連續(xù)縫合間斷打結(jié)吻合動(dòng)脈在小鼠心臟移植中的應(yīng)用

崔得利　譚長(zhǎng)軍　劉　忠

(1大連醫(yī)科大學(xué)附屬第一醫(yī)院普外科　大連　116011;2復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所-上海市器官移植重點(diǎn)實(shí)驗(yàn)室　上?！?00032)

?

連續(xù)縫合間斷打結(jié)吻合動(dòng)脈在小鼠心臟移植中的應(yīng)用

崔得利1▲譚長(zhǎng)軍2▲劉忠1△

(1大連醫(yī)科大學(xué)附屬第一醫(yī)院普外科大連116011;2復(fù)旦大學(xué)附屬中山醫(yī)院肝癌研究所-上海市器官移植重點(diǎn)實(shí)驗(yàn)室上海200032)

【摘要】目的應(yīng)用連續(xù)縫合間斷打結(jié)法吻合動(dòng)脈建立小鼠心臟移植模型。方法近交系C57BL/6j( (H-2b))、BALB/c( (H-2d))小鼠各30只,建立小鼠腹部心臟移植模型 (C57BL/6j→BALB/c),連續(xù)縫合間斷打結(jié)法端側(cè)吻合供體升主動(dòng)脈和受體腹主動(dòng)脈;一針后壁先縫法連續(xù)吻合供體肺動(dòng)脈和受體下腔靜脈。觀察移植心臟搏動(dòng)持續(xù)時(shí)間及受體生存時(shí)間。結(jié)果手術(shù)成功率90%(27/30),總手術(shù)時(shí)間 (91.77±6.70)min,動(dòng)脈吻合時(shí)間 (24.63±1.67)min,靜脈吻合時(shí)間 (15.17±1.80)min。供心中位存活時(shí)間8 天,病理組織學(xué)示典型排斥反應(yīng)。結(jié)論應(yīng)用連續(xù)縫合間斷打結(jié)法吻合動(dòng)脈降低了小鼠心臟移植的手術(shù)難度,效果可靠,值得推廣。

【關(guān)鍵詞】小鼠;動(dòng)物模型;心臟移植;動(dòng)脈吻合

*This work was supported by the General Program of National Natural Science Foundation of China (81373874,81472218).

小鼠心臟移植模型是移植免疫學(xué)研究的重要工具[1],但建模要求高,動(dòng)脈吻合是其中的難點(diǎn)[2],對(duì)于不具備顯微外科基礎(chǔ)的醫(yī)學(xué)生而言難度較大。受器官缺血時(shí)間限制,器官移植動(dòng)物模型多采用連續(xù)縫合法重建血流。但我們?cè)谇捌谟?xùn)練中發(fā)現(xiàn):應(yīng)用經(jīng)典的連續(xù)縫合法建立主動(dòng)脈和腹主動(dòng)脈端側(cè)吻合,開放血流后,大出血和吻合口狹窄發(fā)生率較高,是建模失敗的首要原因。有鑒于此,我們嘗試應(yīng)用連續(xù)縫合間斷打結(jié)法改進(jìn)動(dòng)脈吻合,效果滿意,報(bào)告如下。

材 料 和 方 法

實(shí)驗(yàn)動(dòng)物選擇SPF級(jí)近交系雄性小鼠,C57BL/6j(H-2b)為供體(n=30),BALB/c(H-2d)為受體(n=30),體質(zhì)量22～25 g,購(gòu)于上海斯萊克實(shí)驗(yàn)動(dòng)物有限責(zé)任公司。于無(wú)菌通風(fēng)25℃恒溫環(huán)境中飼養(yǎng),術(shù)前不禁食、不禁水。

器械及試劑16倍手術(shù)顯微鏡 (上海醫(yī)療器械股份有限公司光學(xué)儀器廠),顯微鑷、顯微持針器、顯微剪 (上海金鐘醫(yī)療器械廠),手術(shù)剪 (上海醫(yī)療器械股份有限公司),11-0、8-0、5-0帶線縫合針(上海浦東金環(huán)公司),戊巴比妥鈉(美國(guó)Sigma公司,用生理鹽水稀釋成1%),肝素鈉注射液 (上海第一生化藥業(yè)有限公司,用生理鹽水配成50 IU/mL)。

手術(shù)方法

術(shù)前準(zhǔn)備參考Corry等[3]的手術(shù)方法,建立小鼠腹部心臟移植模型。先受體后供體依次進(jìn)行麻醉,均使用戊巴比妥鈉0.005 mL/g腹腔注射麻醉,并聯(lián)合乙醚輔助。

受體手術(shù)腹正中線剪開腹壁進(jìn)腹,顯露腹主動(dòng)脈及下腔靜脈。11-0線結(jié)扎睪丸動(dòng)脈,8-0線集束結(jié)扎下腔靜脈屬支血管。在腎靜脈下方及髂血管分叉上方各穿1根5-0阻斷線,不打結(jié),備用。

供體手術(shù)顯露下腔靜脈,從下腔靜脈注入冰肝素鹽水2 mL,完成供體肝素化。下腔靜脈放血后迅速剪開胸腔暴露心臟,冰鹽水紗布覆蓋心臟5-0線結(jié)扎上、下腔靜脈。離斷主動(dòng)脈及肺動(dòng)脈,集束結(jié)扎其余血管,剪取心臟放入冰鹽水中保存。

心臟移植阻斷線打活結(jié)阻斷腹主動(dòng)脈及下腔靜脈。11-0縫合針穿過腹主動(dòng)脈前壁,懸吊血管壁,彎顯微剪刀沿縫合針剪一橢圓形切口,同法在下腔靜脈開窗。從冰肝素鹽水中取出供心,置于受體腹部右側(cè),冰鹽水紗布覆蓋。11-0線將供體升主動(dòng)脈兩端分別與受體腹主動(dòng)脈開口兩端 (12點(diǎn)及6點(diǎn)處)縫合,作為兩側(cè)錨定線。11-0線連續(xù)縫合腹主動(dòng)脈左側(cè)壁至對(duì)側(cè)錨定線,共4針,縫合過程預(yù)留“線圈”,周長(zhǎng)約1.3 cm,一側(cè)血管壁縫合結(jié)束后,從中間剪斷“線圈”,相應(yīng)的線頭與線尾打結(jié),打結(jié)時(shí)切忌上提血管以免誤入氣泡(圖1)。

A:Two opposing anchor sutures placed 180° apart;B:Running suture using open loop;C:The loop was cut in between;D:Suture was tied individually as interrupted method.

圖1連續(xù)縫合間斷打結(jié)法示意圖

Fig 1Schematic diagram of continuous-interrupted method

翻轉(zhuǎn)供心至對(duì)側(cè),同法縫合動(dòng)脈右側(cè)壁,縫合結(jié)束前肝素鹽水沖洗管腔,盡量剪短殘余尾線,以免影響靜脈左側(cè)壁吻合。靜脈吻合采用一針后壁先縫法[4]連續(xù)縫合肺動(dòng)脈與下腔靜脈。吻合完畢后,開放血流,溫生理鹽水復(fù)溫,待復(fù)跳規(guī)則后復(fù)位腸管并關(guān)腹(圖2)。

術(shù)后處理與觀察術(shù)后25 w白熾燈照射15～30 min。每天觀察小鼠的活動(dòng)情況并通過觸診判斷供心搏動(dòng)情況,受體存活3天以上且心臟持續(xù)搏動(dòng)則視為手術(shù)成功。術(shù)后未用抗生素及免疫抑制劑,術(shù)后第7天隨機(jī)取5只小鼠的移植心臟行病理組織學(xué)檢查,重點(diǎn)觀察心肌組織損傷及炎性細(xì)胞浸潤(rùn)情況,依據(jù)2005年國(guó)際心肺移植協(xié)會(huì)(ISHLT)制定的心臟移植排斥反應(yīng)標(biāo)準(zhǔn)[5]進(jìn)行分級(jí)。

結(jié)果

心臟移植結(jié)果30例小鼠心臟移植手術(shù)的成功率為90%。總手術(shù)時(shí)間 (91.77±6.70) min,其中動(dòng)脈吻合時(shí)間 (24.63±1.67) min,靜脈吻合時(shí) 間 (15.17±1.80) min。3例手術(shù)失敗,其中1例因麻醉意外死亡，2例因心臟流出道受阻,術(shù)后2 天內(nèi)死亡,未出現(xiàn)心臟灌注不足或動(dòng)脈狹窄等情況。供心中位存活時(shí)間為8天,供心停跳后,受體可長(zhǎng)期生存。

A:Anastomosis was completed;B:After unclamping;C:Anastomotic sites of aorta and inferior vena cava;D:View before abdominal closure.

圖2吻合完畢示意圖

Fig 2Soheme of completed anastomosis

病理組織學(xué)檢查供心存在不同程度的炎性細(xì)胞浸潤(rùn),伴有心肌組織損傷,呈典型排斥反應(yīng),ISHLT分級(jí)為2R級(jí)(圖3)。

A and B:The myocardial area appeared inflammatory cells infiltration,accompanied by myocardial tissue damage in rejection combination after 7 days following surgery (A:100×,B:200×);C and D:The myocardial area was intact in naive C57BL/6j heart (C:100×,D:200×).

圖3心肌部位HE染色

Fig 3HE staining of mycardia

討論

由于器官移植受缺血時(shí)間的限制,小鼠心臟移植的動(dòng)脈吻合多采用連續(xù)縫合法,其主要優(yōu)點(diǎn)是操作簡(jiǎn)便、快速。但我們?cè)谇捌谟?xùn)練中發(fā)現(xiàn)如下缺點(diǎn):(1) 腹主動(dòng)脈開窗口徑在端側(cè)吻合中難以控制[6]。如腹主動(dòng)脈開窗口與供體的升主動(dòng)脈完全匹配,在縫合兩端的錨定線后,兩側(cè)的血管邊緣緊貼在一起,造成血管腔難以顯露,尤其在最后1～2針時(shí)。若吻合口不匹配,在十分狹小的視野內(nèi)連續(xù)縫合針距調(diào)整十分困難。(2) 血管吻合的空間和視野問題。在16倍顯微鏡下,若每次縫針后均拉緊縫線,則最后1～2針經(jīng)常難以確定吻合血管邊緣的空隙。如果采用降落傘法,一側(cè)血管壁縫合結(jié)束后一起拉緊縫線,受腹主動(dòng)脈外膜影響,常導(dǎo)致縫線無(wú)法拉緊甚至斷線。(3) 連續(xù)縫合易導(dǎo)致吻合口狹窄、血栓等不良反應(yīng)[7],成為制約建模成功的主要原因。

Sapountzis等[8]首先報(bào)道在顯微外科血管吻合中使用連續(xù)縫合間斷打結(jié)的方法,操作要點(diǎn)是:在縫合兩端的錨定線后,從一側(cè)開始連續(xù)縫合血管壁,但不拉緊縫線,而是留出較長(zhǎng)的線圈,達(dá)到對(duì)側(cè)錨定線后,中間剪斷線圈,各自線圈的頭尾線分別打結(jié)。我們將此法應(yīng)用于小鼠心臟移植動(dòng)脈吻合(圖4)。

A:Running suture using open loop with the length of about 1.3 cm;B:The loop was cut,and suture was tied individually as interrupted method.

圖4建立縫合線圈

Fig 4Establishment and cut of loop

在應(yīng)用過程中,我們發(fā)現(xiàn)本法的主要優(yōu)點(diǎn)是:(1) 兩側(cè)血管壁都是在充分顯露血管腔的條件下進(jìn)行縫合,最后縫線再分別打結(jié),因此視野清晰,這對(duì)小鼠心臟移植的動(dòng)脈吻合有著重要的意義。(2) 本法解決了吻合血管不匹配的難題。由于最后所有的縫線分別打結(jié),打結(jié)時(shí)調(diào)整縫線偏向就可以糾正偏差。(3)單純間斷縫合法是顯微血管吻合的“金標(biāo)準(zhǔn)”。相對(duì)于小靜脈吻合,動(dòng)脈血管壁順應(yīng)性差、彈性大,更加適用間斷縫[9],但這又與器官移植的缺血時(shí)限相矛盾。連續(xù)縫合間斷打結(jié)法解決了這一矛盾，相對(duì)于間斷縫合,本法連續(xù)運(yùn)針,節(jié)省了中間剪線和換針的時(shí)間。常規(guī)間斷縫合法通常先固定兩端,再縫中點(diǎn),后提中點(diǎn)線,縫合最后2針時(shí)經(jīng)常顯露不佳,而應(yīng)用本法,最后2針縫合時(shí)管腔顯露清楚,縫合更確切。吻合后供受體血管匹配良好,未出現(xiàn)連續(xù)縫合后由于匹配不良引起的吻合口大出血 (圖5)。

A:The left side of aort a was completed;B:The right side of aort a was completed, and the end of sutures must be cut as short as possible to avoid interference of venous anastomosis;C:Anastomoses of aorta and inferior vena cava were completed.

圖5吻合口處血管匹配良好

Fig 5Good matching of anastomosic vessels

本法的不足之處在于:(1)剪斷“線圈”后線尾易糾結(jié)在一起,打結(jié)拉線時(shí)可能會(huì)發(fā)生錯(cuò)誤,將臨近的縫線拉出。(2) 腹主動(dòng)脈右側(cè)壁縫合后殘余的線尾可能會(huì)干擾下腔靜脈左側(cè)壁的縫合,操作過程中應(yīng)加以注意。綜上所述,連續(xù)縫合間斷打結(jié)是建立小鼠腹部心臟移植模型的良好動(dòng)脈吻合方式,簡(jiǎn)單易學(xué),值得推廣。

致謝復(fù)旦大學(xué)附屬中山醫(yī)院麻醉科何義舟主治醫(yī)師指導(dǎo)手術(shù)操作。

參考文獻(xiàn)

[1]彭潤(rùn)生,王春生,楊兆華.小鼠腹部心臟移植模型的顯微外科技巧及并發(fā)癥的預(yù)防[J].復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版),2008,35 (3):411-413.

[2]張善中,譚建明,唐孝達(dá).血管化小鼠異位心臟移植模型[J].中華實(shí)驗(yàn)外科雜志,2006,23 (10):1276.

[3]CORRY RJ,WINN HJ,RUSSELL PS.Primarily vascularized allografts of hearts in mice.The role of H-2D,H-2K,and non-H-2 antigens in rejection[J].Transplantation,1973,16 (4):343-350.

[4]QIAN SG,FUNG JJ,DEMETRIS AV,etal.Orthotopie liver transplantation in the mouse[J].Transplantation,1991,52 (3):562-564.

[5]STEWART S,WINTERS GL,FISHBEIN MC,etal.Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[J].HeartLungTransplant,2005,24 (11):1710-1720.

[6]HUANG LJ,REESE S,DJAMALI A.Contributing factors to complications andsurgical success in mouse kidney transplantation[J].IntBrazJUrol,2012,38 (3):395-403.

[7]TOZZI P,HAYOZ D,RUCHAT P,etal.Animal model to compare the effects of suture technique on cross-sectional compliance on end-to-side anastomoses[J].EurJCardiothoracSurg,2001,19 (4):477-481.

[8]SAPOUNTZIS S,KIRANANTAWAT K,LIM SY,etal.A novel “continuous-interrupted” method for micro-vascular anastomosis[J].Microsurgery,2014,34 (1):82-84.

[9]UMEZAWA H,OGAWA R,NAKAMIZO M,etal.A comparison of microsurgical venous anastomosis techniques[J].NipponMedSch,2015,82 (1):14-20.

The continuous-interrupted method for arterial anastomosis in mouse heart transplantation

CUI De-li1▲, TAN Chang-jun2▲, LIU Zhong1△

(1DepartmentofGeneralSurgery,theFirstAffiliatedHospitalofDalianMedicalUniversity,Dalian116011,LiaoningProvince,China;2LiverCancerInstitute,ZhongshanHospital,FudanUniversity-Shanghai

KeyLaboratoryofOrganTransplantation,Shanghai200032,China)

【Abstract】ObjectiveTo evaluate the feasibility of continuous-interrupted method for arterial anastomoses in mouse model of heart transplantation.MethodsHeterotopic heart transplantations were performed from inbred C57BL/6j( (H-2b)) mouse (n=30) to BALB/c( (H-2d)) mouse (n=30),in which the graft′s ascending aorta was anastomosed end-to-side to the recipient′s aorta by continuous-interrupted sutures,and the pulmonary artery was anastomosed end-to-side to the inferior vena cava by continuous one-suture anastomosis.The survival time of grafts and the duration of cardiac impulse were observed.ResultsThe successful rate of transplantation was 90% (27/30).Total operation time was (91.77±6.70) min with,arterial anastomosis time of (24.63±1.67) min and venous anastomosis time of (15.17±1.80) min.Median beating time of donor heart was 8 days with acute rejection reaction indicated by pathological histology.ConclusionsThe technical difficulties of arterial anastomosis were reduced greatly by continuous-interrupted method, and we recommended this method for mouse heterotopic cardiac transplantation.

【Key words】mouse;animal model;heart transplantation;arterial anastomosis

(收稿日期:2015-06-24;編輯:段佳)

【中圖分類號(hào)】R654.2

【文獻(xiàn)標(biāo)識(shí)碼】A

doi:10.3969/j.issn.1672-8467.2016.02.015

國(guó)家自然科學(xué)基金面上項(xiàng)目(81373874,81472218)

▲Co-first authors

△Corresponding authorE-mail:liuzhong69@sohu.com