建立小鼠腹部心脏移植模型联合尾静脉注射的实践体会(附视频)
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摘要
目的总结建立稳定的小鼠腹部心脏移植模型联合尾静脉注射的实践体会。方法实验练习包括50对供、受体为昆明小鼠的同系移植,40对供、受体为C57BL/6J小鼠的同系移植;正式实验包括10对供、受体为C57BL/6J小鼠的同系移植,30对以Balb/c小鼠为供体、C57BL/6J小鼠为受体的同种异系移植。记录手术过程中每个步骤的时间(包括供体心脏摘取和修整时间、受体血管吻合等)。术后每日观察移植心脏搏动持续时间及受体存活时间,同时记录移植小鼠的尾静脉注射所需时间。同系移植术后30 d、同种异系移植术后7 d行移植心脏病理学检查(各5只)。结果正式实验心脏移植成功率为90%。供体心脏摘取和修整时间为(13.9±0.6)min,受体冷缺血时间为(14.2±1.2)min,血管吻合时间为(34.2±3.1)min,总手术时间为(86.6±5.4)min,术后同系移植小鼠移植心脏存活时间达到100 d以上,术后30 d病理学检查显示仅有少量炎症细胞浸润。同种异系移植小鼠因发生排斥反应,存活时间为(7.2±0.5)d。术后7 d病理学检查示心肌大量炎症细胞浸润,呈急性细胞性排斥反应表现。尾静脉注射超过200次后,成功率达90%。结论本研究成功建立了稳定的小鼠腹部心脏移植模型。同时进行尾静脉注射可有效地利用预实验的小鼠。
Objective To summarize the practice experience of establishing a stable abdominal heart transplantation model combined with tail vein injection in mice. Methods In the preliminary experiment, 50 pairs of donor and recipient Kunming mice received isotransplantation, 40 pairs of donor and recipient C57 BL/6 J mice underwent isotransplantation. In the formal experiment, 10 pairs of donor and recipient C57 BL/6 J mice received isotransplantation,30 pairs of Balb/c mice as the donor and C57 BL/6 J mice as the recipient received allotransplantation. The time of each step of the heart transplantation(including harvesting and dressing of the donor heart, vascular anastomosis of the recipient,etc.) was recorded. The duration of transplanted heart beat and the survival time of the recipient was observed daily after operation. The time required for tail vein injection in the transplanted mice was recorded. Pathological examination of the transplanted heart was performed at 30 d after isotransplantation(n=5) and 7 d after allotransplantation(n=5). Results In the formal experiment, the success rate of heart transplantation was 90%. The harvesting and dressing time of donor heart was(13.9±0.6) min. The cold ischemia time of the recipient was(14.2±1.2) min. The vascular anastomosis time was(34.2±3.1) min. The total operation time was(86.6±5.4) min. Postoperatively, the transplanted heart of the mice undergoing isotransplantation survived longer than 100 d. Pathological examination at postoperative 30 d demonstrated only a slight amount of inflammatory cell infiltration. The survival time of the mice receiving allotransplantation was(7.2±0.5) d due to rejection reaction. At postoperative 7 d, pathological examination showed a large quantity of in?ammatory cells in?ltrating into the myocardium, manifested with acute cellular rejection. The success rate reached90% after over 200 times of tail vein injection. Conclusions In this study, a stable mouse abdominal heart transplantation model is successfully established. The mouse models in the preliminary experiment can be utilized for tail vein injection.
Objective To summarize the practice experience of establishing a stable abdominal heart transplantation model combined with tail vein injection in mice. Methods In the preliminary experiment, 50 pairs of donor and recipient Kunming mice received isotransplantation, 40 pairs of donor and recipient C57 BL/6 J mice underwent isotransplantation. In the formal experiment, 10 pairs of donor and recipient C57 BL/6 J mice received isotransplantation,30 pairs of Balb/c mice as the donor and C57 BL/6 J mice as the recipient received allotransplantation. The time of each step of the heart transplantation(including harvesting and dressing of the donor heart, vascular anastomosis of the recipient,etc.) was recorded. The duration of transplanted heart beat and the survival time of the recipient was observed daily after operation. The time required for tail vein injection in the transplanted mice was recorded. Pathological examination of the transplanted heart was performed at 30 d after isotransplantation(n=5) and 7 d after allotransplantation(n=5). Results In the formal experiment, the success rate of heart transplantation was 90%. The harvesting and dressing time of donor heart was(13.9±0.6) min. The cold ischemia time of the recipient was(14.2±1.2) min. The vascular anastomosis time was(34.2±3.1) min. The total operation time was(86.6±5.4) min. Postoperatively, the transplanted heart of the mice undergoing isotransplantation survived longer than 100 d. Pathological examination at postoperative 30 d demonstrated only a slight amount of inflammatory cell infiltration. The survival time of the mice receiving allotransplantation was(7.2±0.5) d due to rejection reaction. At postoperative 7 d, pathological examination showed a large quantity of in?ammatory cells in?ltrating into the myocardium, manifested with acute cellular rejection. The success rate reached90% after over 200 times of tail vein injection. Conclusions In this study, a stable mouse abdominal heart transplantation model is successfully established. The mouse models in the preliminary experiment can be utilized for tail vein injection.
引文
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[6]林善昌,何巍,周绍昉,等.同种异位不停跳心脏移植模型的建立[J].广西医科大学学报,2008,25(1):75-77.DOI:10.3969/j.issn.1005-930X.2008.01.030.LIN SC,HE W,ZHOU SF,et al.Establishment of allogeneic non-stop beating heart transplantation model[J].J Guangxi Med Univ,2008,25(1):75-77.DOI:10.3969/j.issn.1005-930X.2008.01.030.
[7]陈义发,尹辉,梁宾勇,等.两种异位心脏移植小鼠模型的建立和比较[J].中国实验动物学报,2010,18(1):17-20.CHEN YF,YIN H,LIANG BY,et al.Establishment and comparison of two mouse models of celiac and cervical heterotopic heart transplantation[J].Acta Lab Anim Sci Sin,2010,18(1):17-20.
[8]HASEGAWA T,VISOVATTI SH,HYMAN MC,et al.Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy[J].Nat Protoc,2007,2(3):471-480.
[9]NIIMI M.The technique for heterotopic cardiac transplantation in mice:experience of 3000 operations by one surgeon[J].J Heart Lung Transplant,2001,20(10):1123-1128.
[10]BAXTER MG,MURPHY KL,TAYLOR PM,et al.Chloral hydrate is not acceptable for anesthesia or euthanasia of small animals[J].Anesthesiology,2009,111(1):209-210.DOI:10.1097/ALN.0b013e3181a8617e.
[11]SHEPPARD AM,ZHAO DL,SALVI R.Isoflurane anesthesia suppresses distortion product otoacoustic emissions in rats[J].J Otol,2018,13(2):59-64.DOI:10.1016/j.joto.2018.03.002.
[12]PLENTER RJ,GRAZIA TJ.Murine heterotopic heart transplant technique[J].J Vis Exp,2014(89).DOI:10.3791/51511.
[13]崔得利,谭长军,刘忠.连续缝合间断打结吻合动脉在小鼠心脏移植中的应用[J].复旦学报(医学版),2016,43(2):211-214.DOI:10.3969/j.issn.1672-8467.2016.02.015.CUI DL,TAN CJ,LIU Z.The continuous-interrupted method for arterial anastomosis in mouse heart transplantation[J].Fudan Univ J Med Sci,2016,43(2):211-214.DOI:10.3969/j.issn.1672-8467.2016.02.015.
[14]李轶倞,张娜,田枫.一种简易小鼠尾静脉注射固定法--笼盖压制法[J].中国比较医学杂志,2016,26(10):79-81.DOI:10.3969.j.issn.1671-7856.2016.10.015.LI YL,ZHANG N,TIAN F.A simple fixing method for tail vein injection in mice-cage lid pressing method[J].Chin J Comp Med,2016,26(10):79-81.DOI:10.3969.j.issn.1671-7856.2016.10.015.
[2]周结学,刘东,吴家清,等.儿童心脏死亡器官捐献供者单肾成人肾移植的疗效[J].实用医学杂志,2017,33(1):14-17.DOI:10.3969/j.issn.1006-5725.2017.01.004.ZHOU JX,LIU D,WU JQ,et al.Effect of single kidney transplantation from cardiac death pediatric donors to adult recipients[J].J Pract Med,2017,33(1):14-17.DOI:10.3969/j.issn.1006-5725.2017.01.004.
[3]李巍.小鼠动物模型在移植免疫研究中的重要地位与应用[J].器官移植,2017,8(3):186-189.DOI:10.3969/j.issn.1674-7445.2017.03.002.LI W.Importance and application of mouse organ transplantation models for transplant immunology research[J].Organ Transplant,2017,8(3):186-189.DOI:10.3969/j.issn.1674-7445.2017.03.002.
[4]PLENTER RJ,ZAMORA MR,GRAZIA TJ.Four decades of vascularized heterotopic cardiac transplantation in the mouse[J].J Invest Surg,2013,26(4):223-228.DOI:10.3109/08941939.2012.755238.
[5]BENKE K,SAYOUR AA,MáTYáS C,et al.Heterotopic abdominal rat heart transplantation as a model to investigate volume dependency of myocardial remodeling[J].Transplantation,2017,101(3):498-505.DOI:10.1097/TP.0000000000001585.
[6]林善昌,何巍,周绍昉,等.同种异位不停跳心脏移植模型的建立[J].广西医科大学学报,2008,25(1):75-77.DOI:10.3969/j.issn.1005-930X.2008.01.030.LIN SC,HE W,ZHOU SF,et al.Establishment of allogeneic non-stop beating heart transplantation model[J].J Guangxi Med Univ,2008,25(1):75-77.DOI:10.3969/j.issn.1005-930X.2008.01.030.
[7]陈义发,尹辉,梁宾勇,等.两种异位心脏移植小鼠模型的建立和比较[J].中国实验动物学报,2010,18(1):17-20.CHEN YF,YIN H,LIANG BY,et al.Establishment and comparison of two mouse models of celiac and cervical heterotopic heart transplantation[J].Acta Lab Anim Sci Sin,2010,18(1):17-20.
[8]HASEGAWA T,VISOVATTI SH,HYMAN MC,et al.Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy[J].Nat Protoc,2007,2(3):471-480.
[9]NIIMI M.The technique for heterotopic cardiac transplantation in mice:experience of 3000 operations by one surgeon[J].J Heart Lung Transplant,2001,20(10):1123-1128.
[10]BAXTER MG,MURPHY KL,TAYLOR PM,et al.Chloral hydrate is not acceptable for anesthesia or euthanasia of small animals[J].Anesthesiology,2009,111(1):209-210.DOI:10.1097/ALN.0b013e3181a8617e.
[11]SHEPPARD AM,ZHAO DL,SALVI R.Isoflurane anesthesia suppresses distortion product otoacoustic emissions in rats[J].J Otol,2018,13(2):59-64.DOI:10.1016/j.joto.2018.03.002.
[12]PLENTER RJ,GRAZIA TJ.Murine heterotopic heart transplant technique[J].J Vis Exp,2014(89).DOI:10.3791/51511.
[13]崔得利,谭长军,刘忠.连续缝合间断打结吻合动脉在小鼠心脏移植中的应用[J].复旦学报(医学版),2016,43(2):211-214.DOI:10.3969/j.issn.1672-8467.2016.02.015.CUI DL,TAN CJ,LIU Z.The continuous-interrupted method for arterial anastomosis in mouse heart transplantation[J].Fudan Univ J Med Sci,2016,43(2):211-214.DOI:10.3969/j.issn.1672-8467.2016.02.015.
[14]李轶倞,张娜,田枫.一种简易小鼠尾静脉注射固定法--笼盖压制法[J].中国比较医学杂志,2016,26(10):79-81.DOI:10.3969.j.issn.1671-7856.2016.10.015.LI YL,ZHANG N,TIAN F.A simple fixing method for tail vein injection in mice-cage lid pressing method[J].Chin J Comp Med,2016,26(10):79-81.DOI:10.3969.j.issn.1671-7856.2016.10.015.