【作者】 卢丹；

【导师】 钱静；

【作者基本信息】 扬州大学 ， 中西医结合临床， 2015， 博士

【摘要】 宫颈癌是最常见的妇科恶性肿瘤,其发病机制至今尚未完全阐明。临床上,宫颈癌的治疗方法主要有手术切除、放疗、化疗以及中医中药治疗。最佳的治疗方法则是结合各种疗法的优点,对宫颈癌实施多学科综合治疗。近年来研究表明,磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin, PI3K/AKT/mTOR)信号通路具有调节肿瘤细胞生长、促进细胞周期进展、参与新生血管形成以及使肿瘤细胞产生放化疗拮抗等多种功能,是最主要的抑制肿瘤细胞凋亡的信号通路。在恶性肿瘤的发生过程中存在PI3K/AKT/mTOR信号通路的激活,因此,阻断该信号通路对恶性肿瘤的治疗与预后均具有重要意义。基于PI3K/AKT/mTOR信号通路开发相关靶点抑制剂,通过触发肿瘤细胞凋亡以及联合应用化放疗在宫颈癌多学科综合治疗中发挥重要作用,已成为宫颈癌分子靶向治疗的重要热点。中医药在宫颈癌多学科综合治疗中具有独特作用。紫草素(shikonin)是一类从紫草植物根中提取的萘醌类化合物,化学成分复杂,具有抗肿瘤、抗炎、抗HIV病毒和抗真菌等广泛的药理作用。由于紫草素的毒性较强而其抗肿瘤作用不具特异性,因而未能在临床上被推广应用。近年来,提取或合成高效低毒的紫草素衍生物的研究备受关注β-羟基异戊酰紫草素(p-hydroxyisovalerylshikon, β-HIVS)即是天然存在的紫草素衍生物之一。β-HIVS通过抑制肿瘤细胞生长增殖、诱导肿瘤细胞凋亡、干扰肿瘤细胞生长周期,使其具有开发为新型高效低毒抗肿瘤药物的潜力。本课题通过体内外实验来研究β-HIVS抗宫颈癌的分子作用机制以及与PI3K/AKT/mTOR信号通路的相关性,为临床宫颈癌的中药分子靶向治疗提供新的策略和有效途径。第一部分 PTEN、Survivin、PI3K/AKT/mTOR信号通路与宫颈癌变演化关系的临床研究目的：结合宫颈病变的临床病理学特征,探讨PTEN、Surviving、PI3K、AKT、mTOR、P70S6K在宫颈癌变演化过程中的表达及其临床意义。方法：收集20例宫颈糜烂伴鳞状上皮化生、60例宫颈上皮内瘤变(CIN Ⅰ、CIN Ⅱ、CIN Ⅲ各20例)以及56例宫颈鳞状上皮细胞癌组织标本。应用免疫组织化学(SP法)、实时定量PCR(qRT-PCR)和蛋白质印迹法(Western Blot)检测各组宫颈组织中PTEN、Surviving、 PI3K、AKT、mTOR、P70S6K的表达。选取10例正常宫颈组织作为对照组。结果：1)在宫颈癌变演化过程中,从正常宫颈、宫颈糜烂伴鳞化、宫颈上皮内瘤变到宫颈癌,PTEN阳性表达率逐渐减弱(P<0.05)；而Survivin的表达率则逐渐增高(P<0.05)；PI3K、AKT、mTOR、P70S6K在正常宫颈、宫颈糜烂伴鳞化、宫颈上皮内瘤变以及宫颈癌组织中的阳性表达率均呈逐渐增加趋势(P<0.05)。2) PTEN在宫颈癌中的阳性表达与临床分期、病灶大小有关(P<0.05)；Survivin在宫颈癌中的阳性表达与临床分期、病灶大小和肿瘤分化程度有关(P<0.05)；宫颈癌组织中PI3K、AKT和mTOR的阳性表达率与临床分期、分化程度有关(P<0.05)；P70S6K在宫颈癌中的阳性表达与临床分期有关(P<0.05)。3)PTEN与Survivin在宫颈癌组织中的表达呈显著负相关(r2=-0.613,P<0.01)；P70S6K在宫颈癌组织中的表达与PI3K、AKT、mTOR呈明显正相关(r10=0.418, P<0.01; r11=0.512, P<0.05; r12=0.365, P<0.01); PI3K与AKT、mTOR呈明显正相关(r13=0.438, P<0.01; r14=0.366, P<0.01); AKT与mTOR呈明显正相关(r15=0.351, P<0.01)。4)qRT-PCR检测显示,PTEN mRNA在宫颈癌、CIN Ⅲ组织中的表达显著低于正常宫颈组织(P<0.01)；Survivin mRNA在宫颈癌、CINⅢ组织中明显高于正常宫颈组织(P<0.01); PI3K、AKT、 mTOR和P70S6KmRNA在宫颈癌、CIN Ⅲ组织中均明显高于正常宫颈组织(P<0.01),在CIN Ⅱ组织中的表达也均高于正常宫颈组织(P<0.05)。5) Western Blot检测显示,PTEN蛋白在宫颈癌、CINⅢ、 CIN Ⅱ、CIN Ⅰ组织中的表达显著低于正常宫颈组织(P<0.01)；Survivin蛋白表达在宫颈癌、CINⅢ组织中明显高于正常宫颈组织(P<0.01),在CIN Ⅱ中的表达亦高于正常宫颈组织(P<0.05); PI3K在宫颈癌、CINⅢ、CIN Ⅱ、CIN Ⅰ组织中的表达均明显高于正常宫颈组织(P<0.01); AKT、mTOR和P70S6K蛋白表达在宫颈癌、CINⅢ组织中均明显高于正常宫颈组织(P<0.01),在CIN Ⅱ中的表达也均高于正常宫颈组织(P<0.05)。结论：Survivin、PI3K、AKT、mTOR和P70S6K在宫颈癌变演化过程中存在联合表达,协同参与了宫颈癌的发生及其进展。联合检测为宫颈癌的早期诊断和预后判断提供了新的评价指标,也为宫颈癌的基因治疗提供了新的生物学靶点。第二部分 p-羟基异戊酰紫草素通过PI3K/AKT/mTOR信号通路诱导人宫颈癌Hala细胞凋亡的体外实验研究目的：研究β-羟基异戊酰紫草素(β-HIVS)对人宫颈癌Hela细胞的体外生长抑制作用,探讨β-HIVS促肿瘤细胞凋亡的分子作用机制。方法：以不同浓度β-HIVS作用人宫颈癌Hela细胞,MTT法检测β-HIVS对Hela细胞体外增殖的抑制率；AO/EB染色法荧光显微镜观察Hela细胞凋亡的形态学变化；Annexin/PI双标记法检测Hela细胞凋亡率；流式细胞仪分析Hela细胞周期分布；qRT-PCR和Western blot法检测Hela细胞中PI3K、AKT、mTOR、P70S6K的mRNA和蛋白表达水平变化。结果：1) β-HIVS对Hela细胞体外增殖有显著的抑制作用,呈现明显的量效关系和时间依赖性。2)随着β-HIVS浓度增加,Hela细胞凋亡的数量和形态结构均发生重要改变,细胞凋亡率也逐渐增加。细胞周期分析显示,β-HIVS引起Hela细胞周期分布发生变化,表现为G0/G1细胞数下降,S期细胞数增多,而G2/M期细胞数则无明显变化,细胞阻滞于S期。3)三种不同浓度P-HIVS作用Hela细胞,对PI3K、AKT、mTOR、P70S6K表达水平的影响呈时间和剂量依赖性；药物作用48h后,β-HIVS能显著下调Hela细胞中PI3K、AKT、 mTOR和P70S6K的mRNA表达水平,并下调PI3K、AKT、p-AKT、mTOR、P70S6K的蛋白表达水平。结论：β-HIVS能显著抑制Hela细胞增殖,呈现明显的时间和剂量依赖性。β-HIVS诱导肿瘤细胞发生凋亡可能是通过抑制PI3K/AKT/mTOR信号通路以及降低该通路中相关下游基因蛋白表达水平而发挥其抗癌作用。针对该信号通路的中药分子靶向治疗有望成为宫颈癌治疗的新途径。第三部分 β-羟基异戊酰紫草素通过PI3K/AKT/mTOR信号通路抑制宫颈癌裸鼠移植瘤生长的实验研究目的：建立宫颈癌荷瘤裸鼠动物模型,观察β-HIVS的体内抑瘤效果,探讨其抗肿瘤作用的分子机制。方法：制备宫颈癌荷瘤裸鼠动物模型。获取瘤体组织后进行大体标本观测,评价β-HIVS对裸鼠皮下移植肿瘤的抑制效果；HE染色法观察细胞形态学变化；Tunel染色法检测肿瘤组织内细胞凋亡状态；qRT-PCR、Western blot法检测β-HIVS处理后PI3K、 AKT、mTOR、 p70S6K的mRNA和蛋白表达的变化。结果：1) β-HIVS治疗组宫颈癌移植瘤生长速度明显低于对照组,注射药物后瘤体重量及体积缩小较对照组相比具有统计学差异(p<0.05), β-HIVS治疗组的抑瘤率达到32.80%。2)组织学HE染色显示,β-HIVS治疗组肿瘤细胞发生变性坏死,癌巢结构疏松,核浓缩、碎裂,浓缩的染色质围绕于核周边或形成新月体结构。3) Tunel细胞凋亡原位检测显示,β-HIVS能诱导宫颈癌组织的肿瘤细胞发生凋亡。4) qRT-PCR检测,经β-HIVS治疗后,移植瘤组织中PI3K、AKT、mTOR、P70S6K的mRNA表达均低于对照组(p<0.01)。 Western blot检测,经β-HIVS治疗后,PI3K、AKT、p-AKT、mTOR和P70S6K的蛋白表达水平均明显下降(p<0.01)。结论：β-HIVS通过诱导细胞凋亡、抑制细胞增殖对宫颈癌移植瘤生长具有明显的抑制作用。PI3K/AKT/mTOR信号通路在β-HIVS诱导宫颈癌细胞凋亡中发挥重要作用。更多还原

【Abstract】 Cervical cancer is a common malignant tumor of genital tract, while the pathogenesis is not yet completely understood. Clinically, the treatment options for cervical cancer are surgery, radiotherapy, chemotherapy and traditional Chinese medicine. Among them, the best one for treatment of cervical cancer is the multi-disciplinary comprehensive menthod combined with the advantages of various kinds of options mentioned above. Recent studies indicated that the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway regulates tumor cell proliferation, promotes cell cycle progression, participates in neovascularization, and induces tumor cells to resist radiotherapy and chemotherapy. Moreover, this signaling cascade is a major pathway in suppressing tumor cell apoptosis. Cervical cancer can be treated by triggering tumor cell apoptosis and combined application of radiotherapy and chemotherapy, which is a focus in molecular-targeted therapy of cervical cancer.Traditional Chinese medicine plays a unique role for the treatment of cervical cancer. Shikonin, a naphthoquinone derivative, is a naturally occurring active chemical found in the root of Lithospermum erythrorhizon, and has anti-cancer, anti-inflammation, anti-HIV virus and anti-fungus activities. Shikonin has strong toxicity, but does not exhibit tumor specificity and thus has not been widely used in the clinic. Studies of the extraction or synthesis of high-performance non-toxic shikonin derivatives has received great attention. P-hydroxyisovalerylshikonin (β-HIVS) is a natural shikonin derivative. Moreover, β-HIVS suppressed tumor cell proliferation, induced it apoptosis, and interfered with the cell cycle of tumor cells, highlighting its potential as a novel high-performance non-toxic antitumor medicine. Thus, on the basis of these in vitro and in vivo findings regarding the PI3K/AKT/mTOR signaling pathway, we acquire a deeper understanding of the occurrence, development, treatment and prognosis of cervical cancer, which would be a new strategy for molecular-targeted therapy of cervical cancer.1. Expressions of PTEN,Survivin,PI3K,AKT,mTOR,P70S6K in the progression of cervicalneoplasia and their clinical significances Objective To evaluate the expressions and clinical significances of PTEN, Survivin, PI3K, AKT, mTOR and P70 in the successive steps of progression in cervical lesions.Methods The expression of PTEN, Survivin, PI3K, AKT, mTOR, P70S6K were performed in a total of 146 patients with 10 normal cervical epithelium,20 cervical erosion metaplasia,60 cervical intraepithelial neoplasia (20 CIN Ⅰ,20 CIN Ⅱ,20 CIN Ⅲ), and 56 cervical squamous cell carcinomas were detected by SP immunohistochemistry, qRT-PCR and Western Blot.Results 1) PTEN expression progressively decreased along the continuum from cervical erosion metaplasia to cervical intraepithelial neoplasia and cervical squamous cell carcinoma(P<0.05); Survivin expression were progressively increased(P<0.05).PI3K/AKT/mTOR/P70S6K expression progressively increased along the continuum from normal epithelium to cervical erosion metaplasia,cervical intraepithelial neoplasia and cervical squamous cell carcinoma (P<0.05) 2) Positive PTEN immunostaining was associated with clinical stage(P<0.05), tumor size (P< 0.05) in cervical squamous cell carcinoma. Positive Survivin immunostaining was associated with clinical stage(P< 0.05), tumor size (P<0.05) and the differentiated degree (P<0.05). Positive PI3K/AKT/mTOR immunostaining was associated with clinical stage(P< 0.05), tumor size (P<0.05) in cervical squamous cell carcinoma. Positive P70 immunostaining was associated with clinical stage(P<0.05).3) There was a negative correlation between the expression of PTEN and Survivin in cervical cancer (r2=-0.613, P<0.01). P70S6K positive expression correlated significantly with PI3K,AKT and mTOR in cervical cancer (r10=0.418, P<0.01; r11=0.512, P<0.05; r12=0.365, P<0.01). PI3Kpositive expression correlated significantly with AKT and mTOR (r13=0.438, P<0.01; r14=0.366,P<0.01).AKT positive expression correlated significantly with mTOR(r15=0.351, P<0.01).4) qRT-PCR analysis showed that:compared to those in normal cervix uteri, PTEN mRNA in cervical carcinomas and CINⅢ were significantly decreased (P<0.01), Survivin mRNA in cervical carcinomas and CINⅢ were significantly increased (P<0.01), PI3K/AKT/mTOR/P70S6K mRNA in cervical carcinomas and CIN Ⅲ were significantly increased respectively (P<0.01), PI3K/AKT/mTOR/P70S6K mRNA in CIN Ⅱ were also increased respectively (P<0.05).5) Western Blot analysis showed that:compared to those in normal cervix uteri, PTEN in cervical carcinomas,CINⅢ,CIN Ⅱ and CIN Ⅰ were significantly decreased (P<0.01), Survivin in cervical carcinomas and CIN Ⅲ were significantly increased (P<0.01), Survivin in CIN Ⅱ were also increased (P<0.05), PI3K in cervical carcinomas,CIN Ⅲ,CIN Ⅱ and CIN Ⅰ were significantly increased (P<0.01), AKT/mTOR/P70S6K in cervical carcinomas and CINⅢ were significantly increased respectively (P<0.01), AKT/mTOR/P70S6K in CIN Ⅱ were also increased respectively (P<0.05). Conclusion PTEN,Survivin,PI3K,AKT,mTOR and P70S6K may be valuable biomarkers that are predictive of the progression and prognosis in patients with cervical squamous cell carcinoma. These useful biomarkers may have potential as promising therapeutic targets.2. β-hydroxyisovalerylshikonin induces human cervical cancer cell apoptosis via PI3K/AKT/mTOR signaling Objective To investigate the in vitro proliferation inhibitory effect of β-HIVS on human cervical cancer cells and to identify the mechanism of inducing HeLa cell apoptosis. Methods HeLa cells were treated with β-HIVS and the growth inhibitory effect was detected with MTT assay. Acridine orange/ethidium bromide staining was used for morphological changes in apoptotic HeLa cells. Annexin/propidium iodide staining was applied for HeLa cell apoptotic rate after treatment with P-HIVS. Flow cytometry was employed to analyze apoptosis and cell cycle distribution. qRT-PCR and western blot assays were used to examine PI3K/AKT/mTOR signaling pathway expression.Results 1) P-HIVS inhibited HeLa cell proliferation in a dose-and time-dependent manner.2) With increased concentration of β-HIVS, the apoptotic rate of HeLa cells was increased. Flow cytometry analysis showed that Hela cell cycle distribution changed when treated with β-HIVS. The counts of G0/G1 cells decreased, the cells in S phase increased, while the number of G2/ M phase cells had no obviously changed, which indicated that cell cycle was slightly arrested at S phase.3) Three different concentrations of β-HIVS evidently reduced PI3K, AKT, mTOR and P70S6K mRNA expression levels and PI3K, AKT, β-AKT, mTOR and P70S6K protein expression levels in HeLa cells after 48h in a time- and dose-dependent manner.Conclusion These results indicated that β-HIVS suppressed HeLa cell proliferation in a time-and dose-dependent manner. β-HIVS promoted cervical cancer cell apoptosis by inhibiting the PI3K/AKT/mTOR signaling pathway and suppressing downstream gene expression. Based on the signaling pathway of molecular targeted therapy is expected to develop a new way for cervical cancer treatment.3. In vivo anti-cervical cancer effects of P-hydroxyisovalerylshikonin through PI3K/AKT/mTOR signaling pathwayObjective To establish an animal model of cervical cancer on tumor-burdened nude mice in order to study the potential anti-tumor molecular mechanism of p-HIVS in vivo.Methods An animal model of cervical cancer on tumor-burdened nude mic was established. And then, the macroscopic measurements were taken to evaluate inhibitory effects of p-HIVS treated for the subcutaneously transplanted tumors in nude mice. We detected morphological changes via HE staining, apoptosis status with Tunel staining method. qRT-PCR and Western blot were used to detect the mRNA and protein expressions of PI3K, AKT, mTOR and P70S6K after P-HIVS treatment.Results 1) The growth rate of cervical cancer transplantation tumor that treated by P-HIVS was obviously lower than the control group. Both weight and volume of the tumors in drug injected group were significantly smaller than the control one(p< 0.05). P-HIVS could inhibit the proliferation of human cervical cancer (tumor inhibition rate was 32.80%).2) Histological HE staining showed that the tumor cells of p-HIVS treatment group became degeneration and necrosis, which the nest structure loosened. Cell nuclears turned to pyknosis or karyorrhexis, and the condensed chromatins dispersed around the nucleus or formed the crescentric structures.3) The typical apoptotic morphological changes were found by the Tunel staining method.4) qRT-PCR demonstrated that the mRNA expressions of PI3K, AKT, mTOR and P70S6K were decreased after treated by P-HIVS than the control respectively (p<0.01). Western blot demonstrated that the protein expressions of PI3K, AKT, p-AKT, mTOR and P70S6K were decreased than the control respectively (p<0.05).Conclusion Through inducing tumor cell apoptosis, P-HIVS could obviously inhibit the growth of cervical cancerous tissue in vivo. The PI3K/AKT/mTOR signaling pathway may play an important role in inducing the cervical cancer cells apoptosis that treated by P-HIVS.更多还原