細胞(bao)(bao)(bao)膜是由蛋白(bai)質與(yu)膽固醇等分(fen)(fen)子(zi)鑲嵌在(zai)磷(lin)脂中的典型雙(shuang)(shuang)分(fen)(fen)子(zi)層(ceng)結構。細胞(bao)(bao)(bao)膜仿(fang)生(sheng)(sheng)技術已經成(cheng)功實(shi)(shi)現在(zai)納米(mi)顆(ke)粒(li)以(yi)及(ji)納米(mi)液滴表面(mian)修飾組裝，并實(shi)(shi)現體內的長循環以(yi)及(ji)良好生(sheng)(sheng)物(wu)相容性。典型的細胞(bao)(bao)(bao)膜仿(fang)生(sheng)(sheng)技術是將剝離后的細胞(bao)(bao)(bao)膜直接以(yi)磷(lin)脂雙(shuang)(shuang)分(fen)(fen)子(zi)層(ceng)的形式包被(bei)于(yu)納米(mi)顆(ke)粒(li)或液滴表面(mian)。然而由于(yu)表面(mian)張力作用，細胞(bao)(bao)(bao)膜的磷(lin)脂雙(shuang)(shuang)分(fen)(fen)子(zi)層(ceng)無法直接包被(bei)在(zai)微米(mi)級的氣泡表面(mian)。

本發明采用(yong)反復(fu)液氮冷(leng)凍(dong)(dong)(dong)(dong)-室溫融(rong)(rong)(rong)(rong)化(hua)的(de)(de)方式向(xiang)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)中(zhong)摻雜(za)磷(lin)脂并得到血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)囊(nang)泡(pao)。通(tong)(tong)過(guo)Langmuir-Blodgett膜(mo)(mo)(mo)(mo)天平(ping)測試得到凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)中(zhong)的(de)(de)磷(lin)脂蛋(dan)白質(zhi)分(fen)子在(zai)氣液界面的(de)(de)表面張(zhang)力(li)顯著降低(di)，通(tong)(tong)過(guo)差(cha)適量熱掃描技術(shu)測試表明摻雜(za)后的(de)(de)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)玻璃(li)態轉變溫度降低(di)。利用(yong)超(chao)聲輔助的(de)(de)方式向(xiang)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)囊(nang)泡(pao)中(zhong)鼓入六氟化(hua)硫氣體，在(zai)氣液界面形(xing)成血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)微泡(pao)。通(tong)(tong)過(guo)熒光標記的(de)(de)方式定位(wei)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)蛋(dan)白，并成功示(shi)蹤其(qi)融(rong)(rong)(rong)(rong)合(he)在(zai)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)微泡(pao)。通(tong)(tong)過(guo)對(dui)血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)囊(nang)泡(pao)以及血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)微泡(pao)進行(xing)蛋(dan)白質(zhi)組學(xue)分(fen)析，發現血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)微泡(pao)繼承了血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)囊(nang)泡(pao)61.4%的(de)(de)蛋(dan)白質(zhi)種類，并維持了血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)表面的(de)(de)整合(he)素αIIβ3活(huo)化(hua)的(de)(de)構象。在(zai)大鼠下腔靜(jing)脈(mo)急(ji)慢(man)性血(xue)栓模型實驗中(zhong)，血(xue)小(xiao)(xiao)(xiao)(xiao)板(ban)(ban)(ban)(ban)(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)合(he)物(wu)(wu)微泡(pao)能夠特異性識別急(ji)性血(xue)栓，其(qi)對(dui)于急(ji)性血(xue)栓診斷平(ping)均(jun)信噪比為(wei)12.47 dB，而慢(man)性血(xue)栓為(wei)0.1dB。

細胞膜表面張力調控方(fang)法示意圖；

不同磷脂配方的脂質體對氣液界面表面張力調控效應的表面壓(ya)力與面積等溫(wen)曲線圖；

不(bu)(bu)同蛋(dan)白質(zhi)與(yu)(yu)(yu)磷脂質(zhi)量(liang)比對氣液界面(mian)(mian)(mian)(mian)表(biao)面(mian)(mian)(mian)(mian)張(zhang)力(li)調控(kong)效應圖(tu)：A為牛血清白蛋(dan)白與(yu)(yu)(yu)磷脂在氣液界面(mian)(mian)(mian)(mian)組裝(zhuang)的示意圖(tu)；B為不(bu)(bu)同蛋(dan)白質(zhi)與(yu)(yu)(yu)磷脂質(zhi)量(liang)比的氣液界面(mian)(mian)(mian)(mian)表(biao)面(mian)(mian)(mian)(mian)壓力(li)與(yu)(yu)(yu)面(mian)(mian)(mian)(mian)積等(deng)溫曲線圖(tu)；C為不(bu)(bu)同蛋(dan)白質(zhi)與(yu)(yu)(yu)磷脂質(zhi)量(liang)比的表(biao)面(mian)(mian)(mian)(mian)張(zhang)力(li)值；

實施例：

1、血小板膜的提取：

首先從人、大鼠(shu)、小鼠(shu)、兔、牛、豬的(de)全血中(zhong)選(xuan)取(qu)血小板，所選(xuan)的(de)磷(lin)(lin)脂(zhi)為二(er)棕櫚酸磷(lin)(lin)脂(zhi)酰(xian)膽堿、二(er)硬(ying)脂(zhi)酸磷(lin)(lin)脂(zhi)酰(xian)乙胺醇-聚乙二(er)醇2000、硬(ying)脂(zhi)酸、二(er)硬(ying)脂(zhi)酸磷(lin)(lin)脂(zhi)甘油的(de)組合；然后，在全血提取(qu)出(chu)的(de)血小板中(zhong)，加入終(zhong)濃(nong)度(du)為42 mM的(de)甘露糖、終(zhong)濃(nong)度(du)為1μM的(de)前(qian)列(lie)腺素(su)E1，和磷(lin)(lin)酸酶蛋白酶抑制劑混合物，得到濃(nong)度(du)為2×108

個/mL的血(xue)小(xiao)板(ban)懸(xuan)液(ye)。將血(xue)小(xiao)板(ban)懸(xuan)液(ye)在-80°C冰箱中(zhong)(zhong)冷(leng)凍1小(xiao)時，隨(sui)后(hou)在37°C恒溫水浴(yu)箱中(zhong)(zhong)融(rong)化，反(fan)復三(san)次后(hou)于4°C，12000g條(tiao)件下離(li)心15分鐘得到血(xue)小(xiao)板(ban)膜懸(xuan)液(ye)，并用BCA蛋(dan)白(bai)定(ding)量(liang)(liang)試(shi)劑(ji)盒定(ding)量(liang)(liang)蛋(dan)白(bai)質(zhi)濃度(du)，用磷脂試(shi)劑(ji)盒定(ding)量(liang)(liang)磷脂濃度(du)。

2、磷脂的制備：

磷(lin)(lin)脂(zhi)(zhi)(zhi)按照二棕櫚酸(suan)磷(lin)(lin)脂(zhi)(zhi)(zhi)酰(xian)(xian)膽堿、二硬(ying)脂(zhi)(zhi)(zhi)酸(suan)磷(lin)(lin)脂(zhi)(zhi)(zhi)酰(xian)(xian)乙胺醇(chun)(chun)-聚乙二醇(chun)(chun)2000、硬(ying)脂(zhi)(zhi)(zhi)酸(suan)、二硬(ying)脂(zhi)(zhi)(zhi)酸(suan)磷(lin)(lin)脂(zhi)(zhi)(zhi)甘油(you)的以一(yi)(yi)系列(lie)質(zhi)量比(bi)例溶(rong)解在體積比(bi)為1：1的氯仿(fang)和甲(jia)醇(chun)(chun)中(zhong)(zhong)，于(yu)50℃，100rpm的條件下旋(xuan)蒸直(zhi)到圓底燒(shao)瓶(ping)底部出現一(yi)(yi)層均勻的薄膜(mo)，取下燒(shao)瓶(ping)，真空干(gan)(gan)燥(zao)8小(xiao)(xiao)(xiao)時。往干(gan)(gan)燥(zao)后的燒(shao)瓶(ping)中(zhong)(zhong)加(jia)入磷(lin)(lin)酸(suan)緩(huan)沖液并在50℃、100 w、53 kHZ的條件下水浴超聲得(de)(de)到脂(zhi)(zhi)(zhi)質(zhi)體膜(mo)溶(rong)液，并調節脂(zhi)(zhi)(zhi)質(zhi)體膜(mo)溶(rong)液的PH為7.4。將血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)與脂(zhi)(zhi)(zhi)質(zhi)體在不同(tong)的蛋(dan)白(bai)質(zhi)磷(lin)(lin)脂(zhi)(zhi)(zhi)質(zhi)量比(bi)例下進行混合。將混合物(wu)在液氮和室溫下反復凍融6次(ci)得(de)(de)到血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)凍融復合物(wu)。將血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)或血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)凍融復合物(wu)轉移到置于(yu)冰上的10 mL玻璃瓶(ping)中(zhong)(zhong)，將六氟(fu)化硫氣(qi)體吹入系統(tong)，用探針超聲處理（300 W，5 s開/5 s關）將六氟(fu)化硫氣(qi)體分(fen)(fen)散到水性介質(zhi)中(zhong)(zhong)4個循(xun)環分(fen)(fen)別得(de)(de)到血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)納(na)泡以及(ji)血小(xiao)(xiao)(xiao)板(ban)(ban)(ban)膜(mo)凍融復合物(wu)微泡。

3、蛋白質磷(lin)脂復合物(wu)氣液(ye)界面張力(li)調控：

采(cai)用Langmuir-Blodgett多功能膜天平測量所制備磷脂復合物在氣液界面處的表面張(zhang)力。用微量注射(she)器將20μL磷脂（1mg/mL脂濃(nong)度）氯(lv)(lv)仿溶液涂(tu)在Langmuir-Blodgett多功能膜天平上，使氯(lv)(lv)仿蒸發10分(fen)鐘。將表面的初始面積設置為(wei)282 cm2

，并在(zai)(zai)20℃下以10 mm/min的速率壓縮薄膜，記錄(lu)表(biao)面張力(li)-表(biao)面積曲(qu)線。如圖(tu)2所示(shi)在(zai)(zai)一系列(lie)磷脂配方中，DSPE-PEG2000

的摩爾百分(fen)比為10%時(shi)在氣(qi)液界面具有最低的表面張(zhang)力（3mN/m），DPPC：DPPG：DSPE-PEG2000

的(de)比(bi)例(li)為8：1：1的(de)時候氣(qi)液界(jie)(jie)面的(de)的(de)表面壓力(li)達到69mN/m，因(yin)此(ci)具有最低的(de)氣(qi)液界(jie)(jie)面表面張(zhang)力(li)（2mN/m）。為了探究蛋(dan)(dan)(dan)白質(zhi)與(yu)磷(lin)脂(zhi)在(zai)氣(qi)液界(jie)(jie)面組(zu)裝的(de)規律(lv)，如圖3中的(de)A所示，選用(yong)牛(niu)血清(qing)白蛋(dan)(dan)(dan)白（BSA）作(zuo)為標準蛋(dan)(dan)(dan)白質(zhi)，配置(zhi)好一系列(lie)不同蛋(dan)(dan)(dan)白質(zhi)與(yu)磷(lin)脂(zhi)質(zhi)量比(bi)的(de)磷(lin)脂(zhi)蛋(dan)(dan)(dan)白質(zhi)復(fu)合物，并用(yong)微量注射器將(jiang)20μL磷(lin)脂(zhi)蛋(dan)(dan)(dan)白質(zhi)復(fu)合物（1mg/mL脂(zhi)濃度(du)）氯仿溶液涂在(zai)Langmuir-Blodgett多功能膜天(tian)平上(shang)，使(shi)氯仿蒸發10分鐘。將(jiang)表面的(de)初始(shi)面積設(she)置(zhi)為282cm2

，并在(zai)20℃下(xia)以10 mm/min的(de)(de)速率(lv)壓縮薄膜，記錄表面(mian)(mian)張(zhang)力-表面(mian)(mian)積曲線。如圖3中(zhong)的(de)(de)B以及C所示在(zai)一系列具有(you)(you)不(bu)同質(zhi)量(liang)(liang)比(bi)的(de)(de)磷(lin)脂(zhi)蛋白(bai)質(zhi)復合物中(zhong)當蛋白(bai)質(zhi)比(bi)磷(lin)脂(zhi)的(de)(de)質(zhi)量(liang)(liang)比(bi)為0.02時(shi)具有(you)(you)最(zui)低的(de)(de)氣(qi)液界面(mian)(mian)表面(mian)(mian)張(zhang)力。

4、血小板(ban)膜-磷(lin)脂融合驗證：

分(fen)(fen)別用(yong)(yong)過量的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)DiO和(he)DiI對血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)囊(nang)泡(pao)(pao)(pao)(pao)和(he)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)進行(xing)(xing)染(ran)(ran)色(se)，隨(sui)后(hou)(hou)用(yong)(yong)12000 g，15min，4℃的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)條(tiao)件去(qu)除未嵌(qian)膜(mo)(mo)(mo)(mo)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)染(ran)(ran)料(liao)。按照(zhao)不同(tong)(tong)蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)：磷(lin)(lin)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)添加(jia)兩(liang)種(zhong)膜(mo)(mo)(mo)(mo)材并(bing)在37℃恒溫箱中(zhong)(zhong)(zhong)共孵育30分(fen)(fen)鐘(zhong)(zhong)(zhong)，取出后(hou)(hou)在液(ye)(ye)氮中(zhong)(zhong)(zhong)放(fang)置(zhi)1分(fen)(fen)鐘(zhong)(zhong)(zhong)，隨(sui)后(hou)(hou)放(fang)置(zhi)于37℃融(rong)(rong)(rong)(rong)化，重(zhong)復(fu)(fu)該循(xun)(xun)環5次。凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)循(xun)(xun)環后(hou)(hou)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)溶液(ye)(ye)置(zhi)于冰上(shang)，用(yong)(yong)功率為10 w，頻率為25 Khz的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)探(tan)頭超聲，控制超聲5秒(miao)開時(shi)(shi)(shi)間，5秒(miao)關(guan)時(shi)(shi)(shi)間，總時(shi)(shi)(shi)間為2分(fen)(fen)鐘(zhong)(zhong)(zhong)，得到熒光(guang)(guang)標記血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)囊(nang)泡(pao)(pao)(pao)(pao)。在3 ml的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)石英比(bi)(bi)(bi)(bi)色(se)皿加(jia)入2 ml水和(he)20 ul樣(yang)品，用(yong)(yong)460 nm波長的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)激(ji)光(guang)(guang)作為激(ji)發(fa)光(guang)(guang)，接收480-680 nm的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)發(fa)射光(guang)(guang)。如(ru)圖(tu)(tu)(tu)4中(zhong)(zhong)(zhong)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)A所(suo)示(shi)(shi)(shi)由于DiI與(yu)DiO分(fen)(fen)子(zi)在距離小(xiao)(xiao)于10 nm時(shi)(shi)(shi)會發(fa)生Foster熒光(guang)(guang)共振能量轉(zhuan)移（FRET）現(xian)象，嵌(qian)在磷(lin)(lin)脂(zhi)(zhi)(zhi)雙分(fen)(fen)子(zi)層中(zhong)(zhong)(zhong)疏(shu)水端的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)分(fen)(fen)子(zi)彼此接近(jin)且(qie)小(xiao)(xiao)于10nm可以(yi)(yi)證明血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)與(yu)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)磷(lin)(lin)脂(zhi)(zhi)(zhi)進行(xing)(xing)了相互的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)融(rong)(rong)(rong)(rong)合(he)。如(ru)圖(tu)(tu)(tu)4中(zhong)(zhong)(zhong)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)B所(suo)示(shi)(shi)(shi)，隨(sui)著(zhu)蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)與(yu)磷(lin)(lin)脂(zhi)(zhi)(zhi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)降(jiang)低(di)(di)熒光(guang)(guang)共振轉(zhuan)移現(xian)象逐漸明顯，說明膜(mo)(mo)(mo)(mo)融(rong)(rong)(rong)(rong)合(he)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)程度增(zeng)大。分(fen)(fen)別用(yong)(yong)過量的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)DiO和(he)DiD對血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)囊(nang)泡(pao)(pao)(pao)(pao)和(he)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)進行(xing)(xing)染(ran)(ran)色(se)，隨(sui)后(hou)(hou)用(yong)(yong)12000 g，15 min，4℃的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)條(tiao)件去(qu)除未嵌(qian)膜(mo)(mo)(mo)(mo)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)染(ran)(ran)料(liao)。將染(ran)(ran)色(se)后(hou)(hou)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)囊(nang)泡(pao)(pao)(pao)(pao)以(yi)(yi)及(ji)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)在蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)與(yu)磷(lin)(lin)脂(zhi)(zhi)(zhi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)為0.02時(shi)(shi)(shi)分(fen)(fen)別通(tong)過混合(he)孵育以(yi)(yi)及(ji)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)兩(liang)種(zhong)方式進行(xing)(xing)融(rong)(rong)(rong)(rong)合(he)，通(tong)過流式細胞(bao)術來觀察兩(liang)者融(rong)(rong)(rong)(rong)合(he)效(xiao)率，如(ru)圖(tu)(tu)(tu)4中(zhong)(zhong)(zhong)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)C所(suo)示(shi)(shi)(shi)，當(dang)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)囊(nang)泡(pao)(pao)(pao)(pao)與(yu)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)形成凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)可以(yi)(yi)有(you)效(xiao)融(rong)(rong)(rong)(rong)合(he)兩(liang)種(zhong)組(zu)(zu)分(fen)(fen)。取不同(tong)(tong)膜(mo)(mo)(mo)(mo)蛋(dan)(dan)白(bai)：脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)體(ti)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)微泡(pao)(pao)(pao)(pao)進行(xing)(xing)凍(dong)(dong)(dong)(dong)干(gan)，凍(dong)(dong)(dong)(dong)干(gan)粉進行(xing)(xing)差示(shi)(shi)(shi)量熱掃描分(fen)(fen)析，掃描速(su)度為5℃/min。如(ru)圖(tu)(tu)(tu)5所(suo)示(shi)(shi)(shi)，隨(sui)著(zhu)摻(chan)(chan)雜磷(lin)(lin)脂(zhi)(zhi)(zhi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)比(bi)(bi)(bi)(bi)例增(zeng)加(jia)，血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)玻璃態轉(zhuan)變溫度降(jiang)低(di)(di)，在蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)與(yu)磷(lin)(lin)脂(zhi)(zhi)(zhi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)為0.02時(shi)(shi)(shi)候具有(you)最低(di)(di)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)玻璃態轉(zhuan)變溫度。血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)氣液(ye)(ye)界面(mian)(mian)張力(li)調控。使用(yong)(yong)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)方法向(xiang)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)中(zhong)(zhong)(zhong)摻(chan)(chan)雜不同(tong)(tong)比(bi)(bi)(bi)(bi)例的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)磷(lin)(lin)脂(zhi)(zhi)(zhi)，形成凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)，其(qi)組(zu)(zu)分(fen)(fen)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)構成如(ru)圖(tu)(tu)(tu)6所(suo)示(shi)(shi)(shi)。向(xiang)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)中(zhong)(zhong)(zhong)摻(chan)(chan)雜不同(tong)(tong)比(bi)(bi)(bi)(bi)例的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)磷(lin)(lin)脂(zhi)(zhi)(zhi)，形成的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)在氣液(ye)(ye)界面(mian)(mian)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)表(biao)面(mian)(mian)張力(li)隨(sui)著(zhu)蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)與(yu)磷(lin)(lin)脂(zhi)(zhi)(zhi)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)降(jiang)低(di)(di)而降(jiang)低(di)(di)，如(ru)圖(tu)(tu)(tu)7所(suo)示(shi)(shi)(shi)血(xue)(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)(mo)凍(dong)(dong)(dong)(dong)融(rong)(rong)(rong)(rong)復(fu)(fu)合(he)物(wu)(wu)在蛋(dan)(dan)白(bai)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)與(yu)磷(lin)(lin)脂(zhi)(zhi)(zhi)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)質(zhi)(zhi)(zhi)(zhi)(zhi)(zhi)量比(bi)(bi)(bi)(bi)為0.02時(shi)(shi)(shi)具有(you)最低(di)(di)的(de)(de)(de)(de)(de)(de)(de)(de)(de)(de)氣液(ye)(ye)界面(mian)(mian)表(biao)面(mian)(mian)張力(li)。

5、血小板膜氣液界面組(zu)裝組(zu)分的定(ding)位：

血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)用(yong)Cy5.5進行蛋白標記，用(yong)于(yu)在(zai)(zai)氣液界面組裝過程中進行組分定位。將血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)、血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍融復合物或(huo)血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)和脂(zhi)質(zhi)體(ti)的混合物在(zai)(zai)SF6

氣氛中(zhong)(zhong)超(chao)聲乳化。得(de)(de)到的(de)(de)氣泡(pao)用于流式細胞術分(fen)(fen)析，在FSC/SSC門控中(zhong)(zhong)分(fen)(fen)離所(suo)得(de)(de)氣泡(pao)，并定(ding)量(liang)Q2和Q4區域的(de)(de)Cy5.5熒(ying)光(guang)，如圖(tu)8所(suo)示，相對(dui)(dui)于血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)與脂質體的(de)(de)混(hun)合物(wu)使用血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)凍融復合物(wu)可以(yi)有效將血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)組(zu)分(fen)(fen)組(zu)裝在微米氣泡(pao)上。對(dui)(dui)于熒(ying)光(guang)定(ding)位(wei)分(fen)(fen)析，用Cy5.5標記(ji)血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)組(zu)分(fen)(fen)進行定(ding)向。將所(suo)有囊泡(pao)系統在超(chao)聲處理前(qian)以(yi)12000g離心10分(fen)(fen)鐘，超(chao)聲處理后以(yi)50g離心10分(fen)(fen)鐘，并通過小(xiao)(xiao)動物(wu)體內成像系統（IVIS）成像以(yi)檢(jian)(jian)測(ce)(ce)Cy5.5信(xin)號。超(chao)聲處理后，通過Zetasizer測(ce)(ce)量(liang)原始(shi)混(hun)合物(wu)和離心管中(zhong)(zhong)頂部(bu)或向下部(bu)分(fen)(fen)的(de)(de)粒徑。如圖(tu)9所(suo)示，使用血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)凍融復合物(wu)制備的(de)(de)微泡(pao)中(zhong)(zhong)檢(jian)(jian)測(ce)(ce)到標記(ji)血(xue)小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)的(de)(de)熒(ying)光(guang)分(fen)(fen)子(zi)Cy5.5。

6、制備過程產物形貌及(ji)電位表征：

采用透射電(dian)(dian)子(zi)(zi)顯(xian)微鏡對血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)、血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)、血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)納(na)泡和血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)微泡的(de)形(xing)貌進(jin)行表(biao)(biao)征。將(jiang)所(suo)(suo)有(you)(you)樣品稀釋至0.1 mg/mL（脂質(zhi)濃度(du)）。將(jiang)10μL的(de)血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)和血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)滴落到碳(tan)涂(tu)層銅(tong)(tong)網上(shang)。真空干燥15分鐘(zhong)后(hou)，用1%醋(cu)酸鈾(you)對樣品進(jin)行負染。將(jiang)10μL的(de)PNBs和PMBs在(zai)室溫下滴落在(zai)碳(tan)包(bao)覆(fu)的(de)銅(tong)(tong)網格上(shang)2 h，使其吸附到碳(tan)膜(mo)(mo)(mo)表(biao)(biao)面。使用透射電(dian)(dian)子(zi)(zi)顯(xian)微鏡在(zai)100 kV的(de)加(jia)速電(dian)(dian)壓下檢查所(suo)(suo)有(you)(you)樣品。如圖10所(suo)(suo)示(shi)，血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)（A）與血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)（C）具有(you)(you)典型(xing)(xing)的(de)磷脂雙分子(zi)(zi)層結構，而在(zai)血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)納(na)米泡（B）以及血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)微泡（D）具有(you)(you)典型(xing)(xing)的(de)單分子(zi)(zi)層氣(qi)泡結構。通過Zetasizer測(ce)量血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)、血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)、血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)納(na)泡和血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)微泡的(de)水合粒徑和zeta電(dian)(dian)位，如圖11所(suo)(suo)示(shi)，血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)與血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)納(na)泡具有(you)(you)相似的(de)zeta電(dian)(dian)位，在(zai)摻(chan)雜磷脂后(hou)血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)以及血小(xiao)(xiao)板(ban)(ban)膜(mo)(mo)(mo)凍(dong)(dong)融(rong)復合物(wu)微泡的(de)zeta電(dian)(dian)位有(you)(you)明顯(xian)降低(di)降低(di)為(wei)-47mV。

7、血小板膜(mo)凍融復合物微泡(pao)蛋白表征：

對制備(bei)的(de)(de)(de)血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)與(yu)血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡進行(xing)蛋(dan)白質(zhi)組(zu)學分(fen)(fen)析，如(ru)圖(tu)12所示，血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡繼承了血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)61.7%的(de)(de)(de)蛋(dan)白質(zhi)種類(lei)。將(jiang)制備(bei)的(de)(de)(de)血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡與(yu)FITC標(biao)記(ji)的(de)(de)(de)CD41抗體先渦旋混合(he)(he)(he)2分(fen)(fen)鐘(zhong)，再(zai)放于37°C中(zhong)緩慢攪拌孵育30分(fen)(fen)鐘(zhong)。平行(xing)組(zu)再(zai)用(yong)膜(mo)染料DiI非特異性地(di)染磷脂(zhi)，取出后在200g，2分(fen)(fen)鐘(zhong)的(de)(de)(de)離心(xin)條件將(jiang)微(wei)氣泡與(yu)未結合(he)(he)(he)的(de)(de)(de)抗體分(fen)(fen)離。取微(wei)氣泡稀釋20倍(bei)后滴(di)于帶有凹槽的(de)(de)(de)載玻(bo)片(pian)中(zhong)，再(zai)蓋上(shang)蓋玻(bo)片(pian)，分(fen)(fen)別(bie)用(yong)484nm和559nm的(de)(de)(de)激發(fa)光(guang)源拍(pai)攝共聚焦顯(xian)微(wei)鏡。如(ru)圖(tu)13所示，在血(xue)(xue)(xue)小板(ban)膜(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡中(zhong)來自血(xue)(xue)(xue)小板(ban)膜(mo)的(de)(de)(de)綠色(se)熒光(guang)與(yu)來自脂(zhi)質(zhi)體的(de)(de)(de)紅(hong)色(se)熒光(guang)有效共定位。

8、血小板膜凍融(rong)復合物微泡糖(tang)蛋白αIIbβ3蛋白質(zhi)構象(xiang)分(fen)析：

收集的血(xue)小板靜置在Tyrode緩沖液中（5×105

）。將血(xue)(xue)小(xiao)板(ban)(ban)或(huo)(huo)血(xue)(xue)小(xiao)板(ban)(ban)膜凍融(rong)復(fu)合(he)物(wu)微泡(pao)與(yu)生理鹽水或(huo)(huo)凝血(xue)(xue)酶（0.5 U/mL）在(zai)200μL終體積的(de)Tyrode緩沖液(ye)中(zhong)孵(fu)育(yu)15分(fen)鐘。然后(hou)用(yong)10μL PE-JON/A抗(kang)體在(zai)室溫下黑暗染(ran)色20 min，并在(zai)進行流式分(fen)析(xi)。免疫(yi)共沉淀實(shi)驗中(zhong)用(yong)0.5 U/mL凝血(xue)(xue)酶刺(ci)激(ji)小(xiao)鼠(shu)血(xue)(xue)小(xiao)板(ban)(ban)，加入裂解緩沖液(ye)終止反(fan)應(ying)。然后(hou)將活(huo)化(hua)的(de)血(xue)(xue)小(xiao)板(ban)(ban)裂解物(wu)、血(xue)(xue)小(xiao)板(ban)(ban)裂解物(wu)、血(xue)(xue)小(xiao)板(ban)(ban)膜凍融(rong)復(fu)合(he)物(wu)裂解物(wu)和血(xue)(xue)小(xiao)板(ban)(ban)膜凍融(rong)復(fu)合(he)物(wu)微泡(pao)裂解物(wu)與(yu)抗(kang)β3抗(kang)體（50μg/mL）在(zai)4°C下孵(fu)育(yu)過夜(ye)。次日加入rProtein A/G磁珠吸收免疫(yi)復(fu)合(he)物(wu)。將樣品在(zai)磁架(jia)上分(fen)離，并用(yong)裂解緩沖液(ye)洗(xi)滌6次。使用(yong)SDS-聚丙烯酰胺(an)凝膠電(dian)泳和含有β3和talin-1抗(kang)體的(de)蛋(dan)白(bai)質印跡(ji)分(fen)析(xi)免疫(yi)沉淀。如圖14所示(shi)，血(xue)(xue)小(xiao)板(ban)(ban)膜凍融(rong)復(fu)合(he)物(wu)微泡(pao)表面的(de)糖(tang)蛋(dan)白(bai)αIIbβ3具(ju)有活(huo)化(hua)的(de)構象，并且(qie)其(qi)活(huo)化(hua)不依賴血(xue)(xue)小(xiao)板(ban)(ban)內(nei)的(de)Talin-1蛋(dan)白(bai)。

9、急慢性(xing)血(xue)栓模(mo)型的構建：

用異氟烷麻醉大(da)(da)鼠，開腹(fu)后找到(dao)下腔靜脈，并用一段(duan)長為(wei)4 mm，寬為(wei)2 mm的(de)濾紙條(tiao)泡在(zai)(zai)20%的(de)三氯化(hua)鐵中，取(qu)出后貼(tie)在(zai)(zai)下腔靜脈表面，1分鐘(zhong)后取(qu)走濾紙條(tiao)，并用PBS重(zhong)洗該處，構建大(da)(da)鼠急性血栓(shuan)模型(xing)。在(zai)(zai)慢性血栓(shuan)模型(xing)中，大(da)(da)鼠的(de)傷口縫合后在(zai)(zai)無(wu)菌環(huan)境(jing)下飼(si)養10天，構建慢性血栓(shuan)模型(xing)。

10、急慢性血(xue)栓模(mo)型(xing)的超聲分(fen)子(zi)影像診(zhen)斷(duan)：

超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)造(zao)影設備采用(yong)GE公司(si)生產的(de)(de)(de)LOGIQ E9型(xing)全身應用(yong)超(chao)(chao)(chao)高端(duan)彩超(chao)(chao)(chao)，首先選用(yong)18MHz探(tan)頭(tou)機(ji)械指數MI調節為1.0，在(zai)B模式下(xia)對(dui)(dui)下(xia)腔(qiang)靜脈(mo)進(jin)行(xing)(xing)造(zao)影，對(dui)(dui)血(xue)(xue)栓的(de)(de)(de)大(da)小(xiao)(xiao)及(ji)位置進(jin)行(xing)(xing)判斷，在(zai)多(duo)普勒模式下(xia)對(dui)(dui)血(xue)(xue)栓處(chu)(chu)的(de)(de)(de)血(xue)(xue)流進(jin)行(xing)(xing)造(zao)影。用(yong)5 ml注射器抽取5 ml生理鹽水加入(ru)到血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)氣(qi)泡凍(dong)干粉(fen)中(zhong)，用(yong)力震(zhen)蕩復(fu)溶(rong)形成血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)氣(qi)泡。將血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)氣(qi)泡通過尾靜脈(mo)分別注射入(ru)大(da)鼠的(de)(de)(de)尾靜脈(mo)中(zhong)。再選用(yong)9 MHz的(de)(de)(de)探(tan)頭(tou)，機(ji)械指數MI調節為0.14，在(zai)對(dui)(dui)比(bi)(bi)增強模式（CEUS）下(xia)對(dui)(dui)下(xia)腔(qiang)靜脈(mo)進(jin)行(xing)(xing)造(zao)影，血(xue)(xue)管灌注及(ji)血(xue)(xue)栓靶(ba)向(xiang)時的(de)(de)(de)對(dui)(dui)比(bi)(bi)增強超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)圖(tu)像(xiang)(xiang)分析。連續采集(ji)5分鐘(zhong)內的(de)(de)(de)圖(tu)像(xiang)(xiang)，并使用(yong)MATLAB軟件對(dui)(dui)圖(tu)像(xiang)(xiang)感興趣區域(yu)的(de)(de)(de)信(xin)號(hao)強度(du)進(jin)行(xing)(xing)分析，并根據超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)強度(du)的(de)(de)(de)標尺來確定感興趣區域(yu)的(de)(de)(de)超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)信(xin)號(hao)值(zhi)。如圖(tu)15所示，在(zai)急性(xing)(xing)(xing)(xing)血(xue)(xue)栓模型(xing)中(zhong)，血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)泡能夠有(you)(you)(you)效靶(ba)向(xiang)急性(xing)(xing)(xing)(xing)血(xue)(xue)栓，因此有(you)(you)(you)效低(di)提高急性(xing)(xing)(xing)(xing)血(xue)(xue)栓診(zhen)斷的(de)(de)(de)超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)信(xin)噪比(bi)(bi)。如圖(tu)16所示，在(zai)慢性(xing)(xing)(xing)(xing)血(xue)(xue)栓模型(xing)中(zhong)，血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)泡在(zai)慢性(xing)(xing)(xing)(xing)血(xue)(xue)栓處(chu)(chu)無富集(ji)，其超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)信(xin)噪比(bi)(bi)沒有(you)(you)(you)顯著變化。通過對(dui)(dui)急性(xing)(xing)(xing)(xing)血(xue)(xue)栓以及(ji)慢性(xing)(xing)(xing)(xing)血(xue)(xue)栓進(jin)行(xing)(xing)蘇木精(jing)-伊紅以及(ji)馬松染色分析證明二者組分有(you)(you)(you)明顯差異(yi)，在(zai)急性(xing)(xing)(xing)(xing)血(xue)(xue)栓中(zhong)以纖(xian)維(wei)蛋白為主而在(zai)慢性(xing)(xing)(xing)(xing)血(xue)(xue)栓中(zhong)以膠原為主，血(xue)(xue)小(xiao)(xiao)板膜(mo)(mo)凍(dong)融(rong)(rong)復(fu)合(he)物(wu)微(wei)泡在(zai)二者診(zhen)斷中(zhong)的(de)(de)(de)平均超(chao)(chao)(chao)聲(sheng)(sheng)(sheng)信(xin)噪比(bi)(bi)有(you)(you)(you)顯著性(xing)(xing)(xing)(xing)差異(yi)。

綜上所述，本(ben)發明采用反復(fu)(fu)液氮(dan)冷凍(dong)-室(shi)溫(wen)融(rong)(rong)化(hua)(hua)的方(fang)(fang)(fang)式(shi)(shi)向(xiang)血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)中摻雜磷(lin)脂(zhi)并(bing)得(de)到血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)囊(nang)泡(pao)(pao)。通(tong)過(guo)Langmuir-Blodgett膜(mo)(mo)(mo)(mo)天平測(ce)試得(de)到凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)中的磷(lin)脂(zhi)蛋白(bai)(bai)質分(fen)(fen)子在(zai)(zai)(zai)氣(qi)液界面的表面張力顯著降低，通(tong)過(guo)差適量熱(re)掃描(miao)技術測(ce)試表明摻雜后的血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)玻璃態(tai)轉(zhuan)變溫(wen)度降低。利(li)用超(chao)聲輔助的方(fang)(fang)(fang)式(shi)(shi)向(xiang)血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)囊(nang)泡(pao)(pao)中鼓入六氟化(hua)(hua)硫氣(qi)體，在(zai)(zai)(zai)氣(qi)液界面形成血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡(pao)(pao)。通(tong)過(guo)熒光標記的方(fang)(fang)(fang)式(shi)(shi)定位血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)蛋白(bai)(bai)，并(bing)成功(gong)示蹤(zong)其融(rong)(rong)合(he)(he)(he)在(zai)(zai)(zai)血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡(pao)(pao)。通(tong)過(guo)對血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)囊(nang)泡(pao)(pao)以及血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡(pao)(pao)進行蛋白(bai)(bai)質組學分(fen)(fen)析，發現血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡(pao)(pao)繼承了血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)囊(nang)泡(pao)(pao)61.4%的蛋白(bai)(bai)質種類，并(bing)維持了血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)表面的整合(he)(he)(he)素αIIβ3活化(hua)(hua)的構象。在(zai)(zai)(zai)大鼠下(xia)腔靜脈急(ji)(ji)慢性(xing)血(xue)(xue)(xue)栓模(mo)型實驗(yan)中，血(xue)(xue)(xue)小(xiao)板膜(mo)(mo)(mo)(mo)凍(dong)融(rong)(rong)復(fu)(fu)合(he)(he)(he)物(wu)(wu)微(wei)泡(pao)(pao)能夠特異性(xing)識別急(ji)(ji)性(xing)血(xue)(xue)(xue)栓，其對于急(ji)(ji)性(xing)血(xue)(xue)(xue)栓診斷平均信噪比(bi)為12.47 dB，而(er)慢性(xing)血(xue)(xue)(xue)栓為0.1dB。