劉雪峰，瞿金龍，李文放，林兆奮

·臨床醫(yī)學(xué)·

·論著·

CRISPLD2蛋白對(duì)內(nèi)毒素急性呼吸窘迫綜合征小鼠的保護(hù)作用

劉雪峰，瞿金龍，李文放，林兆奮

目的探討CRISPLD2蛋白對(duì)內(nèi)毒素急性呼吸窘迫綜合征(acute respiratory disease syndrome, ARDS)小鼠的保護(hù)作用。方法采用數(shù)字表法，將120只健康雄性昆明小鼠隨機(jī)分為對(duì)照組(A組，40只)：氣道內(nèi)滴入LPS(5 mg/kg)；LPS預(yù)處理組(B組，40只)：先予小鼠LPS(2 mg/kg)腹腔內(nèi)注射，注射后第8天氣道內(nèi)滴入LPS(5 mg/kg)；重組CRISPLD2處理組(C組，40只)：先予小鼠尾靜脈注射重組 CRISPLD2(50 mg/kg)，6 h后氣道內(nèi)滴入LPS(5 mg/kg)。(1)采用數(shù)字表法將30只小鼠隨機(jī)分成A1組、B1組、C1組(各組10只)，氣道滴入LPS 6 h后處死，測定肺濕干重比(W/D)、通透指數(shù)(LPI)。(2)采用數(shù)字表法將90只小鼠隨機(jī)分成A2組、B2組、C2組(各組20只)，觀察5 d內(nèi)小鼠死亡情況，描繪生存曲線。結(jié)果B、C兩組W/D、LPI及5 d死亡率顯著低于A組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；B組各項(xiàng)檢測結(jié)果低于C組，差異有統(tǒng)計(jì)學(xué)意義(P>0.05)。結(jié)論提升CRISPLD2蛋白血濃度對(duì)內(nèi)毒素所致ARDS具有保護(hù)作用，可降低肺損傷程度，改善肺毛細(xì)血管通透性，降低死亡率。

CRISPLD2蛋白；內(nèi)毒素；急性呼吸窘迫綜合征

急性呼吸窘迫綜合征(acute respiratory disease syndrome, ARDS)多由感染、休克、創(chuàng)傷、大量輸血等病因誘發(fā)，以頑固性低氧血癥為臨床主要表現(xiàn)的急性進(jìn)行性呼吸衰竭[1]，其本質(zhì)是過度炎癥反應(yīng)及多器官功能障礙綜合征在肺部的表現(xiàn)。盡管診治技術(shù)不斷進(jìn)步，但ARDS死亡率仍然居高不下，上海市12家三甲醫(yī)院ICU2001年3月至2002年3月期間ARDS病死率高達(dá)68.5%[2]。CRISPLD2(cysteine-rich secretory protein containing LCCL Domain 2)蛋白是一個(gè)保守的、半胱氨酸富集的、血清濃度較高的LPS結(jié)合蛋白，擁有2個(gè)與LPS結(jié)合的LCCL結(jié)構(gòu)域，并具有較高親和力與LPS[3]結(jié)合，與大鼠肺、腎臟的發(fā)育以及無癥狀的唇裂有關(guān)[4-6]。CRISPLD2是一種分泌型蛋白，人類、小鼠和大鼠的肺臟、小腸、胎盤以及免疫細(xì)胞包括粒細(xì)胞，單核細(xì)胞，T、B、NK細(xì)胞都表達(dá)CRISPLD2[7-8]。本實(shí)驗(yàn)以氣道內(nèi)注射LPS制備的ARDS小鼠為對(duì)象，研究CRISPLD2蛋白對(duì)小鼠模型的保護(hù)作用。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物與模型制備

健康雄性昆明小鼠，體質(zhì)量20～22 g，購自第二軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心，給予標(biāo)準(zhǔn)飲食和飲水。采用氣道內(nèi)滴入LPS(5 mg/kg)制備ARDS小鼠模型，空白對(duì)照組氣道內(nèi)滴入等量生理鹽水。

1.2 主要試劑

LPS購自Sigma公司。重組CRISPLD2蛋白購自上海南方基因中心。其余試劑為國產(chǎn)分析純?cè)噭?/p>

1.3 實(shí)驗(yàn)分組及實(shí)施

1.3.1 分組 采用數(shù)字表法將120只雄性昆明小鼠隨機(jī)分為以下3組：(1)對(duì)照組(A組，40只)：氣道內(nèi)滴入LPS(5 mg/kg)；(2)LPS預(yù)處理組(B組，40只)：先予實(shí)驗(yàn)小鼠LPS(2 mg/kg)腹腔內(nèi)注射，注射后第8天氣道內(nèi)滴入LPS(5 mg/kg)；(3)重組CRISPLD2處理組(C組，40只)：首先給予小鼠尾靜脈注射重組 CRISPLD2(50 mg/kg)，6 h后氣道內(nèi)滴入LPS(5 mg/kg)。

1.3.2 具體實(shí)施 (1)取A、B、C組小鼠各10只，分別作為A1、B1、C1組，往這3組小鼠氣道滴入LPS 6 h斷尾取血測定血漿蛋白含量后處死實(shí)驗(yàn)小鼠，取右肺計(jì)算濕干重比(W/D)，取左肺行支氣管肺泡灌洗，測量肺通透指數(shù)(LPI)(肺泡灌洗液總蛋白含量/血漿總蛋白含量)。(2)A、B、C組每組剩下的30只小鼠分別作為A2、B2、C2組，觀察各組96 h內(nèi)小鼠死亡情況，描繪生存曲線。

1.4 統(tǒng)計(jì)學(xué)處理

檢測數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，以SPSS 18.0統(tǒng)計(jì)軟件進(jìn)行分析。2組之間的比較采用t檢驗(yàn)或秩和檢驗(yàn)；多組之間采用方差分析或者秩檢驗(yàn)。P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 W/D比值、LPI指數(shù)

實(shí)驗(yàn)結(jié)果表明LPS預(yù)處理組與重組CRISPLD2蛋白處理組的W/D比值和LPI明顯低于LPS組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。LPS預(yù)處理組的W/D和LPI值低于重組CRISPLD2蛋白處理組，但差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表1。

表1 CRISLPD2蛋白對(duì)內(nèi)毒素急性呼吸窘迫綜合征小鼠肺水腫的影響(x±s)

注：ARDS為急性呼吸窘迫綜合征；與A1組比較aP<0.05

2.2 實(shí)驗(yàn)小鼠5 d生存率

數(shù)據(jù)分析表明LPS預(yù)處理組與重組CRISPLD2蛋白處理組的實(shí)驗(yàn)小鼠5 d生存率明顯高于LPS組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。LPS預(yù)處理組的5 d生存率高于重組CRISPLD2蛋白處理組，但差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見圖1。

注：與重組CRISPLD2蛋白組比較P<0.05

3 討論

ARDS實(shí)質(zhì)是肺毛細(xì)血管通透性損傷，其發(fā)病誘因中全身性感染及肺部感染約占50%以上，因肺部感染直接損傷所引起的ARDS約占70%[9-10]。因此筆者選用氣道滴入LPS(5 mg/kg)直接引起肺部損傷制備ARDS小鼠模型，結(jié)果表明小鼠肺W/D、LPI符合ARDS表現(xiàn)，與文獻(xiàn)報(bào)道一致[11]，提示動(dòng)物模型制備成功。

目前認(rèn)為CRISPLD2蛋白是一種保守的與肺、腎發(fā)育相關(guān)的分泌型蛋白，可由肺、腸、免疫細(xì)胞等多種組織細(xì)胞分泌?，F(xiàn)有研究發(fā)現(xiàn)LPS可刺激NK細(xì)胞、單核細(xì)胞、PBMC以及粒細(xì)胞等多種免疫細(xì)胞增加CRISPLD2分泌；同時(shí)與其他應(yīng)急反應(yīng)蛋白相比，小鼠腹腔注射小劑量非毒性劑量LPS以后，12 h內(nèi)血清CRISPLD2濃度快速下降，24 h后其濃度又大幅反彈，此時(shí)可高于正常血清1～2倍以上，并維持2周之久[12]，因此實(shí)驗(yàn)分組中的LPS預(yù)處理組選擇實(shí)驗(yàn)小鼠小劑量LPS腹腔內(nèi)注射后第8天行氣道內(nèi)滴入LPS。實(shí)驗(yàn)研究還發(fā)現(xiàn)CRISPLD2可與LPS結(jié)合，影響LPS與TLR4受體結(jié)合，從而發(fā)揮反饋調(diào)控機(jī)制，減少炎癥介質(zhì)的釋放，降低全身性炎癥反應(yīng)[12]。

本實(shí)驗(yàn)通過內(nèi)毒素直接引起肺部損傷的小鼠模型為基礎(chǔ)，分別通過LPS預(yù)處理、重組CRISPLD2蛋白預(yù)注射進(jìn)行提前干預(yù)，發(fā)現(xiàn)通過LPS預(yù)先腹腔內(nèi)注射或重組CRISPLD2蛋白直接靜脈注射提高小鼠血清CRISPLD2蛋白濃度能夠減輕內(nèi)毒素所致肺損傷程度，改善預(yù)后。

本實(shí)驗(yàn)結(jié)果中LPS預(yù)處理組W/D、LPI、5 d死亡率低于重組CRISPLD2處理組，原因考慮與2種干預(yù)方式所升高的小鼠血清CRISPLD2蛋白濃度差異相關(guān)。通過增加實(shí)驗(yàn)制模前重組CRISPLD2預(yù)注射劑量能否再次降低內(nèi)毒素所致ARDS小鼠的死亡率以及將重組CRISPLD2蛋白由預(yù)注射改為實(shí)驗(yàn)制模后的不同時(shí)間點(diǎn)注射是否能起到同樣改善預(yù)后的作用尚需進(jìn)一步研究。

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(本文編輯：張陣陣)

Protective effect of CRISPLD2 protein on acute respiratory disease syndrome induced by lipopolysaecharide in mice

LiuXuefeng,QuJinlong,LiWenfang,LinZhaofen

Objective To investigate the protective effect of CRISPLD2 protein on acute respiratory disease syndrome (ARDS) induced by lipopolysaecharide (LPS) in mice.Methods Healthy male Kunming mice were randomly divided into 3 groups: the control group (or group A) which received lipopolysaecharide via the intratracheal instillation at a dosage of 5 mg/kg, the LPS preconditioning group (or group B) which had intraperitoneal injection of LPS at a dosage of 2mg/kg and at day 8 received LPS via intratracheal instillation also at a dosage of 5mg/kg. The recombinant CRISPLD2 preconditioning group (or group C) which was injected with recombinant CRISPLD2 at a dosage of 50mg/kg via cauda vein, and then at hour 6 received LPS via intratracheal instillation at the same dosage. (1)Thirty mice were randomly divided into group A1, group B1 and group C1, each consisting of 10 animals. The mice were sacrificed 6 hours after LPS intratracheal instillation. Then, W/D and LPI were measured. (2) Ninety mice were randomly divided into group A2, group B2 and group C2, each consisting of 30 animals. The death of the animals in each group was closely observed and the survival curve was described.Results W/D, lung index and mortality after 5 days in group B and C were significantly lower, as compared with those of group A (P<0.05). The detected data of various indexes in group B were lower than those in group C (P>0.05).Conclusion Increased serum concentration of CRISPLD2 could obviously produce protective effect on ARDS induced by LPS, lighten injury on the lungs and improve permeability of pulmonary capillary and decrease mortality.

CRISPLD2; Endotoxin; Lipopolysaecharide; Acute respiratory disease syndrome

[參考文獻(xiàn)]

上海市衛(wèi)生和計(jì)劃生育委員會(huì)項(xiàng)目(2012210)

200003 上海，第二軍醫(yī)大學(xué)附屬長征醫(yī)院急救科

R563

A

10.3969/j.issn.1009-0754.2017.01.008

(DepartmentofCriticalCare,ChangzhengHospital,Shanghai200003,China)

2016-12-30)