目的:研究对映-贝壳杉烷型二萜Leukamenin E对人早幼粒白血病HL-60细胞分化的诱导作用。方法:采用台盼蓝染色法、吉姆萨染色法、NBT还原力测定法、荧光颗粒吞噬法及流式细胞术分别检测0（空白对照）、0.3、0.6、0.9、1.2μmol/L的Leukamenin E及1.2μmol/L全反式维甲酸（ATRA）作用于HL-60细胞24、48、72、96 h后对细胞数目的影响,以及作用72 h后细胞核形态、NBT还原能力（以NBT阳性细胞率计）、细胞吞噬能力（以荧光探针P的荧光强度计）以及细胞表面抗原CD11b表达的改变情况。结果:与空白对照比较,0.31.2μmol/L的Leukamenin E及1.2μmol/L的ATRA作用48、72、96 h后HL-60细胞数目减少,且作用72 h后带状核细胞和分叶状核细胞数目增加（P<0.01）,细胞NBT阳性细胞率和细胞表面CD11b表达水平增加（P<0.01）,P荧光强度增强。结论:Leukamenin E可诱导HL-60向成熟粒细胞分化,与白血病分化治疗剂ATRA具有相似的分化诱导特点。

以甲基丙烯酰氯、三乙胺和荧光素反应得到荧光素甲基丙烯酸酯(FMA),将其与聚N-(2-羟丙基)甲基丙烯酰胺(HPMA)以物质的量之比1∶10混合并通过引发剂偶氮二异丁腈(AIBN)引发聚合反应,生成带有荧光探针的聚合物P(HPMA-FMA).采用台盼蓝排染法评估了该聚合物的细胞毒性,荧光显微镜和流式细胞仪观察和检测了全反式维甲酸(ATRA)诱导HL-60细胞分化过程中,P(HPMA-FMA)被细胞吞噬后的荧光示踪效应.结果表明:P(HPMA-FMA)的细胞毒性极低,当P(HPMA-FMA)的质量浓度为4～16 mg/mL时对细胞增殖无影响;当其质量浓度为30 μg/mL时即可满足荧光显微镜定性示踪观察和流式细胞术定量检测所需要的荧光强度.

以N-（2-羟丙基）甲基丙烯酰胺（HPMA），N-（3-氨基丙基）甲基丙烯酰胺（APMA）和全反式维甲酸（ATRA）为原料，采用自由基溶液聚合法设计合成P（HPMA-APMA）-ATRA，并用核磁共振氢谱对该化合物进行结构表征. 相比于单体ATRA，聚合物的水溶性显著增加，同时可通过胞吞作用进入细胞. 3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐（MTT）法评估聚合物和单体ATRA对人早幼粒白血病细胞HL-60生长的抑制作用，流式细胞术检测两者对HL-60细胞周期分布及细胞表面抗原CD11b表达的影响，进一步结合氯化硝基四氮唑蓝（NBT）还原法评估聚合物诱导HL-60细胞分化的能力. 结果显示，聚合物比单体ATRA具有更强的细胞生长抑制活性，其IC 50 值分别为1.03和4.09 μmol/L；聚合物还具有更高的G 0 /G 1 期细胞阻滞效应，1.2 μmol/L时，聚合物比单体ATRA的G 0 /G 1 期细胞率高出17.7%；同样，0.4 μmol/L聚合物与2.4 μmol/L单体ATRA诱导HL-60的NBT还原能力相当，0.8 μmol/L聚合物与2.4 μmol/L单体ATRA诱导HL-60细胞表面抗原CD11b表达相当，表明聚合物比单体ATRA具有更强的诱导HL-60细胞向粒细胞分化的能力，其药效增强3~4倍.

T he trypan blue staining is used to detect the proliferation inhibition effect of Leukamenin E on human leukemia HL-60 cells . The changes in morphology of cell and its nucleus are detected under inverted microscope and fluorescence microscope observation . Cell cycle arrest and apoptosis of HL-60 cells induced by Leukamenin E are studied by flow cytometry and AO/EB double staining fluorescence . Results show that Leukamenin E could inhibit the grow th of the HL-60 cells in a low concentration for a short time and also make strong lethal effect at high concentration (2.1~2.7 μmol· L -1 ) for long time (>36h) in a time-and dose-dependent manner ;Leukamenin E induces significantly cell cycle arrest in S phase in HL-60 cells with different concentrations range from 1.5~2.7 μmol·L -1 for 24 h or 48 h;the compound also induces significant apoptosis ( P< 0.01 ) in HL-60 cells in a time- and dose-dependent manner ;compared with control group , renal nucleation and polynucleation come as a very pleasant surprise in treated HL-60 cells .

The influences of heparin-like compounds on tumor metastasis are reviewed in three aspects:(1) the influence of the compounds on the enzymes relevant to tumor metastasis;(2) the influence of the compounds on the adhesion of tumor cells to endothelial cells and platelets;(3) the influence of the compounds on the angiogenesis of tumor.Heparin-like compounds with low anticoagulant activity are promising to be developed into a kind of novel tumor metastasis inhibitors.

The metastasis of tumor cells is mainly defined as the breakthrough of two layers of barrier composed of extracellular matrix and basement membrane,and heparanase found in animal cells,is the only endogenous glucosidase which can degrade the side chain of heparan sulfate proteoglycan in extracellular matrix.The studies show a close relationship among heparanase,tumor metastasis and angiogenesis.This article focuses on the influence of heparanase on tumor metastasis and angiogenesis,and discusses the development status and significance of heparanase inhibitors.

良好的GMP意识能够保证药品质量,通过加强人员培训;结合质量管理,强化GMP意识;建立严格的自检制度;坚持人本管理等,从而使企业员工获得良好的GMP意识。