【作者】 郭清；

【导师】 吴小华；

【作者基本信息】 河北医科大学 ， 外科学， 2009， 博士

【摘要】 目的:卵巢上皮性癌(简称卵巢癌)是妇科肿瘤领域中最具有挑战性的疾病,其死亡率居女性生殖道恶性肿瘤之首。卵巢癌高死亡率主要是因为该病在确诊时70%的患者是晚期,疾病的范围已经超过卵巢扩散到腹腔和腹膜后淋巴结,而晚期卵巢癌的五年生存率则长期徘徊在30%左右。与其他多种类型的癌不同,卵巢癌的主要转移途径不是通过血管,而是腹腔转移。由于在腹腔的广泛种植和转移常常引起严重腹水和肠梗阻,所以卵巢癌的腹腔转移是直接引起患者死亡的主要原因。因此深入了解调控卵巢癌腹腔选择性转移的机制十分重要,将对卵巢癌的治疗产生深远的影响。卵巢癌的腹腔播散转移特性除了与其解剖和机械的原因有关外,还主要是由于腹腔液中、腹水中存在和腹膜间皮细胞产生的趋化因子、生长因子、粘附因子等发挥了重要的作用。卵巢癌生长晚期,大网膜和腹膜是常见的转移部位。卵巢癌转移的目的地腹膜间皮细胞(human peritoneal mesohelial cells, HPMC)是脱落的肿瘤细胞腹腔播散时遇到的第一道防线,具有多种生理和病理功能。本课题组前期的研究发现,正常卵巢上皮无CXCL12及CXCR4蛋白表达,正常腹膜和腹腔液有CXCL12的表达,卵巢癌组织有CXCL12和CXCR4表达。目前已知CXCR4是趋化因子CXCL12的唯一生理受体,CXCL12又是CXCR4的唯一生理性配体,两者之间有非常高的亲和力。本课题拟通过构建稳定表达CXCR4卵巢癌细胞株及人腹膜间皮细胞的原代培养,到体外细胞实验,最后动物实验来证实这样由浅入深,层次递进的模式来探讨CXCL12-CXCR4生物轴在卵巢癌生长转移中的作用,从而更好的理解卵巢癌腹腔转移的机制,为抗卵巢癌腹腔转移的治疗寻找新的途径。方法:本课题分为三部分:1稳定表达CXCR4的卵巢癌细胞株的建立与鉴定及其增殖、迁移、侵袭能力的体外实验研究:首先将载体pReceiver-M02和目的基因CXCR4连接构建真核表达重组质粒pReceiver-M02-CXCR4,经双酶切和测序鉴定,结果证实正确。采用阳离子脂质体介导的转染方法,将高表达CXCR4的真核表达重组质粒pReceiver-M02-CXCR4及载体pReceiver-M02分别转染卵巢癌细胞SKOV3,经G418筛选并且通过单细胞分离技术获得了数个单细胞,转染成功的细胞经RT-PCR,Western blot和免疫细胞化学方法鉴定。将转染质粒、转染载体及未转染的三种卵巢癌细胞SKOV3进行体外培养。采用四甲基偶氮唑蓝(MTT)比色法进行增殖实验,判断在无牛血清培养液的生长条件下,不同浓度的CXCL12对三种细胞增殖的影响以及CXCR4中和抗体和CXCR4拮抗剂AMD3100的抑制作用;以Transwell侵袭小室为模型,应用Matrigel探讨不同浓度的CXCL12对三种细胞迁移、侵袭的影响以及CXCR4中和抗体和CXCR4拮抗剂AMD3100的抑制作用。2人腹膜间皮细胞的原代培养及其对卵巢癌细胞黏附、迁移、侵袭力影响的体外实验研究:术中取无盆腹腔炎症、正常患者的大网膜。用0.25%胰酶+0.02%乙二胺四乙酸(EDTA)消化大网膜组织,去除红细胞后培养;显微镜观察细胞形态变化;HE染色观察细胞形态并计算细胞纯度;扫描电子显微镜((SEM)观察细胞超微结构;免疫细胞化学法鉴定;粘附、迁移、侵袭体外实验观察腹膜间皮细胞对卵巢癌细胞生长转移能力的影响。3荷人卵巢癌细胞裸鼠腹腔移植瘤模型的建立及实验研究:转染质粒、转染载体和未转染的三种卵巢癌细胞进行体外培养。将裸鼠随机分为三组,每组各12只,分别将4×106个三种细胞注入每组裸鼠腹腔,自注入细胞后第二天起,将每组裸鼠又随机分为对照组和处理组,对照组腹腔内再注入生理盐水,处理组腹腔内再注入CXCR4拮抗剂AMD3100,同时每组均腹腔内注射CXCL12 0.2 ml,浓度为100ng/ml,每2天注射一次,连用2周共7次。裸鼠自然死亡后,观察腹腔成瘤数、体积、腹水量和存活期的变化。结果:1稳定表达CXCR4的卵巢癌细胞株的建立与鉴定及其增殖、迁移、侵袭能力的分析:目的基因CXCR4与载体pReceiver-M02的连接成功构建真核表达重组质粒pReceiver-M02-CXCR4后,进行DNA测序,结果证明顺序正确,其序列与GeneBank中人CXCR4基因(NM003467)的标准序列完全一致。确定G418的筛选浓度为600μg/mL。14 d后转染的卵巢癌细胞绝大部分死亡形成细胞岛,经有限稀释法获得了数个单细胞,将单细胞进行扩大培养。G418抗性筛选一个月后细胞仍继续生长,转染细胞的形态和生长速度未发生明显变化,伸展速度较未转染细胞慢。④将获得的稳定表达CXCR4卵巢癌细胞株通过RT-PCR、Western blot、免疫细胞化学三种方法进行鉴定,三种方法均表明CXCR4目的基因在卵巢癌细胞内表达,我们选取的11个单细胞扩大培养,通过上述三种方法鉴定,结果8个单细胞扩大培养后有CXCR4的阳性表达,转染效率平均为73%(8/11)。同时将转染真核表达重组质粒pReceiver-M02-CXCR4和转染载体pReceiver-M02的卵巢癌细胞分别命名为SKOV3/CXCR4和SKOV3/neg细胞。⑤MTT法观察100ng/ml CXCL12单独作用可促进SKOV3/CXCR4细胞的增殖,且能被10μg/ml CXCR4中和抗体和1μg/ml CXCR4拮抗剂AMD3100所抑制(F=256.89,P=0.000),100ng/ml CXCL12加入SKOV3/neg和SKOV3细胞中,增殖未发生明显变化,表明CXCL12-CXCR4生物轴相互作用方可促进卵巢癌细胞的增殖。⑥在无血清条件下,10ng/ml CXCL12促进SKOV3/CXCR4细胞的迁移;而100ng/ml CXCL12使迁移的细胞数明显增多,表明CXCL12对SKOV3/CXCR4细胞的迁移有明显的催化性,并随CXCL12浓度的增加迁移细胞数增多;10μg/ml CXCR4中和抗体和1μg/ml和CXCR4的拮抗剂AMD3100能够抑制100ng/ml CXCL12对卵巢癌细胞的趋化性迁移(F=702.6,P=0.003)。对于SKOV3/neg及SKOV3细胞,无论加入多大浓度的CXCL12都不能促进细胞的迁移(P>0.01),进一步表明CXCL12-CXCR4生物轴相互作用可促进卵巢癌细胞迁移,同时也表明CXCL12与CXCR4相互作用的相对特异性。⑦对于SKOV3/CXCR4细胞来说,当下室为无血清的培养液时,穿过Matrigel的细胞数很少(25.00±8.42),10ng/ml CXCL12作用后穿过Matrigel的细胞数目增多,100ng/ml CXCL12刺激后,穿过Matrigel的细胞数明显增多(56.00±9.23),两者相比有显著性差异(F=16.65,P=0.005),表明CXCL12对SKOV3/CXCR4细胞穿过Matrigel的侵袭性有趋化作用,并且随着CXCL12浓度的增加穿过Matrigel的细胞数增多;10μg/ml CXCR4中和抗体和1μg/ml CXCR4拮抗剂AMD3100,能够抑制CXCL12对卵巢癌细胞的趋化性侵袭;对于SKOV3/neg及SKOV3细胞,无论加入多大浓度的CXCL12穿过Matrigel的细胞数无明显变化,各组之间比较,无显著性差异(P>0.01)。2人腹膜间皮细胞的原代培养及其对卵巢癌细胞黏附、迁移和侵袭力影响的分析:分离培养的人腹膜间皮细胞为多角形,汇合时呈铺路石样排列,培养细胞的纯度达95%以上。SEM下见细胞表面大量的微绒毛。免疫细胞化学法显示:细胞角蛋白、波形蛋白抗原阳性,白细胞CD45、第Ⅷ因子相关抗原阴性,鉴定符合腹膜间皮细胞的特点。④氢化可的松(0.5% )、胰岛素(20μg/ml)对腹膜间皮细胞的生长有明显的促进作用。⑤免疫细胞化学法证实腹膜间皮细胞中表达CXCL12、间皮素,不表达CXCR4。⑥黏附、迁移、侵袭实验显示腹膜间皮细胞对卵巢癌细胞的转移能力有增强作用。3 CXCL12-CXCR4生物轴对荷人卵巢癌裸鼠腹腔移植瘤生长的影响:三组荷瘤裸鼠自然死亡后解剖腹腔显示无论是对照组还是处理组,均在腹腔形成移植瘤,成瘤率100%。各组裸鼠腹腔均有腹水形成,部分有血性腹水,腹水量相比没有显著性差异(F=48.09,P >0.05)。所有裸鼠盆腔、腹腔、网膜、肠系膜、腹壁多处有肿瘤结节,部分有肝脏、脾脏、子宫双附件转移,心脏、肾脏、肺脏未见转移。④三组裸鼠对照组的平均生存期相比有显著性差异(Z=-2.40,P<0.05),接种SKOV3/CXCR4细胞裸鼠处理组的平均生存期长于对照组,两者相比具有显著性差异(Z=-2.29,P=0.02)。⑤将腹腔内的移植瘤剪下,计数并称取重量。接种SKOV3/CXCR4细胞裸鼠的对照组腹腔移植瘤的平均个数为87.67±1.86 n,平均重量量是3.74±0.11 g,CXCR4拮抗剂AMD3100治疗组腹腔移植瘤的平均个数为80.17±4.58 n,平均重量是2.83±0.10 g,两者相比具有显著性差异(P<0.05);接种SKOV3/neg细胞裸鼠对照组腹腔内移植瘤的平均个数为79.17±2.32 n,平均重量为2.82±0.13 g,CXCR4拮抗剂AMD3100治疗组腹腔移植瘤的平均个数为79.83±3.25 n,平均重量为2.76±0.11 g,两者相比无显著性差异(P>0.05);接种SKOV3细胞裸鼠对照组腹腔内移植瘤的平均个数为78.16±1.17 n,平均重量为2.82±0.12 g,CXCR4拮抗剂AMD3100治疗组腹腔移植瘤的平均个数为77.67±3.33 n,平均重量为2.84±0.11 g,两者相比无显著性差异(P>0.05);三组裸鼠对照组腹腔移植瘤的个数和重量相比有显著性的差异(P<0.05)。结论:1稳定表达CXCR4的卵巢癌细胞株构建成功,并通过RT-PCR、Westernblot、免疫细胞化学三种方法鉴定,CXCL12-CXCR4生物轴在体外细胞水平可促进卵巢癌细胞增殖、迁移、侵袭。2胰酶+EDTA消化法是一种简单、有效的分离腹膜间皮细胞的方法。腹膜间皮细胞中表达CXCL12、间皮素,不表达CXCR4,腹膜间皮细胞对卵巢癌细胞黏附、迁移、侵袭有增强作用。3荷人卵巢癌细胞裸鼠腹腔移植瘤模型建立成功,并在体内动物实验水平进一步说明CXCL12-CXCR4生物轴可促进卵巢癌细胞增殖、侵袭能力。总结实验,我们可以推断,阻断CXCL12-CXCR4生物轴相互作用或某些细胞因子的表达可为肿瘤治疗提供新的途径。更多还原

【Abstract】 Objective: Epithelial ovarian cancer (EOC) is the most common female malignancy and is the leading cause of death from gynecological malignancies. The hyper-mortality from the ovarian cancer dues to the majority of patients are diagnosed with advanced stage when tumor is already spreaded to abdominal cavity and retroperitoneal lymph node. Despite of aggressive treatment, 5-year survival rate remains about 30%. Unlike other cancers, major route of metastasis of ovarian cancer is not by blood vessel. The growth and metastasis in peritoneal cavity can produce ascites and intestinal obstruction, so peritoneal metastasis of EOC is major cause of mortality. Understanding the mechanisms of ovarian cancer cell adhesion, migration and invasion is very important. Metastasis of ovarian cancer in peritoneum is related with chemotactic factor, growth factor and adhesion factor in ascites beside anatominal and mechanical agent. In 2001, Muller studied chemotactic factor and receptor in breast cancer and melanoma, which made people realize chemotactic metastasis is regulated by CXCL12-CXCR4 biological axis. This study will investigate that CXCL12-CXCR4 biological axis and human peritoneal mesothelial cells’influence is on peritoneal cavity metastasis of EOC. We firstly constructed the epithelial ovarian cancer cell strain which expresses CXCR4 protein stably and explored that CXCR4’s influence was to proliferation, migration, invasion of ovarian cancer cells from the study in vitro, then isolated human peritoneal mesothelial cells and verified the expression of some cell factors in human peritoneal mesothelial cells, and studied the effect of peritoneal mesothelial cells to ovarian cancer cell SKOV3 in invasion, adhesion and migration. At last, we established the animal model that were injected tumor cells through intraperitoneal pathway in nude mice, when they died naturally, we dissected abdominal cavity and observed neoplasm quantity and weight, the amount of ascites, the mean survival time, the effect of AMD3100. This is useful to better understand biologic behavior of ovarian cancer, and may provide basis in resisting metastasis in ovarian cancer treatment.Method: This project was divided into three parts:1 Establishment and identification of the EOC cell strain which expresses CXCR4 protein stably and its effect to cell growth and metastasis. We firstly established the eukaryotic expression recombinant plasmid pReceiver-M02- CXCR4, and identified by enzyme cutting and sequencing. The eukaryotic expression recombinant plasimid pReceiver-M02-CXCR4 and vector pReceiver-M02 were transfected into SKOV3 by lipofectamine-mediated gene transfection method, and gained positive cell clone by G418 screening and single cell isolation techniques. Cells that were transfected were examined by RT-PCR, Western Blot, and immmunocytochemistry methods. SKOV3 transfected with plasmid, SKOV3 transfected with vector, SKOV3 were cultured in vitro. MTT was used to analyze effect of different concentration of CXCL12 on three cell lines proliferation in serum-free growing conditions, and to determine inhibition of neutralizing CXCR4 antibody or antagonist AMD3100. Transwell and Matrigel were used to evaluate effect of various concentrationof CXCL12 on three ovarian cell lines’migration and invasion, as well as inhibition of neutralizing CXCR4 antibody or antagonist AMD3100.2 Primary culture of human peritoneal mesothelial cells and study of the impact of peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in invasion adhesion and migration. Human greater omenta was digested with 0.25%trypsin-0.02% ethylene diaminetetraacetic acid (EDTA), and cultured after removing red blood cells. Morphologic change was detected by inverted microscope during cellculture. Morphologic types were observed by HE staining and the purity was calculated. The ultrastructure was observed by scanning electron microscopy (SEM). Isolated cells were characterized by immunocytochemical analysis. To verify the expression of some cell factors in human peritoneal mesothelial cells by immunocytochemical analysis. Invasion, adhesion and migration of SKOV3 to human peritoneal mesothelial were detected by adhesion assay, Transwell Chamber invision assay and migration assay.3 Establishment and research of animal model that were injected tumor cells through intraperitoneal pathway in nude mice SKOV3 transfected with plasmid, SKOV3 transfected with vector and SKOV3 were cultured. Nude mice were divided into three groups at random, every group had twelve mice. 4×106 cells were injected into abdnominal cavity of nude mice respectively. After two days, every group was divided into control group and treatment group at random. Control group was injected PBS, and treatment group was injected AMD3100. When they died naturally, we dissected abdominal cavity and observed neoplasm, quantity and weight, the amount of ascites and the mean survival time.Result:1 Establishment and identification of the EOC cell strain which expresses CXCR4 protein stably and its effect to cell growth and metastasis:①After costruction and sequence of eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4, the result confirms that the order of connection of gene and vector is correct and the sequence of gene is coincidence with CXCR4 of GeneBank.②Screening concentration of G418 is 600μg/ml.③After fourteen days, most of ovarian cancer cells tansfected with plasmid and vector died and formed cell sites. We gained several single cells by limiting dilution assay. The shape and growth velocity of the cells that were transfected had no marked change, but their stretch speed became slow.④We identified the epithelial ovarian cancer cells which express CXCR4 protein stably by RT-PCR, Western blot, immunocytochemistry methods. All these indicate that CXCR4 is positive in ovarian cancer cells transfected with plasmid. Transfection efficiency is about 73%. At the same time, we named ovarian cancer cells that transfected with eukaryotic expression recombinant plasimid pReceiver-M02-CXCR4 and vector pReceiver-M02 as SKOV3/CXCR4 and SKOV3/neg cells.⑤Under serum-free suboptimal condition, 100ng/ml CXCL12 can promote the proliferation of SKOV3/CXCR4 cells (F=256.89, P<0.05), and this effect can be inhibited by 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. 100ng/ml CXCL12 can not promote the proliferation of SKOV3/neg and SKOV3 cells.⑥Under serum-free suboptimal condition, with regard to SKOV3/CXCR4 cells, 10ng/ml CXCL12 can promote cell migration, 100ng/ml CXCL12 can also promote cell migration (F=702.6, P<0.05). This enhancing effect of CXCL12 on cell migration is increased with increasing concentration of CXCL12, and is strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. With regard to SKOV3/neg and SKOV3 cell, CXCL12 can not promote cell migration, regardless of the concentration of CXCL12.⑦Matrigel is reconstituted artificial basel membrane, which have the shape and function of internal basel membrame. Under serum-free suboptimal condition, with regard to SKOV3/CXCR4 cells, they displayed minimal invasiveness through Matrigel when CXCL12 was no present in the lower chamber of transwell (25.00±8.42). 10ng/ml CXCL12 promoted cell invasion across Matrigel. The number of invading cells through Matrigel in the prensnce of 100ng/ml CXCL12 was significantly higher than that of 10ng/ml CXCL12 (56.00±9.23) (F=16.65, P<0.05). The enhancing effect of CXCL12 on cell invasion was increased with increasing concentration of CXCL12, and is strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or antagonist AMD3100. With regard to ovarian cancer cell SKOV3/neo and SKOV3 cell, CXCL12 can not promote cell invasion, regardless of the concentration of CXCL12. Every group had no significant difference (P>0.05) .2 Primary culture of human peritoneal mesothelial cells and study the impact peritoneal mesothelial cells to human ovarian carcinoma cell line SKOV3 in invasion adhesion and migration:①Cultured cells were multipolar and presented a cobblestone-like appearance when they reached confluence, with a purity of 95%.②SEM verified the abundant microvilli on the surface of the cells.③Immunocytochemical studies showed positive staining for cytokeratin and vimentin, but negative staining for white blood cell CD45 antigen and factorⅧassociated antigen. All the characters of the isolated cells were coincided with mesothelial cells.④Hydrocortisone (0.5%) and insulin (20μg/ml) had an exact effect to stimulate the growth of mesothelial cells.⑤Expression of CXCL12, MSLN in human peritoneal mesothelial cells were verified. The study also imformed that CXCR4 was negative in human peritoneal mesothelial cells.⑥Adhesion assay confirmed that human peritoneal mesothelial cells promoted the adhesion of SKOV3. Invision and migration assay confirmed that human peritoneal mesothelial cells promotes the invasion and migration of SKOV3.3 Effects of CXCL12-CXCR4 axis can the growth in skov3 ovarian cancer cells peritoneal Xengraft-bearing mice:①The rate of tumor formation is 100%.②Nude mice have ascites in every group. The quantity of ascites have no significant difference(F=48.09,P >0.05).③All nude mice have tumors in peritoneal cavity, gastrocolic omentum and mesentery. Liver, spleen, uterus and annexa had some metastasis. Heart, kidney, lung had no metastasis.④The mean survival time of the nude mice that were injected SKOV3/CXCR4 cells is 15.00±0.90 days in control group, 17.50±1.64 days in treatment group, two groups had significant dfference (Z=-2.40, P<0.05). The mean survival time of the nude mice that were injected SKOV3/neg cells is 16.83±1.33 days in control group, 17.00±0.89 days in treatment group, two groups had no significant difference (F=-2.29, P>0.05). The mean survival time of the nude mice that were injected SKOV3 cells is 17.20±1.90 days in control group, 17.17±1.94 days in treatment group, two groups had no significant difference (P>0.05). The mean survival time of the nude mice of the three groups in control group had significant difference.⑤The number of the metastatic tumors in nude mice that were injected SKOV3 was 78.16±1.17, which of significantly lower than that of nude mice that were injected SKOV3/CXCR4 cells 87.67±1.86 (P<0.05), and which have no obvious difference with nude mice that were injected SKOV3/neg cells 79.17±2.32 (P>0.05). The weight of the metastatic tumors in nude mice that were injected SKOV3 cells was 2.82±0.12, which was significantly lower than that of nude mice that were injected SKOV3/CXCR4 cells 3.74±0.11 (P<0.05), and which had no obvious difference with nude mice that were injected SKOV3/neg cells 2.82±0.13 (P>0.05).Conclusion:1 Establishment of the epithelial ovarian cancer cell which expresses CXCR4 protein stably is successful, and which is identified by RT-PCR, Western blot, immunocytochemistry. CXCL12-CXCR4 can promote proliferation, migration, invasion of ovarian cancer cells in vitro.2 Trypsin-EDTA enzymatic disaggregation method is a simple, effective and repetitive protocol for isolation of human peritoneal mesothelial cells. CXCL12, MSLN are positive and CXCR4 is negative in human peritoneal mesothelial cells. HPMC can promote proliferation, migration, invasion of ovarian cancer cell.3 Establishment of animal model that are injected tumor cells through intraperitoneal pathway in nude mice is successful. Ovarian cancer cells can promote cell proliferation, migration in vivo.These data indicate that interfering with interaction of CXCL12 and CXCR4 will improve treatment of patients with ovarian cancer.更多还原