kanrofsky scores in a mean of 250 days follow-up in the study group, and 19 patients GVHD and 4 patients respectively in a mean of 440 days follow-up in the control group. There was a significant difference for acute and chronic GVHD and life quality between the two groups.</p><p><b>CONCLUSIONS</b>Addition of anti-thymocyte globulin to the FBC conditioning regimen had no effect on stem cells engraftment but could decrease acute and chronic GVHD and improve patients life quality.</p>

Experimental study on NK cells promoting donor marrow engraftment and hematopoietic reconstitution after MHC haploidentical BMT in mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of donor-derived NK cells added to pretreatment conditioning regimen on hematopoietic reconstitution after MHC haplotype-mismatched BMT in mice.</p><p><b>METHODS</b>Murine model of MHC haplotype-mismatched BMT was established by using BALB/c(H-2d) x C57BL/6(H-2b) (CB6F(1)(H-2d/b)) mouse as recipient, and C57BL/6(H-2b) mouse as donor. Fifty recipient mice were divided into 5 groups. The mice in the first three groups were each infused 1 x 10(6), 5 x 10(5), 2 x 10(5)/mouse donor-derived NK cells, respectively before TBI ((60)Co, 9.0 Gy) and then conditioned with TBI, followed by infusion of C57BL/6(H-2b) mice bone marrow cells four hours later. The mice in the fourth group received TBI only, and in the fifth group, TBI and BMT at the some doses as the first three groups. Hematopoietic reconstitution, survival time, body weight, histopathology of the recipients were followed up.</p><p><b>RESULTS</b>(1) Survival time was (5.15 +/- 0.66) days for the fourth group, and > 30 days for the other 4 groups. (2) Leukocyte and platelet counts at day 10 after BMT were (0.99 +/- 0.22) x 10(9)/L and (61.0 +/- 7.27) x 10(9)/L respectively for the fifth group and (2.01 +/- 0.21) x 10(9)/L, (101.50 +/- 16.34) x 10(9)/L; (1.98 +/- 0.29) x 10(9)/L, (99.50 +/- 16.41) x 10(9)/L and (1.97 +/- 0.21) x 10(9)/L, (98.0 +/- 16.19) x 10(9)/L for the first three groups, respectively. Histopathology displayed no GVHD in all the groups.</p><p><b>CONCLUSION</b>Donor-derived NK cells could promote hematopoietic reconstitution after MHC haplotype-mismatched BMT in mice.</p>

Treatment of malignant hematologic diseases by peripheral blood stem cell transplantation combined with halotype lymphocyte infusion / 中国实验血液学杂志

In order to observe the curative and side effects in malignant hematologic diseases treated with autologous peripheral blood stem cell transplantation (auto-PBSCT) combined with halotype lymphocyte infusion, auto-PBSCs were mobilized, harvested and stored at -196 degrees C from patients in first CR or PR with intensive chemotherapy (Ara-C 1.0 g/m(2) x 5 days or cyclophosphamide 60 mg/kg x 2 days) and G-CSF. Unpurged auto-PBSCs were infused when patients received the conditioning regimen with busulfan, total irradiation or cyclophosphamide. Halotype lymphocytes [mean 5.0 x 10(7)/kg, (4.5 - 6.5) x 10(7)/kg] irradiated with 7.5 Gy were infused to patients when WBCs were more than 1 x 10(9)/L. Hematopoietic recovery and survival of patients were observed. The results showed that in 12 cases accepted this protocol, five patients with acute non-lymphocytic leukemia got to durable remission, of which 2 had durable remission of more than 50 months. One of three patients with non-Hodgkin's lymphoma IVb reached durable remission, and two relapsed and died on 4 and 6 months after treatment, respectively. Two CML patients were also achieved durable remission. One patient with multiple myeloma relapsed on 36 months later, but he still survived disease-free with treatment of thalidomide. In a follow-up survey of 25 months, the disease-free survival was 83%. No severe side effects were observed except platelet delayed recovery after halotype lymphocyte infusion. STR-PCR analysis showed that infused donor lymphocytes disappeared in 3 recipients at 72 hours after infusion. It is concluded that auto-PBSCT combined with halotype lymphocyte infusion could decrease the relapse of malignant hematologic diseases and improve the effect of auto-PBSCT. Recovery of platelet, however, could be delayed by halotype lymphocyte infusion.