PD-1 expression, mRNA level and cytotoxicity changes in CD19CAR-T cells / 中华血液学杂志

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.

Methodological Research on Latex Enhanced Immune Turbidimetric Method for the Detection of Urine Free Hemoglobin / 现代检验医学杂志

Objective To establish the test way of latex enhanced immune turbidimetric method for the concentration detection of urine free hemoglobin on the automatic biochemical analyzer,and evaluated the detection performance.Methods Prepared the detection reagents of latex enhanced immune turbidimetry method for detection of urine free hemoglobin with materials such as hemoglobin HBA0 antibody (8.5 mg/ml),80 nm carboxylic olystyrene latex microspheres (estapor,nanometer microspheres) and so on.Then,seted the project parameters on TBA120 automatic biochemical analyzer and proceeded the calibrations.Evaluated the detection performance by accuracy,precision,linear range,sensitivity,specificity and other indicators.Lastly,established the biological reference interval for urine free hemoglobin of healthy people in the region and verified it.Results To seted the project parameters on TBA120 automatic biochemical analyzer and passed the calibration.The calibration fitting curve had a smooth trend and no obvious deviation with each measuring point.In the recovery tests,added standard liquid with different concentrations of 100 mg/L and 500 mg/L to the samples with low values,the recovery rate was 95.3％ and 102.7％ respectively.Same to the samples with high values,the recovery rate was 104.2％ and 103.5％ respectively.In the precision test,samples with low and high value had a precision CV of 6.52％ and 4.18％ respectively.The linear range was 0 ～ 1 100 mg/L,analysis sensitivity was 2.8 mg/L.In the interference test,found that,samples had no obvious disturbance to the test with lower concentrations of free bilirubin,combined with bilirubin,vitamin C,animal hemoglobin lower than 342 μmol/L,342 μmol/L,0.03 g/L,500 mg/L respectively,while had significant interference when the concentration of chyle was higher than 870 FTU.Their study had established the biological reference interval of healthy people,male was 0～ 13.3 mg/L and female 0 ～ 17.1 mg/L,there was no significant difference between them by a t test (P＞0.05).Conclusion The latex enhanced immune turbidimetric method for the detection of urine free hemoglobin has the performance of testing clinical urine specimen,which not only solved the lackage problem of specificity for occult blood in the urine dry chemistry test and realized batch automation quantitative detection.The experimental data of this study provides a theoretical basis for the application of the method in other types of clinical samples.

Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells / 中华血液学杂志

Objective: To Evaluation the effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells (CD19-CAR-T) in vitro. Methods: Five patients with high PD-1 expression in peripheral blood and five healthy volunteers were selected. These peripheral blood mononuclear cells were used as the source of T cells to prepare CD19-CAR-T cells. Different doses (72, 36, 18 μg/ml) of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 μg/ml Nivolumab and CD19-CAR-T cells of healthy volunteers were used as controls. CCK-8, lactate dehydrogenase (LDH) cytotoxicity assay and ELASA were used to detect the proliferation capacity, the specific cytotoxicity and the inflammatory factor secretion. Results: ①T cells from patients with high expression of PD-1 as the source of CD19-CAR-T cells did not affect transfection rate compared with that of healthy volunteers [(32.80±7.22)% vs (35.10±5.84)%, t=-0.554, P=0.593]. ②Incubation of CD19-CAR-T cells with 72 μg/ml Nivolumab did not affect CD19-CAR-T cell proliferation, but its cytotoxicity was significantly higher than that of CD19-CAR-T cells alone or patients' T cells +72 μg/ml Nivolumab (all P<0.001), there was no significant difference in the killing activity between the 72 μg/ml and 36 μg/ml Nivolumab treated CD19-CAR-T cells on Pfeiffer cells (P=0.281, 0.267, respectively), and they were all higher than those of 18 μg/ml Nivolumab treated CD19-CAR-T cells (all P<0.001). ③Different doses of PD-1 inhibitor Nivolumab combined with CD19-CAR-T cells does not affect the secretion of IFN-γ and IFN-α (all P>0.05). Conclusion: Combination of 36 μg/ml PD-1 inhibitor and CD19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.

Protective effect and underlying mechanism of cordycepin on non-alcoholic fatty liver in ob/ob mice / 药学学报

This study is designed to investigate the protective effect and mechanism of cordycepin on nonalcoholic fatty liver in ob/ob mice. Twelve-week-old male ob/ob mice were divided into 5 groups according to their body weight and blood glucose, and C57BL/6J mice were used in the control group. The animals were orally administered with cordycepin for 7 weeks. Body weight and food intake were measured once a week. Blood were collected from ophthalmic venous and biochemical indexes were determined at the 2nd and 4th week. Insulin tolerance test was performed at the 5th week. After 7 weeks of administration, liver tissues were collected to determine the contents of triglycerides and total cholesterol, and pro-inflammatory cytokines. Liver histology was performed by hematoxylin-eosin and oil-red O staining. Total RNA were extracted from liver tissues and the levels of lipid metabolism-related and inflammation-related genes were detected by real time PCR. Cordycepin effectively reduced the blood lipids level and improved liver function. Nevertheless, it did not improve insulin resistance in ob/ob mice. Cordycepin significantly reduced the contents of triglycerides and cholesterol, and the levels of pro-inflammatory cytokines in liver tissues. Moreover, cordycepin remarkably suppressed the expression of genes related to lipids synthesis and inflammation. These results indicate that cordycepin may improve non-alcoholic fatty liver in ob/ob mice, and the underlying mechanism may be associated with decreased expression of genes related to lipids synthesis and inflammation.

Suppression effect of MaiShu on formation of atherosclerotic plaque of apolipoprotein E knock-out mice / 药学学报

The research aimed to investigate the suppression effect of MaiShu which contains hawthorn, hippophae, medlar, phytosterols (β-sitosterol, stigmasterol and campesterol), β-glucan and lycopeneon formation of atherosclerotic plaque in apolipoprotein E knock-out (ApoE-/-) mice. Liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MC) methods were used to analyze the main chemical composition of MaiShu.Atherosclerotic mice models were established by high-fat diet. The mice were administrated with MaiShu (1, 2, 4 g·kg-1·d-1) or other contrast materials by intragastric route for 10 weeks continuously. At the end of administration, the blood of mice was collected for tests of the serum total cholesterol (TC), total triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) level. Atherosclerotic lesions in aorta and aortic root were assessed by calculating the relative area of lesions (oil red O stained). Intravital fluorescence microscopic system was used to evaluate the leukocyte-endothelial adhesion in mesenteric artery of mice by detecting the rolling velocity of white blood cells (WBC). Collagenous fibers and macrophages in lesions were detected by sirius red staining and immunological histological chemistry to evaluate the atherosclerotic plaque stability. Results showed that MaiShu contains various flavonoids (9.5%), phytosterols (23.8%) and polysaccharides (8.9%). The serum lipid level of model animals was significantly higher than the control animals. Serum TC level was decreased by MaiShu (4 g·kg-1, P-1) compared to model (P-1, P-1, P-1, P-1, P-1, P-1) and macrophages were decreased (2, 4 g·kg-1) compared to model. These results demonstrate that MaiShu can obviously decrease the serum lipid levels and the risk of leukocyte-endothelial adhesion in ApoE-/- mice. The effect of MaiShu may be associated with the decrease of macrophages in plaque.

Evaluation of dose-related effects of 2', 3', 5'-tri-O-acetyl-N6-(3-hydroxylaniline)adenosine using NMR-based metabolomics / 药学学报

2', 3', 5'-Tri-O-acetyl-N6-(3-hydroxylaniline)adenosine (WS070117) is a derivative compound of natural product cordycepin. It has significant lipids regulating activity and low toxicity which has been proved by in vitro and in vivo experiments. In this study, 1H NMR-based metabolomics was used to investigate the dose-related effects of WS070117 on hyperlipidemia of high-fat-fed hamsters. The hyperlipidemic hamsters were administrated with six different doses of WS070117, including 3, 12, 50, 100, 200 and 400 mg x kg(-1) x d(-1). 1H NMR spectra of hamster serum were visually and statistically analyzed using two multivariate analyses: principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). As a result, WS070117-treated groups showed dose-related regulation of metabolites associated with lipid metabolism, choline metabolism and glucose metabolism. The dose of 3 mg x kg(-1) x d(-1) of WS070117 only exhibited a little lipids regulating activity. However, the doses of 12 and 50 mg x kg(-1) x d(-1) of WS070117 both regulated the contents of metabolites to reverse significantly toward normal levels. When the dose of WS070117 reached 100 mg x kg(-1) x d(-1), it was more effective than positive control drugs. The work suggested that NMR-based metabolomics might be a valuable approach to evaluate dose-related effects of lipids regulating compounds.

Culturing surplus poor-quality embryos to blastocyst stage have positive predictive value of clinical pregnancy rate

Clinical reproductive centers produce large amounts of surplus poor-quality embryos annually, how to maximize the use of these embryos, and which of them have the potential to develop into blastocyst stage and influencing factors were lack of systematic research. To investigate the fate of surplus poor-quality embryos which were cultured to obtain blastocyst, determine the factors which may influence the blastulation, and discuss their application in predicting of the pregnancy outcomes. Day 3 [D3] after embryo transfer and freezing, surplus poor-quality embryos from IVF/ICSI cycles were cultured to blastocyst by the sequential method, then the blastulation outcomes were observed. Focusing on the blastulation rate of those embryos with different number cells and different embryonic grade; and last the relationship between the pregnancy outcomes of remained poor-quality embryos with successful blastulation or failed blastulation groups were studied. Of 127 patients with 569 poor-quality in vitro cultured embryos, there were formation of 248 blastocysts from 91 patients [43.59%], which lead to development of 138 high-quality blastocysts [24.25%]. With the increase in cells number of D 3 blastomeres, the blastulation rate gradually increased, that, 7-cell blastomeres blastulation rate was the highest [70.59%], and 8-cell blastomeres is a little below [70.37%]; while the embryonic levels and blastulation rate did not show this positive relationship. The clinical pregnancy rate and implantation rate of those who had successful blastulation [67.03% and 42.39%] were higher than of those who failed to develop to blastocyst [p=0.039]. Day 3 poor-quality embryos with successful blastulation or with failed blastulation had predictive value on pregnancy outcomes. For embryo transfer 7-8 cells grade III-IV embryo is better than 4-5 cells grade I-II embryo, in case of lack good-quality embryos

Clinical and cytogenetic analysis of 45,X/46,XY individuals / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze main clinical manifestations and cytogenetic characteristics of patients with a 45,X/46,XY karyotype.</p><p><b>METHODS</b>G-banding karyotype analysis was carried out. PCR was performed to detect azoospermia factor (AZF) microdeletion in adult male patients and sex-determining region on Y chromosome (SRY) gene in all patients. Clinical phenotype and genetic characteristics were summarized.</p><p><b>RESULTS</b>Among the 9 individuals with 45,X/46,XY, there have been 7 males and 2 females. Six out of the 7 males have manifested primary infertility, which included 5 with azoospermia, 1 with oligospermia, and 1 with hypospadia. Three of the 6 infertile patients were found to have AZF microdeletions. Two females showed typical Turner syndrome. All of the 9 cases were SRY-positive.</p><p><b>CONCLUSION</b>The 45,X/46,XY karyotype may result in a range of phenotypes. No correlation has been found between clinical manifestations and proportion of mosaicism cells for their peripheral blood karyotypes. As phenotypically normal male patients may produce sperm, infertile patients should undergo further examination at the molecular level.</p>

A 1H NMR based metabonomics approach to progression of coronary atherosclerosis in a hamster model / 药学学报

To obtain a better understanding of the progression of atherosclerosis and identify potential biomarkers, proton nuclear magnetic resonance spectroscopy (1H NMR)-based metabonomics was used to study the metabolic changes in the plasma of hamster fed with a high-fat/cholesterol diet. Plasma samples were collected at different time points during the progression of atherosclerosis and individual proton NMR spectra were visually and statistically assessed using multivariate analyses. NMR results for all samples showed a time-dependent development from physiological to pathophysiological status during atherosclerosis. Analysis of the identified biomarkers of atherosclerosis suggests that lipid and amino acid metabolisms are significantly disturbed, together with inflammation, oxidative stress, following cholesterol overloading. The results enriched our understanding of the mechanism of atherosclerosis and demonstrated the effectiveness of the NMR-based metabonomics approach to study such a complex disease.

Primary study of skin wound healing in rats for Loropetalum chinens / 中国中药杂志

<p><b>OBJECTIVE</b>Cutaneous wound is a common health problem of humans. Loropetalum chinens, a medicinal plant, is widely used to treat wounds among the people. The research aims to observe whether L. chinens can promote the rats' wounds healing process, isolate the extracts primarily and commit the wound healing selection, which provide work basis for wound healing research of L. chinens.</p><p><b>METHOD</b>First we analyzed the possible components with HC-MS/MS, then committed our wound healing experiments for L. chinens in the rat incision wound model and excision wound model, which are commonly used worldwide. After that, we carried on the preliminary isolation of the L. chinens and we screened the heal-promoting effects of the isolations in incision wound model.</p><p><b>RESULT</b>L. chinens significantly accelerates the wound healing of rat's skin, shortens the healing period, enhances the healing intensity and promotes the cell proliferation and blood vessels formation around the wounds. The isolations, which are petroleum ether layer, ethyl acetate layer and n-butyl alcohol layer, exert heal-promoting effects. It indicates that the possible morphon that promotes wound healing may exist in these three components, with small polar.</p><p><b>CONCLUSIONS</b>L. chinens possesses strong wound healing promoting effects, and the active constituent, with small polar, exists in petroleum ether layer, ethyl acetate layer and n-butyl alcohol layer, and we should focus on these three layers when carrying on further studies.</p>

Decline of semen quality and increase of leukocytes with cigarette smoking in infertile men

Previous researches about the effect of smoking on semen quality are contradictory, and the mechanism behind the harmful effect of smoking on semen quality still remains unclear until today. The objectives of this study are evaluation of the relationship between smoking and fertility, investigation of the effects of cigarette smoking on sperm parameters and detection of presence of leukocytes within the semen of idiopathic infertile men from Northeastern China. A retrospective study of 1512 infertile patients who visited affiliated hospitals of Jilin University from 2007-2010 were enrolled in this study. Patients were assigned into one non-smoking and one smoking group which was divided into mild, moderate and heavy subgroups. Sperm parameters [including leukocytes] and sperm morphology analysis were performed using standard techniques. Compared with non-smokers, smokers had a significant decrease in semen volumes [p=0.006], rapid progressive motility [p=0.002] and sperm viability [p=0.019]; moreover, smokers had a significant increase in the levels of immotile sperms [p=0.005] and semen leukocytes [p=0.002]; pH and sperm concentration were not statistically significant [p=0.789 and p=0.297 respectively]. Sperm motion parameters were all lower in the smokers except for beat-cross frequency [Hz] [BCF]. Further, the percentage of normal morphology sperm was decreased significantly in smokers [p=0.003], the sperm morphology was worse with increasing degree of smoking. These findings suggest that smoking leads to a significant decline in semen quality and higher levels of leukocytes, thus smoking may affects the fertilization efficiency

Effects of humanized interleukin 21 on anti-leukemic activity of cytokine induced killer cells and the mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.</p><p><b>METHODS</b>Mononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.</p><p><b>RESULTS</b>After IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-α increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-α increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.</p><p><b>CONCLUSION</b>Humanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.</p>

Y chromosome microdeletions in severe oligospermia men with varicocele / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate Y chromosome microdeletions in severe oligospermia men with varicocele.</p><p><b>METHODS</b>We randomly selected 100 cases of severe oligospermia with left varicocele (sperm concentration <5 x 10(6)/ml, group 1), 100 cases of mild oligospermia with left varicocele (sperm concentration 10 -20 x 10(6)/ml, group 2), 100 cases of idiopathic infertility with severe oligospermia (group 3), 100 cases of idiopathic infertility with moderate oligospermia (group 4) and 30 normal fertile men as controls (group 5). We used polymerase chain reaction (PCR) technology to screen 9 sequence tagged sites (STS) of the AZF a, b and c regions and detect Y chromosome microdeletions.</p><p><b>RESULTS</b>AZF microdeletions were found in 19 patients in group 1 (19%) and 11 in group 3 (11%), with a higher rate in the former than in the latter, but not in the other three groups.</p><p><b>CONCLUSION</b>Screening of Y chromosome microdeletions should be performed before the treatment of severe spermatogenesis with varicocele.</p>

Synthesis and biological evaluation of tetrahydrocoptisine quaternary ammonium compounds / 药学学报

The goal of treatment of metabolic syndrome is the prevention of diabetes and cardiovascular events. A series of novel tetrahydrocoptisine quaternary ammonium compounds were prepared to evaluate their action of hypoglycemia and hypolipidemia for finding the therapeutic agents of metabolic syndrome. Starting from the coptisine hydrochloride (2), fifteen target compounds were synthesized by reduction and substitution of the 7-N position. All of the target compounds were characterized by 1H NMR and HR-MS. Their hypoglycemic activities were evaluated in HepG2 cell and hypolipidemic activities of compounds with better hypoglycemic activity were tested further in vivo. Results indicated that compounds 5, 7, 8 and 9 exhibited better hypoglycemic activities in vitro and compounds 5 and 8 exhibited good hypolipidemic activities in high-fat-diet (HFD) induced hyperlipidemia mice and (or) hamsters. However, the activity is not as good as simvastatin.

Lipid-lowering effect of cordycepin (3'-deoxyadenosine) from Cordyceps militaris on hyperlipidemic hamsters and rats / 药学学报

3'-Deoxyadenosine, so-called cordycepin, is a bioactive component of the fungus Cordyceps militaris. It has been known to exhibit multiple-biological effects including: modulation of immune response, inhibition of tumor growth, hypotensive and vasorelaxation activities, and promoting secretion of adrenal hormone. To investigate its lipid-lowering effect, hyperlipidemic hamsters and rats fed by high-fat diet were both administered orally with cordycepin extracted from Cordyceps militaris for four weeks. The levels of lipids in hamsters and rats were measured enzymatically before and after the administration of cordycepin (12.5, 25 and 50 mg x kg(-1)). The results suggested that levels of serum total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C) increased markedly in the two animal models by feeding high-fat diet. Meanwhile, cordycepin reduced levels of serum TC, TG, LDL-C, VLDL-C as well as LDL-C/HDL-C (high density lipoprotein cholesterol) and TC/HDL-C ratios. In concert with these effects, an increase in lipoprotein lipase (LPL) and hepatic lipase (HL) activity afforded by cordycepin was considered to contribute to the regulation on lipid profiles. Furthermore, no toxicity of cordycepin was observed by intragastric administration at the maximal tolerant dose in ICR mice for 14 days. The exact lipid-lowering effect of cordycepin needs further investigation.

Establishment of iron overloaded bone marrow model in vitro and its impact on hematopoiesis / 中国实验血液学杂志

This study was to establish an iron overload bone marrow (BM) model by co-culturing the mononuclear cells from BM with iron, and investigate its hematopoiesis changes. The iron overload model was set up by adding different concentration of ferric citrate (FAC) into the mononuclear cells from BM and culturing for different time, and the model was confirmed by detecting labile iron pool (LIP). Then the apoptosis of hematopoietic cells, ability of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) and percentage of the CD34(+) cells of the BM cells all were determined. The changes of these indexes were tested after the iron-overloaded BM was treated with deferasirox (DFO). The results showed that after BM cells were cultured with FAC at different concentrations for different time, the LIP increased in time-and concentration-dependent manners. The intracellular LIP reached maximum level when cultured at 400 µmol/L of FAC for 24 hours. The detection of BM cell hematopoietic function found that the apoptotic rate of the FAC-treated cells (24.8 ± 2.99%) increased significantly, as compared with normal control (8.9 ± 0.96%)(p < 0.01). The ability of hematopoietic colony forming in FAC-treated cells decreased markedly, as compared with normal control (p < 0.05). The percentage of CD34(+) cells of FAC-treated cells (0.39 ± 0.07%) also decreased significantly, as compared with normal control (0.91 ± 0.12%)(p < 0.01). And these changes could be alleviated by adding DFO. It is concluded that the iron-overloaded model has been set by adding iron into the mononuclear cells from BM in vitro, and the hematopoietic function of iron-overloaded BM is deficient. These changes can be alleviated by removing the excess iron from the BM cells through treating with DFO. These findings would be helpful to further study the mechanism of iron-overload on the hematopoiesis of BM and also useful to find the way to treat iron-overload patients with hematopoietic disorders.

Clinical significance of leukemia stem/progenitor cell related gene expression in acute leukemia / 中国实验血液学杂志

This study was aimed to detect the expression of leukemia stem/progenitor cell (LSPC) related genes (ABCB1, BMI-1, HOXB4) in the patients with acute leukemia, and to explore its clinical significance in acute leukemia. Bone marrow samples were collected from de novo acute leukemia patients (41 cases), patients with complete remission (CR, 16 cases) and the patients with non-malignant hematologic diseases (10 cases) respectively. And the expressions of ABCB1, BMI-1, HOXB4 genes were detected by comparative real-time quantitative PCR (RQ-PCR) with SYBR Green assay. The results showed that the expressions of ABCB1, BMI-1, HOXB4 were not detected in the patients with non-malignant hematologic diseases, but were higher (relative expressive level: 4.26 ± 2.26, 3.72 ± 1.91, 3.74 ± 2.38) in de novo acute leukemia patients and lower (relative expressive level: 2.14 ± 1.47, 2.07 ± 0.99, 1.47 ± 0.89) in the acute leukemia patients with CR (p < 0.05). The expressions of LSPC related genes were lower (relative expressive level: 1.77 ± 1.29, 2.09 ± 1.26, 1.78 ± 1.49) in the patients acquired CR/partial remission (PR) than those in the patients not acquired CR/PR (relative expressive level: 7.23 ± 1.78, 3.96 ± 0.92, 4.48 ± 2.57) (p < 0.01). Univariate analysis revealed that there were more cases with the expression of LSPC immunophenotype (CD34(+)CD38(-)CD96(+) and CD34(+)CD38(-)CD123(+)) and more hyperleukocytosis cases in patients with any higher expression of LSPC related gene (p < 0.05). Analysis of multiple parameters discovered larger significance (p < 0.01). It is concluded that there is a good relationship between LSPC related genes (ABCB1, BMI-1, HOXB4) and LSPC immunophenotype. The expression of LSPC-related genes is higher in de novo acute leukemia patients, and lower in patients acquired CR/PR. The patients with higher expressed LSPC-related genes display worse response to chemotherapy, lower CR/PR rate and higher leukocytosis, the analysis of multiple parameters may be a good method for assessing the therapeutic efficacy/prognosis of acute leukemia.

In vitro effect of iron overload on bone marrow cell function by inducing the reactive oxygen species / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the in vitro effect of iron overload on the generation of reactive oxygen species (ROS) and of bone marrow (BM) cell function.</p><p><b>METHODS</b>BM mononuclear cells (BMMNCs) were cultured with ferric citrate (FAC) at different concentrations and for different time to create iron overload and confirmed by the detection of cellular labile iron pool (LIP). The changes of ROS, apoptosis, hematopoietic colony formation (CFU-E, BFU-E, CFU-GM and CFU-mix) and the percentage of the CD34 + cells percentage were analyzed. The differences of these index were tested after the iron overload treated with deferasirox (DFO) or antioxidants (N-acetyl-L-cysteine, NAC).</p><p><b>RESULTS</b>1) When BMMNCs were cultured with FAC, the LIP was found to increase in a time and concentration dependent manner. The intracellular LIP reached maximum at 400 micromol/L of FAC for 24 hours. 2) The ROS of total cells, leukocytes and erythrocytes increased to 1.77, 1.75 and 2.12 fold respectively compared with that of normal control when cells were cultured at 400 micromol/L of FAC for 24 hours . DFO and NAC could reduce the ROS efficiently (P<0.05). 3) The apoptotic rates of the FAC treated cells [(24.80 +/- 2.99)%] increased significantly compared with that of normal control [(8.90 +/- 0.96)%]. The capacity of hematopoietic colony formation in FAC treated cells decreased markedly compared with that of normal control (P<0.05). The percentage of CD34+ cells of FAC treated cells [(0.39 +/- 0.07)%] also decreased significantly compared with that of normal control [(0.91 +/- 0.12)%]. And these changes could be recovered by addition of NAC or DFO.</p><p><b>CONCLUSION</b>Iron overload can affect the hematopoiesis by inducing the generation of ROS and this damage could be corrected by removing the excess iron and ROS of the BM cells. These findings might improve the treatment of dyshematopoiesis in patients with iron overload.</p>

Hydroxysafflor yellow A up-regulates HIF-1alpha via inhibition of VHL and p53 in Eahy 926 cell line exposed to hypoxia / 药学学报

In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression.

Inhibitory action of hydroxysafflor yellow A on inflammatory signal transduction pathway related factors in rats with cerebral cortex ischemia / 药学学报

Hydroxysafflor yellow A (HSYA) is a main active monomer purified from Carthamus tinctorius L. The research is to study the inhibitory effect of HSYA on the inflammatory signal transduction pathway related factors which were induced by permanent cerebral ischemia in rats. By using the successive administration at a 30 min interval of HSYA and the rats permanent focal cerebral ischemia model established by a intraluminal suture occlusion method. After cerebral artery occlusion 3, 6, 12 and 24 h, cortex was removed for the next experiments. Western blotting was used to detect the expression of p65 protein and the phospho-IkappaB-alpha (pIkappaB-alpha) in the cytoplasm and nucleus. Nuclear factor-kappaB (NF-kappaB) DNA binding activity was measured by Trans-AM transcription factor assay kits. mRNA expression of cytokines TNF-alpha, IL-1beta, IL-6 and IL-10 was measured by the RT-PCR method. The result showed that intravenous injection of HSYA (10 mg x kg(-1)) to rats after cerebral occlusion, the p65 translocation activity and the phosphorylation of IkappaB-alpha were significantly inhibited. At the same time, HSYA suppressed p65 binding activity and the transcriptional level of pro-inflammatory cytokines including TNF-alpha, IL-1beta and IL-6, and promoted the mRNA expression of anti-inflammatory cytokine IL-10. In conclusion, the anti-cerebral ischemic mechanism of HSYA may be due to its inhibition of NF-kappaB activity and the mRNA expression of cytokines in the inflammatory transduction pathway.