1、纳米金-表阿霉素复合体的体外抗肿瘤作用赵晓旭 潘运龙 胡杨志 覃莉 丁晖 巫青【摘要】 【目的】 观察纳米金-表阿霉素复合体(EPI-AuNP)能否抑制人脐静脉内皮细胞(HUVEC)?人肝癌细胞(HepG2)的增殖?【方法】 采用化学合成法制备 EPI-AuNP,通过紫外-可见吸收光谱?荧光淬灭实验?动态光散射及 Zeta 电位变化对其进行鉴定?体外实验分为 AuNP 处理组?EPI 处理组?EPI-AuNP 处理组和空白对照组?将 HUVEC?HepG2 细胞分别接种于 96孔板,培养 24 h 后各组分别加入 AuNP 溶液?EPI 溶液?EPI-AuNP 溶液和无血清培养液 200 L,
2、继续培养 24 h 后:MTT 比色法检测 HUVEC?HepG2 细胞生存率;紫外-可见分光光度法检测各细胞内 EPI 的积聚量?【结果】 紫外-可见吸收光谱显示:AuNP 的最大吸收峰在 520 nm 处,而 EPI-AuNP在 525 nm 处?EPI (100 mg/L)荧光强度为 195.2 7.5;EPI-AuNP 为16.4 5.0,P=0.000?AuNP 的平均粒径及 Zeta 电位分别为:(14.34 0.75) nm?(-21.19 0.64)mV;EPI-AuNP 为:(18.54 1.84)nm?(-15.34 0.72)mV,P0.01?体外实验:MTT 比色法结果
3、显示 EPI 处理组HUVEC?HepG2 细胞的生存率分别为(29.25 1.59)%?(71.10 4.16)%;EPI-AuNP 处理组:(21.29 1.51)%?(43.82 2.21)%,P=0.000?【结论】 成功合成 EPI-AuNP 复合体,体外实验证实其对 HUVEC?HepG2 细胞均具有增殖抑制作用? 【关键词】 纳米金-表阿霉素复合体;HUVEC;HepG2 细胞Abstract: 【Objective】 To observe the in vitro antitumor effects of epirubicin-nanogold compounds (EPI-A
4、uNP). 【Methods】 EPI-AuNP was prepared by chemosynthesis and investigated using UV-Vis spectrophotometer, fluorescence studies, dynamic light scattering,and zeta potential. Human umbilical vein endothelial cells (HUVEC) and HepG2 cells were divided into 4 groups: AuNP treatment group, EPI treatment g
5、roup, EPI-AuNP treatment group and control group. After seeded in 96-well plate and cultured for 24 h separately, HUVEC and HepG2 cells were treated with 200 L of AuNP,EPI,EPI-AuNP,and serum-free medium, respectively. Inhibition effect of each group on the HUVEC and HepG2 cells was assessed using MT
6、T colorimetric method. UV-Vis spectrophotometer was applied to detect the cells epirubicin accumulation of different groups. 【Results】 A red shift in the SPR band maxima in the EPI-AuNP spectrum(max525 nm) as compared with the spectrum of AuNP alone (max520 nm);The fluorescence intensity of EPI (100
7、 mg /L) was (195.2 7.5) and EPI-AuNP was (16.4 5.0),P=0.000. The hydrodynamic diameter of AuNP was (14.34 0.75) nm while EPI-AuNP was (18.54 1.84) nm. Meanwhile, the zeta potential of AuNP was (-21.19 0.64) mV while EPI- AuNP was (-15.34 0.72) mV,P0.01. The HUVEC survival rate of EPI-AuNP treatment
8、group (21.29 1.51)% was lower than the EPI group (29.25 1.59)%. The HepG2 cells survival rate of EPI-AuNP treatment group (43.82 2.21)% was lower than the EPI group (71.10 4.16)%, P0.01. 【Conclusions】 EPI-AuNP has been synthesized and indicated enhanced drug potency in vitro by MTT assay.Key words:
9、epirubicin-nanogold compounds; HUVEC; HepG2 cellsSnake venoms contain toxins that interfere in the mechanism of haemostasis and thrombosis. Some of them affect platelet aggregation and/or blood coagulation. The ability of procoagulant venom proteins that can activate the coagulation cascade at speci
10、fic steps enables their use in diagnostic determination of coagulation factors. Factor(F) is one of the key components in the blood coagulation cascade,which can be activated to form activated Factor (Fa) by Fa, in the presence of Ca2+, phospholipid, and Factor a (Fa); or by Factor a (Fa), in the pr
11、esence of Ca2+ and tissue factor. The activation results from the cleavage site of the Arg52- Ile53 bond in the heavy chain of human F and release of a 52-residue activation peptide. And the snake venom activators of F are non-physiological enzymes, which can convert F into Fa, in the presence of Ca
12、2+ at the same cleavage site of Arg52-Ile531. The venoms of Daboia russelli siamensis have been reported to contain strong F-activating components. Because of their biological activities, some of these venom proteins are useful for basic studies of hemostasis, and for pharmacological and clinical ap
13、plications. Although we have known that the venom of Daboia russelli siamensis also contains some hemostatic fractions,studies about the hemostatic effect and procoagulant mechanism of Daboia russelli siamensis venom are minimal. In the present investigation,We purified the F-activating fraction,Fe-
14、1 from Daboia russelli siamensis (Myanmar) venom,determined the physical and chemical prope-rties,and investigated the mechanism of hemostasia of Fe-1.1 Materials and Methods1.1Snake venomThe lyophilized venom of Daboia russelli siamensis (Myanmar) was purchased from Guangzhou Medical College (Guang
15、zhou, China) and stored in a desiccator.1.2ReagentsCM-Sephadex C-50 and SuperdexTM 75 were purchased from Pharmacia (Uppasala, Sweden). Human fibrinogen and thrombin were supplied from Sigma(St Louis,MO,USA). F,Fa, prothrombin,Fa chromogenic substances and prothrombin chro-mogenic substances were fr
16、om Hyphen BioMed. RVV- was purchased from Enzyme Research Laboratoties (Global Coag, USA). All other reagents were of analytical grade and obtained from commercial sources (Guangzhou, China).1.3Purification of Fe-1Daboia russelli siamensis (Myanmar) venom (1.0 g) was dissolved in 0.5 mol/L ammonium
17、acetate (pH 5.8) and applied to a CM-Sephadex C-50 column (2.6 cm80 cm). The fractions were eluted with 0.1 mol/L ammonium acetate buffer (pH 5.8) containing a linear NaCl gradient (0.5-1 mol/L) at a flow rate of 4 tubes/h. The peak containing highest hemostatic activity was chromatographed on a Sup
18、erdexTM column (2.0 cm100 cm) equilibrated with 0.02 mol/L sodium phosphate buffer (pH 7.4). Elution was performed with the same buffer. The fraction with hemostatic activity was again applied on a SuperdexTM column. All steps were carried out at room temperature and monitored at 280 nm. All fractio
19、ns in each step were tested for hemostatic activity as further described.1.4Melecular weight and isoelectric pointSDS-PAGE was performed according to the method of Laemmli2. The molecular weight standards were MBP-G-galactosidase(175 000),MBP-paramyosin (83 000), glutamic dehydrogenase (62 000), ald
20、olase (47 500), triosephosphate isomerase (32 500), G-lactoglobulin A(25 000), lysozyme(16 500), and aprotinin(6 500). Isoelectrofocusing_PAGE was carried out with a pH gradient of 4.65-9.6 generated by ampholine; Amersham Biosciences, Uppsala, Sweden), as described by Zhang et al3.1.5Mass spectrome
21、tric analysisMALDI-MS anaylysis was assayed on a Autoflex MALDI-TOF-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectr-ometer,Bruker)equipped with nitrogen laser wave-length of 337 nm and the matrix was CCA as previously described4. Protein mass spectra were obtained in t
22、he positive ion mode at an acceleration voltage of 20 kV, extraction voltage of 23 kV.1.6N-terminal sequence determinationAccording to the method of Kennedy5, 16 g of Fe-1 sample was applied on a 12.5% Tricine- SDS-PAGE and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. After s
23、taining with Coomassie brilliant blue, the protein band of interest was cut out. N-terminal amino acid sequencing of Fe-1 was determined by automated Edman degradation using a AB1491A protein sequencer (PE) and compared with other protein sequences at the National Center for Biotechnology Informatio
24、n (NCBI) database using the BLAST service.1.7Measurement of hemostatic activitiesFour different groups were established at various concentration intervals. Each concentration was also tested 6 times. The Fe-1 groups were given of the doses of 6.00, 3.00, 1.50, 0.75, and 0.38 mg/mL. Thrombin was used
25、 as a positive control with the dose of 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 U/mL. RVV- was used as another positive control with the dose of 1200.00, 600.00, 300.00, 150.00, 75.00, 37.50, and 18.75 ng/mL. Normal saline was used as a negative control. The hemostatic activities of thro
26、mbin and Fe-1 were measured according to the method of the coagulation time determined by Williams6. The citrated plasma (100 L) was incubated with 100 L of 0.01 mol/L Tris-HC1 buffer (pH 7.3) containing 0.15 mol/L NaCl at 37 for 3 min. 100 L of 0.05 mol/L CaCl2 plus thrombin (100 L) or Fe-1 (100 L)
27、 were added to the pre-incubation mixture respectively.The clotting time of the plasma was then recorded.1.8Effect of Fe-1 on human blood factor XThe activation of purified human F was followed by using the Fa differential chromogenic substance7. Purified human blood factor X (100 U) was incubated a
28、t 37 in 50 mmol/L Tris-HCl (pH 7.4) containing 0.1 mol/L NaCl, 0.01 mol/L CaCl2, and 12 g of Fe-1 in a total reaction volume of 500 L. Aliquots (50 L) were taken at 5, 10, 15, 20, 25, 30, 35, 40, and 45 min intervals, and 50 L of FXa differential chromogenic substance solution dissolved in 50 mmol/L
29、 Tris-HCl (pH 7.4) containing 0.1 mol/L NaCl (final concentration of 0.2 mmol/L) was added. The absorbance was recorded at 405 nm. Fa was used as a positive control, and normal saline was used as a negative control. In addition, at 3, 7, 15, 30, 45, 60, 90, and 120 min intervals, aliquots (20 L) wer
30、e removed from Fe-1 group and analyzed by SDS-PAGE.1.9Effect of Fe-1 on human prothrombinUsing the method of Hofmann8,the activation of the purified prothrombin was followed by using the thrombin differential chromogenic substance. Purified human prothrombin (6.67 g/L) was incubated at 37 in 50 mmol
31、/L, Tris-HCl (pH 7.4) containing 0.1 mol/L NaCl and 10 mmol/L CaCl2 with Fe-1(12 g). Aliquots (50 L) were removed at various times. 450 L of thrombin differential chromogenic substance solution 5 mg/mL, dissolved in 50 mmol/L Tris-HCl (pH 7.4) containing 0.1 mol/L NaCl was added into the aliquots. T
32、he absorbance was recorded at 405 nm. Thrombin was used as a positive control, and normal saline was used as a negative control.1.10Effect of Fe-1 on fibrinogenFe-1 (20 L, concentrations were 6.00, 3.00, 1.50, 0.75, and 0.38 mg/mL, respectively) and 200 L of human fibrinogen solution (3 mg/mL) in 50
33、 mmol/L Tris-HCl buffer (pH 7.4, containing 0.1 mol/L NaCl and 10 mmol/L CaCl2) were mixed and incubated at 37 . The clotting time was recorded. Thrombin was used as a positive control, and normal saline was used as a negative control.1.11Effect of heat on venom stabilityFe-1 (20 L, 1 mg/mL) was pre
34、incubated respectively at 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, and 85 for 15 min. The samples were cooled on ice. The activation of purified human F was followed by using the Fa differential chromogenic substance as described above. The samples untreated by Fe-1 was used as control. These
35、 values were expressed as percentages relative to control9. Each temperature was tested 3 times.1.12Effect of pH on venom stabilityFe-1 (1 L, 1 mg/mL) was preincubated at 37 for 30 min in different buffers (pH 5.5-12), and the activation of purified human factor was determined as described above. Th
36、e samples untreated by Fe-1 was used as control. These values were expressed as percentages relative to control9. Each pH was also tested 3 times.1.13Effect of Ca2+ on Fe-1 activationFe-1 (1 L, 1 mg/mL) was preincubated at 37 for 30 min in Tris-Hcl buffer (0.05 mol/L,pH 7.4) containing various conce
37、ntrations of Ca2+ (0-15 mmol/L),and the activation of purified human factor was determined as described above.The samples by untreated FVe-1 was used as control. These values were expressed as percentages relative to control9.1.14Effect of inhibitors on Fe-1 activationFe-1 (1 g) was incubated with s
38、everal inhibitors: EDTA(5 mmol/L), PMSF(1 mmol/L), DTT(5 mmol/L), and aprotinin (0.05 mmol/L) at 37 for 30 min, respectively. The activation of purified human factor X was determined as described above. Fe-1 untreated was used as control. These values were expressed as percentages relative to control9.1.15Statistical analysisData are presented as meanSEM. Statistical significance was determined by Students t-test. Value with a P-value0.05 was considered to be statistically significant.
