阿糖胞苷纳米粒的制备及其释药规律研究

采用乳化聚合法制备阿糖胞苷纳米粒，研究其体内外释药特性。结果表明阿糖胞苷纳米粒体外释药规律符合双指数方程，有明显的缓释作用。在家兔体内的药物动力学过程符合二室模型，与阿糖胞苷注射剂相比，t1／2β和MRT延长，CL降低，表明阿糖胞苷纳米粒可显著延长阿糖胞苷在体内存留时间，具有明显的缓释特征。     

红景天苷对阿糖胞苷诱导的骨髓间充质干细胞凋亡的影响

本研究旨在探讨红景天苷（salidroside）对阿糖胞苷（Ara-C）诱导的人骨髓间充质干细胞（humanbonemes-enchymalstemcells,hBMMSCs）凋亡影响及其机制。体外分离培养hBMMSC,流式细胞术鉴定免疫表型,特异性染色鉴定hBMMSC体外向成骨、成脂诱导分化能力,MTT法检测细胞增殖情况。实验分4组：对照组、Ara-C组、sali-droside组、Ara-C＋salidroside组;流式细胞术检测细胞凋亡变化;RT-PCR法检测BCL-2、BAXmRNA表达。结果表明,体外成功分离培养hBMMSC;细胞表达CIM4、CD29和HLA-ABC,不表达CD34、CD45和HLA．DR;成骨、成脂诱导21d,茜素红染色可见钙化结节,油红0染色有质滴出现;Ara-C对红景天苷处理的hBMMSC的增殖抑制程度较未经红景天苷处理的hBMMSC明显减低（P〈0．05）;在凋亡方面,Ara-C作用48h后hBMMSC的凋亡率显著高于对照组（P〈0．05）,BCL-2基因表达下调,BAX表达上调;而中剂量红景天苷处理的细胞凋亡率较Ara-C组降低,BCL-2基因表达上调,BAX表达下调（P〈0．05）。结论：红景天苷可抑制Ara-C诱导的hBMMSC凋亡,其机制可能与BCL-2／BAX表达调控有关。     

胞苷脱氨酶基因对小鼠大剂量化疗的保护作用

Objective:To explore the feasibility of transfecting cytidine deaminase(CD)gene into mouse bone marrow cells in order to observe the drug resistance of high dose Ara-C and improve the tolerance of myelosuppression following combination chemotherapy.Methods:Human cytidine deaminase gene was transfected into mice bone marrow cells by retroviral vector.Resistant colony-forming unit granulocyte-macrophage(CFU-GM)assay was performed after the transfected mice bone marrow cells treated by the Ara-C.DNA was extracted from mice bone marrow cells.The drug resistant gene in mice bone marrow cells after transfection was detected by PCR.Results:Bone marrow cells of lhe donor mice cultured with lhe retroviral producer cells showed the drug resistant colonies and resistance to Ara-C,so did accept mice transplanted with the CD gene(CFU-GM of donor mice was 52%,X2=124.62,P＜0.01:accept mice was 54%,X2=126.26.P＜0.01,both compared with the contrast group).The animal survival rate was significantly higher in gene transfected group than that of the control(X2=7.42.P＜0.01).CD gene of transfected bone marrow cells was confirmed by PCR.Conclusion:CD gene can be transfected into bone marrow cells of mice efficiently and increase the drug resistance to Ara-C.     

Results of treatment with an intensive induction regimen using idarubicin in combination with cytarabine and etoposide in children with acute myeloblastic leukemia

We report results achieved in our institution with a study opened in July 1990 (similar to the German AML-BFM-87 in which daunorubicin was replaced by idarubicin in the induction phase and cranial preventive radiotherapy was omitted) and closed in December 1994, for the treatment of newly diagnosed acute myeloblastic leukemia (AML), without prior malignancies except for myelodysplasia.This evaluation included 68 patients, whose mean age was 6 years (range: 1 month–16 years). Thirty-nine were boys and 29 were girls. Complete remission rate was 80.9% (55/68), death on induction rate was 14.7% and induction failure rate was 4.4%. At median follow up of 38 months (range: 12–66 months), the 4-year event-free survival (EFS) estimate was 0.428 (S.E.: 0.062), event-free interval (EFI) estimate was 0.529 (S.E.: 0.07) and overall survival (OS) estimate was 0.44 (S.E.: 0.071).We conclude that idarubicin in combination with cytarabine and etoposide is a highly effective regimen for induction in children with AML. Although preventive cranial irradiation was not delivered, we have observed only one combined CNS relapse. Finally, we corroborate that in this setting two definite risk groups may be identified in children with AML.     

Inv(16) acute myeloid leukemia cells show an increased sensitivity to cytosine arabinoside in vitro

Abstract: Karyotype represents the major independent prognostic factor for response and remission duration in acute leukemia. In particular, it has been reported that acute myeloid leukemia (AML) patients with inv(16) abnormality show a better prognosis, especially in case of treatment with high-dose Ara-C (HD Ara-C) containing regimens. In this study we aimed at testing whether leukemic cells from patients showing the inv(16) were more sensitive to Ara-C in vitro, compared to AML blasts from patients with normal karyotype or chromosomal abnormalities other than t(15;17) or t(8;21). We analyzed blast cells from 30 patients who were diagnosed and treated in our institution. The IC₅₀ of Ara-C, as tested by the XTT colorimetric assay, was significantly lower in cases with inv(16) (18.5±15.88 μmol/l vs. 38±14.6 μmol/l, in cases with other abnormalities, p = 0.01). This result was confirmed by a higher incorporation of [³H]-Ara-C into DNA (p = 0.02 and p = 0.001 compared to samples with normal and abnormal karyotype, respectively). All the same, Ara-C induced apoptosis was significantly increased in cells from patients with inv(16). Our data suggest a possible interaction between the molecular background of inv(16) and a modification of intracellular metabolism of Ara-C, and could thus provide a rationale for HD-Ara-C-based schedules for patients with inv(16) AML.     

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.     

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines

1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase. Altered membrane transport is also a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell, lipophilic prodrugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double bonds were esterified at the 5' position on the sugar moiety of ara-C. The compounds were tested in two pairs of ara-C resistant leukemic cell lines (murine L1210 and rat BCLO and their resistant variants L4A6 and Bara-C, respectively) and two pairs of cell lines with a resistance to gemcitabine, another deoxycytidine analog (human ovarian cancer A2780 and murine colon cancer C26-A and their resistant variants AG6000 and C26-G, respectively). L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the parent cell lines, ara-C was more active, but in the resistant variants several of the analogs were more active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were inactive. Structure-activity relation analysis showed that ara-C derivatives with shorter acyl chains and more double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and longer lasting than for the parent drug.     

Chromosomal rearrangement in Down syndrome with acute myeloid leukemia

The incidence of acute leukemia in children with Down syndrome (DS) is high as compared to general population. Recent findings have demonstrated that DS children with acute myeloid leukemia (AML) have the highest event free survival rates with high dose cytosine arabinoside (Ara-C). We present 3 year-old DS female child with AML-M5, whose chromosomal analysis revealed constitutional t(21;21) alongwith del(5)(q31q33) and a unique translocation t(16;20)(q13;q12). After chemotherapy, child achieved complete clinical remission. Karyotype analysis of remission marrow showed disappearance of abnormal clone of der(20) t(16;20)(q13;q12), del(5q) indicating cytogenetic remission too. This case alongwith supportive literature indicate that pediatric DS-AML is a distinct biologic sub-group differs from that of non-DS-AML with respect to chemosensitivity.     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

Pretreatment leukaemia cell drug resistance is correlated to clinical outcome in acute myeloid leukaemia

In 85 adult patients diagnosed with acute myeloid leukaemia (AML) and treated at the same institution during a 5-yr period, the clinical significance of in vitro cellular drug resistance to the anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as the nucleoside analogue cytarabine (Ara-C) was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. In 59 patients of whom 40 were treated by the combination of Acla and Ara-C we found that leukaemia cell drug resistance towards Acla was higher (by a factor 2.80) in patients who failed to enter complete remission (CR) after the first cycle of induction chemotherapy as compared to patients who entered complete remission. The relationship was significant in univariate as well as multivariate analysis (p=0.02 and 0.03, respectively). By contrast, no in vitro single drug resistance values were consistently correlated to other parameters of clinical outcome (overall CR rate, overall survival (OS), or continuous complete remission (CCR)), whereas the combined Acla and Ara-C drug resistance profile (Acla/Ara-C DRP) was of prognostic significance to overall survival of all 85 patients (p=0.004) as well as to the CCR of 39 complete responders (p=0.04). These findings remained statistically significant in multivariate analyses correcting for other variables influencing clinical outcome including patient age, leukocyte count, karyotype, FAB-subtype, and presence/absence of secondary AML. We conclude that the in vitro drug resistance of leukaemia cells at time of disease presentation appears to be independent of prognostic significance to short- and long-term clinical outcome in AML.