摘要
第一部分聚乙二醇修饰的柔红霉素脂质体治疗小鼠白血病模型的研究
目的探讨聚乙二醇(polyethylene glycol,PEG)修饰的柔红霉素(Daunorubicin,DNR)脂质体(PL-DNR)治疗小鼠白血病模型的疗效及毒副作用。
方法采用薄层水化法和主动载药法制备PL-DNR,测定脂质体药物的理化指标,进行体外L1210细胞增殖抑制实验,体内实验主要包括L1210实体瘤模型、P388肿瘤腹水模型、L615肿瘤血液系统模型治疗实验和药物对小鼠心肌毒性实验,心肌毒性实验采用原位末端标记法(TUNEL)标记凋亡心肌细胞的方法。
结果应用激光粒径散射仪测量PL-DNR粒径大小在(110±10)nm,柔红霉素药物包封率在94.21%。L1210实体瘤模型治疗实验中PL-DNR组抑瘤率为77.59%,DNR组为57.56%,两组间有统计学差异(P＜0.05)。DNR组心肌细胞凋亡指数明显高于PL-DNR组(P＜0.05)。P388肿瘤腹水模型、L615肿瘤血液系统模型治疗实验中均观察到PL-DNR组相比对照组都能改善治疗效果,并有效延长小鼠生存期。
结论聚乙二醇修饰的柔红霉素脂质体在L1210荷瘤鼠模型、P388肿瘤腹水模型、L615肿瘤血液系统模型中的治疗效果均好于柔红霉素组,并且降低了毒副作用。
第二部分CpG ODN与Poly(I:C)在肿瘤免疫中联合应用的研究
目的选择两种新型的生物型免疫刺激剂CpG ODN和poly(I:C)联合作为MUC-1多肽抗原佐剂,通过体外及体内多种免疫功能指标检测,探讨二者联合应用的免疫增效作用,并制备抗MUC-1的单克隆抗体。
方法体外实验中用CpG ODN和poly(I:C)单独或联合与正常小鼠脾细胞混合培养,24小时后检测细胞培养上清中细胞因子分泌情况;体内实验中用CpG ODN和poly(I:C)单独或者联合作为MUC1多肽抗原的佐剂免疫小鼠,通过ELISA方法检测免疫血清中抗体效价,以及应用细胞毒性T淋巴细胞杀伤实验方法,对各组间免疫水平的差别进行评价。采用单克隆抗体制备方法制备抗MUC-1的单克隆抗体。
结果CpG ODN与poly(I:C)联合应用能体外有效诱导脾细胞分泌IL-12p40和TNF-α。体内实验联合组小鼠血清中TNF-α和IFN-γ水平显著升高,IL-4水平明显降低。小鼠血清中抗MUC1多肽抗体效价各组间无明显差异,但细胞毒性T淋巴细胞(CTL,cytotoxicity T lymphocyte)杀伤实验显示联合组能有效杀伤靶细胞。成功制备出一株效价比达到1/106的抗MUC-1单克隆抗体。
结论CpGODN和poly(I:C)联合作为可溶性多肽MUC1的免疫佐剂,与单用相比能有效地增强机体的抗肿瘤免疫应答反应。
Objective To explore anti-tumor effects and toxicity responses of pegylated liposomal daunorubicin(PL-DNR) for curing leukemia in mouse model.Methods PL-DNR was prepared using methods of dry lipid hydration and remote loading,then physiochemical index was analysed.The in vitro cytotoxicity experiment and the in vivo therapeutic trial using animal model were performed.The apoptotic cardiomyocytes were detected using the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling method(TUNEL).Results Average diameter of PL-DNR was(110±10)nm.The encapsulation efficiency was 94.21%.The growth of tumors in PL-DNR and DNR therapy group was obviously suppressed,which reached 77.59%and 57.56%respectively with a significant difference between them (P＜0.05).Compared with PL-DNR group,DNR group had significantly higher index of apoptotic cardiomyocytes(P＜0.05).The same results can be obtained in the therapeutic models of P388 and L615.Conclusion In treatment of the leukemia three models in mice,pegylated liposomal daunorubicin can improve therapeutic effects and reduce cardiomyocyte apoptosis in daunorubicin-induced cardiomyopathy.
Objective poly(I:C) and CpG ODN can activate immunological reactions respectively.In the experiment,we combinate CpG ODN and poly(I:C) as the adjuvant of tumor associated antigen MUC-1 to oberserve whether enhanced stimulation of innate immunological response and adaptive immunological response can be induced.Methods In vitro,murine splenocytes were incubated with poly(I:C) alone,CpG ODN alone or the combination of the two agents.Then the levels of cytokine production of splenocytes were examined including Interleukin(IL)-12p40, tumor necrosis factor-α(TNF-α),IFN-γand IL-4 by the method of ELISA.In vivo,to detect if there is synergetic effect of the combination of two immunological stimulator as adjuvant of MUC-1 peptide,C57BL/6 were immunized with MUC1 plus poly(I:C) and/or CpG ODN.Then the serum levels of cytokines were detected including.We use the CTL test to evaluate the cellular immune response,and use the serum level of MUC-1 antibody to evaluate the humoral immune response.At last we ultilize several mice to produce the monoclonal antibody of MUC-1.Results In vitro,the combination of CpG ODN and poly(I:C) demonstrated synergetic effect for the increased levels of IL-12p40,TNF-αproduction.The same result can be observed in vivo experiment,that is,immunization of mice with the combination of poly(I:C) and CpG ODN demonstrated synergy for the increased serum levels of IL-12p40,TNF-α. Then we obtained a strain of monoclonal anti-MUC-1 antibody with high titer. Conclusions The combination of poly(I:C) and CpG ODN as the adjuvant of peptide MUC1,resulted in an enhanced innate and adaptive immune response that could be used for tumor vaccination or immunotherapy.
引文
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