馬 穎,何援利

(南方醫(yī)科大學珠江醫(yī)院婦產科,廣州 510280)

子宮內膜異位裸鼠模型的建立及血管生成的觀察

馬 穎,何援利△

(南方醫(yī)科大學珠江醫(yī)院婦產科,廣州 510280)

目的建立子宮內膜異位(EMs)裸鼠模型,觀察異位病灶血管生成情況。方法建立EMs裸鼠皮下種植和腹腔注射模型,觀察異位病灶的形態(tài)特點,用免疫組織化學法檢測血管內皮生長因子(VEGF)及微血管密度。結果皮下種植組10只,成模9只,腹腔注射組10只,成模8只。異位病灶呈現增生期子宮內膜特點,VEGF表達陽性,并有微血管形成。結論成功建立EMs裸鼠皮下種植及腹腔注射模型,異位病灶新生血管較多,該模型可用于人EMs血管生成及抗血管生成治療的研究。

子宮內膜異位癥;血管內皮生長因子類;血管生成;微血管密度

子宮內膜異位(endometriosis,EM s)是生育期婦女常見病,其病因和發(fā)病機制不明[1]。異位內膜組織及其周圍組織新血供的建立和維持,是異位病灶種植、存活和EMs發(fā)生的基本條件[2-4]。本研究通過建立EMs裸鼠模型,檢測血管生成,尋找理想的動物模型。

1 材料與方法

1.1 實驗動物 SPF級飼養(yǎng)的BALB/c雌性裸鼠20只,6～10周齡,體質量18～22 g,5只1籠,給予滅菌水及飼料,每周添加營養(yǎng)的攝入。

1.2 標本收集 子宮內膜取自行子宮切除的EM s患者,年齡41～45歲,月經正常,術前6個月未使用激素類藥物。子宮內膜刮取后剪成碎塊,置入無菌DM EM培養(yǎng)基中,取材1 h內接種。

1.3 模型建立 予水合氯醛0.1 mL/10 g腹腔注射麻醉。皮下種植組將裸鼠腹部皮膚切開0.5 cm,將內膜碎塊埋植入皮下,縫合皮膚。腹腔注射組以7號針頭吸取含子宮內膜碎屑液,以腹中線臍下為穿刺點,注入裸鼠腹腔1 mL。術后觀察裸鼠傷口及生活情況,每周檢查皮下結節(jié)3次;裸鼠于注射14 d

后頸椎脫臼處死,將異位病灶放入甲醛中固定、石蠟包埋。

1.4 免疫組織化學染色 將異位病灶均進行4 μ m厚5張連續(xù)切片,1張行HE染色,4張用SP試劑盒行免疫組織化學染色。結果用自動圖像分析軟件分析。微血管密度:CD34染色的病理片中與相鄰的微血管、平滑肌有明顯界限的棕色內皮細胞或內皮細胞簇為1條微血管。管腔及紅細胞均不作為判定微血管的指標;出現分支、結構上不完全獨立者視為1條微血管。由兩位經過專門培訓的病理醫(yī)師在盲法下進行微血管密度的判定。

1.5 統(tǒng)計學處理 采用SPSS12.0統(tǒng)計軟件進行統(tǒng)計學處理,計量資料的均數比較采用Fisher精確概率法,以P＜0.05為差異有統(tǒng)計學意義。

2 結 果

2.1 模型建立情況 20只裸鼠無死亡例數。腹腔注射組成模8只,病灶多位于腹壁、腸系膜和肝表面,約2.0 mm×2.0 mm×2.0 mm大小,呈清亮水泡樣或實質結節(jié)樣,表面有小血管爬行;皮下種植組成模9只,腹部切口均于術后3 d愈合,約于第12天形成約2.0 mm×2.0 mm×2.0 mm結節(jié)樣突起結構,見封 3 圖 1、2。

2.2 形態(tài)觀察結果 兩組病灶光鏡下觀察均呈增生期子宮內膜表現,腺上皮及間質細胞生長,腺體形成,形成腺腔,周圍有炎細胞及間質細胞浸潤;病灶周圍可見新生毛細血管形成、結締組織增生及玻璃樣變性。

2.3 免疫組織化學檢測結果 VEGF陽性為胞質棕色顆粒,腺上皮、間質及血管內皮均有表達;CD34陽性定位于血管內皮細胞,呈棕黃色,微血管有的形成管腔,有的僅為單個內皮細胞或內皮細胞簇,見封 3圖3、4。

3 討 論

3.1 EMs裸鼠模型的建立 鼠類與人類有99%的相同基因,價格便宜,易于飼養(yǎng)操作,觀察周期短,故成為理想的模型動物。裸鼠是T淋巴細胞功能缺陷動物,其細胞免疫力低下,可保留移植物組織學、遺傳學以及生物化學特性,因此裸鼠是理想的研究EMs的模型動物[5]。Masuda等[6]于 1994年成功建立皮下種植EMs裸鼠模型,并觀察到新生的腺體及間質。Ozawa等[7]提出裸鼠的腹腔種植模型同樣具有成模及治療的有效性,并檢測到VEGF的分泌。本研究嘗試了兩種造模途徑,均獲較高的成功率,光鏡下觀察到裸鼠體內種植病灶形態(tài)與人子宮內膜異位病灶極其相似。腹腔注射組3例失敗,可能是由于內膜組織碎屑太小,影響其生存和種植;皮下種植組2例失敗,可能是種植切口處線結松弛,子宮內膜組織遺失。

3.2 VEGF的表達及意義 異位內膜細胞的黏附、血管形成、侵襲生長是EMs形成的基礎[8],VEGF在其中起到了重要的作用[9-11]。EM s的病因至今不明,但經血逆流學說已經得到了諸多研究的支持與證實,隨經血逆流的子宮內膜組織必須首先與盆腹膜或臟器黏附后,才可在局部形成新生血管網,進而發(fā)生浸潤及生長。VEGF則能特異性地促進內皮細胞增殖,并可提高血管的通透性,促進細胞外基質降解,因此VEGF的過表達可能是導致EMs發(fā)生的重要因素,其與脫落內膜的侵襲與轉移及EMs的發(fā)生、發(fā)展都有密切的關系[12-16]。裸鼠皮下種植及腹腔注射模型均是理想的研究人EM s的動物模型,在研究血管生長及抗血管治療中有較好的應用價值。

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The establishment of nude mouse endometriosis model and the observation of angiogenesis

Ma Ying,He Yuanli△
(Department of Gynecology and Obstetrics,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China)

ObjectiveTo establish the nude mouse endometriosis model and observe the angiogenesis.MethodsThe nude mouse endometriosis models were established by abdominal cavity injection and subcutaneous implantation.The ectopic focus were detected by microscopy.The expression of VEGF and microvessel density were detected by immunohistochemistry.Results9 and 8 mouse were accomplished in subcutaneous implantation group and in abdominal cavity injection group respectively.The ectopic lesions were observed the endometrium-liked structrue with gland and substance in microscope.All of the ectopic lesions were VEGF positive and were to form many microvessel.ConclusionIt is suggested that the nude mice endometriosis model of abdominal cavity injection and subcutaneous implantation are safe and available.It is a kind of ideal materials to endometriosis animal model.

endometriosis;vascular endothelial growth factors;angiogenesis;microvessel density

10.3969/j.issn.1671-8348.2011.04.020

A

1671-8348(2011)04-0357-02

△通訊作者,Tel:13318807468;E-mail:heyuanli310@yahoo.com.cn。

2010-06-15

2010-11-09)

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