沈俊，袁平，唐俊明，楊建業(yè)，李興元，張蕾，趙繼先，張煥鑫，薛仕珍，馮怡，王家寧

· 論著 ·

Meox1在心肌梗死大鼠心肌中的表達情況

沈俊1,2，袁平2，唐俊明1，楊建業(yè)1，李興元1，張蕾1，趙繼先2，張煥鑫2，薛仕珍2，馮怡2，王家寧1,2

目的 分析同源盒基因Meox1在心肌梗死大鼠心肌中的表達情況。方法 清潔級雄性SD大鼠20只，隨機分入心肌梗死組和假手術組，每組10只。心肌梗死組采用結扎左冠狀動脈前降支構建心肌梗死模型，手術過程死亡2只。假手術組僅穿線不結扎。術后7 d處死大鼠，制作石蠟切片，HE染色以及Masson染色觀察心肌組織變化，免疫組化法檢測Meox1的表達水平。結果 HE染色結果：心肌梗死組可見梗死區(qū)心肌細胞顯著減少，核碎裂，核消失，被大量排列紊亂的結締組織代替，可見大量粒細胞、單核細胞浸潤。梗死邊緣區(qū)可見心肌細胞代償性肥大，橫紋模糊或消失，心肌間隙水腫增寬，部分心肌纖維溶解、斷裂，可見炎性細胞浸潤、成纖維細胞增生。梗死遠離區(qū)及假手術組大鼠心肌形態(tài)正常，結構清晰，心肌纖維排列整齊。Masson染色結果，非梗死區(qū)染色呈紅色為正常心肌組織，藍色為膠原纖維。假手術組及梗死遠離區(qū)心肌細胞排列整齊緊密，細胞間隙散在分布少量膠原纖維；心肌梗死組梗死區(qū)可見細胞間隙大量膠原纖維沉積，梗死邊緣區(qū)的細胞間隙也有大量的膠原纖維沉積。免疫組化結果，梗死區(qū)及梗死邊緣區(qū)的心肌細胞中可見棕黃色顆粒沉積，有Meox1表達；在梗死遠離區(qū)的正常心肌細胞中未見Meox1的表達；假手術組心肌細胞中亦未見Meox1的表達。假手術組及梗死遠離區(qū)毛細血管內皮細胞中未見Meox1的表達；心肌梗死組梗死區(qū)及梗死邊緣區(qū)毛細血管內皮細胞中Mexo1表達不明顯。結論 在心肌梗死大鼠心肌組織中Meox1表達增高，提示其可能參與心肌梗死后心肌重塑。

心肌梗死；同源盒基因Meox1；表達

在我國冠狀動脈粥樣硬化性心臟病（冠心?。┦侵饕劳鲈蛑唬募」Ｋ溃∕I）后1年內死亡率較高[1]，梗死和非梗死區(qū)細胞外基質的異常集聚，導致組織結構改變、組織硬度增加，心肌廣泛重構，最終引起心功能不全[2-8]。因此尋求有效治療靶點非常必要。同源盒基因Meox1是控制發(fā)育的主要基因，對心血管系統(tǒng)發(fā)育及疾病的發(fā)生有調控作用[9]。本研究通過結扎SD大鼠冠狀動脈前降支制備心肌梗死模型，采用HE染色、Masson染色及免疫組化的方法觀察心肌梗死后心肌組織變化及Meox1的表達情況。

1 材料與方法

1.1 實驗動物、主要材料與儀器 清潔級雄性SD大鼠20只由湖北醫(yī)藥學院實驗動物中心提供，重量180～230 g。飼養(yǎng)條件：溫度23℃，濕度95%，12 h明暗交替，大鼠專用飼料喂養(yǎng)。實驗過程嚴格遵守倫理學準則。Meox1抗體（美國abcom公司）、DAB顯色試劑盒（武漢博士德生物工程有限公司），RM2245型病理切片機、H11220型烘片機、H11210型攤片機（德國Leica公司）、BX-51型光學顯微鏡（日本Nikon公司）。手術器械包（手術剪、彎頭和直頭止血鉗、彎形鑷子、眼科剪、顯微持針器、開胸器、手術線、干棉花、酒精棉、紗布、頭皮針、注射器、三通管、棉線），6-0無菌醫(yī)用非吸收性縫合線無損傷縫合針（杭州富陽醫(yī)用縫合針線廠）。將20只大鼠隨機分入心肌梗死組和假手術組，每組10只，制作心肌梗死模型死亡2只。

1.2 方法

1.2.1 心肌梗死動物模型的構建 按照Kumar等[10]的改進方法，實驗前大鼠禁食2 h以上，水合氯醛（300 mg/kg）進行腹腔注射麻醉，以有無翻正反射作為大鼠麻醉與否的評定標準。固定大鼠，手術區(qū)剃毛后，剪開皮膚，分離頸部肌肉，暴露氣管，用氣管穿刺針進行氣管插管。插管成功后接小動物呼吸機輔助呼吸，潮氣量30～50 ml/kg，呼吸頻率55～65 次/min，呼吸比2:1。連接心電圖機測肢體Ⅱ導聯心電圖。在三、四肋間心尖搏動最強點（大約為平左前肢與左胸骨2 mm處）切開皮膚，鈍性分離第三、四肋骨的肋間肌，止血鉗撐開肋間隙擴大手術視野。小心地撕開心包膜暴露心臟，在心臟收縮的一瞬間，用食指與拇指擠出心臟，快速從左心耳下方2.0～3.0 mm入針，進針深度為1.5～2.0 mm，然后快速還納心臟，于左心耳和肺動脈圓錐與心尖連線中點處（約為左冠狀動脈走行處）以6-0無創(chuàng)縫合線將留置軟管一起進行縫扎，結扎時力度要適中，結扎缺血區(qū)心肌組織變白，同時心電圖對應的導聯抬高。假手術組僅開胸和分離冠狀動脈，但是不結扎。觀察心臟跳動情況，實驗中若出現心律失常，則在腹腔注射少量的利多卡因。結扎后逐層縫合胸腔，并保持預留留置針通暢以防止氣胸。待大鼠呼吸穩(wěn)定后，放入恢復籠內飼養(yǎng)。腹腔注射3～5 ml生理鹽水補液后，觀察并及時清除氣管痰液。待大鼠蘇醒后肌肉注射青霉素鈉以防感染。

1.2.2 組織的獲得和處理 手術后7 d，麻醉并處死大鼠，開胸暴露心臟，將接有生理鹽水的輸液針頭扎在右心耳，用眼科剪將心尖剪一個約2 mm小口，生理鹽水灌洗心腔，沖洗干凈后用4%多聚甲醛沖洗心腔2 h。取出心臟，去除心房。將心室置于4%多聚甲醛固定24 h，于結扎線處水平將心室切成1 mm薄片，置于包埋盒中流水沖洗過夜，常規(guī)脫水、透明、浸蠟、包埋等處理。使用切片機連續(xù)切片，厚度為5 μm，并按順序編號。

1.2.3 HE染色 具體步驟如下：①切片常規(guī)二甲苯脫蠟，梯度酒精水化，用去離子水洗1～2 min；②蘇木精染色8～10 min，自來水洗1～2 min；③體積分數為1%的鹽酸-酒精分化1～2 s（顯微鏡下觀察效果），自來水洗10～15 s；④體積分數為l%的氨水或肥皂水返藍20～40 s，自來水洗1 min；⑤伊紅染色4～5 min，自來水洗1 min；⑥切片依次入體積分數為95%乙醇5～10 s，無水乙醇3次，每次5～10 s，二甲苯3次，每次1～2 min；⑦中性樹脂封片，鏡下觀察。

1.2.4 Masson染色 步驟：①切片常規(guī)二甲苯脫蠟，梯度酒精水化，去離子水洗1～2 min；②用配制的Weigert鐵蘇木素染色5～10 min；③體積分數為1%的鹽酸-酒精分化1～2 s，自來水洗10～15 s；④Masson藍化液返藍20～40 s，自來水洗1 min，去離子水洗1 min；⑤麗春紅染色5～10 min；⑥蒸餾水：弱酸溶液=2：1比例配置弱酸工作液，用弱酸工作液洗1 min；⑦磷鉬酸溶液洗1～2 min，弱酸工作液洗1 min；⑧苯胺藍染色1～2 min，弱酸工作液洗1 min；⑨切片依次入體積分數為95%乙醇5～10 s，無水乙醇3次，每次5～10 s，二甲苯3次，每次1～2 min；⑩中性樹脂封片，鏡下觀察。

1.2.5 免疫組化分析 具體步驟如下：①切片常規(guī)二甲苯脫蠟，梯度酒精水化，用去離子水洗1～2 min；②微波修復抗原15 min，3%H2O2孵育5～10 min，PBS洗3次，每次5 min；③10%馬血清封閉，室溫1 h；④加一抗：Meox1兔多克隆抗體（1:100），4℃濕盒過夜，PBS洗3次，每次5 min；⑤加二抗：鼠抗兔多克隆抗體（1:100），室溫1 h，PBS洗3次，每次5 min；⑥DAB染色（現配現用），PBS洗3次，每次5 min；⑦蘇木精染色8～10 min，自來水洗1～2 min；⑧體積分數為1%的鹽酸-酒精分化1～2 s（顯微鏡下觀察效果），自來水洗10～15 s；⑨體積分數為1%的氨水或肥皂水返藍20～40 s，自來水洗1 min；⑩切片依次入體積分數為95%乙醇5～10 s，無水乙醇3次，每次5～10 s；二甲苯透明3次，每次1～2 min；?中性樹脂封片。

2 結果

2.1 兩組HE染色結果比較 HE染色結果：心肌梗死組可見梗死區(qū)心肌細胞顯著減少，心肌纖維凝固性壞死，核碎裂，核消失，被大量排列紊亂的結締組織代替，肌漿均質紅染或呈不規(guī)則粗顆粒狀，可見大量粒細胞、單核細胞浸潤（圖1）。梗死邊緣區(qū)可見心肌細胞代償性肥大，肌橫紋模糊或消失，心肌間隙水腫增寬，部分心肌纖維溶解、斷裂，可見炎性細胞浸潤、成纖維細胞增生。梗死遠離區(qū)及假手術組（圖1）大鼠心肌形態(tài)正常，結構清晰，心肌纖維排列整齊有序，肌纖維間無成纖維細胞聚集、增生現象。

圖1 大鼠心肌組織HE染色結果（A：假手術組；B：心肌梗死組梗死區(qū)；C：心肌梗死組梗死邊緣區(qū)；D：心肌梗死組梗死遠離區(qū)，×200）

2.2 兩組Masson染色結果比較 Masson染色結果，非梗死區(qū)染色呈紅色為正常心肌組織，藍色為膠原纖維。假手術組及梗死遠離區(qū)可見紅色的心肌細胞排列整齊緊密，細胞間隙內有散在分布少量藍色膠原纖維；心肌梗死組可見梗死區(qū)細胞間隙大量膠原纖維沉積，梗死邊緣區(qū)紅色的細胞間隙有大量的膠原纖維沉積（圖2）。

2.3 Meox1在心肌細胞中的表達情況 在梗死區(qū)及梗死邊緣區(qū)的心肌細胞中可見棕黃色顆粒沉積，提示Meox1在心肌細胞中表達；在梗死遠離區(qū)的正常心肌細胞中未見Meox1的表達；假手術組心肌細胞中亦未見Meox1的表達（圖3）。

2.4 Meox1在毛細血管內皮細胞中的表達情況 假手術組及梗死遠離區(qū)毛細血管內皮細胞中未見Meox1表達；心肌梗死組梗死區(qū)及梗死邊緣區(qū)毛細血管內皮細胞中Mexo1表達不明顯，其動態(tài)變化可能在心肌梗死后基質重塑中起作用（圖4）。

圖2 大鼠心肌組織Masson染色結果（A：假手術組；B：心肌梗死組梗死區(qū)；C：心肌梗死組梗死邊緣區(qū)；D：心肌梗死組梗死遠離區(qū)，×200）

圖3 Meox1在心肌細胞中的表達情況（A：假手術組；B：心肌梗死組梗死區(qū)；C：心肌梗死組梗死邊緣區(qū)；D：心肌梗死組梗死遠離區(qū)，×200）

3 討論

心肌梗死后伴心室重構，梗死和非梗死區(qū)細胞外基質的異常集聚，導致組織結構破壞、組織硬度增加，引起左心腔擴大及心功能不全[11-16]。心肌梗死后的心室重構是一個慢性過程，研究表明[17-24]，細胞外基質的合成及降解、腎素-血管緊張素-醛固酮系統(tǒng)、轉化生長因子β、金屬蛋白酶、Micro RNA及各種轉錄生長因子等都參與了心肌纖維化的過程。

同源盒蛋白是廣泛存在于真核生物的一類轉錄調控因子[25-27]。同源盒結構域高度保守，通過螺旋-轉折-螺旋結構模式與啟動子或增強子的序列結合，激活或抑制靶基因的轉錄[28-30]。同源盒基因Meox所編碼的蛋白質也屬于轉錄因子[31,32]。近年關于同源盒基因的報道較多，而關于Meox1在心血管系統(tǒng)的作用卻較少。王書美等[32]通過建立心臟特異表達Meox1的轉基因小鼠，發(fā)現Meox1在心臟過表達引起擴張型心肌病。

圖4 Meox1在毛細血管內皮細胞中的表達情況（A：假手術組；B：心肌梗死組梗死區(qū)；C：心肌梗死組梗死邊緣區(qū)；D：心肌梗死組梗死遠離區(qū)，×200）

通過免疫組化染色發(fā)現大鼠心肌梗死后心肌組織中表達Meox1，在心肌梗死區(qū)及梗死邊緣區(qū)毛細血管內皮細胞中表達，而遠離區(qū)及正常內皮細胞中無表達。還發(fā)現心肌梗死區(qū)及梗死邊緣區(qū)心肌細胞亦表達Meox1，而遠離區(qū)及正常心肌細胞則無Meox1表達。推測：梗死區(qū)及梗死邊緣區(qū)心肌細胞及血管內皮細胞為缺血缺氧細胞，缺血缺氧誘導Meox1表達，而心肌梗死后Meox1表達短暫升高或與心肌細胞的存活相關，其機制不清。本研究表明，Meox1在心肌梗死大鼠的心臟血管內皮細胞及心肌細胞中表達，提示其參與心肌梗死后的心肌重構，具體機制需更深入的研究。

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本文編輯：姚璐，田國祥

Changes in the expression of Meox1 after acute myocardial infarction in rats

SHEN Jun*, YUAN Ping, Tang Jun-ming, YANG Jian-ye, LI Xing-yuan, Zhang Lei, ZHAO Ji-xian, ZHANG Huan-xin, XUE Shi-zhen, FENG Yi, WANG Jia-ning.*Institute of Clinical Medicine and Department of Cardiology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.

Objective To analyze the expression of Meox1 in myocardium in rats with acute myocardial infarction (AMI). Methods Male SD rats (n=20) were randomly divided into AMI group and sham-operation group (each n=10). The model of AMI was established by ligation of left anterior descending branch of coronary artery in AMI group (2 rats died during the procedure), and sham-operation group was given the same procedure except of ligation. The rats were killed 7 d after the procedure for preparing paraffin sections, and changes of myocardial tissue were observed after HE staining and Masson staining and expression of Meox1 was detected by using immunohistochemistry technique. Results The results of HE staining showed that cardiomyocyte decreased significantly at infarction zone, and there were karyorrhexis, nuclear disappearing, disorganized connective tissue in quantity, and severe infiltration of granulocytes and monocytes in AMI group. The compensatory hypertrophy and cross striatation blurring or disappearing of cardiomyocytes, widened myocardial gap edema, fibrinolysis and fragmentation of partial myocardial fibers, infiltration of inflammatory cells and proliferation of fibroblast were observed at infarction marginal zone. The normal myocardial histology, clear structure and aligned myocardial fibers were observed at infarction remote zone and in sham-operation group. The results of Masson staining showed that red was normal myocardial tissue and blue was collagen fibers at non-infarction zone. The cardiomyocytes were aligned and tight, and there were less collagen fibers in intercellular space in sham-operation group and at infarction remote zone. There was deposition of collagen fibers in quantity in intercellular space and at infarction zone and infarction marginal zone in AMI group. The results of immunohistochemistry technique showed that there was deposition of brown granules and Meox1 expression in cardiomyocytes at infarction zone and infarction marginal zone, and there was no Meox1 expression in normal cardiomyocytes at infarction remote zone and in sham-operation group. There was no Meox1 expression observed in capillary endothelial cells in sham-operation group and at infarction remote zone, and which was not significant at infarction zone and infarction marginal zone in AMI group. Conclusion The expression of Meox1 increases in myocardial tissue in AMI rats, which indicates that Meox1 expression may take part in the myocardial remodeling after AMI.

Myocardial infarction; Homeobox gene Meox1; Expression

R541.4

A

1674-4055(2016)11-1329-04

國家自然科學基金(81270221)

1442000 十堰,湖北醫(yī)藥學院附屬人民醫(yī)院臨床醫(yī)學研究所;2442000 十堰,湖北醫(yī)藥學院附屬人民醫(yī)院心臟病中心1病區(qū)

王家寧,E-mail:rywjn@vip.163.com

10.3969/j.issn.1674-4055.2016.11.13