人原發(fā)性胃惡性淋巴瘤裸小鼠原位移植模型的建立

1、    作者：脫帥 劉秋珍 張寧 朱希偉 王明耀    【摘要】  目的 為探討胃惡性淋巴瘤的發(fā)病機(jī)制和治療方法提供理想的動(dòng)物模型。方法 采用人胃惡性淋巴瘤術(shù)中原發(fā)灶和肝轉(zhuǎn)移灶新鮮瘤組織植入裸小鼠胃黏膜下層，觀察原位移植成瘤率，移植瘤的侵襲和轉(zhuǎn)移率，進(jìn)行形態(tài)學(xué)、染色體核型和流式細(xì)胞分析。結(jié)果 13例人胃惡性淋巴瘤標(biāo)本9例移植成功。依據(jù)WHO新分類標(biāo)準(zhǔn)，從中篩選出1株人胃原發(fā)性非霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植肝轉(zhuǎn)移模型HGBL9903，1株人胃原發(fā)性非霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植模型HGBL9904和1株人

2、胃原發(fā)性霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植模型HGBL9902。免疫組織化學(xué)示CD19、CD20、CD22、CD45和CD79a均陽(yáng)性。染色體眾數(shù)范圍7587條，流式細(xì)胞分析示DI值1.231.47，均為異倍體。3株模型分別傳至83代、87代和89代，共移植裸鼠785只；自第3代起腫瘤的移植生長(zhǎng)率和液氮凍存復(fù)蘇成活率均為100%。HGBL9903肝轉(zhuǎn)移率為100%，脾轉(zhuǎn)移率為36.7%，淋巴結(jié)轉(zhuǎn)移率為57.6%。人胃惡性淋巴瘤在裸鼠胃壁內(nèi)自主侵襲性生長(zhǎng)，侵襲破壞胃壁各層組織結(jié)構(gòu)。發(fā)生血液(肝，脾)轉(zhuǎn)移，淋巴轉(zhuǎn)移和腹腔內(nèi)種植轉(zhuǎn)移。結(jié)論 HGBL9904、HGBL9903和HGBL9902三株人胃

3、原發(fā)性惡性淋巴瘤裸鼠原位移植模型，移植瘤的組織病理學(xué)、超微結(jié)構(gòu)、DNA含量測(cè)定及染色體核型分析結(jié)果與來源人胃惡性淋巴瘤細(xì)胞相一致。完整的模擬了人胃惡性淋巴瘤患者的自然臨床過程。 【關(guān)鍵詞】  移植模型 動(dòng)物 胃腫瘤 惡性淋巴瘤 腫瘤移植 裸鼠     Establishment of orthotopic transplantation model of human primary gastric malignant lymphoma in nude mice     【Abstract】

4、60; Objective  To establish ideal animal models for exploring pathogenesis and experimental therapy of primary gastric malignant lymphoma.Methods  Histologically fresh primary and liver metastatic lymphoma tissues removed from patients with primary gastric malignant lymphoma

5、 were transplanted into gastric submucosa of nude mice to observe tumorgenecity, invasion and metastasis for analysis of morphological characteristics, karyotype and flow cytometry. Results  Successful transplantation of human primary gastric malignant lymphoma was seen in 9 out of 13 case

6、s. According to the new classification standards of WHO on malignant lymphoma, there screened three strains of models, ie, orthotopic transplantation model (HGBL9903) of human primary gastric malignant lymphoma (nonHodgkin's B cell) in nude mice, metastasis model (HGBL9904) of human primary gast

7、ric malignant lymphoma (nonHodgkin's B cell) in nude mice and orthotopic transplantation model (HGBL9902) of human primary gastric malignant lymphoma (Hodgkin's B cell) in nude mice. Immunohistochemistry showed positive CD19, CD20, CD22, CD45 and CD79a, with the number of chromosome for 7587

8、. Flow cytometry showed that DNA index (DI) ranged from 1.23 to 1.47, ie, heteroploid. Three models were passaged for 83, 87 and 89 generations respectively, where 785 nude mice were used for transplantation. Both the growth rate and resuscitation rate of liquid nitrogen cryopreservation of the tran

9、splanted tumors were 100% from the third generation. In HGBL9903, liver metastasis rate, spleen metastasis rate and lymph node metastasis rate were 100%, 36.7% and 57.6%, respectively. The transplanted tumors spontaneously grew in the stomach of nude mice to infiltrate and disrupt every layer of the

10、 gastric wall, with hematogenous metastasis (eg, liver and spleen involved), lymph node metastasis and celiac planting metastasis occurred. Conclusions  Three successfully established orthotopic transplantation models (HGBL9904, HGBL9903 and HGBL9902) of human primary gastric malignant lym

11、phoma in nude mice are consistent with the original human gastric malignant lymphoma in aspects of histopathological and ultrastructural features, DNA content and chromosomal karyotype and hence completely simulate the natural clinical process of human primary gastric malignant lymphoma.   

12、; 【Key words】  Transplantation model, animal; Gastric lymphoma;  Malignant lymphoma;  Tumor transplantation;  Nude mice    為深入探討胃惡性淋巴瘤的生物學(xué)行為，我們將人胃惡性淋巴瘤新鮮組織塊移植于裸鼠胃黏膜下層，成功地建立了1株人原發(fā)性胃非霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植肝轉(zhuǎn)移模型HGBL9903；1株人原發(fā)性胃非霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植模型HGBL9904；1株

13、人原發(fā)性胃霍奇金B(yǎng)細(xì)胞惡性淋巴瘤裸鼠原位移植模型HGBL9902。現(xiàn)報(bào)道如下。     1  材料與方法    1.1  實(shí)驗(yàn)動(dòng)物    BALB/Cnu/nu裸小鼠由中國(guó)醫(yī)學(xué)科學(xué)院腫瘤研究所動(dòng)物室提供。鼠齡35周，體重1720 g，雌雄兼用，在無特殊病原體(specific pathogen free,SPF)的條件下飼養(yǎng)。    1.2  標(biāo)本來源    人胃惡性淋巴瘤新鮮標(biāo)本取自我院和遼寧省腫瘤醫(yī)院，共13例。

14、其中非霍奇金B(yǎng)細(xì)胞彌漫性小裂細(xì)胞型4例，非霍奇金B(yǎng)細(xì)胞彌漫性大細(xì)胞型5例，非霍奇金淋巴細(xì)胞型扭曲細(xì)胞2例，霍奇金大B細(xì)胞型2例。    1.3  標(biāo)本處理    無菌切取的人胃惡性淋巴瘤原發(fā)灶、肝轉(zhuǎn)移灶活體瘤組織小塊1 cm3，置入RPMI1640培養(yǎng)液中，4 保存轉(zhuǎn)運(yùn)到無菌室，清除非瘤組織，將其剪切成1 mm×1 mm×1 mm瘤組織小塊，供移植用。    1.4  原位移植    裸鼠在戊巴比妥鈉(30 mg/kg)靜脈注射麻醉下，取上腹正中切口在胃竇

15、部沿小彎側(cè)切開胃壁3 mm，切口深達(dá)黏膜下層，用100無損傷縫合線將2塊1 mm×1 mm×1 mm瘤組織塊固定于胃壁黏膜下層，70絲線縫合腹膜，50絲線縫皮，術(shù)后繼續(xù)飼養(yǎng)，自由進(jìn)食，每周觀察2次。    1.5  傳代    當(dāng)荷瘤裸鼠處于瀕死狀時(shí)，用頸椎脫位術(shù)處死，取出移植瘤。一部分瘤組織以原代移植方法行鼠間連續(xù)原位傳代(每代35只不等)；余下瘤組織液氮凍存?zhèn)溆谩Ｗ匀凰劳龅暮闪雎闶?，均解剖觀察其發(fā)生腫瘤的侵襲和轉(zhuǎn)移。    1.6  觀察項(xiàng)目    1

16、.6.1  解剖學(xué)和組織學(xué)檢查    全部荷瘤裸鼠均經(jīng)詳細(xì)解剖學(xué)檢查，測(cè)量腫瘤體積，觀察侵襲周圍臟器情況；觀察頸部、幽門、賁門和肝門淋巴結(jié)，以及各臟器有無腫瘤轉(zhuǎn)移。腫瘤組織，區(qū)域淋巴結(jié)和各臟器組織均經(jīng)10 ml/L甲醛固定，石蠟切片，蘇木精伊紅染色，整個(gè)胃和肝臟切開全部包埋于石蠟后，于不同平面切片1020張，光鏡觀察胃和肝臟轉(zhuǎn)移情況。    1.6.2  免疫組織化學(xué)染色    采用LSAB法，對(duì)全部胃腫瘤或有器官轉(zhuǎn)移的切片應(yīng)用CD19、CD20、CD22、CD45、CD79a抗體染色，所用試劑均購(gòu)自Dako公司。    1.6.3  電鏡觀察    移植瘤組織以2.5%戊二醛和1%鋨酸雙重固定，Epon812包埋，超薄切片，鈾鉛染色后PHILIPSCM10透射電鏡觀察。    1.6.4  流式細(xì)胞分析1    采用美國(guó)FACS420型流式細(xì)胞儀對(duì)正常胃黏膜細(xì)胞、移植瘤細(xì)胞進(jìn)行DNA含量測(cè)定。