申星,熊?chē)?guó)林,謝玲,郭玲玲,李明,趙艷芳,王磊,洪劍,楊萌,趙黎,彭瑞云,王勇,楊曉明,羅慶良,余祖胤,葛常輝,邢爽
近年來(lái),To ll樣受體(To ll-like recep to r,TLR)信號(hào)通路的調(diào)控已成為研究熱點(diǎn)[1],目前已研發(fā)出多種TLR激動(dòng)劑用于抗感染、抗腫瘤以及治療自身免疫性疾病[2]?;诖碳LR、調(diào)節(jié)核轉(zhuǎn)錄因子κB(NF-κB)信號(hào)通路發(fā)揮抗輻射損傷作用的研究發(fā)現(xiàn)了一系列新的輻射防護(hù)劑[3-5],其中以TLR5激動(dòng)劑的作用最強(qiáng),能同時(shí)防護(hù)骨髓和腸道的輻射損傷,對(duì)重度以上骨髓型放射病有較好的輻射防護(hù)作用[5]。TLR5廣泛表達(dá)于樹(shù)突細(xì)胞、巨噬細(xì)胞以及腸黏膜固有層內(nèi)皮細(xì)胞的表面,能夠被細(xì)菌鞭毛蛋白或其衍生物CBLB502識(shí)別,并激活NF-κB信號(hào)通路,從而抑制細(xì)胞凋亡、抵抗機(jī)體氧化損傷[6]。CBLB502是經(jīng)過(guò)改構(gòu)的鞭毛蛋白衍生物,高效且免疫原性低,研究表明其對(duì)致死劑量照射的小鼠和獼猴均有顯著的保護(hù)作用[5,7-8]。為進(jìn)一步探索CBLB502的輻射防護(hù)作用,本研究以恒河猴為模型動(dòng)物,采用輻射防護(hù)效果較好的WR-2721(氨磷汀,阿米福汀)[9]作為陽(yáng)性對(duì)照藥物,觀察了不同劑量CBLB502對(duì)7.0Gy60Co γ射線全身照射恒河猴的防護(hù)作用,旨在為CBLB502的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。
1.1 實(shí)驗(yàn)動(dòng)物 30只健康成年恒河猴購(gòu)自北京協(xié)爾鑫生物資源研究所,雌雄各半,體重5.0±1.0kg[動(dòng)物質(zhì)量合格證號(hào):SCXK-(京)2005-0005]。動(dòng)物飼養(yǎng)于軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心普通級(jí)猴實(shí)驗(yàn)室內(nèi),24h負(fù)壓通風(fēng)換氣,室溫18~24℃,相對(duì)濕度40%~70%,10h日光燈照明,14h黑暗,每天沖洗清潔籠具2次。動(dòng)物到達(dá)后單籠單養(yǎng),除飼以軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心配制的猴用標(biāo)準(zhǔn)飼料外,適當(dāng)給予新鮮水果蔬菜等加強(qiáng)營(yíng)養(yǎng),消毒涼水置籠內(nèi)自由飲用。動(dòng)物飼養(yǎng)設(shè)施合格證號(hào):SYXK-(軍)2007-004。動(dòng)物實(shí)驗(yàn)均按照軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物倫理委員會(huì)要求執(zhí)行。
1.2 分組 依據(jù)性別、體重和外周血白細(xì)胞計(jì)數(shù)將入選的30只恒河猴分為對(duì)癥治療組、WR-2721組和CBLB502 2.5、10、40μg/kg組,每組6只。此外,預(yù)備3只體重為6~12kg的健康成年恒河猴用作對(duì)癥治療中輸血時(shí)的供血猴。
1.3 給藥方法 CBLB502注射液由北京放射醫(yī)學(xué)研究所研制,批號(hào)20100613,4.0m g/支(1.0m l),為無(wú)色澄明水溶液。WR-2721由大連美羅大藥廠生產(chǎn),白色凍干粉劑,400m g/瓶,批號(hào)53090902。不同劑量CBLB502和WR-2721均于照射前0.5h一次肌肉注射,對(duì)癥治療組肌肉注射等體積生理鹽水(0.3m l/kg)。
1.4 模型制備與對(duì)癥支持治療 采用軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所的60Co γ射線放射源,照射前10m in用3%戊巴比妥鈉(30m g/kg)靜脈注射麻醉恒河猴,將麻醉猴固定于特制照射木箱內(nèi)行7.0Gy一次全身雙側(cè)照射,照射距離為4.75m,照射劑量率為13.00~13.52cGy/m in。為防止照射動(dòng)物在觀察期內(nèi)過(guò)早死亡影響實(shí)驗(yàn)結(jié)果,依實(shí)驗(yàn)動(dòng)物倫理及福利要求,受試動(dòng)物在相同原則下均給予綜合對(duì)癥治療,主要措施包括抗感染、抗出血、輸血、維持水電解質(zhì)和酸堿平衡、補(bǔ)充營(yíng)養(yǎng)等。腸道滅菌:自照前3d開(kāi)始,給予慶大霉素16萬(wàn)U+甲硝唑0.4g口服,1次/d,連續(xù)3d。預(yù)防感染:動(dòng)物外周血白細(xì)胞數(shù)低于1×109/L時(shí)開(kāi)始使用抗生素(青霉素80萬(wàn)U/d,肌肉注射,頭孢噻肟鈉1.0g/d,靜脈注射),根據(jù)病情及時(shí)調(diào)整、更換,連續(xù)3d外周血白細(xì)胞數(shù)超過(guò)1×109/L時(shí)停藥。條件輸血:動(dòng)物外周血小板(PLT)低于50×109/L時(shí),隔天一次靜脈輸注新鮮全血30m l,輸注前均經(jīng)15Gy60Co γ射線照射。
1.5 觀察指標(biāo) 觀察照射后動(dòng)物一般體征并檢測(cè)外周血象,取照射后1d的外周血細(xì)胞和照射后39d的骨髓細(xì)胞進(jìn)行造血細(xì)胞集落培養(yǎng)。觀察動(dòng)物存活情況,死亡動(dòng)物即時(shí)解剖、存活動(dòng)物實(shí)驗(yàn)結(jié)束時(shí)采用安樂(lè)死方式處死,取胸骨、淋巴結(jié)、小腸進(jìn)行組織病理學(xué)檢查,上述臟器和組織用中性福爾馬林固定,常規(guī)制片、切片和染色后光鏡下觀察。
1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)分析,采用Orig in Pro 9.0軟件包作圖。計(jì)量資料以±s表示,血小板減少癥持續(xù)時(shí)間和生存率組間差異比較采用Kap lan-Meier檢驗(yàn)。外周血細(xì)胞數(shù)比較采用重復(fù)測(cè)量的方差分析(repeated m easures data of ANOVA),造血細(xì)胞集落數(shù)比較采用單因素方差分析(one-w ay ANOVA),進(jìn)一步兩兩比較采用LSD-t檢驗(yàn)或Dunnett t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 7.0Gy γ射線全身照射猴損傷程度 7.0Gy60Co γ射線全身照射后,各組實(shí)驗(yàn)猴造血功能均嚴(yán)重受損,外周血淋巴細(xì)胞數(shù)迅速下降,照射后48h降至0.5×109/L左右,并于照射后第7天開(kāi)始陸續(xù)出現(xiàn)發(fā)熱癥狀,最高體溫達(dá)40.4℃,發(fā)熱期間動(dòng)物食欲減退甚至拒食,體重明顯減輕。病程極期,各組實(shí)驗(yàn)猴外周血白細(xì)胞、中性粒細(xì)胞和血小板數(shù)均明顯減少,發(fā)生血細(xì)胞減少癥。對(duì)癥治療猴于照射后17d開(kāi)始出現(xiàn)死亡,其中4只猴在照射后3周內(nèi)死于嚴(yán)重感染和出血并發(fā)癥,存活時(shí)間為19.4±1.7d,40d時(shí)存活數(shù)僅為2只。對(duì)死亡猴進(jìn)行組織病理學(xué)分析見(jiàn)骨髓腔空虛,廣泛出血,造血細(xì)胞顯著減少;脾臟廣泛出血,紅髓白髓分界不清,淋巴細(xì)胞明顯減少,脾小體結(jié)構(gòu)消失;腸黏膜剝脫、出血,黏膜上皮細(xì)胞嗜酸性變,間質(zhì)細(xì)胞減少,淋巴細(xì)胞固縮等。根據(jù)一般臨床表現(xiàn)、造血損傷程度和存活情況,參考急性放射病的分型分度標(biāo)準(zhǔn)[10],本實(shí)驗(yàn)條件下,7.0Gy γ射線全身照射猴發(fā)生了重度骨髓型急性放射病。
2.2 CBLB502對(duì)7.0Gy γ射線照射猴存活和體征的影響 7.0Gy60Co γ射線照射后各組實(shí)驗(yàn)猴均出現(xiàn)發(fā)熱癥狀,體重呈下降趨勢(shì)。CBLB502 2.5、10、40μg/kg組發(fā)熱動(dòng)物數(shù)少于對(duì)癥治療組,除其中1只實(shí)驗(yàn)猴在照射后36d死于皰疹病毒引發(fā)的感染外,其他動(dòng)物全部存活。WR-2721組照射后17d因外周血小板嚴(yán)重減少死亡1只,對(duì)癥治療組照射后40d內(nèi)死亡4只,死亡動(dòng)物存活時(shí)間為19.4±1.7d(表1)。該結(jié)果表明,照射前0.5h單次肌肉注射CBLB502可明顯提高7.0Gy γ射線全身照射所致重度急性放射病實(shí)驗(yàn)猴的存活率,改善其生存質(zhì)量。
表1 7.0Gy γ射線全身照射恒河猴體征及存活情況(n=6)Tab. 1 Signs and survival of Rhesus monkey irradiated with 7.0Gy γ-ray (n=6)
2.3 CBLB502對(duì)7.0Gy γ射線照射猴外周血小板數(shù)的影響 CBLB502治療組血小板數(shù)在照射后早期有一過(guò)性下降,在照射后2~7d明顯低于對(duì)癥治療組(P<0.05),各組照射猴外周血小板數(shù)自照射后第5天開(kāi)始急劇下降,第13~15天達(dá)最低值,CBLB502 10μg/kg和40μg/kg組最低值明顯高于對(duì)癥治療組(P<0.05,表2),CBLB502 40μg/kg組血小板減少和缺乏(血小板數(shù)<50×109/L,為臨床輸血指征)持續(xù)時(shí)間明顯低于CBLB502 2.5μg/kg組和WR-2721組(P<0.05,表3)。WR-2721組血小板數(shù)變化趨勢(shì)與
表2 CBLB502對(duì)7.0Gy γ射線照射恒河猴外周血細(xì)胞數(shù)最低值的影響(×109/L,±s)Tab. 2 Effects of CBLB502 on peripheral b lood nad irs of Rhesus monkey irrad iated with 7.0Gy γ-ray (×109/L, ±s)
表2 CBLB502對(duì)7.0Gy γ射線照射恒河猴外周血細(xì)胞數(shù)最低值的影響(×109/L,±s)Tab. 2 Effects of CBLB502 on peripheral b lood nad irs of Rhesus monkey irrad iated with 7.0Gy γ-ray (×109/L, ±s)
(1)P<0.05 com pared with IR group; (2)P<0.05 com pared with WR-2721 group. PLT. Platelets; WBC. White b lood cells; NEUT. Neutrophil;LYM. Lym phocytes
Hem ocyte IR WR-2721 CBLB502 (μg/kg)2.5 10 40 PLT 14.7±9.4 28.8±9.3 33.6±8.9 47.5±13.0(1) 53.7±38.2(1)WBC 0.2±0.1 0.4±0.1 0.4±0.2 0.6±0.2(1) 0.8±0.7(1)NEUT 0.01±0.01 0.03±0.03 0.02±0.02 0.09±0.07 0.13±0.15(1)(2)LYM 0.19±0.14 0.31±0.10 0.15±0.03(2) 0.33±0.10(1) 0.25±0.12
表3 CBLB502對(duì)7.0Gy γ射線照射恒河猴外周血血小板數(shù)的影響(±s)Tab. 3 Effects of CBLB502 on peripheral blood p latelet count of Rhesus monkey irrad iated with 7.0Gy γ-ray (±s)
表3 CBLB502對(duì)7.0Gy γ射線照射恒河猴外周血血小板數(shù)的影響(±s)Tab. 3 Effects of CBLB502 on peripheral blood p latelet count of Rhesus monkey irrad iated with 7.0Gy γ-ray (±s)
(1)P<0.05 com pared with WR-2721 group; (2)P<0.05 com pared with CBLB502 2.5μg/kg g roup. Recovery initiation tim e w as the tim e point when PLT>100×109/L began to occur. Since 4/6 Rhesus monkeys in the IR group died during the crisis period, the duration of the PLT nad ir w as no t ca lcu lated.
Group Du ration of PLT nad ir (d)PLT recovery in itiation (d)<100×109/L <50×109/L WR-2721 5.4±1.3 3.2±1.3 20.2±2.3 CBLB502 2.5μg/kg 12.2±5.8(1)4.8±3.9 21.0±2.0 CBLB502 10μg/kg 5.0±4.0(1)1.3±1.9(1)(2) 21.3±8.7 CBLB502 40μg/kg 3.8±2.8(1)1.3±1.6(1)(2) 15.5±8.3
CBLB502 2.5μg/kg組相似,恢復(fù)稍快于對(duì)癥治療組。照射后39d各組血小板數(shù)相當(dāng),達(dá)照射前水平的56%~76%(圖1A)。
2.4 CBLB502對(duì)7.0Gy γ射線照射猴外周血白細(xì)胞數(shù)的影響 CBLB502 10μg/kg和40μg/kg組照射猴外周血白細(xì)胞數(shù)最低值均明顯高于對(duì)癥治療組(P<0.05,表2),且開(kāi)始恢復(fù)時(shí)間提前(圖1B)。CBLB502 40μg/kg組白細(xì)胞數(shù)在照射后13d明顯高于WR-2721組和對(duì)癥治療組(P<0.05),15~17d明顯高于對(duì)癥治療組(P<0.01),其白細(xì)胞數(shù)減少和缺乏(白細(xì)胞數(shù)<1.0×109/L)持續(xù)時(shí)間明顯低于CBLB502 2.5μg/kg組和對(duì)癥治療組(P<0.05,表4,圖1B)。中性粒細(xì)胞和淋巴細(xì)胞數(shù)變化趨勢(shì)(圖1C、D)與白細(xì)胞基本一致,CBLB502 40μg/kg組照射后15d
表4 CBLB502對(duì)7.0Gy γ射線全身照射恒河猴外周血白細(xì)胞的影響(±s)Tab. 4 Effects of CBLB502 on peripheral b lood WBC count of Rhesus m onkey irrad iated with 7.0Gy γ-ray (±s)
表4 CBLB502對(duì)7.0Gy γ射線全身照射恒河猴外周血白細(xì)胞的影響(±s)Tab. 4 Effects of CBLB502 on peripheral b lood WBC count of Rhesus m onkey irrad iated with 7.0Gy γ-ray (±s)
Group Du ration of WBC nad ir (d)WBC recovery in itiation (d)<2.0×109/L <1.0×109/L WR-2721 16.2±3.1 12.0±3.2 21.4±2.6 CBLB502 2.5μg/kg 17.8±2.6 12.8±3.3 22.6±2.2 CBLB502 10μg/kg 16.3±3.3 10.5±5.2 21.0±2.8 CBLB502 40μg/kg 12.5±6.4(1) 7.0±5.8(1)(2) 20.0±2.8
(1)P<0.05 com pared with WR-2721 group; (2)P<0.05 com pared with CBLB502 2.5μg/kg group. Recovery initiation tim e was the tim e point when WBC>2.0×109/L began to occur. Since 4/6 Rhesus monkeys in IR group died during crisis period, duration of WBC nadir w as no t ca lcu lated中性粒細(xì)胞數(shù)明顯高于對(duì)癥治療組和WR-2721組(P<0.05),CBLB502 40和10μg/kg組照后各時(shí)間點(diǎn)的淋巴細(xì)胞絕對(duì)值相當(dāng),且在照射后17d均明顯高于對(duì)癥治療組(P<0.01,P<0.05)。該結(jié)果提示,照射前0.5h單次肌肉注射CBLB502可促進(jìn)7.0Gy γ射線全身照射恒河猴外周血白細(xì)胞數(shù)恢復(fù),以40μg/kg組效果最佳,其療效優(yōu)于WR-2721。
圖1 CBLB502對(duì)7.0Gy γ射線全身照射恒河猴外周血細(xì)胞數(shù)的影響Fig. 1 Effect of CBLB502 on peripheral b lood cells coun t of the Rhesus monkeys irrad iated with 7.0Gy γ-ray A. Plate let; B. Leucocyte; C. Neu troph il; D. Lym phocyte; E. Eryth rocyte; F. Reticu locyte
2.5 CBLB502對(duì)7.0Gy γ射線照射猴外周血紅細(xì)胞和網(wǎng)織紅細(xì)胞(RET)數(shù)的影響 恒河猴外周血紅細(xì)胞數(shù)在照射后緩慢下降,極期時(shí)CBLB502 10、40μg/kg組紅細(xì)胞數(shù)較其他各組略低(圖1E)。外周血中RET數(shù)可以間接反映受照射機(jī)體造血功能損傷程度及恢復(fù)情況,檢測(cè)結(jié)果表明:7.0Gy γ射線照射后9 d內(nèi)各組RET絕對(duì)值無(wú)明顯差異,對(duì)癥治療組RET低值持續(xù)至第21天才開(kāi)始逐漸恢復(fù),而CBLB502治療組外周血RET于照射后9~15d有小幅升高且高于同期對(duì)癥治療組,第15天后CBLB502 2.5μg/kg組與對(duì)癥治療組恢復(fù)速度相當(dāng),但CBLB502 10、40μg/kg組迅速上升并明顯高于同期CBLB502 2.5μg/kg組、對(duì)癥治療組及WR-2721組(P<0.05,圖1F)。該結(jié)果提示,照射前0.5h單次肌肉注射CBLB502可加速7.0Gy γ射線全身照射恒河猴造血功能的恢復(fù)。
2.6 CBLB502對(duì)7.0Gy γ射線照射猴骨髓和外周血造血細(xì)胞集落形成的影響 照射后1d外周血造血祖細(xì)胞集落培養(yǎng)結(jié)果顯示,CBLB502治療組巨核細(xì)胞集落形成單位(co lonyform ing unit-m egakaryocyte,CFU-MK)、紅系爆式集落形成單位(bu rst-fo rm ing un it-eryth roid,BFU-E)和粒細(xì)胞-巨噬細(xì)胞集落形成單位(colonyform ing unit-granulocyte m acrophage,CFU-GM)集落數(shù)均有所增加,但是受樣本數(shù)量所限,僅CBLB502 2.5μg/kg組CFU-GM集落數(shù)優(yōu)于對(duì)癥治療組、WR-2721組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05,表5)。第39天骨髓造血集落培養(yǎng)結(jié)果顯示,各組集落生成數(shù)相當(dāng),提示此時(shí)存活照射猴的造血損傷均已得到恢復(fù)(表6)。
表5 7.0Gy γ射線照射后1d各組外周血細(xì)胞集落形成單位數(shù)(/2×104單個(gè)核細(xì)胞,±s)Tab. 5 Colony counting in peripheral b lood of the anim als 1-day after radiation with 7.0Gy γ-ray (/2×104 m ononuclear cells, ±s)
表5 7.0Gy γ射線照射后1d各組外周血細(xì)胞集落形成單位數(shù)(/2×104單個(gè)核細(xì)胞,±s)Tab. 5 Colony counting in peripheral b lood of the anim als 1-day after radiation with 7.0Gy γ-ray (/2×104 m ononuclear cells, ±s)
CFU-MK. Co lony form ing unit-m egakaryocyte; BFU-E. Burstform ing unit-erythroid; CFU-E. Colony form ing unit erythroid; CFU-GM.Colony form ing unit-granulocyte m acrophage; CFU-M ix. M ixed colonyfo rm ing unit. (1)P<0.05 com pared with CBLB502 2.5μg/kg g roup
Group CFU-MK BFU-E CFU-E CFU-GM CFU-M ix IR 0.6±0.90.5±1.00.1±0.3 4.1±2.8(1)0.1±0.3 WR-2721 0.3±0.40.8±0.9 0.0 5.8±2.8(1) 0.0 CBLB502 2.5μg/kg 1.4±0.70.4±0.40.5±0.9 14.8±6.7 0.5±0.4 CBLB502 10μg/kg 2.0±2.02.9±3.9 0.0 7.2±4.1(1) 0.0 CBLB502 40μg/kg 1.1±0.33.3±3.3 0.0 9.1±5.2 0.6±0.9
2.7 CBLB502對(duì)7.0Gy γ射線全身照射恒河猴對(duì)癥治療的影響 統(tǒng)計(jì)各組動(dòng)物接受對(duì)癥治療的用藥情況發(fā)現(xiàn),CBLB502 40μg/kg組有2只動(dòng)物照射后外周血小板數(shù)一直>100×109/L,不需輸血或接受止血藥治療,另4只動(dòng)物接受止血藥治療時(shí)間較WR-2721組明顯縮短(P<0.05),且輸血量較WR-2721組及CBLB502 2.5μg/kg組明顯減少(P<0.05,表7)。該結(jié)果提示,照射前0.5h單次肌肉注射40μg/kg CBLB502可明顯縮短7.0Gy γ射線全身照射恒河猴接受抗生素和止血藥治療的時(shí)間并減少輸血量。
2.8 各組7.0Gy照射猴組織病理學(xué)改變 組織病理學(xué)觀察顯示,照射前0.5h單次肌肉注射CBLB502可明顯減輕7.0Gy60Co γ射線全身照射后骨髓、腸道、腸系膜淋巴結(jié)等組織的損傷,其中CBLB502 10μg/kg和40μg/kg組的改善作用更為突出,主要表現(xiàn)為骨髓、腸道、腸系膜淋巴結(jié)組織結(jié)構(gòu)良好,細(xì)胞數(shù)量增加,血管淤血、出血減輕或消失。
表6 7.0Gy γ射線照射后39d各組骨髓血細(xì)胞集落形成單位數(shù)(/2×104單個(gè)核細(xì)胞,±s)Tab. 6 Colony counting in bone m arrow of the anim als 39 days after radiation with 7.0Gy γ-ray (/2×104 mononuclear cells, ±s)
表6 7.0Gy γ射線照射后39d各組骨髓血細(xì)胞集落形成單位數(shù)(/2×104單個(gè)核細(xì)胞,±s)Tab. 6 Colony counting in bone m arrow of the anim als 39 days after radiation with 7.0Gy γ-ray (/2×104 mononuclear cells, ±s)
CFU-MK. Colony form ing unit-m egakaryocyte; BFU-E. Burstform ing unit-erythroid; CFU-E. Co lony form ing unit erythroid; CFUGM. Colony form ing unit-granulocyte m acrophage; CFU-M ix. M ixed co lony-form ing unit. Since 4/6 Rhesus monkeys from IR group died during crisis period, colony counting was not done to bone m arrow
Group CFU-MK BFU-E CFU-E CFU-GM CFU-M ix WR-2721 8.9±2.814.2±1.95.6±1.994.8±16.05.2±3.1 CBLB502 10μg/kg9.1±1.613.5±1.53.8±1.2102.6±3.72.4±0.4 CBLB502 10μg/kg7.2±1.514.8±3.44.9±1.792.5±13.64.5±2.1 CBLB502 40μg/kg6.5±1.514.1±5.73.9±1.682.4±14.23.6±1.7
表7 各組對(duì)癥治療情況(±s)Tab. 7 Sym p tom atic therap ies of experim en tal g roup s (±s)
表7 各組對(duì)癥治療情況(±s)Tab. 7 Sym p tom atic therap ies of experim en tal g roup s (±s)
(1)P<0.05 com pared with WR-2721 group; (2)P<0.05 com pared with CBLB502 2.5μg/kg group. Since 4/6 Rhesus monkeys from IR group died du ring crisis period, sym p tom atic therapy du ration w as no t calcu lated
Item WR-2721 CBLB502 (μg/kg)2.5 10 40 An tibio tics treatm en t du ration (d) 20.2±2.6 20.6±4.2 19.3±3.1 20.3±4.6 Hem ostatic treatm ent duration (d) 8.3±3.0 9.6±2.9 6.7±4.2 4.3±3.7(1)To ta l transfusion vo lum e (m l) 95.0±22.6 75.0±33.2 20.0±15.5(1)(2) 37.5±56.8(1)(2)
骨髓:對(duì)癥治療組4只恒河猴在21d內(nèi)死亡,組織病理學(xué)觀察見(jiàn)骨髓腔空虛,廣泛出血,造血細(xì)胞顯著減少,殘留的造血細(xì)胞以成熟細(xì)胞為主,胞核固縮,并見(jiàn)大量含鐵血黃素沉積(圖2A)。WR-2721組可見(jiàn)骨髓腔少量出血,造血細(xì)胞增生,以粒細(xì)胞為主,其結(jié)構(gòu)損傷較對(duì)癥治療組有所改善(圖2B)。CBLB502 40μg/kg組骨髓均見(jiàn)造血細(xì)胞增生活躍,形態(tài)良好,核分裂象多見(jiàn),出血減少(圖2C)。該結(jié)果提示,CBLB502預(yù)防給藥對(duì)7.0Gy γ射線全身照射猴的骨髓組織有明顯保護(hù)作用。
腸道:對(duì)癥治療組恒河猴腸黏膜脫落、出血,腸腔散在細(xì)胞碎片,黏膜上皮細(xì)胞可見(jiàn)嗜酸性變,間質(zhì)細(xì)胞減少,淋巴細(xì)胞固縮,隱窩未見(jiàn)明顯增生(圖3A)。WR-2721組存活猴腸上皮排列整齊、結(jié)構(gòu)完整,隱窩和淋巴細(xì)胞增生,有少量出血,其結(jié)構(gòu)損傷較對(duì)癥治療組減輕(圖3B)。CBLB502 40μg/kg組恒河猴腸黏膜上皮細(xì)胞排列整齊,隱窩明顯增生,淋巴細(xì)胞數(shù)目增加,間質(zhì)細(xì)胞豐富、形態(tài)正常(圖3C),其結(jié)構(gòu)損傷較對(duì)癥治療組和WR-2721組明顯改善。
圖2 7.0Gy照射猴骨髓病理組織學(xué)觀察(HE ×400)Fig. 2 Histopatho logical observation of bone m arrow of Rhesus m onkey irrad iated with 7.0Gy γ-ray (HE ×400)A. Anim al in IR g roup and d ied on 21d; B. Anim al in WR-2721 g roup; C. Anim al in CBLB502 40μg/kg g roup
圖3 7.0Gy照射猴小腸病理組織學(xué)觀察(HE ×200)Fig. 3 Histopatho log ical observation of sm all in testine of Rhesus m onkey irrad iated with 7.0Gy γ-ray (HE ×200)A. An im a l in IR g roup and d ied on 21d; B. An im a l in WR-2721 g roup; C. Anim a l in CBLB502 40μg/kg g roup
腸系膜淋巴結(jié):對(duì)癥治療組腸系膜淋巴結(jié)皮質(zhì)變薄,皮髓分界不清,淋巴細(xì)胞顯著減少,核固縮、溶解,可見(jiàn)含鐵血黃素沉積(圖4A)。WR-2721組腸系膜淋巴結(jié)皮髓質(zhì)可見(jiàn)少量淤血、出血,淋巴細(xì)胞壞死和增生并存,可見(jiàn)核碎片和核分裂結(jié)構(gòu),其結(jié)構(gòu)損傷較對(duì)癥治療組有所改善(圖4B)。CBLB502 40μg/kg組恒河猴腸系膜淋巴結(jié)出血減少,皮髓質(zhì)分界清晰,淋巴細(xì)胞增生活躍(圖4C),其結(jié)構(gòu)損傷較對(duì)癥治療組和WR-2721組明顯改善。
圖4 7.0Gy照射猴腸系膜淋巴結(jié)病理組織學(xué)觀察(HE ×200)Fig. 4 Histopathological observation of m esenteric lym ph node of Rhesus monkey irradiated with 7.0Gy γ-ray (HE ×200)A. Anim al in IR g roup and d ied on 21d; B. Anim al in WR-2721 g roup; C. Anim al in CBLB502 40μg/kg g roup
本研究以7.0Gy60Co γ射線全身照射恒河猴為模型動(dòng)物,觀察了CBLB502對(duì)重度骨髓型急性放射病的防護(hù)效果。對(duì)動(dòng)物體征、存活率與存活時(shí)間、造血功能的損傷與重建、非造血器官及組織的損傷和修復(fù)等方面進(jìn)行比較分析發(fā)現(xiàn):7.0Gy γ射線全身照射后,對(duì)癥治療組恒河猴在3周內(nèi)死亡4只,死亡動(dòng)物存活時(shí)間為19.4±1.7d;所有動(dòng)物均出現(xiàn)不同程度的發(fā)熱,體溫最高達(dá)40.4℃;照射前0.5h單次肌肉注射CBLB502可明顯提高動(dòng)物活存率,其中2.5μg/kg CBLB502提高存活的效果與30m g/kg WR-2721相同(均存活5/6);CBLB502 10、40μg/kg組恒河猴全部存活,發(fā)熱動(dòng)物數(shù)減少,體重下降幅度較對(duì)癥治療組有所改善。照前0.5h單次肌肉注射2.5、10、40μg/kg CBLB502均可明顯促進(jìn)7.0Gy γ射線全身照射恒河猴造血功能恢復(fù),升高外周血白細(xì)胞、血小板和中性粒細(xì)胞數(shù)的最低值,縮短外周血細(xì)胞數(shù)最低值持續(xù)時(shí)間并使其開(kāi)始恢復(fù)時(shí)間提前。此外,CBLB502可明顯減輕7.0Gy60Co γ射線照射后骨髓、淋巴結(jié)和腸道等主要造血、免疫和消化器官的組織損傷。照射后39d骨髓造血細(xì)胞集落培養(yǎng)并未觀察到CBLB502可促進(jìn)造血細(xì)胞集落的生成,可能是因?yàn)楣撬铇颖静杉瘯r(shí)間較晚,此時(shí)各組動(dòng)物的造血功能均已恢復(fù)正常。本研究結(jié)果顯示,抗輻射損傷作用以CBLB502 40μg/kg組最佳,10μg/kg CBLB502仍具有較好的輻射防護(hù)效果;經(jīng)比較發(fā)現(xiàn),照前0.5h單次肌肉注射40μg/kg CBLB502對(duì)7.0Gy γ射線全身照射恒河猴的輻射防護(hù)作用明顯優(yōu)于國(guó)際公認(rèn)的輻射防護(hù)劑WR-2721(照前0.5h單次肌內(nèi)注射30m g/kg)。
TLR是最先被發(fā)現(xiàn)的模式識(shí)別受體,在人類(lèi)固有免疫和獲得性免疫應(yīng)答中發(fā)揮著重要作用[11-12],迄今為止在人體內(nèi)已發(fā)現(xiàn)其11個(gè)家族成員,且不同的TLR有其相應(yīng)的配體。目前研究發(fā)現(xiàn),TLR2/6、TLR5以及TLR9的激動(dòng)劑均能發(fā)揮輻射防護(hù)作用[2-5],其中細(xì)菌鞭毛蛋白是TLR 5已知的唯一配體。CBLB502是利用大腸埃希菌制備的重組蛋白,包含了沙門(mén)菌鞭毛蛋白完整的N末端和C末端保守的結(jié)構(gòu)域,不但可在常溫下長(zhǎng)時(shí)間保存而不喪失藥物活性,而且與鞭毛蛋白具有同樣的穩(wěn)定性,毒性和免疫原性卻大為較低[5]。利用TLR5基因敲除小鼠進(jìn)行研究發(fā)現(xiàn),CBLB502可通過(guò)TLR5依賴途徑激活NF-κB信號(hào)通路發(fā)揮輻射防護(hù)效應(yīng),其具體機(jī)制包括:①誘導(dǎo)IAP、Bc l-2表達(dá),抑制輻射所致的細(xì)胞凋亡;②誘導(dǎo)超氧化物歧化酶2的表達(dá),促進(jìn)機(jī)體清除氧自由基;③誘導(dǎo)多種具有輻射防護(hù)作用的細(xì)胞因子如G-CSF和IL-6的表達(dá),刺激白細(xì)胞和血小板的生成[5]。此外,不同于造血刺激生長(zhǎng)因子類(lèi)輻射防護(hù)劑,CBLB502在保護(hù)造血系統(tǒng)的同時(shí),還對(duì)消化系統(tǒng)有明確的輻射防護(hù)作用。目前,CBLB502的抗氧化損傷作用已在小鼠腎臟缺血再灌注損傷研究中得到證實(shí)[13]。由于對(duì)放射性腸道上皮損傷有顯著的治療作用,CBLB502也被用于頭頸部放療所致的皮炎及口腔黏膜炎的實(shí)驗(yàn)治療,可減輕輻射所致的上皮細(xì)胞損傷,且不降低腫瘤細(xì)胞對(duì)輻射的敏感性[14]。值得關(guān)注的是,有報(bào)道稱(chēng)CBLB502對(duì)放射性間質(zhì)性肺炎和肺纖維化也有明顯的預(yù)防作用[15]。相信隨著研究的不斷深入,CBLB502作為安全、有效的新型輻射防護(hù)劑,可擁有更加廣闊的應(yīng)用前景。
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