本文選題：鼻咽腫瘤 + 動物模型��； 參考：《華中科技大學(xué)》2007年碩士論文

【摘要】： 目的:以人鼻咽癌CNE1細胞系為例建立的balb/c裸鼠肌肉內(nèi)移植瘤模型,觀察高分化細胞系CNE1在體內(nèi)的生物學(xué)行為及生長侵襲能力,為下一步研究鼻咽高分化癌亞臨床灶在活體內(nèi)的顯像建立合適的動物模型。此動物模型的移植瘤需無包膜,腫瘤細胞較易向周圍組織侵襲,以便于我們對移植瘤周圍正常組織中侵襲的單個腫瘤細胞的生物學(xué)特性的研究。 材料:人鼻咽癌CNE1細胞株(LMP1表達陽性);胎牛血清;RPMI-1640培養(yǎng)基、胰蛋白酶;LMP1( CS1-4)單克隆抗體;pEGFP-C1質(zhì)粒;梭華-SofastTM轉(zhuǎn)染試劑;質(zhì)粒抽提試劑盒(V-gene);SABC二抗試劑盒;4-6周齡balb/c裸鼠;TJ-TY-500型全自動圖像分析系統(tǒng);超凈細胞培養(yǎng)實驗室及SPF層流動物房。 方法:體外培養(yǎng)LMP1陽性表達的CNE1細胞系,細胞爬片后固定,免疫熒光證實LMP1的表達清情況。pEGFP-C1質(zhì)粒體外轉(zhuǎn)染CNE1細胞系,篩選穩(wěn)定表達綠色熒光蛋白的細胞克隆并擴大培養(yǎng),做流式細胞儀和熒光顯微鏡檢測。動物分為三組,分別將帶有pEGFP基因的細胞株、不帶有EGFP基因的細胞株擴增后制成濃度為1.0×107細胞懸液,將不帶EGFP基因的細胞株于半小時內(nèi)分別種植于兩組balb/c裸鼠臀大肌或背部皮下;將帶EGFP基因的細胞株于半小時內(nèi)種植于第三組balb/c裸鼠臀大肌內(nèi)。定期觀察并測量移植瘤大小,待有裸鼠瀕臨死亡時處死全部裸鼠取標本,4%多聚甲醛固定后石蠟包埋,做連續(xù)石蠟切片,第三組裸鼠相鄰切片分別做冰凍切片、蘇木素-伊紅(HE)和免疫組化染色。圖像分析以及熒光顯微鏡觀察并比較腫瘤細胞浸潤及轉(zhuǎn)移情況。 結(jié)果:皮下移植瘤潛伏期長,生長較慢,腫瘤體積小,腫瘤周圍可見明顯包膜包繞;肌肉移植瘤腫瘤細胞向周圍組織侵襲明顯,腫瘤組織和周圍正常組織界限不清,周圍淋巴結(jié)和遠處轉(zhuǎn)移率較皮下移植瘤高。組織切片可見肌肉組織內(nèi)癌巢形成,并且有癌細胞侵入肌細胞現(xiàn)象;不帶有EGFP基因的兩組細胞株進行比較,皮下移植瘤平均潛伏期為(15.40±2.46),腫瘤倍增時間為(3.01±0.32),平均瘤重(1.13±0.15),淋巴結(jié)和肝臟轉(zhuǎn)移率低(41.7%)。而肌肉組移植瘤體內(nèi)平均潛伏期(11.92±2.02d)和瘤體平均倍增時間(2.32±0.24d)明顯縮短(P0.01),平均瘤重明顯增加(1.32±0.17g,P0.05),淋巴結(jié)和肝臟轉(zhuǎn)移率明顯增高(41.7%/100%,P 0.05),而成瘤率無顯著差異(83.3%/100%,P0.05)。 EGFP在裸鼠體內(nèi)穩(wěn)定表達,且不影響細胞在體內(nèi)的增值與轉(zhuǎn)移,移植瘤重量為(1.33±0.11g),潛伏期為(10.98±2.21),倍增時間為(2.29±0.62),與未傳染EGFP組無顯著統(tǒng)計學(xué)差異。冰凍切片在熒光顯微鏡下直接觀察,較容易直接區(qū)分腫瘤區(qū)與非腫瘤區(qū),并能發(fā)現(xiàn)向周圍侵襲的腫單個腫瘤細胞,其敏感性及特異性明顯優(yōu)于HE染色和免疫組化方法。 結(jié)論:鼻咽癌高分化細胞系CNE1的裸鼠肌肉移植瘤模型在體內(nèi)的生長侵襲能力強,腫瘤周圍無包膜形成,為下一步研究亞臨床灶在活體內(nèi)的顯像建立了較為理想的動物模型。 EGFP體外轉(zhuǎn)染CNE1細胞并建立裸鼠移植瘤模型,對研究鼻咽癌的體內(nèi)侵襲行為具有一定的優(yōu)勢。
[Abstract]:Objective : To investigate the biological behavior and invasion ability of the high - differentiated cell line CNE1 in vivo and to establish a suitable animal model for the development of high - differentiated carcinoma sub - clinical range in vivo . The transplanted tumor of this animal model needs no envelope , and the tumor cells are more vulnerable to the surrounding tissues in order to study the biological characteristics of the single tumor cells that are invasive in the normal tissues around the transplanted tumor .


Materials : Human nasopharyngeal carcinoma CNE1 cell line ( LMP1 expression - positive ) ; fetal bovine serum ; RPMI - 1640 medium , trypsin ; LMP1 ( CS1 - 4 ) monoclonal antibody ; shuttle - SofastTM transfection reagent ; plasmid extraction kit ( V - gene ) ; SABC two - antibody kit ; 4 - 6 week - old balb / c nude mouse ; TJ - TY - 500 type full - automatic image analysis system ; Ultra clean cell culture laboratory and SPF laminar flow animal room .


Methods : CNE1 cell line was cultured in vitro . The expression of LMP1 was confirmed by immunofluorescence . The expression of LMP1 was confirmed by immunofluorescence . The cell line with EGFP gene was isolated and cultured in half an hour . The cell line with EGFP gene was implanted in the gluteus maximus of the third group of balb / c nude mice .


Results : The latent period of subcutaneous transplantation tumor was long , the growth was slow , the tumor volume was small , the tumor around the tumor was significantly shortened ( P0.01 ) . The average latent period of tumor was ( 15.40 鹵 2.46 ) , the tumor doubling time was ( 3.01 鹵 0.32 ) , the lymph node and liver metastasis rate were significantly increased ( 41.27 % / 100 % , P0.05 ) , but the rate of tumor formation was not significantly different ( 83.3 % / 100 % , P0.05 ) .


EGFP was stably expressed in nude mice without affecting the proliferation and metastasis of cells in vivo . The weight of transplanted tumor was ( 1.33 鹵 0.11 g ) . The incubation period was ( 10.98 鹵 2.21 ) . The doubling time was ( 2.29 鹵 0.62 ) .


Conclusion : The nude mouse muscle transplantation tumor model of high differentiation cell line CNE1 in nasopharyngeal carcinoma has strong invasion ability in vivo and no envelope formation around the tumor , which provides an ideal animal model for the next study of the imaging of subclinical foci in vivo .


EGFP in vitro transfection of CNE1 cells and the establishment of nude mice transplantation tumor model has some advantages to the study of invasive behavior in patients with nasopharyngeal carcinoma .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R-332;R739.63

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