【作者】 张晓燕；

【导师】 徐丛剑； 孙红； 陈钧； 康玉；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 博士

【摘要】 卵巢癌是女性生殖系统最常见的恶性肿瘤之一,其死亡率居妇科恶性肿瘤之首位。虽然近年来在手术及药物治疗方面取得很大进展,但5年生存率一直徘徊在25%-30%。其原因之一在于卵巢癌起病隐匿,缺乏有效的早期诊断措施,当临床确诊时70%以上的患者已处于晚期阶段。此时手术难以清除所有病灶,放疗效果有限,所以在治疗过程中化疗占据了重要地位。但是传统化疗又有很大的缺憾,即药物分布缺乏选择性,很大一部分集中在非病灶区,导致“真正”的有效剂量大大降低,而且带来严重的剂量限制性毒副作用。这就使得传统化疗受到很大限制,以致疾病进展或复发,生存率徘徊不前。因此,积极开发精确的科学的个体化靶向治疗策略,在提高药物疗效的同时降低毒副作用,已成为肿瘤研究的重要任务。靶向治疗具有很强的目的性,能够特异性的针对肿瘤某些特定分子,在提高药物对肿瘤细胞杀伤力的同时,可以显著减少药物对其它无关组织的毒副作用,所以在肿瘤的生物治疗中具有相当的优势。按照靶向源动力可将靶向给药系统分为被动靶向和主动靶向两类。其中,被动靶向药物,如普通纳米粒和脂质体药物进入体内首先被单核巨噬系统摄取,随着载体的逐步降解,药物缓慢释放。药物多集中到肝、脾等器官,很难到达其它靶部位。与此相比,主动靶向制剂用修饰过的药物载体作为“导弹”,将药物特异性输送到靶细胞,可使药物在靶区浓集。然而采用酯化或化学修饰方法构建的系统对药物理化性质要求较高,很可能影响化疗药物的活性。应用较多的单抗介导的靶向治疗,多由肿瘤相关抗原或细胞因子制备。由于靶抗原在动物体内是肿瘤特异性的,而在人体内仅具有肿瘤相关性,使得实际应用中特异性较差,而且单抗具有较强的免疫原性和种属选择性,而人源化抗体的制备又较为复杂。目前受体介导的靶向治疗研究所选择的目标受体多在性腺以外的正常组织也有分布,就卵巢癌而言,仍缺乏足够的选择性。理想的抗肿瘤靶向给药系统应该是仅作用于拟定的肿瘤细胞靶点,而不作用于正常细胞的相同靶点。为了克服上述缺陷,本课题基于卵巢癌病因学和生化特征,结合受体靶向治疗和纳米粒载体各自的优势,构建了一种新型的卵泡刺激素受体(Folliclestimulating hormone receptor,FSHR)介导的卵巢癌主动靶向治疗系统—卵泡刺激素多肽修饰的纳米粒给药系统(Follicle stimulating hormone peptide modifiednanoparticulate system,FSHP-NP),以克服目前治疗所遇障碍。该新型治疗系统将经聚乙二醇(Polyethylene glycol,PEG)表面修饰的纳米粒作为药物载体,可显著提高载药量和药物运送能力,并增加药物稳定性,减少单核巨噬系统的吞噬。卵巢是促性腺激素的靶器官,FSHR多局限性分布于生殖系统,且在卵巢表面生发上皮(Ovarian surface epithelium,OSE)、卵巢癌细胞系及组织都有表达。因此将卵泡刺激素结合片段作为靶向头基,可通过FSHR特异性介导的内吞方式,选择性的递送更多药物进入靶细胞,提高“真正”有效剂量,降低毒副反应。该新型给药系统在国内外均未见报道,具有较高的创新性。为了评价将FSHR作为卵巢癌治疗靶点的可行性,本文第一部分采用免疫细胞化学、免疫组织化学以及Western blot方法对如下细胞系和组织中FSHR和促黄体激素受体(Luteinizing hormone receptor,LHR)的表达情况进行了检测:几种常见人卵巢癌细胞系、中国仓鼠卵巢细胞、人肝癌细胞系;人卵巢癌组织;人卵巢癌裸鼠模型的瘤体及重要器官组织。结果证实人卵巢癌细胞系Caov-3和OVCAR-3,56.67%和30.00%的人卵巢癌组织,以及裸鼠Caov-3皮下移植瘤都表达有FSHR和LHR。除了子宫卵巢以外,FSHR和LHR在裸鼠心、肝、脾、肺和肾组织中均未见表达,人肝癌细胞系BEL-7402和人卵巢癌细胞系SKOV-3也呈阴性表达。这些结果以及既往文献报道都提示促性腺激素受体的局限性分布,提示将其用作卵巢癌治疗靶点是可行的。本文第二部分旨在找寻能够与卵巢癌细胞特异性结合且对细胞无促增殖作用的FSH结合片段,用作给药系统的靶向头基。首先合成并用生物素和异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记FSHβ链第1-15、33-53、51-65、81-95氨基酸片段,然后采用非竞争酶联免疫吸附实验(Enzyme-linkedimmunosorbnent assay,ELISA)对这些多肽与FSHR的亲和力进行了检测,并用荧光显微镜和流式细胞术对其特异性加以验证,最后通过MTT实验检测了其对人卵巢癌细胞生长的影响。结果表明这4条多肽均能够识别并结合FSHR,其中,FSHβ33-53和FSHβ81-95的结合能力相对较强,能够特异性识别FSHR阳性的Caov-3细胞,且对人卵巢癌细胞系Caov-3的生长也没有显著影响。因此,本课题将选择FSHβ33-53和FSHβ81-95作为靶向头基进行初步研究。本文第三部分为FSHP-NP的构建和表征。将马来酰亚胺聚乙二醇-聚乳酸(Maleimide-PEG-PLA)和甲氧基聚乙二醇-聚乳酸(Methoxyl PEG-polylactic acid,MPEG-PLA)以1:9的比例,采用复乳/溶媒蒸发法制备得到纳米粒(Nanoparticle,NP)。通过NP表面的马来酰亚胺基分别与FSHβ33-53和FSHβ81-95的巯基进行共价连接,得到FSHP33-NP和FSHP81-NP。所制备的NP平均数粒径在100nm以下,Zeta电位为-25 mV左右。NP表面经多肽修饰后粒径并没有显著增加。X射线光电子能谱分析表明纳米粒表面的N元素主要来自于表面所修饰的多肽,证实纳米粒表面成功连接了靶向头基FSHβ33-53和FSHβ81-95。为了评价FSHP-NP的递药特性,第四部分制备了载6-香豆素和载紫杉醇(Paclitaxel,PTX)的NP以及FSHP-NP,并建立了相应的高效液相色谱(Highperformance liquid chromatography,HPLC)分析方法。采用6-香豆素作为荧光探针,通过荧光显微镜和流式细胞术观察并测定载6-香豆素的FSHP33-NP和FSHP81-NP对FSHR表达阴性和阳性的卵巢癌细胞的靶向作用。同时采用HPLC测定了Caov-3细胞对载PTX的FSHP33-NP和FSHP81-NP的摄取能力。细胞摄取实验结果表明,FSHR表达阳性的Caov-3细胞对FSHP33-NP和FSHP81-NP的摄取显著高于FSHR表达阴性的SKOV-3细胞;Caov-3细胞对FSHP33-NP和FSHP81-NP的摄取显著高于未修饰FSHP的普通NP。而且摄取呈现时间、浓度和温度依赖性。三种纳米粒的靶向效率依次为:FSHP33-NP＞FSHP81-NP＞NP。为了实现对卵巢癌的靶向治疗,第五部分以PTX为模型药物,制备了包载PTX的FSH多肽修饰的纳米粒,并对其进行了体内外的疗效学考察。首先采用MTT方法检测了其对人卵巢癌细胞的抑制作用;其次建立人卵巢癌裸鼠皮下移植瘤模型,分为生理盐水、市售PTX、NP-PTX、FSHP33-NP-PTX和FSHP81-NP-PTX五组,考察了其抑瘤效果,及对肿瘤细胞周期的影响。体外检测结果表明四组药物对Caov-3细胞的抑制效应依次为:FSHP33-NP-PTX＞FSHP81-NP-PTX＞NP-PTX＞PTX。FSH多肽修饰后纳米粒的IC50比普通纳米粒低2倍左右,比游离PTX低10倍左右。当累计给予30 mg/kg体重的PTX时,靶向治疗组裸鼠瘤体的细胞周期明显受到阻滞,体积抑瘤率和重量抑瘤率均为69%左右,分别是普通纳米粒和市售PTX的2倍和3.5倍左右。上述结果表明本部分构建的PTX靶向制剂无论在体内外都具有较强的抗肿瘤作用,提示这种主动靶向给药系统能够提高相同剂量化疗药物的抑瘤效果。综上所述,本课题构建的这种新型的FSHP-NP药物递送系统,能够通过FSHR介导的特异性内吞,选择性的递送更多药物进入卵巢癌细胞,实现高效低毒的目的。本课题为改善卵巢癌治疗现状奠定了坚实的实验基础。更多还原

【Abstract】 Ovarian cancer is one of the most common gynecologic malignancies and is the leading cause of death from gynecologic malignancies.In recent years,great development has taken place in the way of operation and medication.However,the 5 year survival rate always fluctuates at the level of 25%-30%.One of the reasons lies in that it is hard to be diagnosed at an early time,which is partly due to a shortage of the effective and in-time diagnosing measures.The majority of ovarian cancer patients are diagnosed at an advanced stage,at which time cytoreductive surgery combination chemotherapy is the routine therapeutic approach.However,neither cytoreductive surgery nor radiation treatment is effective to control this vicious disease thoroughly. Therefore,chemotherapy is to take an important role in the course of treatment. Nevertheless,there exist some inevitable limitations to the measure of systemic chemotherapy。That is,the distribution of chemotherapeutic drugs has no selectivity and specificity,that is to say,majority of chemotherapeutic drugs locate at the non-focal sites,which lowers the real dose of chemotherapeutic drugs and besides results in serious dose limiting side effects.Based on these limitations that listed above, the disease can not be effectively prevented and survival rate can not be greatly improved.In that case,the problem of how to improve the efficacy of medication on one part and lower side effect,i.e.,to explore targeted therapy with precise selectivity has become one of the most important subject in the field of cancer research.Targeted therapy has a high degree of selectivity,which is competent in recognition and binding with the specific molecule of tumor focus.Not only can it improve the killing effect of drugs on tumor cells but also the side effect of drugs to other unrelated organs can be greatly lowered.Therefore,targeted therapy is superior to other kind of tumor biological treatment.By targeted power,target-oriented drug delivery system can be categorized into two kinds,active and passive targeting preparations.By comparing the two kinds,passive targeting preparation is hard to achieve at the targeted foci.For instance,nanoparticles and liposomes are firstly endocytosed by macrophage-monocyte system and with the degradation of carriers and the release of drugs,the majority of drugs are concentrated to the organs as liver and spleen. However,by using the cartier with surface modification,active targeting preparations are capable to deliver drugs to targeting cells so as to concentrate drugs to the targeting area.While there still a problem needs solving,that is the lipidization or chemical delivery system has a high requirement to the physico chemical characteristics of drugs. At present,monoclonal antibody-oriented delivery system is commonly used,which is prepared by tumor associated antigen or cytokine.Due to the fact that the antigen is tumor-specific in laboratory animals and tumor-associated in human body,the specificity in clinical practice is low.What is more,the preparation of humanized fusion antibody is quite complicated.In the recent research of receptor mediated targeting treatment,most receptors chosen express in some normal tissues other than gonad.So for ovarian cancer,its selectivity is still not high enough.The ideal antitumor target-oriented drug delivery system should only aim at those tumor cells of interested,rather than those normal cells with the same targeting sites.To solve the problems listed above,our project constructed a novel target-oriented drug delivery system for ovarian cancer based on the etiology and biochemical characteristics of ovarian cancer.That was follicle stimulating hormone peptide modified nanoparticulate system(FSHP-NP),which was mediated by follicle stimulating hormone receptor(FSHR).The aim of our project was to overcome the limitations of current treatment of ovarian cancer.This novel therapeutic system used nanoparticles modified with polyethylene glycol(PEG) as drug carrier.In this way endocytosis of macrophage-monocyte system could be decreased.And the drug delivery capacity was enhanced.In addition,physico chemical characteristics of drugs were overshaded instead of nanoparticle properties.Ovary is the target organ of gonadotropin,whose receptor,FSHR,usually localized in the reproductive system.But it is reported that ovarian surface epithelium(OSE),most ovarian cancer cell lines and tissues express FSHR mRNA and protein.In this case,we selected follicle stimulating hormone peptide(FSHP) as targeting head,which could deliver more drugs to the interested sites with a high selectivity so as to enhance efficacy and minimize side effect.This process was implemented by FSHR mediated specific endocytosis.In order to evaluate the feasibility of using FSHR as the target of ovarian cancer therapy,the first part of this paper detected the expression of FSHR and luteinizing hormone receptor(LHR) by immunocytochemistry,immunohistochemistry and Western blot in several human ovarian cancer cell lines,Chinese hamster ovary cells, human hepatic cancer cell line,human ovarian cancer tissues,human ovarian cancer xenografts and main organs of nude mice.The results showed that FSHR and LHR positively expressed in human ovarian cancer cell lines Caov-3 and OVCAR-3,human ovarian cancer tissues(FSHR is 56.67%;LHR is 30.00%),and Caov-3 xenografts of nude mice.Except uterus and ovary,the expression of FSHR and LHR were negative in heart,liver,spleen,lung and kidney of nude mice.Besides,human hepatic cancer cell line BEL-7402 and human ovarian cancer cell line SKOV-3 were negative,either. Both our results and previous literatures have indicated that the distribution of gonadotropin receptor was relative specific in body and thus demonstrated it was feasible to use it as the therapeutic target of ovarian cancer treatment.The aim of the second part was find out the very FSH binding fragment that can recognized FSHR with great specificity in ovarian cancer cells and have no stimulating effect on cellular growth.We firstly prepared FSH 13 1-15,33-53,51-65 and 81-95 amino acid fragments and labeled with biotin or fluorescein isothiocyanate(FITC). And then the affinity between FSHP and FSHR was analyzed using enzyme-linked immunosorbnent assay(ELISA),fluorescence microscopy and flow cytometry.Finally, their influence on ovarian cancer cells growth was detected by MTT assay.The results showed that the four polypeptide fragments all recognized and bound with FSHR in Caov-3 cells,of which FSHβ33-53 and FSHβ81-95 fragments had the ability to specifically recognize Caov-3 cells with a higher affinity.What is more,the four polypeptide fragments had no significant influence on the growth of human ovarian cancer cell line Caov-3.Based on the above,FSHβ33-53 and FSHβ81-95 was chosen as the targeting head in our research.In the third part,FSHP-modified nanoparticles were prepared by incorporating maleimide into one end of PEG-PLA copolymer and using its thiol group binding reactivity to conjugate with FSHβ33-53 and FSHβ81-95.The mean size of the well-prepared nanoparticles was below 100 rim,and its zeta potential was about -25 mV.Even after the surface modification with FSHP,the size didn’t increase significantly,either.X-ray photoelectron spectroscopy showed that N element of the surface of NP mainly came from those peptides modified,which demonstrated that FSHβ33-53 and FSHβ81-95 were covalently coupled to the surface of nanoparticles.In order to evaluate the capacity of FSHP-NP for drug delivery into ovarian cancer cells,in the fourth part,a lipophilic fluorescent probe with high sensitivity,6-coumarin, and the chemotherapeutic drug,paclitaxel(PTX) were incorporated into nanoparticles. The concentrations of 6-coumarin and PTX in FSHR positive Caov-3 cells and FSHR negative SKOV-3 cells were determined by fluorescence microscopy,flow cytometry, and high performance liquid chromatography(HPLC).The results showed that,by comparing with SKOV-3 cells,Caov-3 cells had a higher uptake for FSHP33-NP and FSHP81-NP,and the uptake amount of both FSHP33-NP and FSHP81-NP was significantly higher than NP with no FSHE Besides,the uptake of FSHP33-NP and FSHP81-NP was the time-,concentration- and temperature-dependent.The drug-targeting efficiency of the nanoparticles was ranked as the followings: FS HP33-NP＞FSHP81 -NP＞NP.In the fifth part,the chemotherapeutic drug,PTX,was incorporated into FSHP-NP and its preliminary evaluation was performed in vitro and in vivo.The inhibitory effects of PTX loaded FSHP-NP on human ovarian cancer cells were determined by MTT assay.To analyze antitumor effect in vivo,nude mice models of human ovarian cancer were established and randomly divided into five groups administrated with physiological saline,commercial PTX,NP-PTX,FSHP33-NP-PTX and FSHP81-NP-PTX,respectively.The inhibitory effects of the four groups on Caov-3 cells ranked as the following:FSHP33-NP-PTX＞FSHP81-NP-PTX＞NP-PTX＞PTX. The IC50 of FSHP-NP-PTX was two times lower than NP-PTX,and ten times lower than free PTX.When the accumulated amount of PTX was 30 mg per kg body weight, cell cycles of tumor xenografts were significantly arrested.And the inhibition rates of tumor volume and weight were both about 69%in FSHP-NP-PTX group,about 2 times higher than NP-PTX and 3.5 times higher than commercial PTX.The results showed that PTX loaded FSHP-NP had an enhanced anti-tumor effect both in vitro and in vivo,and the efficacy of chemotherapeutic drugs in the same dose could be improved with the help of FSHP-NP drug delivery system.In conclusion,the novel FSHP-NP drug delivery system,constructed in this project, was capable of delivering more drugs into FSHR positive cells with a high selectivity through specific endocytosis mediated by FSHR.It facilitated the concentration of drugs to foci so as to enhance antitumor efficacy of chemotherapeutic drugs and minimize side effect in unrelated normal organs.Thus,our research established a strong experimental foundation for the treatment against ovarian cancer.更多还原