本文關(guān)鍵詞： 上皮細(xì)胞 角質(zhì)細(xì)胞 原代培養(yǎng) 宮頸 宮頸上皮內(nèi)瘤變 角質(zhì)細(xì)胞 原代培養(yǎng) 角蛋白 宮頸上皮內(nèi)瘤變 生物學(xué)行為 增殖 腫瘤標(biāo)志物 MMP-2 侵襲　出處：《山東大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：宮頸癌是威脅女性生命的首位生殖系統(tǒng)惡性腫瘤,近年來,隨著宮頸癌篩查的有效開展,世界范圍內(nèi)宮頸癌的發(fā)病率和死亡率已經(jīng)大幅下降,而宮頸癌前病變即宮頸上皮內(nèi)瘤變(Cervical intraepithelial neoplasia, CIN)的檢出率卻越來越高。研究發(fā)現(xiàn),宮頸癌發(fā)生發(fā)展過程中存在較長的、可逆轉(zhuǎn)的癌前病變期,包括宮頸不典型增生和宮頸原位癌,它反映宮頸癌發(fā)生發(fā)展中的連續(xù)過程。宮頸上皮不典型增生表現(xiàn)為宮頸上皮層內(nèi)出現(xiàn)異型細(xì)胞,即細(xì)胞大小不一,形狀不規(guī)則,細(xì)胞核增大濃染,核漿比例增大,核分裂象增多,甚至有病理性核分裂,細(xì)胞極性紊亂等。根據(jù)細(xì)胞異常的程度及上皮累積范圍又可將CIN分為三級,即CIN1級(輕度不典型增生),Ⅱ級(中度不典型增生)和Ⅲ級(重度不典型增生和原位癌)。根據(jù)對CIN的自然病史的研究,絕大多數(shù)的低級別CIN(即CINI)會自然消退,只有0.3%-1%會發(fā)展為浸潤癌,而未經(jīng)治療的高級別CIN(即CIN II和CINIII)中20%-45%將發(fā)展為原位癌或浸潤癌,故高級別CIN為潛在的宮頸癌前病變。由于CIN發(fā)展形成宮頸浸潤癌的病程較長大約10年,因此,對CIN的患者及時早期發(fā)現(xiàn)、積極有效治療是降低宮頸癌發(fā)病率的關(guān)鍵。目前認(rèn)為人乳頭瘤病毒(human papillomavirus, HPV)感染是CIN和宮頸癌發(fā)生的始動因素,在99%以上的宮頸癌及CIN中存在HPV DNA. HPV可分為高危型和低危型兩大類,低危型多導(dǎo)致疣類病變和CIN1,而高危型則多導(dǎo)致CIN2-3及宮頸癌的發(fā)生。但是HPV不能進(jìn)行常規(guī)的體外培養(yǎng),而高危型HPV引起的疾病是一個漫長的過程。因此,對攜帶HPV的宮頸上皮內(nèi)瘤變細(xì)胞進(jìn)行原代培養(yǎng)并對其生物學(xué)特性進(jìn)行研究,有利于更好的了解HPV感染宮頸上皮細(xì)胞的重要的意義,為高級別CIN和宮頸癌的進(jìn)一步研究提供理論依據(jù)和實驗基礎(chǔ)。 目前,正常宮頸上皮細(xì)胞(Normal uterine cervix, NUC)及宮頸癌細(xì)胞的原代培養(yǎng)國內(nèi)外已屢見報道,但是對于高級別CIN細(xì)胞這一潛在癌前病變的培養(yǎng)及其的生物學(xué)特性的研究國內(nèi)外鮮有報道。眾所周知,癌細(xì)胞的生物學(xué)特性主要包括：①形態(tài)特征：細(xì)胞異型性如癌細(xì)胞大小形態(tài)不一,核質(zhì)比顯著高于正常細(xì)胞；核形態(tài)不一,并可出現(xiàn)巨核、雙核或多核現(xiàn)象；核分裂像常增多。②生理生化特征：細(xì)胞接觸抑制喪失；具有遷移性和侵襲性等。但是,對于高級別CIN這一癌前期病變細(xì)胞卻僅從形態(tài)學(xué)特征方面進(jìn)行了描述,對于其生理生化特征卻未見報道。單從宮頸癌細(xì)胞和高級別CIN細(xì)胞的形態(tài)特征來看,二者均具有異型性,故很難從形態(tài)學(xué)上對二者進(jìn)行區(qū)分。但是癌細(xì)胞增殖活性強(qiáng),具有非接觸性抑制、遷移性及侵襲性,與人類腫瘤侵襲有關(guān)的MMP-2亦明顯升高。高級別CIN細(xì)胞是否亦具有上述特性?高級別CIN細(xì)胞為何可以局限于上皮內(nèi)多年或消失而不發(fā)生轉(zhuǎn)移?基于此,本項目擬對高級別CIN細(xì)胞進(jìn)行原代培養(yǎng),探索一種快速、簡便、有效的培養(yǎng)方法,同時對其生物學(xué)特性進(jìn)行體外研究,為宮頸癌及其癌前病變的進(jìn)一步研究提供理論及實驗依據(jù)。 第一部分：一種新的正常人宮頸上皮細(xì)胞原代培養(yǎng)技術(shù)的建立 研究目的： 1.提供一種新型培養(yǎng)基,既能促進(jìn)細(xì)胞的貼壁,又能減少成纖維細(xì)胞的污染。 2.從培養(yǎng)器皿方面,探索一種能協(xié)同促進(jìn)宮頸上皮細(xì)胞貼壁的方法。 3.探索一種高純度的宮頸上皮細(xì)胞的體外培養(yǎng)的方法。 研究方法： 1.取人正常宮頸上皮組織,用Ⅰ型膠原酶消化分解后得到宮頸上皮細(xì)胞懸液。 2.將細(xì)胞分別接種到以下培養(yǎng)瓶中：①未經(jīng)鼠尾膠原包被,僅含無血清培養(yǎng)基(keratinocyte serum-free medium, K-SFM);②未經(jīng)鼠尾膠原包被,含5%胎牛血清的K-SFM;③用鼠尾膠原包被,僅含K-SFM;④用鼠尾膠原包被,含5%胎牛血清的K-SFM. 3.待細(xì)胞貼壁后,首次換液,將培養(yǎng)基全部換成K-SFM. 4.采用細(xì)胞免疫熒光檢測宮頸上皮基層角蛋白(Keratin, K)5、K14和K19的表達(dá)。 結(jié)果： 1.在含5%胎牛血清的培養(yǎng)基中,細(xì)胞的貼壁速度較無血清的培養(yǎng)基快(約30%和10%)。 2.用鼠尾膠原I型包被的培養(yǎng)瓶中,細(xì)胞的貼壁速度較未經(jīng)鼠尾膠原包被的培養(yǎng)瓶中快(約60%和30%)。 3.聯(lián)合使用含5%胎牛血清的培養(yǎng)基和經(jīng)鼠尾膠原Ⅰ型包被的培養(yǎng)瓶是最理想的培養(yǎng)宮頸上皮細(xì)胞的方法。 4. K5, K14和K19幾乎在所有細(xì)胞的細(xì)胞漿中均呈陽性表達(dá),但K5表達(dá)很弱。 結(jié)論： 1.低濃度胎牛血清可以提高上皮細(xì)胞的貼壁能力,而無血清培養(yǎng)基抑制成纖維細(xì)胞的生長。 2.聯(lián)合使用5%胎牛血清的培養(yǎng)基和經(jīng)鼠尾膠原Ⅰ型包被的培養(yǎng)瓶是一種新穎、簡便、有效的人宮頸上皮細(xì)胞的培養(yǎng)方法。 3.我們的方法可以從成人宮頸上皮組織中快速得到大量高純度的正常宮頸上皮細(xì)胞。 第二部分：人高級別宮頸上皮內(nèi)瘤變細(xì)胞的原代培養(yǎng)和鑒定研究目的： 建立一種人小塊宮頸瘤變組織來源的自然感染HPV的高級別宮頸上皮內(nèi)瘤變細(xì)胞的體外培養(yǎng)方法；為高級別上皮內(nèi)瘤變細(xì)胞生物學(xué)特性的研究提供實驗基礎(chǔ)。 研究方法： 1.取小塊人宮頸瘤變組織,取出一小部分行HPV DNA分型檢測。 2.將剩余瘤變組織用I型膠原酶消化分解后制成類細(xì)胞懸液(未經(jīng)細(xì)胞篩過濾)。 3.將類細(xì)胞懸液接種到含5%胎牛血清+K-SFM的、經(jīng)鼠尾膠原包被的培養(yǎng)瓶中。 4.一組細(xì)胞培養(yǎng)3天后首次換液并更換K-SFM,收集未貼壁細(xì)胞并重新種植到新的培養(yǎng)瓶中繼續(xù)培養(yǎng)3天。另一組細(xì)胞培養(yǎng)6天后首次換液并更換K-SFM. 5.將第三代細(xì)胞收集后行HPV DNA分型檢測。 6.通過細(xì)胞免疫熒光檢測宮頸上皮基底層K14和K19,宮頸干細(xì)胞K17和P63的表達(dá),對CIN細(xì)胞進(jìn)行鑒定。 結(jié)果： 1.細(xì)胞培養(yǎng)的第1、3和6天,分別有30%、60%和80%的高級別CIN細(xì)胞貼壁,未貼壁細(xì)胞在第3天重新收集并繼續(xù)培養(yǎng)3天后仍然有30%的細(xì)胞貼壁,但由于細(xì)胞密度較低很難長滿培養(yǎng)瓶并傳代。 2.傳代后高級別CIN細(xì)胞仍然保持其大小不等、形狀異常的形態(tài),沒有明顯分化現(xiàn)象。 3.傳代后高級別CIN細(xì)胞的HPV分型檢測結(jié)果同原代一致。 4.K14和K19幾乎在所有NUC細(xì)胞和高級別CIN細(xì)胞的胞漿中表達(dá)；但是,K17和P63僅在高級別CIN細(xì)胞的胞漿中表達(dá)。 結(jié)論： 1.我們將“組織塊培養(yǎng)法”與“酶消化法”相結(jié)合制備了類細(xì)胞懸液,不僅克服了“組織塊培養(yǎng)法”中細(xì)胞因移動受到限制而造成的生長緩慢,而且節(jié)省細(xì)胞過濾過程中丟失的細(xì)胞,獲得了更多的CIN細(xì)胞,克服了CIN培養(yǎng)過程中因標(biāo)本過小而造成的困難。 2.高級別CIN細(xì)胞培養(yǎng)的第6天可作為最佳首次換液時間。 3.我們建立了一種從小塊宮頸瘤變組織獲取自然攜帶HPV的高級別宮頸上皮內(nèi)瘤變細(xì)胞的簡單而實用的培養(yǎng)方法,為CIN病變的體外研究提供了實驗基礎(chǔ)。 第三部分：人高級別宮頸上皮內(nèi)瘤變細(xì)胞生物學(xué)特性的體外研究研究目的： 通過與NUC細(xì)胞及宮頸癌Caski細(xì)胞的對比,觀察人高級別CIN細(xì)胞的生物學(xué)行為如生長增殖能力、腫瘤標(biāo)志物的表達(dá)及遷徙和侵襲能力等,探討高級別CIN細(xì)胞的生物學(xué)特性,為宮頸癌前病變及宮頸癌的進(jìn)一步研究及治療提供理論及實驗依據(jù)。研究方法： 1.應(yīng)用MTT實驗測定高級別CIN細(xì)胞及對照組NUC細(xì)胞和宮頸癌Caski細(xì)胞的生長曲線。 2.應(yīng)用細(xì)胞接觸抑制試驗檢測高級別CIN細(xì)胞的生長活性。當(dāng)細(xì)胞生長到約80%-90%融合時,繼續(xù)培養(yǎng)3-5天,觀察細(xì)胞有無接觸性抑制現(xiàn)象。 3.應(yīng)用流式細(xì)胞技術(shù),檢測高級別CIN細(xì)胞及對照組NUC細(xì)胞和Caski細(xì)胞的生長周期。 4.應(yīng)用DNA倍體分析技術(shù),檢測高級別CIN細(xì)胞及對照組NUC細(xì)胞和Caski細(xì)胞的異倍體比例。 5.采用細(xì)胞免疫熒光技術(shù),檢測高級別CIN細(xì)胞及其對照組NUC細(xì)胞和Caski細(xì)胞中腫瘤標(biāo)志物Ki-67和p16的表達(dá)情況。 6.在酶聯(lián)免疫分析實驗中,應(yīng)用競爭性抑制法測定高級別CIN細(xì)胞及其對照組NUC細(xì)胞和Caski細(xì)胞分泌性及細(xì)胞內(nèi)基質(zhì)金屬蛋白酶2(matrix metalloproteinase2,MMP-2)水平；應(yīng)用雙抗體夾心法測定基質(zhì)金屬蛋白酶抑制因子2(Tissue inhibitors of metalloproteinase2, TIMP-2)的水平。 7.應(yīng)用Transwell小室技術(shù),檢測高級別CIN細(xì)胞及其對照組NUC細(xì)胞和Caski細(xì)胞的遷移和侵襲能力。 8. SPSS13.0統(tǒng)計軟件對數(shù)據(jù)進(jìn)行分析：生長曲線用重復(fù)測量資料的方差分析；計量資料統(tǒng)計結(jié)果以均數(shù)士標(biāo)準(zhǔn)差表示,三組之間的差異比較采用方差分析。 結(jié)果： 1.高級別CIN細(xì)胞、NUC細(xì)胞和宮頸癌Caski細(xì)胞的生長速度明顯不同(P0.01),Caski細(xì)胞的生長速度明顯快于高級別CIN細(xì)胞和NUC細(xì)胞(P0.01),高級別CIN細(xì)胞的生長速度明顯快于NUC細(xì)胞(P0.01)。高級別CIN細(xì)胞生長曲線的延緩期明顯縮短,Caski細(xì)胞的生長曲線沒有明顯的延緩期。 2.高級別CIN細(xì)胞貼壁生長匯合成單層后即停止生長,存在接觸抑制現(xiàn)象；Caski細(xì)胞貼壁生長匯合成單層后繼續(xù)生長,接觸抑制現(xiàn)象消失。 3.細(xì)胞周期檢測實驗顯示S期細(xì)胞比率(SPF):Caski細(xì)胞組高級別CIN細(xì)胞組NUC細(xì)胞組(P0.01)。 4.DNA倍體分析實驗中,高級別CIN細(xì)胞組和Caski細(xì)胞組異倍體細(xì)胞所占比例均明顯高于NUC細(xì)胞組(P0.01),但高級別CIN細(xì)胞組和Caski細(xì)胞組之間異倍體細(xì)胞所占比例無顯著性差異(P=0.392)。 5.細(xì)胞免疫熒光顯示,在幾乎所有NUC細(xì)胞、高級別CIN細(xì)胞和Caski細(xì)胞的細(xì)胞核中,均可見Ki-67呈彌漫的強(qiáng)陽性染色。p16在NUC細(xì)胞、高級別CIN細(xì)胞和Caski細(xì)胞的細(xì)胞核和細(xì)胞漿中均呈陽性表達(dá)。 6. Caski細(xì)胞組分泌性MMP-2的含量明顯高于NUC細(xì)胞組和高級別CIN細(xì)胞組(P0.05),NUC細(xì)胞組與高級別CIN細(xì)胞組之間無顯著性差異(P0.05),三組細(xì)胞之間細(xì)胞內(nèi)MMP-2的含量無顯著性差異(P0.05)；三組細(xì)胞之間分泌性和細(xì)胞內(nèi)TIMP-2的含量及MMP-2/TIMP-2的比值無顯著性差異(P0.05)。 7. Transwell體外遷移和侵襲實驗均表明：高級別CIN細(xì)胞的遷移能力明顯優(yōu)于NUC細(xì)胞,但顯著低于Caski細(xì)胞(P0.01)；高級別CIN細(xì)胞有侵襲能力,但明顯低于Caski細(xì)胞(P0.001),NUC細(xì)胞無明顯侵襲能力。 結(jié)論： 1.高級別CIN細(xì)胞的生長活力較NUC細(xì)胞明顯增強(qiáng),但仍然具有接觸抑制現(xiàn)象。 2.高級別CIN細(xì)胞與宮頸癌Caski細(xì)胞在增殖的過程中均出現(xiàn)了大量的異倍體細(xì)胞,說明高級別CIN細(xì)胞的增殖能力較強(qiáng)；細(xì)胞周期中S期細(xì)胞比率的升高,進(jìn)一步說明了高級別CIN細(xì)胞具有較強(qiáng)的生長活力。 3.腫瘤標(biāo)志物Ki-67和p16在NUC細(xì)胞、高級別CIN細(xì)胞和Caski細(xì)胞均呈陽性表達(dá),說明它們可能僅僅是細(xì)胞增殖的標(biāo)記物。 4.高級別CIN細(xì)胞及其對照組細(xì)胞內(nèi)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細(xì)胞；同時,高級別CIN細(xì)胞具有較弱的侵襲能力,進(jìn)一步說明了分泌性MMP-2在侵襲中的作用；而高級別CIN病變基底膜的完整性,說明在原位癌到侵襲癌的演進(jìn)過程中,除了細(xì)胞本身的惡性潛能外,實質(zhì)與間質(zhì)之間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錛，

本文編號：1545297