人高级别宫颈上皮内瘤变细胞的原代培养及其生物学特性的体外研究

发布时间：2018-02-28 02:08

  本文关键词： 上皮细胞 角质细胞 原代培养 宫颈 宫颈上皮内瘤变 角质细胞 原代培养 角蛋白 宫颈上皮内瘤变 生物学行为 增殖 肿瘤标志物 MMP-2 侵袭　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：宫颈癌是威胁女性生命的首位生殖系统恶性肿瘤,近年来,随着宫颈癌筛查的有效开展,世界范围内宫颈癌的发病率和死亡率已经大幅下降,而宫颈癌前病变即宫颈上皮内瘤变(Cervical intraepithelial neoplasia, CIN)的检出率却越来越高。研究发现,宫颈癌发生发展过程中存在较长的、可逆转的癌前病变期,包括宫颈不典型增生和宫颈原位癌,它反映宫颈癌发生发展中的连续过程。宫颈上皮不典型增生表现为宫颈上皮层内出现异型细胞,即细胞大小不一,形状不规则,细胞核增大浓染,核浆比例增大,核分裂象增多,甚至有病理性核分裂,细胞极性紊乱等。根据细胞异常的程度及上皮累积范围又可将CIN分为三级,即CIN1级(轻度不典型增生),Ⅱ级(中度不典型增生)和Ⅲ级(重度不典型增生和原位癌)。根据对CIN的自然病史的研究,绝大多数的低级别CIN(即CINI)会自然消退,只有0.3%-1%会发展为浸润癌,而未经治疗的高级别CIN(即CIN II和CINIII)中20%-45%将发展为原位癌或浸润癌,故高级别CIN为潜在的宫颈癌前病变。由于CIN发展形成宫颈浸润癌的病程较长大约10年,因此,对CIN的患者及时早期发现、积极有效治疗是降低宫颈癌发病率的关键。目前认为人乳头瘤病毒(human papillomavirus, HPV)感染是CIN和宫颈癌发生的始动因素,在99%以上的宫颈癌及CIN中存在HPV DNA. HPV可分为高危型和低危型两大类,低危型多导致疣类病变和CIN1,而高危型则多导致CIN2-3及宫颈癌的发生。但是HPV不能进行常规的体外培养,而高危型HPV引起的疾病是一个漫长的过程。因此,对携带HPV的宫颈上皮内瘤变细胞进行原代培养并对其生物学特性进行研究,有利于更好的了解HPV感染宫颈上皮细胞的重要的意义,为高级别CIN和宫颈癌的进一步研究提供理论依据和实验基础。 目前,正常宫颈上皮细胞(Normal uterine cervix, NUC)及宫颈癌细胞的原代培养国内外已屡见报道,但是对于高级别CIN细胞这一潜在癌前病变的培养及其的生物学特性的研究国内外鲜有报道。众所周知,癌细胞的生物学特性主要包括：①形态特征：细胞异型性如癌细胞大小形态不一,核质比显著高于正常细胞；核形态不一,并可出现巨核、双核或多核现象；核分裂像常增多。②生理生化特征：细胞接触抑制丧失；具有迁移性和侵袭性等。但是,对于高级别CIN这一癌前期病变细胞却仅从形态学特征方面进行了描述,对于其生理生化特征却未见报道。单从宫颈癌细胞和高级别CIN细胞的形态特征来看,二者均具有异型性,故很难从形态学上对二者进行区分。但是癌细胞增殖活性强,具有非接触性抑制、迁移性及侵袭性,与人类肿瘤侵袭有关的MMP-2亦明显升高。高级别CIN细胞是否亦具有上述特性?高级别CIN细胞为何可以局限于上皮内多年或消失而不发生转移?基于此,本项目拟对高级别CIN细胞进行原代培养,探索一种快速、简便、有效的培养方法,同时对其生物学特性进行体外研究,为宫颈癌及其癌前病变的进一步研究提供理论及实验依据。 第一部分：一种新的正常人宫颈上皮细胞原代培养技术的建立 研究目的： 1.提供一种新型培养基,既能促进细胞的贴壁,又能减少成纤维细胞的污染。 2.从培养器皿方面,探索一种能协同促进宫颈上皮细胞贴壁的方法。 3.探索一种高纯度的宫颈上皮细胞的体外培养的方法。 研究方法： 1.取人正常宫颈上皮组织,用Ⅰ型胶原酶消化分解后得到宫颈上皮细胞悬液。 2.将细胞分别接种到以下培养瓶中：①未经鼠尾胶原包被,仅含无血清培养基(keratinocyte serum-free medium, K-SFM);②未经鼠尾胶原包被,含5%胎牛血清的K-SFM;③用鼠尾胶原包被,仅含K-SFM;④用鼠尾胶原包被,含5%胎牛血清的K-SFM. 3.待细胞贴壁后,首次换液,将培养基全部换成K-SFM. 4.采用细胞免疫荧光检测宫颈上皮基层角蛋白(Keratin, K)5、K14和K19的表达。 结果： 1.在含5%胎牛血清的培养基中,细胞的贴壁速度较无血清的培养基快(约30%和10%)。 2.用鼠尾胶原I型包被的培养瓶中,细胞的贴壁速度较未经鼠尾胶原包被的培养瓶中快(约60%和30%)。 3.联合使用含5%胎牛血清的培养基和经鼠尾胶原Ⅰ型包被的培养瓶是最理想的培养宫颈上皮细胞的方法。 4. K5, K14和K19几乎在所有细胞的细胞浆中均呈阳性表达,但K5表达很弱。 结论： 1.低浓度胎牛血清可以提高上皮细胞的贴壁能力,而无血清培养基抑制成纤维细胞的生长。 2.联合使用5%胎牛血清的培养基和经鼠尾胶原Ⅰ型包被的培养瓶是一种新颖、简便、有效的人宫颈上皮细胞的培养方法。 3.我们的方法可以从成人宫颈上皮组织中快速得到大量高纯度的正常宫颈上皮细胞。 第二部分：人高级别宫颈上皮内瘤变细胞的原代培养和鉴定研究目的： 建立一种人小块宫颈瘤变组织来源的自然感染HPV的高级别宫颈上皮内瘤变细胞的体外培养方法；为高级别上皮内瘤变细胞生物学特性的研究提供实验基础。 研究方法： 1.取小块人宫颈瘤变组织,取出一小部分行HPV DNA分型检测。 2.将剩余瘤变组织用I型胶原酶消化分解后制成类细胞悬液(未经细胞筛过滤)。 3.将类细胞悬液接种到含5%胎牛血清+K-SFM的、经鼠尾胶原包被的培养瓶中。 4.一组细胞培养3天后首次换液并更换K-SFM,收集未贴壁细胞并重新种植到新的培养瓶中继续培养3天。另一组细胞培养6天后首次换液并更换K-SFM. 5.将第三代细胞收集后行HPV DNA分型检测。 6.通过细胞免疫荧光检测宫颈上皮基底层K14和K19,宫颈干细胞K17和P63的表达,对CIN细胞进行鉴定。 结果： 1.细胞培养的第1、3和6天,分别有30%、60%和80%的高级别CIN细胞贴壁,未贴壁细胞在第3天重新收集并继续培养3天后仍然有30%的细胞贴壁,但由于细胞密度较低很难长满培养瓶并传代。 2.传代后高级别CIN细胞仍然保持其大小不等、形状异常的形态,没有明显分化现象。 3.传代后高级别CIN细胞的HPV分型检测结果同原代一致。 4.K14和K19几乎在所有NUC细胞和高级别CIN细胞的胞浆中表达；但是,K17和P63仅在高级别CIN细胞的胞浆中表达。 结论： 1.我们将“组织块培养法”与“酶消化法”相结合制备了类细胞悬液,不仅克服了“组织块培养法”中细胞因移动受到限制而造成的生长缓慢,而且节省细胞过滤过程中丢失的细胞,获得了更多的CIN细胞,克服了CIN培养过程中因标本过小而造成的困难。 2.高级别CIN细胞培养的第6天可作为最佳首次换液时间。 3.我们建立了一种从小块宫颈瘤变组织获取自然携带HPV的高级别宫颈上皮内瘤变细胞的简单而实用的培养方法,为CIN病变的体外研究提供了实验基础。 第三部分：人高级别宫颈上皮内瘤变细胞生物学特性的体外研究研究目的： 通过与NUC细胞及宫颈癌Caski细胞的对比,观察人高级别CIN细胞的生物学行为如生长增殖能力、肿瘤标志物的表达及迁徙和侵袭能力等,探讨高级别CIN细胞的生物学特性,为宫颈癌前病变及宫颈癌的进一步研究及治疗提供理论及实验依据。研究方法： 1.应用MTT实验测定高级别CIN细胞及对照组NUC细胞和宫颈癌Caski细胞的生长曲线。 2.应用细胞接触抑制试验检测高级别CIN细胞的生长活性。当细胞生长到约80%-90%融合时,继续培养3-5天,观察细胞有无接触性抑制现象。 3.应用流式细胞技术,检测高级别CIN细胞及对照组NUC细胞和Caski细胞的生长周期。 4.应用DNA倍体分析技术,检测高级别CIN细胞及对照组NUC细胞和Caski细胞的异倍体比例。 5.采用细胞免疫荧光技术,检测高级别CIN细胞及其对照组NUC细胞和Caski细胞中肿瘤标志物Ki-67和p16的表达情况。 6.在酶联免疫分析实验中,应用竞争性抑制法测定高级别CIN细胞及其对照组NUC细胞和Caski细胞分泌性及细胞内基质金属蛋白酶2(matrix metalloproteinase2,MMP-2)水平；应用双抗体夹心法测定基质金属蛋白酶抑制因子2(Tissue inhibitors of metalloproteinase2, TIMP-2)的水平。 7.应用Transwell小室技术,检测高级别CIN细胞及其对照组NUC细胞和Caski细胞的迁移和侵袭能力。 8. SPSS13.0统计软件对数据进行分析：生长曲线用重复测量资料的方差分析；计量资料统计结果以均数士标准差表示,三组之间的差异比较采用方差分析。 结果： 1.高级别CIN细胞、NUC细胞和宫颈癌Caski细胞的生长速度明显不同(P0.01),Caski细胞的生长速度明显快于高级别CIN细胞和NUC细胞(P0.01),高级别CIN细胞的生长速度明显快于NUC细胞(P0.01)。高级别CIN细胞生长曲线的延缓期明显缩短,Caski细胞的生长曲线没有明显的延缓期。 2.高级别CIN细胞贴壁生长汇合成单层后即停止生长,存在接触抑制现象；Caski细胞贴壁生长汇合成单层后继续生长,接触抑制现象消失。 3.细胞周期检测实验显示S期细胞比率(SPF):Caski细胞组高级别CIN细胞组NUC细胞组(P0.01)。 4.DNA倍体分析实验中,高级别CIN细胞组和Caski细胞组异倍体细胞所占比例均明显高于NUC细胞组(P0.01),但高级别CIN细胞组和Caski细胞组之间异倍体细胞所占比例无显著性差异(P=0.392)。 5.细胞免疫荧光显示,在几乎所有NUC细胞、高级别CIN细胞和Caski细胞的细胞核中,均可见Ki-67呈弥漫的强阳性染色。p16在NUC细胞、高级别CIN细胞和Caski细胞的细胞核和细胞浆中均呈阳性表达。 6. Caski细胞组分泌性MMP-2的含量明显高于NUC细胞组和高级别CIN细胞组(P0.05),NUC细胞组与高级别CIN细胞组之间无显著性差异(P0.05),三组细胞之间细胞内MMP-2的含量无显著性差异(P0.05)；三组细胞之间分泌性和细胞内TIMP-2的含量及MMP-2/TIMP-2的比值无显著性差异(P0.05)。 7. Transwell体外迁移和侵袭实验均表明：高级别CIN细胞的迁移能力明显优于NUC细胞,但显著低于Caski细胞(P0.01)；高级别CIN细胞有侵袭能力,但明显低于Caski细胞(P0.001),NUC细胞无明显侵袭能力。 结论： 1.高级别CIN细胞的生长活力较NUC细胞明显增强,但仍然具有接触抑制现象。 2.高级别CIN细胞与宫颈癌Caski细胞在增殖的过程中均出现了大量的异倍体细胞,说明高级别CIN细胞的增殖能力较强；细胞周期中S期细胞比率的升高,进一步说明了高级别CIN细胞具有较强的生长活力。 3.肿瘤标志物Ki-67和p16在NUC细胞、高级别CIN细胞和Caski细胞均呈阳性表达,说明它们可能仅仅是细胞增殖的标记物。 4.高级别CIN细胞及其对照组细胞内MMP-2和TIMP-2的含量及其比值相似,但分泌性MMP-2的含量低于Caski细胞；同时,高级别CIN细胞具有较弱的侵袭能力,进一步说明了分泌性MMP-2在侵袭中的作用；而高级别CIN病变基底膜的完整性,说明在原位癌到侵袭癌的演进过程中,除了细胞本身的恶性潜能外,实质与间质之间的相互作用起了重要的作用。
[Abstract]:Cervical cancer is a threat to the lives of women's reproductive system malignant tumor, in recent years, with the effective screening of cervical cancer worldwide, the morbidity and mortality of cervical cancer has dropped significantly, and cervical precancerous lesions cervical intraepithelial neoplasia (Cervical intraepithelial, neoplasia, CIN) the detection rate is increasing. The study found that the presence of a long process of development of cervical carcinoma, can reverse precancerous lesions, including cervical dysplasia and cervical carcinoma in situ, it reflects the continuous process of the occurrence and development of cervical cancer. Cervical dysplasia appears as atypical cells of cervical epithelial layer, cell size and shape the irregular, hyperchromatic nuclei increased, karyoplasmic ratio increased, mitotic activity, and pathological mitosis, cell polarity disorder. And according to the degree of abnormal epithelial cells and accumulation CIN were divided into three grades, namely grade CIN1 (mild dysplasia), grade II (moderate dysplasia) and class III (severe dysplasia and carcinoma in situ). Based on the natural history of CIN, most of the low level of CIN (CINI) will naturally fade, only 0.3%-1% the development of invasive cancer, and without a high level of CIN treatment (CIN, II and CINIII) in 20%-45% progression to carcinoma in situ or invasive carcinoma, so the high level CIN for cervical precancerous lesions. The potential of CIN development and the formation of invasive cervical cancer long duration of about 10 years, therefore, for CIN patients in a timely manner early discovery and effective treatment is the key to reduce the incidence of cervical cancer. The human papilloma virus (human, papillomavirus, HPV) infection is the initiating factor of CIN and cervical cancer. HPV DNA. HPV can be divided into high-risk and low-risk type two categories of cervical cancer and CIN in more than 99%. Low Risk causes warts lesions and CIN1, and the high risk type in CIN2-3 and cervical cancer. But HPV can not be cultured in vitro, and high-risk disease caused by HPV is a long process. Therefore, to carry HPV cervical intraepithelial neoplasia cells were primary cultured and raise study on the biological characteristics, is conducive to better understand the HPV infection of cervical epithelial cells of great significance, provide theoretical and experimental basis for further research on the high level of CIN and cervical cancer.
At present, the normal cervical epithelial cells (Normal uterine cervix, NUC) and primary cervical cancer cells at home and abroad have been repeatedly reported, but the study is rarely reported at home and abroad for the biological characteristics of cultivation of this high level of potential cancer CIN cells and precancerous lesions. As is known to all, the main biological characteristics of cancer cells including: the morphological characteristics: pleomorphic cells such as cancer cell size and shape, karyoplasmic ratio was significantly higher than that in normal cells; nuclear morphology, and the emergence of giant nuclei, with two or more nuclei; caryocinesia often increased. The physiological and biochemical characteristics: cell loss of contact inhibition with migration and invasion;. However, for the high level of CIN in the precancerous cells but only from the morphological characteristics were described for its physiological and biochemical characteristics have not been reported. The single from cervical cancer cells and high levels of CIN cells. Sign, two have atypical, so it is very difficult from the morphology of the two distinction. But the proliferation of cancer cells, with non contact inhibition, migration and invasiveness, and human tumor invasion in MMP-2 increased significantly. Whether the high level of CIN cells also has the characteristics of high level? Why CIN cells can be limited to years or disappear without intraepithelial metastasis? Based on this, the project intends to the high level of CIN cells were cultured to explore a rapid, simple and effective training methods, at the same time on the biological characteristics of research, which provides theoretical and experimental basis for further study of cervical cancer and precancerous lesions.
Part one: a new technique for primary culture of normal human cervical epithelial cells
The purpose of the study is:
1. provides a new type of medium, which can not only promote the adhesion of cells, but also reduce the pollution of fibroblasts.
2. to explore a method to promote the adherence of cervical epithelial cells in terms of culture utensils.
3. to explore an in vitro culture of a highly purified cervix epithelial cell.
Research methods:
1. the normal cervical epithelial tissue was obtained, and the cervical epithelial cell suspension was obtained after digestion and decomposition of type I collagenase.
2. cells were inoculated into the culture flask: without coated by rat tail collagen, only containing serum-free medium (keratinocyte serum-free, medium, K-SFM); II without coated by rat tail collagen, K-SFM containing 5% fetal bovine serum; the rat tail collagen coating, containing only K-SFM 4 use; rat tail collagen coated, K-SFM. containing 5% fetal bovine serum
3. after the cell was adhered to the wall, the liquid was changed for the first time, and all the medium was changed to K-SFM.
4. the expression of Keratin (K) 5, K14 and K19 were detected by cell immunofluorescence.
Result锛，

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