發(fā)布時(shí)間：2018-09-09 19:38

【摘要】：惡性腫瘤嚴(yán)重威脅著人類的健康和生命,對(duì)于腫瘤發(fā)病機(jī)制及抗腫瘤藥物的開發(fā)已經(jīng)投入了大量的人力、財(cái)力和物力。順鉑(cisplatin, CDDP)是目前臨床上化療的首選藥物之一,具有抗癌活性譜廣,殺傷作用強(qiáng)等優(yōu)點(diǎn),然而研究表明：鉑類抗腫瘤藥物屬周期非特異性藥物,半衰期長(zhǎng),溶解度低,細(xì)胞毒性大,無(wú)靶向性,大大限制了其在臨床上長(zhǎng)期和高劑量的使用。因此,研究和開發(fā)低毒高效的順鉑類似物,一直是藥物修飾領(lǐng)域的研究熱點(diǎn)。 隨著材料科學(xué)技術(shù)的發(fā)展和應(yīng)用,高分子聚合物作為藥物載體的研究越來(lái)越多,基于腫瘤組織內(nèi)部特有的高通透性和滯留效應(yīng)(EPR效應(yīng)),高分子藥物載體通過(guò)與藥物的相互作用,可以提高藥物的水溶性,改善藥物的藥代動(dòng)力學(xué)特性,調(diào)控藥物在組織中的分布,靶向給藥,降低藥物的毒副作用或藥源性疾病等,對(duì)于提高藥物的安全性和穩(wěn)定性具有很高的應(yīng)用價(jià)值�？莶菅挎邨U菌發(fā)酵來(lái)源的γ型聚谷氨酸(γ-PGA)就是一種良好的藥物載體,它具有結(jié)構(gòu)單一、制備工藝簡(jiǎn)單、良好的生物相容性和生物可降解性、良好的緩釋和控釋性能等優(yōu)點(diǎn),近年來(lái),以y-PGA作為載體的研究備受關(guān)注。 基于以上研究背景,為開發(fā)一種半衰期長(zhǎng)、毒性低、具被動(dòng)靶向和緩釋效果的順鉑類似物,我們將順鉑巧妙地連接到生物發(fā)酵合成的γ-聚谷氨酸藥物載體上,制備順鉑-聚谷氨酸復(fù)合物(γ-PGA-CDDP),在實(shí)驗(yàn)室原有研究基礎(chǔ)上,進(jìn)一步研究該復(fù)合物的性質(zhì)；將其與臨床常用化療藥物進(jìn)行比較；并制備γ-聚谷氨酸-天冬氨酸復(fù)合物(Glutamine Aspartate Polymers, GAP460)以提高藥物載量。具體研究?jī)?nèi)容和結(jié)果分為以下兩個(gè)方面： (1)枯草芽孢桿菌發(fā)酵制備γ-PGA,通過(guò)優(yōu)化反應(yīng)條件,制備γ-PGA-CDDP復(fù)合物,并借助紅外吸收光譜和核磁共振的方法對(duì)復(fù)合物進(jìn)行表征鑒定。為系統(tǒng)研究y-PGA-CDDP復(fù)合物的性質(zhì)和生物學(xué)活性,將該復(fù)合物與臨床常用化療藥物順鉑、卡鉑和奧沙利鉑進(jìn)行比較：以Bcap-37、BEL-7404和SH-SY5Y三株人源腫瘤細(xì)胞為例比較四種藥物的細(xì)胞毒性和半抑制濃度(IC50)；模擬藥物體外釋放曲線；以正常昆明小鼠和BALB/cA荷瘤小鼠為動(dòng)物模型,比較藥物作用后的抑瘤效果、體重變化、存活率；白細(xì)胞(WBC)、血小板(Platelet, BP)等血液生化指標(biāo)；肌酐(CRE, CREAP)、尿素氮(BUN, UREA)、髓質(zhì)過(guò)氧化物酶(MPO)、丙二醛(MDA)、還原型谷胱甘肽(GSH)、谷胱甘肽過(guò)氧化物酶(GSH-Px)等腎臟毒性指標(biāo),結(jié)果證明：γ-PGA-CDDP復(fù)合物在細(xì)胞水平上不僅可以顯著抑制Bcap-37等三株腫瘤細(xì)胞的增殖,而且大大降低了細(xì)胞毒性,表現(xiàn)為IC50值明顯高于順鉑,與奧沙利鉑相當(dāng),略低于卡鉑；在BALB/cA荷瘤模型動(dòng)物體內(nèi),γ-PGA-CDDP復(fù)合物可以有效抑制體內(nèi)腫瘤的生長(zhǎng),相同實(shí)驗(yàn)條件下,其抑制作用效果與順鉑相當(dāng),優(yōu)于卡鉑和奧沙利鉑,同時(shí)藥物的毒副作用大大降低；在昆明小鼠體內(nèi),γ-PGA-CDDP復(fù)合物對(duì)于動(dòng)物的損傷最小,順鉑等化療藥物作用后,均可不同程度的引起體內(nèi)WBC和Platelet數(shù)目下降、CRE和BUN含量升高、MPO和MDA上調(diào)、GSH和GSH-Px下調(diào),而γ-PGA-CDDP復(fù)合物作用后,動(dòng)物體內(nèi)各生化指標(biāo)均保持穩(wěn)定,腎組織氧化性損傷顯著改善,在與臨床化療藥物的比較中體現(xiàn)出明顯的優(yōu)勢(shì)。 (2)γ-PGA的藥物載量偏低,高劑量藥物治療時(shí)載體用量較大,另外,y-PGA在體內(nèi)的代謝過(guò)程可能導(dǎo)致神經(jīng)遞質(zhì)前體的累積,本實(shí)驗(yàn)室研究發(fā)現(xiàn),γ-PGA-CDDP復(fù)合物的載藥量只有14.6%。為解決上述問(wèn)題,通過(guò)酰胺化反應(yīng),制備GAP460復(fù)合物藥物載體,并以順鉑為例,研究順鉑-聚谷氨酸-天冬氨酸復(fù)合物(PACC)的性質(zhì)和功能,結(jié)果證明：GAP460復(fù)合物藥物載體在37℃、pH6.0-7.4的環(huán)境下保持穩(wěn)定,溫度的升高、偏酸或偏堿的環(huán)境均可使GAP60復(fù)合物發(fā)生不同程度的降解；急性毒性實(shí)驗(yàn),單次靜脈注射GAP460復(fù)合物4.0g/kg動(dòng)物存活率為100%,單次靜脈給予2.0g/kg,動(dòng)物的體重,體內(nèi)白細(xì)胞(WBC)、血小板(BP),血清谷丙轉(zhuǎn)氨酶(GPT)、谷草轉(zhuǎn)氨酶(GOT)、肌酐(CREAP)和尿素氮(UREA)的含量均保持穩(wěn)定,藥物載體安全無(wú)毒；相同實(shí)驗(yàn)條件下,GAP460復(fù)合物的藥物載量為38.52%,約為γ-PGA藥物載體的3倍,藥物載量的提高可大大減少載體的用量,并改善藥物的控釋效果；PACC復(fù)合物不僅在體外可以顯著抑制Bcap-37等腫瘤細(xì)胞的增殖,IC50值明顯高于游離順鉑,而且可以有效抑制BALB/cA荷瘤裸鼠體內(nèi)腫瘤的生長(zhǎng)；同時(shí),PACC復(fù)合物大大降低了體內(nèi)毒副作用,用藥后動(dòng)物體重?zé)o明顯下降,腎臟功能和腎臟氧化性損傷得到明顯改善。 綜上所述,本文將γ-PGA-CDDP復(fù)合物與臨床常用化療藥物開展系統(tǒng)比較研究,并引入天冬氨酸分子對(duì)聚谷氨酸藥物載體進(jìn)行改造,為高分子藥物載體和γ-PGA-CDDP復(fù)合物的臨床應(yīng)用提供了有益的探索。 癌癥是危害人類健康甚至危及生命的重要疾病之一,而惡性腫瘤的侵襲和轉(zhuǎn)移又是癌癥預(yù)防和治療的難點(diǎn)。據(jù)統(tǒng)計(jì),乳腺癌的發(fā)病率占全身各種惡性腫瘤的7-10%,并呈逐年上升的趨勢(shì),乳腺癌的預(yù)防和治療已成為研究熱點(diǎn)。乳腺腫瘤的發(fā)生和發(fā)展是一個(gè)復(fù)雜的多基因參與的生物學(xué)過(guò)程,每個(gè)階段均由不同的基因和信號(hào)分子調(diào)控。近年來(lái),以分子分析技術(shù)為基礎(chǔ)的腫瘤分型系統(tǒng)的建立,為腫瘤的分類提供了更多的信息,50余種組織和細(xì)胞特異性的乳腺癌發(fā)病動(dòng)物模型相繼建立,腫瘤的早期檢出率和治愈率不斷提高,患者死亡率逐漸下降。 乳腺腫瘤研究的動(dòng)物模型多借助于轉(zhuǎn)基因小鼠,主要研究策略是在組織特異性啟動(dòng)子的誘導(dǎo)下,通過(guò)SV40、Ras、Neu等原癌基因誘導(dǎo)小鼠發(fā)生乳腺癌,觀察腫瘤的特征和性質(zhì),并研究相關(guān)基因的功能,但是建立在同源重組基礎(chǔ)上的轉(zhuǎn)基因動(dòng)物并不能完全模擬人體內(nèi)乳腺癌的發(fā)病環(huán)境和特點(diǎn)。在新型乳腺腫瘤發(fā)病模型的研究中,以RCAS逆轉(zhuǎn)錄病毒載體為基礎(chǔ)的RCAS-TVA系統(tǒng)備受關(guān)注借助病毒侵染,誘導(dǎo)單個(gè)細(xì)胞突變,從而誘導(dǎo)腫瘤發(fā)生的方法逐漸被認(rèn)可。 Twist基因是存在于人類染色體上的重要轉(zhuǎn)錄因子,上世紀(jì)90年代的研究表明：Twist基因在胚胎發(fā)育過(guò)程中對(duì)于中胚層的形成具有重要作用。Twist基因與腫瘤轉(zhuǎn)移的關(guān)系于2004年被首次報(bào)道,它可以通過(guò)促進(jìn)上皮-間葉轉(zhuǎn)化而促進(jìn)腫瘤細(xì)胞的侵襲和轉(zhuǎn)移,此外,Twist還與細(xì)胞周期及凋亡、血管形成和腫瘤細(xì)胞耐藥性有關(guān),但目前的研究大多集中在細(xì)胞生物學(xué)水平,隨著Twist條件性敲除轉(zhuǎn)基因小鼠的建立,Twist與腫瘤轉(zhuǎn)移的關(guān)系越來(lái)越多的被關(guān)注。 為克服傳統(tǒng)轉(zhuǎn)基因動(dòng)物模型的弱點(diǎn),真實(shí)模擬體內(nèi)腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移的生物學(xué)環(huán)境,并在動(dòng)物體內(nèi)深入研究腫瘤相關(guān)基因的功能,本文建立基于RCAS-TVA系統(tǒng)的小鼠乳腺癌發(fā)病模型,并以轉(zhuǎn)錄因子Twist為對(duì)象,研究該基因在腫瘤發(fā)生、發(fā)展和轉(zhuǎn)移過(guò)程中的重要作用,主要研究?jī)?nèi)容和結(jié)果如下： (1) RCAS-CRE-IRES-PyMT逆轉(zhuǎn)錄病毒質(zhì)粒載體的構(gòu)建與病毒的制備 設(shè)計(jì)CRE作用元件(Cre重組酶基因)和原癌基因Polyomavirus Middle T Antigen (PyMT)兩段基因插入序列,并以內(nèi)部核糖體進(jìn)入位點(diǎn)Internal Ribosomal Entry Site (IRES)作為L(zhǎng)inker連接,以逆轉(zhuǎn)錄病毒質(zhì)粒載體RCAS-Y為基礎(chǔ),通過(guò)分子生物學(xué)手段,構(gòu)建RCAS-CRE-IRES-PyMT重組子質(zhì)粒載體。該重組子質(zhì)粒載體的特點(diǎn)是：借助Long Terminal Repeat (LTR)啟動(dòng)子序列和內(nèi)部核糖體進(jìn)入位點(diǎn)IRES序列的共同作用,在同一條mRNA上分別同時(shí)表達(dá)CRE和PyMT兩個(gè)基因,并以Western Blot方法檢測(cè)基因表達(dá)。結(jié)果表明：病毒載體可同時(shí)表達(dá)CRE和PyMT,且IRES序列前后,兩種蛋白表達(dá)水平相當(dāng),確保了CRE和PyMT生物學(xué)功能的發(fā)揮；采用細(xì)胞轉(zhuǎn)染和超速離心的方法收集RCAS-CRE-IRES-PyMT病毒,測(cè)定病毒滴度為1010/ml。 (2)基于RCAS-TVA系統(tǒng)的小鼠乳腺癌發(fā)病模型的建立及性質(zhì) 通過(guò)MMTV-TVA轉(zhuǎn)基因小鼠與Rosa26R-LacZ報(bào)告基因小鼠的雜交,在F2代獲取Rosa26R-LacZR/+MMTV-TVA+/-小鼠,并以此為模型,借助乳腺導(dǎo)管原位注射的方式,誘發(fā)小鼠產(chǎn)生藍(lán)色乳腺腫瘤,通過(guò)檢測(cè)目的基因的特異性表達(dá)、腫瘤的發(fā)生和生長(zhǎng)曲線、腫瘤發(fā)展各階段的細(xì)胞表面標(biāo)志物的變化,系統(tǒng)研究該動(dòng)物模型的性質(zhì)和特征。結(jié)果表明：建立在RCAS-TVA系統(tǒng)的理論基礎(chǔ)上,逆轉(zhuǎn)錄病毒通過(guò)特異性的侵染乳腺組織管腔內(nèi)皮細(xì)胞,在誘發(fā)小鼠乳腺腫瘤的同時(shí),實(shí)現(xiàn)了目的基因的條件性敲除,為基因功能研究奠定了基礎(chǔ)。實(shí)體瘤組織發(fā)生于病毒侵染后的第7-9周,并于第10周后出現(xiàn)侵襲和轉(zhuǎn)移特性；該模型中腫瘤細(xì)胞的特征與傳統(tǒng)轉(zhuǎn)基因動(dòng)物模型不同,更加真實(shí)的模擬了人體內(nèi)腫瘤發(fā)生環(huán)境。 (3)轉(zhuǎn)錄因子Twist在小鼠乳腺癌肺轉(zhuǎn)移中的作用初步研究 通過(guò)MMTV-TVA轉(zhuǎn)基因小鼠與TwistF/F轉(zhuǎn)基因小鼠的雜交,在F2代獲取TwistF/F-MMTV-TVA+/-小鼠,并以此為模型,借助乳腺導(dǎo)管原位注射的方式,誘發(fā)小鼠產(chǎn)生乳腺腫瘤,通過(guò)檢測(cè)腫瘤的發(fā)生、生長(zhǎng)和轉(zhuǎn)移,研究Twist基因與乳腺腫瘤的關(guān)系。結(jié)果表明：Twist基因?qū)δ[瘤的發(fā)生和生長(zhǎng)沒(méi)有影響,但是對(duì)腫瘤的轉(zhuǎn)移具有促進(jìn)作用；Twist可以通過(guò)調(diào)控EMT過(guò)程中相關(guān)標(biāo)志物基因的變化而促進(jìn)乳腺腫瘤的肺轉(zhuǎn)移。 本文通過(guò)建立基于RCAS-TVA系統(tǒng)的小鼠乳腺癌發(fā)病模型,并以轉(zhuǎn)錄因子Twist為對(duì)象,研究該基因在腫瘤轉(zhuǎn)移中的功能,為乳腺腫瘤動(dòng)物模型的建立和乳腺腫瘤轉(zhuǎn)移的相關(guān)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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R-332;R737.9

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