单抗F(ab′)2段导向抗肝癌阿霉素免疫毫微球的制备及其体外杀伤癌细胞作用

目的制备人肝癌特异性单克隆抗体HAb18的F(ab′)2片段导向抗肝癌阿霉素(ADR)白蛋白(HSA)免疫毫微球(HAb18 F(a b′)2-ADR-HSA-NP),并研究其体外靶向肝癌细胞和杀伤肝癌细胞的作用.方法采用乳化高温固化法制备阿霉素白蛋白毫微球(ADR-HSA-NP)后,应用改进的N-琥珀酰亚胺基-3-(2-吡啶二硫)丙酸酯(SPDP)法制备HAb18 F(ab′)2-ADR- HSA-NP.在光镜和电镜下观察体外HAb18 F(ab′)2-ADR-HSA-NP和ADR-HSA-NP与肝癌细胞株SMMC-7721的结合特性,并用3H-TdR法测定两种微球体外杀伤肝癌细胞株SMMC -7 721的作用.结果在荧光染色后,HAb18 F(ab′)2-ADR-HSA-NP表面发出明亮黄绿色荧光,而ADR-HSA-NP未见荧光.HAb18 F(ab′)2-ADR-HSA-NP能结合肝癌细胞株SMMC-7721,且能呈剂量依赖地有效杀伤肝癌细胞株SMMC-7721,而ADR-HSA-NP 不能结合和明显杀伤肝癌细胞株SMMC-7721.两种微球均不能结合和杀伤人大肠癌细胞株SW 1116.结论 HAb18 F(ab′)2-ADR-HSA-NP具良好的体外特异性结合并杀伤人肝癌细胞.     

阿霉素免疫毫微粒对多药耐药肝癌的体内杀伤作用

目的 探讨阿霉素免疫毫微粒对多药耐药(MDR)肝癌的体内杀伤作用及其可能的机制.方法 用人肝癌细胞株耐阿霉素亚株(SMMC-7721/ADM)制成肝癌裸鼠模型,分别使用阿霉素免疫毫微粒、阿霉素毫微粒及阿霉素,检测三者的肿瘤抑制率:同时检测三者在肿瘤组织中的药物浓度.结果 阿霉素免疫毫微粒有比普通阿霉素毫微粒及阿霉素更强的体内逆转MDR的功能:阿霉素免疫毫微粒比阿霉素毫微粒及阿霉素在肿瘤中的药物浓度明显高.结论 免疫毫微粒有体内逆转MDR的功能,其可能机制是免疫毫微粒在体内可与肿瘤细胞紧密结合,所载药物释放后形成肿瘤组织内有较高的药物浓度.

Abstract：

Objective To observe the effect of the immunonanoparticles loaded with adriamycin in reversing multidrug resistance (MDR) in liver cancer in a nude mouse model and explore the possible mechanisms. Methods The cytotoxicity of adriamycin, adriamycin-loaded nanoparticles, and adriamycin-loaded immunonanoparticles was assessed in a nude mouse model bearing implant tumors of adriamycin-resistant hepatoma cell line SMMC-7721/ADM. The concentration of adriamycin in the tumor tissue was determined. Results Adriamycin-loaded immunonanoparticles showed significantly stronger cytotoxicity against the implant tumors of SMMC-7721/ADM than adriamycin-loaded nanoparticles and adriamycin.Administration of adriamycin-loaded immunonanoparticles resulted in significantly higher drug concentrations in the tumor tissue than adriamycin-loaded nanoparticles and adriamycin. Conclusion Adriamycin-loaded immunonanoparticles may reverse the MDR of liver cancers in vivo probably resulting from the close binding of the particles with the tumor cells to produce a high local concentration of adriamycin in the tumors.     

单抗F（ab‘）2段导向抗肝癌阿霉素免疫毫微球的制备及其体外杀伤癌细胞作用

目的：制备人肝癌特异性单克隆抗体HAb18的F（ab‘)2片段导向抗肝癌阿霉素（ADR）白蛋白（HSA）免疫毫微球（HAb18F(ab‘)2-ADR-HSA-NP)，并研究其体外靶向肝癌细胞和杀伤肝癌细胞的作用。方法：采用乳化高温固化法制备阿霉素白蛋白毫微球（ADR－HSA－NP）后，应用改进的N－琥珀酰亚胺基－3－（2－吡啶二硫），丙酸酯（SPDP）法制备HAb18F(ab‘)2-ADR-HSA-NP。在光镜和电镜下观察体外HAb18 F(ab‘)2-ADR-HSA-NP和ADR－HSA－NP与肝癌细胞株SMMC－7721的结合特性，并用3H－TdR法测定两种微球体外杀伤肝癌细胞株SMMC－7721的作用。结果：在荧光染色后，HAb18F(ab‘2)-ADR-HSA-NP表面发出明亮黄绿色荧光，而ADR－HSA－NP未见荧洮，HAb18F(ab‘)2-ADR-HSA-NP能结合肝癌细胞株SMMC－7721，且能呈剂量依赖地有效杀伤肝癌细胞株SMMC－7721，而ADR－HSA－NP不能结合和明显杀伤肝癌细胞株SMMC－7721，两种微球均不能结合和杀伤人大肠癌细胞株SW1116。结论：HAb18F(ab‘)2-ADR-HSA-NP具良好的体外特异性结合并杀伤人肝癌细胞。     

抗人肝癌免疫毫微球的制备及其抗癌效果观察

目的 制备抗人肝癌免疫毫微球,观察其活性及抗癌效果.方法 抗人肝癌单克隆抗体HAb18通过异型双功能交联剂SPDP与载阿霉素(ADR)人血清白蛋白毫微球[HSA(ADR)-NS]偶联,制成抗人肝癌免疫毫微球HAb18-HSA(ADR)-NS,使用凝集试验及免疫荧光检测其活性,光镜和电镜下观察其与人肝癌细胞株SMMC-7721特异性结合.MTT法检测该免疫毫微球的体外杀伤性.于人肝癌裸鼠模型上分别使用HAbl8-HSA(ADM)-NS、HSA(ADM)-NS及ADM,检测3者的肿瘤抑制率.结果 HAb18-HSA(ADM)-NS具有单抗活性,能与肝癌细胞特异结合;其体外杀伤SMMC-7721细胞IC50值为44.6μg/ml,与HSA(ADM)-NS(345.5μg/ml)及ADM(365.5μg/ml)相比,明显降低;体内肿瘤抑制率比HSA(ADM)-NS及ADM明显增强(P＜0.001).结论 HAb18-HSA(ADM)-NS具有免疫活性,对肝癌细胞有主动靶向性,体内外均具有比HSA(ADM)-NS及ADM更强的抗癌效果.     

抗人肝癌免疫毫微粒的制备及体外免疫学性质的鉴定

目的研究抗人肝癌免疫毫微粒的制备以及对靶细胞的杀伤作用。方法使用异型双工能SPDP对人肝癌以及载阿霉素（ADR）和人血清蛋白微粒进行偶联,通过实验对其的免疫特性进行测试。检测的方法使用MTT法,这种方法的检测对免疫毫微粒是否能够对体外具备杀伤的作用十分有效。检测后在人肝癌老鼠模型上使用HAb18-HSA（ADM）-NS、HSA（ADM）-NS和ADM,来测试其对肝癌的抑制情况。结果 HAb18抗体可以偶联到毫微粒上,对体外杀伤的细胞值为45.5μg/mL,与ADM的366.1μg/mL比较下,明显了降低。免疫毫微粒能够和靶细胞结合并且对靶细胞具有杀伤和选择功能。结论偶联的方法适用制备肝癌免疫毫微粒,并且免疫毫微粒在对抗肝癌治疗上有着明显的效果,值得推广使用。     

阿霉素对人肝癌细胞SMMC-7721抑制作用的研究

目的:研究阿霉素对人肝癌细胞SMMC-7721生长的影响。方法:应用MTT法、流式细胞术及透射电镜技术观察阿霉素对SMMC-7721细胞生长的作用及细胞周期和形态学改变。结果:阿霉素明显抑制人肝癌细胞SMMC-7721的生长(P相似文献   

阿霉素人血清白蛋白微球的制备及其性质的初步研究

目的 制备载阿霉索人血清白蛋白微球(ADR-HSA-MS)，并研究其药剂学性质和体外对肿瘤细胞的细胞毒作用。方法 以阿霉索(ADR)、人血清白蛋白(HSA)为材料，采用乳化高温固化法制备ADR-HSA-MS；采用光镜、电镜技术观察ADR-HSA-MS的形态；采用胃蛋白酶消化法、动态透析法分别测定ADR-HSA-MS的载药量，体外释药性质；采用MTT比色分析法测定ADR-HSA-MS对人肝癌株SMMC-7721细胞的细胞毒作用。结 ADR-HSA-MS圆球状，表面较光滑，平均粒径为1．2μm，外观为红色；ADR-HSA-MS表观载药量为2．73％，有效载药量为1．69％，释药呈持续缓慢状态，至5d时，其累积释药分数达50％，最大累积释药分数为65％；ADR-HSA-MS与SMMC-7721人肝癌细胞孵育4d后，对SMMC-7721细胞具明显杀伤作用，在0．3～2．5μg／ml ADR质量浓度范围内呈一定剂量依赖性，其杀伤曲线与游离ADR接近。结论 采用乳化高温固化法能制备出具缓释性质的载阿霉素人血清白蛋白微球。     

表阿霉素对人肝癌SMMC-7721细胞bcl-2蛋白表达的影响

目的 探讨表阿霉素诱导SMMC-7721细胞凋亡的可能分子机制。方法 采用流式细胞仪间接免疫荧光法检测表阿霉素作用于人肝癌SMMC-7721细胞后bcl-2蛋白的表达。结果 随着表阿霉素的浓度增加，作用于SMMC-7721细胞时间的延长，细胞bcl-2蛋白的表达逐渐降低，SMMC-7721细胞bcl-2蛋白的表达与表阿霉素作用浓度EY时间呈负相关。结论 Bcl-2基因表达下调可能是表阿霉素诱导人肝癌SMMC-7721细胞凋亡的重要机制之一。     

重组腺病毒载体转染p16基因对肝癌细胞的生长抑制作用

目的：通过腺病毒转染p16基因观察对肝癌SMMC-7721细胞的杀伤作用。方法：克隆人p16基因全长cDNA序列，构建携带p16基因的增殖缺陷型重组腺病毒p16(AdV-p16)，以AdV-p16感染人肝癌SMMC-7721细胞，观察p16基因的表达及其对细胞的生长抑制或杀伤作用。结果：AdV．p16能介导外源基因p16在肝癌SMMC-7721细胞系中高效表达。在感染AdV-p16后随重复感染度(MOI)升高p16基因阳性表达率升高，四甲基偶氮唑蓝染色法(MTT)实验结果表明，SMMC-7721细胞的生长受抑制。结论：腺病毒能够介导p16基因在肝癌细胞中高效表达，促进细胞周期阻滞和诱导肿瘤细胞凋亡。     

单抗F(ab′)2段导向抗肝癌阿霉素免疫毫微球的制备及其体外杀伤癌细胞作用

目的　制备人肝癌特异性单克隆抗体 HAb18的 F(ab′) 2 片段导向抗肝癌阿霉素 (ADR)白蛋白(HSA)免疫毫微球 (HAb18F(ab′) 2 - ADR- HSA- NP) ,并研究其体外靶向肝癌细胞和杀伤肝癌细胞的作用。方法　采用乳化高温固化法制备阿霉素白蛋白毫微球 (ADR- HSA- NP)后 ,应用改进的 N-琥珀酰亚胺基 - 3- (2 -吡啶二硫 )丙酸酯 (SPDP)法制备 HAb18F(ab′) 2 - ADR- HSA- NP。在光镜和电镜下观察体外 HAb18F(ab′) 2 - ADR- HSA- NP和 ADR- HSA- NP与肝癌细胞株 SMMC- 772 1的结合特性 ,并用 3H- Td R法测定两种微球体外杀伤肝癌细胞株SMMC- 772 1的作用。结果　在荧光染色后 ,HAb18F(ab′) 2 - ADR- HSA - NP表面发出明亮黄绿色荧光 ,而 ADR-HSA- NP未见荧光。 HAb18F(ab′) 2 - ADR- HSA- NP能结合肝癌细胞株 SMMC- 772 1,且能呈剂量依赖地有效杀伤肝癌细胞株 SMMC- 772 1,而 ADR- HSA- NP不能结合和明显杀伤肝癌细胞株 SMMC- 772 1。两种微球均不能结合和杀伤人大肠癌细胞株 SW1116。结论　 HAb18F(ab′) 2 - ADR- HSA - NP具良好的体外特异性结合并杀伤人肝癌细胞     

Preparation and in vitro killing effect of adriamycin-loaded immunonanosphere against hepatoma led by F (ab')2 Fragment of monoclonal antibodies]

OBJECTIVE: To study the preparation method and in vitro killing effect of adriamycin (ADR)-loaded human serum albumin (HSA) immunonanosphere (HAb18 F(ab')2-ADR-HSA-NP) against hepatoma led by F(ab')2 fragment of human hepatoma specific monoclonal antibody HAb18. METHODS: After ADR loaded HSA nanosphere (ADR-HSA-NP) was prepared in the emulsifying high temperature solidifying way, HAb18 F(ab')2-ADR-HSA-NP was prepared using the modified N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) method. In vitro binding characters of HAb18 F(ab')2-ADR-HSA-NP and ADR-HSA-NP and hepatoma cell SMMC-7721 were observed under optical microscopy and electronic microscopy. In vitro effects of killing hepatoma cell SMMC-7721 of two microspheres were determined using the method of 3H-TdR. RESULTS: The surfaces of HAb18 F(ab')2-ADR-HSA-NP gave out bright yellow-green fluorescence after it was dyed with fluorescent agent, whereas ADR-HSA-NP did not give out fluorescence. HAb18 F(ab')2-ADR-HSA-NP could integrate with hepatoma cell SMMC-7721 and effectively killed hepatoma cell SMMC-7721 with dose dependence, but ADR-HSA-NP could not obviously integrate and kill SMMC-7721. Neither of the two microspheres could bind and kill human large intestine cancer cell SW1116. CONCLUSION: HAb18 F(ab')2-ADR-HSA-NP has a good character for in vitro specific targeting to bind and kill human hepatoma cell.     

Internalization of monoclonal antibodies against human hepatoma in the target cells]

The monoclonal antibodies against human hepatoma, HAb23, HAb18 and HAb8, were linked with 5 nm colloid gold. They were used to incubate with the target cells, QGY-7703 and SMMC-7721 human hepatoma cell lines, at 4 degree C for 1 hour. The incubated hepatoma cells were divided into 6 groups and then were put into 37 degree C water incubator for 0, 5, 10, 30, 60 and 120 minutes respectively. After washing, the target cells were fixed with Karnovsky's fixative and embedded in Epon 812. There were four intracellular routings for HAb 23, HAb18 and HAb8 to enter the QGY-7703 and SMMC-7721 hepatoma cells: (1) Coated pits coated: The colloid gold-antibodies which clustered in the specialised regions of the surface membrane were invaginated rapidly into the cells to form coated vesicles. (2) Enclosed invagination: One or two colloid gold-antibodies which were attached on the surface membrane were invaginated into the cell by endocytosis. (3) Microvilli involved routing: The microvilli which were adsorbed by the colloid gold-antibodies were broken and then were phagocytized by the cells. (4) Routing via glycocalyx-like material: The glycocalyx-like material which was clustered by the colloid gold-antibodies was invaginated during endocytosis.     

丹皮酚增强顺铂对人肝癌细胞SMMC-7721的增殖抑制作用

目的探讨丹皮酚和顺铂联合应用对人肝癌细胞SMMC07721的增殖抑制作用。方法用不同浓度的丹皮酚、顺铂和两药联用处理人肝癌细胞SMMC-7721,采用四甲基偶氮唑盐（MTT）法测定药物在体外对人肝癌SMMC07721细胞的增殖抑制作用,应用两药相互作用指数（CDI）进行联合用药分析。结果丹皮酚（7．81-250mg／L）和顺铂（0．625～20mg／L）单独应用均对人肝癌SMMC-7721细胞有抑制作用,呈现剂量效应关系。丹皮酚与顺铂联合应用后对人肝癌SMMC-7721细胞的抑制率明显大于单药,有显著的协同杀伤作用。结论丹皮酚与顺铂联合应用具有明显的协同抗肝癌细胞株SMMC-7721增殖的作用。     

土大黄苷对人肝癌细胞SMMC-7721增殖和分化的影响

目的探讨土大黄苷(rhaponticin)对人肝癌细胞SMMC-7721细胞增殖和分化的影响并初步探讨其作用机制。方法采用MTT法检测土大黄苷对人肝癌细胞SMMC-7721细胞增殖抑制率,镜下观察细胞形态改变,测定各组细胞SOD水平。结果不同浓度土大黄苷对SMMC-7721细胞的增殖抑制率随浓度和时间依赖性(P<0.01),并且药物浓度在100-250μmol/L的范围内显著抑制肝癌细胞SMMC-7721增殖;药物在200μmol/L作用24h后,显微镜下可观察到肝癌细胞SMMC-7721的细胞形态和结构向正常肝细胞方向逆转;肝癌细胞在药物浓度为200μmol/L下作用12h、24h和48h后,各时间组肝癌细胞中SOD活力逐渐上升(P<0.05)。结论土大黄苷在体外可呈浓度和时间依赖性抑制人肝癌细胞SMMC-7721细胞增殖并促进其分化。其作用机制可能与提高SOD的活性有关。     

全反式维甲酸增强携单纯疱疹病毒胸苷酸激酶基因的超声微泡对SMMC-7721细胞的旁观者效应

目的观察经全反式维甲酸(all-trans retinoic acid,ATRA)诱导后,单纯疱疹病毒胸苷酸激酶/丙氧鸟苷系统(HSV-TK/GCV)对人肝癌细胞SMMC-7721旁观者效应的影响。方法免疫组化法观察经全反式维甲酸诱导前后SMMC-7721细胞连接蛋白Cx32的表达;将含有HSV-TK基因的质粒通过静电吸附在脂质微泡表面上,采用超声辐照转染并用倒置显微荧光镜及RT-PCR观察TK基因的转入及表达;MTT法观察经全反式维甲酸诱导后HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应。结果经全反式维甲酸诱导后SMMC-7721细胞膜上Cx32表达明显增加,而胞质内仅少量表达;通过超声辐照后HSV-TK基因可顺利转入SMMC-7721细胞中并稳定表达;与对照组相比,经全反式维甲酸诱导组可明显提高HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应(P<0.05)。结论全反式维甲酸可提高SMMC-7721细胞中连接蛋白Cx32的表达且能正确定位于细胞膜上,这可能与其能增强HSV-TK/GCV系统对肝癌细胞SMMC-7721的旁观者效应有关。     

Biological Effects of Millimeter Wave Irradiation on Human Hepatoma Cell Line

The biological effects of 36.11GHz millimeter-wave irradiation atlow power densities (1, 2 and 3mw/cm~2) were studied on human hepatomacell line SMMC-7721 using scanning electron microscopy, histochemicalquantitative analysis of DNA by texture analysis system and cell cloningtechnique. After the irradiation, the surface structures of SMMC-7721cells were morphologically injured and both DNA content of interphasecells and clonogenic capacity of living cells were decreased significantly.     

白毛藤提取物对肝癌SMMC-7721细胞增殖及c-myc基因表达的抑制作用

目的观察白毛藤水提液对人肝癌SMMC-7721细胞增殖及c-myc基因mRNA表达水平的影响,探讨中药白毛藤的抗癌作用机制。方法用不同浓度白毛藤水提取物处理肝癌SMMC-7721细胞,观察细胞形态变化,用台盼兰染色并作细胞计数,cck-8试剂盒检测不同药物浓度组的细胞增殖抑制情况。RT-PCR检测c-myc基因mRNA表达水平。结果倒置显微镜下可见给药组细胞数目明显减少,且随药物浓度增加其抑制效果增强。WST-8法显示,在药物浓度为2,4,8,16 mg/ml时,细胞增殖明显受到剂量依赖性抑制,其抑制率分别为24.37%,50.07%,66.14%,83.23%。细胞中c-myc基因mRNA表达水平降低,并与药物浓度呈明显的量效关系。结论白毛藤可显著抑制人肝癌SMMC-7721细胞的增殖,下调c-myc基因mRNA表达,这可能是白毛藤的抗癌作用的部分机制。