白细胞靶向心肌超声造影技术评价心脏移植后急性排斥反应的研究

Evaluation of acute cardiac transplant rejection with targeted myocardial contrast echocardiography

目的 探讨白细胞靶向心肌超声造影技术对心脏移植术后急性排斥反应及其程度的诊断价值.方法 将实验大鼠随机分为4组,行腹腔异位心脏移植术.A组为同系移植,供、受者均采用健康雄性SD大鼠;B、C、D组为同种移植,供者采用健康雄性SD大鼠,受者采用健康雄性Wistar大鼠.B、C组分别于移植前3 d起每天腹腔注射环孢素A（CsA）10 mg·kg-1·d-1和3 mg·kg-1·d-1,A、D组不给予CsA治疗.每组抽取成功建立腹部异位心脏移植模型的受者各8只.术后第3天,将声诺维造影剂经颈内静脉持续注入大鼠体内,应用心肌超声造影技术观察移植心脏,分别获取注入造影剂20 s时的心肌灌注造影图像和5 min时的白细胞靶向心肌造影图像,利用图像分析仪分别测定每只受者的造影图像灰阶值（GS20s、GS5m）和靶向灰阶值（Gstarget,为GS5m与GS20s之差）.造影观察完毕后处死大鼠,行病理学检查,HE染色确定移植心肌排斥反应程度;免疫组织化学法检测移植心肌组织内CD3＋T淋巴细胞浸润程度,并将其分别与各组的GS20s、GS5m和Gstatget做相关性分析.结果 心肌灌注造影显示各组GS20s之间无明显差异（P＞0.05）;白细胞靶向造影显示各组GS5m之间存在梯度,差异有统计学意义（P＜0.05）;且各组Gstarget之间的差异也有统计学意义（P＜0.01）.移植心肌组织病理学检测显示：A组为0～Ⅰ级排斥反应,B组为Ⅰ～Ⅱ级排斥反应,C组为Ⅱ～Ⅲ级排斥反应,D组为Ⅲ～Ⅳ级排斥反应.免疫组织化学检测显示：从A组到D组,移植心肌组织中CD3＋T淋巴细胞计数逐渐增多.相关性分析显示：CD3＋T淋巴细胞计数与Gstarget呈正相关（r=0.86,P＜0.001）,与GS5m有相关性,与GS20s无相关性.结论 白细胞靶向心肌超声造影技术能无创、有效的评价心脏移植术后的急性排斥反应程度.

Objective To discuss the value of leukocyte-targeted myocardial contrast echocardiography （MCE） as a tool in observing the degree of acute rejection after heart transplantation. Methods Abdominal heterotopic cardiac transplantation was performed on 32 rats successfully, among which 8 isografts served as group A, and groups B, C and D involved 8 allografts respectively. The rats in groups B and C were treated with cyclosporine A （CsA） at a high dose （10mg· kg-1 · day-1 ）, a low dose （3 mg · kg-1 · day-1 ） from 3rd day before transplantation respectively.The rats in groups A and D were untreated with CsA. MCE was performed during continuous intravenous SonoVue injection postoperatively on the third day after operation. We performed 2 types of MCE： perfusion imaging and leukocyte-targeted imaging. The images were obtained at 20 s and 5 min after injection of contrast agent. The value of the contrast image grayscale （GS） was measured by image analyzer （GS20s, GS5 min）. GStarget was calculated as the GS5min minus the GS20s in the same rat.Postmortem histology was performed after observation. The degree of myocardial rejection was determined by HE-stained graft myocardium. Immunohistochemistry was performed to quantify the CD3-positive cells, and correlation analysis was performed between CD3-positive cell count and GS20s,GS5min, GStarget. Results Perfusion imaging showed no significant difference in myocardial GS20s of each group. Leukocyte-Targeted imaging exhibited a clear gradient in these groups （P＜0. 05）. There was significant difference in GStarget of each group （P＜0. 001）. Postmortem histology showed 0- Ⅰ grade rejection in group A, Ⅰ -Ⅱ grade rejection in group B, Ⅱ-Ⅲ grade rejection in group C, Ⅲ-Ⅳ grade rejection in group D. Immunohistochemistry revealed the CD3-positive cell infiltration was increased in turn from the group A to the group D. There was a significantly positive correlation between the CD3-positive cell count and GStarget （ r = 0. 86, P ＜ 0. 001 ）. Conclusion Leukocyte-targeted contrast echocardiography may thus offer a noninvasive and effective ultrasound imaging technique for detecting the degree of acute cardiac transplant rejection.