Ⅰ顺铂-聚谷氨酸复合物药物修饰系统的建立及应用 Ⅱ基于RCAS-TVA系统的小鼠乳腺癌发病模型的建立及应用

发布时间：2018-09-09 19:38

【摘要】：恶性肿瘤严重威胁着人类的健康和生命,对于肿瘤发病机制及抗肿瘤药物的开发已经投入了大量的人力、财力和物力。顺铂(cisplatin, CDDP)是目前临床上化疗的首选药物之一,具有抗癌活性谱广,杀伤作用强等优点,然而研究表明：铂类抗肿瘤药物属周期非特异性药物,半衰期长,溶解度低,细胞毒性大,无靶向性,大大限制了其在临床上长期和高剂量的使用。因此,研究和开发低毒高效的顺铂类似物,一直是药物修饰领域的研究热点。随着材料科学技术的发展和应用,高分子聚合物作为药物载体的研究越来越多,基于肿瘤组织内部特有的高通透性和滞留效应(EPR效应),高分子药物载体通过与药物的相互作用,可以提高药物的水溶性,改善药物的药代动力学特性,调控药物在组织中的分布,靶向给药,降低药物的毒副作用或药源性疾病等,对于提高药物的安全性和稳定性具有很高的应用价值。枯草芽孢杆菌发酵来源的γ型聚谷氨酸(γ-PGA)就是一种良好的药物载体,它具有结构单一、制备工艺简单、良好的生物相容性和生物可降解性、良好的缓释和控释性能等优点,近年来,以y-PGA作为载体的研究备受关注。基于以上研究背景,为开发一种半衰期长、毒性低、具被动靶向和缓释效果的顺铂类似物,我们将顺铂巧妙地连接到生物发酵合成的γ-聚谷氨酸药物载体上,制备顺铂-聚谷氨酸复合物(γ-PGA-CDDP),在实验室原有研究基础上,进一步研究该复合物的性质；将其与临床常用化疗药物进行比较；并制备γ-聚谷氨酸-天冬氨酸复合物(Glutamine Aspartate Polymers, GAP460)以提高药物载量。具体研究内容和结果分为以下两个方面： (1)枯草芽孢杆菌发酵制备γ-PGA,通过优化反应条件,制备γ-PGA-CDDP复合物,并借助红外吸收光谱和核磁共振的方法对复合物进行表征鉴定。为系统研究y-PGA-CDDP复合物的性质和生物学活性,将该复合物与临床常用化疗药物顺铂、卡铂和奥沙利铂进行比较：以Bcap-37、BEL-7404和SH-SY5Y三株人源肿瘤细胞为例比较四种药物的细胞毒性和半抑制浓度(IC50)；模拟药物体外释放曲线；以正常昆明小鼠和BALB/cA荷瘤小鼠为动物模型,比较药物作用后的抑瘤效果、体重变化、存活率；白细胞(WBC)、血小板(Platelet, BP)等血液生化指标；肌酐(CRE, CREAP)、尿素氮(BUN, UREA)、髓质过氧化物酶(MPO)、丙二醛(MDA)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)等肾脏毒性指标,结果证明：γ-PGA-CDDP复合物在细胞水平上不仅可以显著抑制Bcap-37等三株肿瘤细胞的增殖,而且大大降低了细胞毒性,表现为IC50值明显高于顺铂,与奥沙利铂相当,略低于卡铂；在BALB/cA荷瘤模型动物体内,γ-PGA-CDDP复合物可以有效抑制体内肿瘤的生长,相同实验条件下,其抑制作用效果与顺铂相当,优于卡铂和奥沙利铂,同时药物的毒副作用大大降低；在昆明小鼠体内,γ-PGA-CDDP复合物对于动物的损伤最小,顺铂等化疗药物作用后,均可不同程度的引起体内WBC和Platelet数目下降、CRE和BUN含量升高、MPO和MDA上调、GSH和GSH-Px下调,而γ-PGA-CDDP复合物作用后,动物体内各生化指标均保持稳定,肾组织氧化性损伤显著改善,在与临床化疗药物的比较中体现出明显的优势。 (2)γ-PGA的药物载量偏低,高剂量药物治疗时载体用量较大,另外,y-PGA在体内的代谢过程可能导致神经递质前体的累积,本实验室研究发现,γ-PGA-CDDP复合物的载药量只有14.6%。为解决上述问题,通过酰胺化反应,制备GAP460复合物药物载体,并以顺铂为例,研究顺铂-聚谷氨酸-天冬氨酸复合物(PACC)的性质和功能,结果证明：GAP460复合物药物载体在37℃、pH6.0-7.4的环境下保持稳定,温度的升高、偏酸或偏碱的环境均可使GAP60复合物发生不同程度的降解；急性毒性实验,单次静脉注射GAP460复合物4.0g/kg动物存活率为100%,单次静脉给予2.0g/kg,动物的体重,体内白细胞(WBC)、血小板(BP),血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)、肌酐(CREAP)和尿素氮(UREA)的含量均保持稳定,药物载体安全无毒；相同实验条件下,GAP460复合物的药物载量为38.52%,约为γ-PGA药物载体的3倍,药物载量的提高可大大减少载体的用量,并改善药物的控释效果；PACC复合物不仅在体外可以显著抑制Bcap-37等肿瘤细胞的增殖,IC50值明显高于游离顺铂,而且可以有效抑制BALB/cA荷瘤裸鼠体内肿瘤的生长；同时,PACC复合物大大降低了体内毒副作用,用药后动物体重无明显下降,肾脏功能和肾脏氧化性损伤得到明显改善。综上所述,本文将γ-PGA-CDDP复合物与临床常用化疗药物开展系统比较研究,并引入天冬氨酸分子对聚谷氨酸药物载体进行改造,为高分子药物载体和γ-PGA-CDDP复合物的临床应用提供了有益的探索。癌症是危害人类健康甚至危及生命的重要疾病之一,而恶性肿瘤的侵袭和转移又是癌症预防和治疗的难点。据统计,乳腺癌的发病率占全身各种恶性肿瘤的7-10%,并呈逐年上升的趋势,乳腺癌的预防和治疗已成为研究热点。乳腺肿瘤的发生和发展是一个复杂的多基因参与的生物学过程,每个阶段均由不同的基因和信号分子调控。近年来,以分子分析技术为基础的肿瘤分型系统的建立,为肿瘤的分类提供了更多的信息,50余种组织和细胞特异性的乳腺癌发病动物模型相继建立,肿瘤的早期检出率和治愈率不断提高,患者死亡率逐渐下降。乳腺肿瘤研究的动物模型多借助于转基因小鼠,主要研究策略是在组织特异性启动子的诱导下,通过SV40、Ras、Neu等原癌基因诱导小鼠发生乳腺癌,观察肿瘤的特征和性质,并研究相关基因的功能,但是建立在同源重组基础上的转基因动物并不能完全模拟人体内乳腺癌的发病环境和特点。在新型乳腺肿瘤发病模型的研究中,以RCAS逆转录病毒载体为基础的RCAS-TVA系统备受关注借助病毒侵染,诱导单个细胞突变,从而诱导肿瘤发生的方法逐渐被认可。 Twist基因是存在于人类染色体上的重要转录因子,上世纪90年代的研究表明：Twist基因在胚胎发育过程中对于中胚层的形成具有重要作用。Twist基因与肿瘤转移的关系于2004年被首次报道,它可以通过促进上皮-间叶转化而促进肿瘤细胞的侵袭和转移,此外,Twist还与细胞周期及凋亡、血管形成和肿瘤细胞耐药性有关,但目前的研究大多集中在细胞生物学水平,随着Twist条件性敲除转基因小鼠的建立,Twist与肿瘤转移的关系越来越多的被关注。为克服传统转基因动物模型的弱点,真实模拟体内肿瘤的发生、发展和转移的生物学环境,并在动物体内深入研究肿瘤相关基因的功能,本文建立基于RCAS-TVA系统的小鼠乳腺癌发病模型,并以转录因子Twist为对象,研究该基因在肿瘤发生、发展和转移过程中的重要作用,主要研究内容和结果如下： (1) RCAS-CRE-IRES-PyMT逆转录病毒质粒载体的构建与病毒的制备设计CRE作用元件(Cre重组酶基因)和原癌基因Polyomavirus Middle T Antigen (PyMT)两段基因插入序列,并以内部核糖体进入位点Internal Ribosomal Entry Site (IRES)作为Linker连接,以逆转录病毒质粒载体RCAS-Y为基础,通过分子生物学手段,构建RCAS-CRE-IRES-PyMT重组子质粒载体。该重组子质粒载体的特点是：借助Long Terminal Repeat (LTR)启动子序列和内部核糖体进入位点IRES序列的共同作用,在同一条mRNA上分别同时表达CRE和PyMT两个基因,并以Western Blot方法检测基因表达。结果表明：病毒载体可同时表达CRE和PyMT,且IRES序列前后,两种蛋白表达水平相当,确保了CRE和PyMT生物学功能的发挥；采用细胞转染和超速离心的方法收集RCAS-CRE-IRES-PyMT病毒,测定病毒滴度为1010/ml。 (2)基于RCAS-TVA系统的小鼠乳腺癌发病模型的建立及性质通过MMTV-TVA转基因小鼠与Rosa26R-LacZ报告基因小鼠的杂交,在F2代获取Rosa26R-LacZR/+MMTV-TVA+/-小鼠,并以此为模型,借助乳腺导管原位注射的方式,诱发小鼠产生蓝色乳腺肿瘤,通过检测目的基因的特异性表达、肿瘤的发生和生长曲线、肿瘤发展各阶段的细胞表面标志物的变化,系统研究该动物模型的性质和特征。结果表明：建立在RCAS-TVA系统的理论基础上,逆转录病毒通过特异性的侵染乳腺组织管腔内皮细胞,在诱发小鼠乳腺肿瘤的同时,实现了目的基因的条件性敲除,为基因功能研究奠定了基础。实体瘤组织发生于病毒侵染后的第7-9周,并于第10周后出现侵袭和转移特性；该模型中肿瘤细胞的特征与传统转基因动物模型不同,更加真实的模拟了人体内肿瘤发生环境。 (3)转录因子Twist在小鼠乳腺癌肺转移中的作用初步研究通过MMTV-TVA转基因小鼠与TwistF/F转基因小鼠的杂交,在F2代获取TwistF/F-MMTV-TVA+/-小鼠,并以此为模型,借助乳腺导管原位注射的方式,诱发小鼠产生乳腺肿瘤,通过检测肿瘤的发生、生长和转移,研究Twist基因与乳腺肿瘤的关系。结果表明：Twist基因对肿瘤的发生和生长没有影响,但是对肿瘤的转移具有促进作用；Twist可以通过调控EMT过程中相关标志物基因的变化而促进乳腺肿瘤的肺转移。本文通过建立基于RCAS-TVA系统的小鼠乳腺癌发病模型,并以转录因子Twist为对象,研究该基因在肿瘤转移中的功能,为乳腺肿瘤动物模型的建立和乳腺肿瘤转移的相关基因功能的研究提供了新思路。
[Abstract]:Malignant tumor is a serious threat to human health and life. A lot of manpower, financial and material resources have been invested in the development of tumor pathogenesis and anti-tumor drugs. Cisplatin (CDDP) is one of the first choice drugs in clinical chemotherapy. It has a broad spectrum of anticancer activity and strong killing effect. However, studies have shown that platinum is an anti-tumor drug. Tumor drugs are non-specific drugs with long half-life, low solubility, high cytotoxicity and non-targeting, which greatly limit their long-term and high-dose use in clinic.
With the development and application of materials science and technology, more and more researches on polymer as drug carriers have been carried out. Based on the unique high permeability and retention effect (EPR effect) in tumor tissues, polymer drug carriers can improve the water-solubility of drugs, improve the pharmacokinetic properties of drugs, and regulate drug delivery by interacting with drugs. Distribution of drugs in tissues, targeted drug delivery, reduction of toxic and side effects or drug-induced diseases are of great value in improving the safety and stability of drugs. Bacillus subtilis fermentation-derived gamma-polyglutamic acid (gamma-PGA) is a good drug carrier, which has a single structure, simple preparation process and good performance. In recent years, y-PGA has attracted much attention due to its biocompatibility, biodegradability, good sustained and controlled release properties.
Based on the above research background, in order to develop a kind of cisplatin analogue with long half-life, low toxicity, passive targeting and sustained-release effect, we cleverly linked cisplatin to the gamma-polyglutamic acid drug carrier synthesized by bio-fermentation to prepare the cisplatin-polyglutamic acid complex (gamma-PGA-CDDP). On the basis of the original research in the laboratory, we further studied the cisplatin-polyglutamic acid The properties of the complex were compared with those of commonly used chemotherapeutics, and the compound of glutamine Aspartate Polymers (GAP460) was prepared to increase the drug loading.
(1) Gamma-PGA-CDDP complex was prepared by Bacillus subtilis fermentation and characterized by infrared absorption spectroscopy and nuclear magnetic resonance spectroscopy. In order to study the properties and biological activities of y-PGA-CDDP complex systematically, the complex was prepared with cisplatin, carboplatin and octreotide, which were commonly used in clinical chemotherapy. Thaliplatin was compared with Bcap-37, BEL-7404 and SH-SY5Y human tumor cells as examples to compare the cytotoxicity and semi-inhibitory concentration (IC50) of the four drugs, simulate the drug release curve in vitro, and compare the tumor inhibition effect, weight change and survival rate in normal Kunming mice and BALB/cA tumor-bearing mice. Blood biochemical indexes such as white blood cell (WBC), platelet (BP), creatinine (CRE, CREAP), urea nitrogen (BUN, UREA), myeloperoxidase (MPO), malondialdehyde (MDA), reduced glutathione peroxidase (GSH-Px) and other renal toxicity indicators, the results showed that: gamma-PGA-CDDP complex at the cellular level can not only be significant. Inhibiting the proliferation of Bcap-37 and other three tumor cells, and greatly reducing the cytotoxicity, IC50 value was significantly higher than cisplatin, similar to oxaliplatin, slightly lower than carboplatin; in BALB/cA tumor-bearing model animals, gamma-PGA-CDDP complex can effectively inhibit the growth of tumor in vivo, under the same experimental conditions, its inhibitory effect and cisplatin. Platinum is comparable to carboplatin and oxaliplatin, and the toxicity and side effects of the drugs are greatly reduced. In Kunming mice, the damage of gamma-PGA-CDDP complex to animals is the smallest. After the effect of cisplatin and other drugs, the number of WBC and latelets in vivo can be decreased, the contents of CRE and BUN are increased, MPO and MDA are up-regulated, GSH and GSH-Px are down-regulated. After the treatment with gamma-PGA-CDDP complex, the biochemical indexes in vivo remained stable, and the oxidative damage of renal tissue was significantly improved, which showed obvious advantages compared with the clinical chemotherapy drugs.
(2) The drug loading of gamma-PGA is low, and the dosage of carrier is high in high-dose drug therapy. In addition, the metabolic process of y-PGA in vivo may lead to the accumulation of neurotransmitter precursors. Our laboratory study found that the drug loading of gamma-PGA-CDDP complex is only 14.6%. To solve these problems, the drug carrier of GAP460 complex was prepared by amidation reaction. Taking cisplatin as an example, the properties and functions of cisplatin-polyglutamic acid-aspartic acid complex (PACC) were studied. The results showed that the drug carrier of GAP460 complex remained stable at 37 C and pH 6.0-7.4. With the increase of temperature, the GAP60 complex could be degraded in varying degrees in acidic or alkaline environment. The survival rate of animals injected with 4.0g/kg GAP460 complex by pulse injection was 100%. The body weight, WBC, BP, GPT, GOT, CREAP and UREA were all stable and the drug carrier was safe and non-toxic under the same experimental conditions. The drug loading was 38.52%, about 3 times that of gamma-PGA carrier. The increase of drug loading could greatly reduce the dosage of carrier and improve the controlled-release effect of the drug. PACC complex not only significantly inhibited the proliferation of Bcap-37 and other tumor cells in vitro, but also significantly inhibited the IC50 value of BALB/cA tumor-bearing nude mice compared with free cisplatin. At the same time, the PACC complex greatly reduced the toxicity and side effects in vivo, the weight of animals did not decrease significantly, renal function and renal oxidative damage were significantly improved.
To sum up, this paper systematically compares gamma-PGA-CDDP complex with commonly used chemotherapeutic drugs, and introduces aspartate molecule to modify the polyglutamate drug carrier, which provides a useful exploration for the clinical application of polymer drug carrier and gamma-PGA-CDDP complex.
Cancer is one of the most important diseases endangering human health and even life, and the invasion and metastasis of malignant tumors are the difficulties in cancer prevention and treatment. According to statistics, the incidence of breast cancer accounts for 7-10% of all kinds of malignant tumors in the body, and it is increasing year by year. The prevention and treatment of breast cancer has become a research hotspot. In recent years, the establishment of tumor typing system based on molecular analysis technology has provided more information for tumor classification. More than 50 kinds of animal models of tissue and cell-specific breast cancer have been established. Following the establishment, the early detection rate and cure rate of tumor increase continuously, and the mortality of patients gradually decreases.
Animal models for breast cancer research mostly rely on transgenic mice. The main research strategy is to induce mice to develop breast cancer by tissue-specific promoters, such as SV40, Ras, Neu and other proto-oncogenes, to observe the characteristics and properties of tumors, and to study the function of related genes. However, transgenic animals based on homologous recombination are the main research strategies. RCAS-TVA system based on RCAS retroviral vectors has attracted much attention in the study of new breast cancer models. Viral infection has been used to induce single cell mutation and thus induce tumorigenesis.
Twist gene is an important transcription factor on human chromosomes. Studies in the 1990s showed that Twist gene plays an important role in the formation of mesoderm during embryonic development. The relationship between Twist gene and tumor metastasis was first reported in 2004. It can promote tumor cells by promoting epithelial-mesenchymal transformation. In addition, Twist is also associated with cell cycle and apoptosis, angiogenesis and tumor cell resistance. However, most of the current studies focus on cell biology. With the establishment of Twist conditioned knockout transgenic mice, the relationship between Twist and tumor metastasis has attracted more and more attention.
In order to overcome the weakness of traditional transgenic animal models, simulate the biological environment of tumor occurrence, development and metastasis in vivo, and study the function of tumor-related genes in vivo, a mouse model of breast cancer based on RCAS-TVA system was established, and the transcription factor Twist was used as the target to study the genesis of tumor. The main contents and results of the development and transfer process are as follows:
(1) construction of RCAS-CRE-IRES-PyMT retroviral vector and preparation of virus.
Two insertion sequences of CRE acting element (Cre recombinase gene) and proto-oncogene Polyomavirus Middle T Antigen (PyMT) were designed. The internal ribosome entry site, Internal Ribosomal Entry Site (IRES), was used as a linker to construct RCAS-CRE-IRES-based retroviral plasmid vector RCAS-Y by molecular biological means. The recombinant plasmid vector of PyMT was characterized by simultaneous expression of CRE and PyMT genes on the same mRNA by the interaction of the promoter sequence of Long Terminal Repeat (LTR) and the IRES sequence of the internal ribosome entry site. The expression of the two genes was detected by Western Blot method. Both CRE and PyMT could be expressed at the same time, and the expression level of the two proteins was similar before and after IRES sequence, which ensured the biological function of CRE and PyMT. RCAS-CRE-IRES-PyMT virus was collected by cell transfection and ultracentrifugation, and the viral titer was 1010/ml.
(2) establishment and characterization of a mouse breast cancer model based on RCAS-TVA system.
Rosa26R-LacZR/+MMTV-TVA+/-mice were obtained in F2 generation by hybridization of MMTV-TVA transgenic mice with Rosa26R-LacZ reporter gene mice. The model was used to induce blue breast tumors in mice by in situ injection of MMTV-TVA+/-mice into the mammary duct. Specific expression of the target gene, tumor genesis and growth curve, and tumor development were detected. The results showed that, on the basis of RCAS-TVA system, retrovirus could induce mouse breast tumors by specific infection of endothelial cells in the lumen of mammary gland, and at the same time, conditional knockout of the target gene was achieved. Solid tumors occur at the 7th to 9th week after viral infection and exhibit invasion and metastasis characteristics after the 10th week.
(3) the role of transcription factor Twist in lung metastasis of breast cancer in mice
TwistF/F-MMTV-TVA+/-mice were hybridized with TwistF/F transgenic mice. TwistF/F-MMTV-TVA+/-mice were obtained in F2 generation. The model was used to induce breast tumors in mice by in situ mammary duct injection. The relationship between Twist gene and breast tumors was studied by detecting the occurrence, growth and metastasis of tumors. It has no effect on the occurrence and growth of breast tumors, but promotes the metastasis of breast tumors. Twist can promote the lung metastasis of breast tumors by regulating the changes of related marker genes during EMT.
In this paper, a mouse model of breast cancer based on RCAS-TVA system was established, and Twist, a transcription factor, was used to study the function of Twist in tumor metastasis.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R-332;R737.9

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