本文選題：前B細(xì)胞克隆增強(qiáng)因子 + 急性呼吸窘迫綜合征��； 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：背景：ARDS是ICU中的常見(jiàn)病和多發(fā)病，各種因素如肺部感染、膿毒血癥、重癥急性胰腺炎、創(chuàng)傷、輸血等均可導(dǎo)致ARDS的發(fā)生，是機(jī)體過(guò)度炎癥反應(yīng)在肺部的表現(xiàn)。隨著醫(yī)學(xué)的不斷發(fā)展，ARDS的發(fā)生率和死亡率較以前明顯降低，，但形式仍不容樂(lè)觀，因此，ARDS發(fā)病機(jī)制和治療手段研究成為眾多學(xué)者們關(guān)注的熱點(diǎn)，現(xiàn)有研究也顯示ARDS的發(fā)生發(fā)展機(jī)制十分復(fù)雜，涉及眾多的信號(hào)通路及細(xì)胞因子。 前B細(xì)胞克隆增強(qiáng)因子(Pre-B-Cell Colony Enhancing Factor)，又稱為內(nèi)脂素(visfatin)，或煙酰胺磷酸核糖轉(zhuǎn)移酶(Nampt)，是近年來(lái)發(fā)現(xiàn)的主要由成熟脂肪細(xì)胞和激活的炎癥細(xì)胞分泌，全身多個(gè)器官均可表達(dá)的炎癥因子。目前有體外細(xì)胞實(shí)驗(yàn)研究發(fā)現(xiàn)炎癥刺激可以使肺泡上皮細(xì)胞、肺微動(dòng)脈內(nèi)皮細(xì)胞等大量表達(dá)PBEF，而減少PBEF的表達(dá)則可減輕上述細(xì)胞由炎癥刺激引起的相關(guān)損害，這表明PBEF可能涉及包括肺微血管損傷、肺泡上皮細(xì)胞損傷、通透性增加等在內(nèi)有關(guān)ARDS的多個(gè)病理生理過(guò)程，同時(shí)也有學(xué)者通過(guò)對(duì)犬類、鼠類的不同ARDS模型進(jìn)行研究后認(rèn)為PBEF是ARDS的潛在生物標(biāo)志，因此PBEF可能成為研究ARDS的發(fā)病機(jī)制和治療ARDS的新靶點(diǎn)。 目的：以油酸尾靜脈注射法建立大鼠ARDS模型，并行藥物干預(yù)，觀察前B細(xì)胞克隆增強(qiáng)因子對(duì)ARDS大鼠肺組織病理學(xué)評(píng)分、濕干重比及動(dòng)脈血氧分壓的影響；對(duì)ARDS大鼠肺泡灌洗液中TNF-α及IL-1β含量的影響；對(duì)ARDS大鼠肺組織TNF-α、IL-1β mRNA和ICAM-1、VCAM-1mRNA及蛋白表達(dá)以及NF-κB p65蛋白表達(dá)的影響；通過(guò)肺組織病理學(xué)評(píng)分、濕干重比、炎癥因子及粘附因子表達(dá)及肺泡灌洗液中炎癥因子含量的變化，結(jié)合已有的體外實(shí)驗(yàn)所觀察到的現(xiàn)象，試圖證實(shí)前B細(xì)胞克隆增強(qiáng)因子在ARDS中的作用，為進(jìn)一步研究ARDS的發(fā)病機(jī)制和病理生理過(guò)程以及治療措施提供新的思路和依據(jù)。 方法：40只成年健康雄性SD大鼠，體質(zhì)量（214±26）g，按隨機(jī)數(shù)字法分為空白對(duì)照組(C組)、模型組(OA組)、藥物干預(yù)組(D組)、溶媒對(duì)照組(S組)，OA組、D組及S組大鼠以油酸尾靜脈注射法復(fù)制ARDS模型，劑量為0.15ml/kg，D組及S組大鼠于造模前24小時(shí)、12小時(shí)及半小時(shí)腹腔內(nèi)注射藥物，D組以10mg/kg腹腔內(nèi)注射PBEF抑制劑FK866，S組注射FK866等體積溶媒二甲亞砜。通過(guò)觀察大鼠呼吸頻率、皮膚及口唇顏色和活動(dòng)能力等初步判定造模是否成功。造模成功6小時(shí)后以3.5%水合氯醛以10ml/kg腹腔內(nèi)注射麻醉，麻醉成功后先分離出氣管，剖腹從腹主動(dòng)脈處抽動(dòng)脈血行血?dú)夥治鰴z測(cè)氧分壓（mmHg），開(kāi)胸觀察肺組織顏色及形態(tài)，結(jié)扎右肺門，行氣管切開(kāi)置入導(dǎo)管后行左肺肺泡灌洗術(shù)，并收集肺泡灌洗液，離心取上清液備用，測(cè)定炎癥因子TNF-α、IL-1β含量（ng/L）；收集完畢后取材，右肺上葉立即固定于福爾馬林中，行病理學(xué)、免疫組化檢測(cè)，右肺中葉行濕干重比檢測(cè)，右肺下葉立即凍存于-80℃冰箱或者液氮中備用，一部分以RT-PCR及RT-qPCR方法檢測(cè)炎癥因子TNF-α、IL-1β及粘附因子ICAM-1、VCAM-1mRNA的表達(dá)，另一部分以Western Blot方法檢測(cè)NF-κB p65蛋白的表達(dá)。 結(jié)果： 1.行為學(xué)及外表觀察 C組大鼠一般情況好，較為活潑，無(wú)毛發(fā)豎立等現(xiàn)象，口唇及皮膚呈現(xiàn)粉紅色，無(wú)呼吸困難；OA組、D組及S組大鼠造模后短時(shí)間內(nèi)即出現(xiàn)口唇及皮膚紫紺，全身毛發(fā)豎立，活動(dòng)減少，呼吸頻率顯著增加，與C組比較差異均有統(tǒng)計(jì)學(xué)意義（p0.001）：OA組與S組呼吸頻率最高，但兩者比較差異沒(méi)有統(tǒng)計(jì)學(xué)意義（p0.05），D組呼吸頻率較OA組與S組低，差異有統(tǒng)計(jì)學(xué)意義（均p0.05），各組均無(wú)死亡。 2.動(dòng)脈血氧分壓 C組大鼠動(dòng)脈血氧分壓為（103.2±11.2）mmHg，與其他各組比較差異有統(tǒng)計(jì)學(xué)意義（均p0.001），D組動(dòng)脈血氧分壓為（73.8±6.8）mmHg，與OA組及S組比較差異有統(tǒng)計(jì)學(xué)意義（p0.05），OA組動(dòng)脈血氧分壓為（59.6±6.7）mmHg，S組動(dòng)脈血氧分壓為（59.8±11.0）mmHg，OA組及S組比較差異無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。 3.肺組織病理學(xué)及濕干重比 C組大鼠肺組織在肉眼下觀察呈粉紅色，未見(jiàn)明顯充血、出血及腫脹，光鏡下觀察為正常肺組織結(jié)構(gòu)；OA組大鼠肺組織在肉眼觀察下呈暗紅色、腫脹，可見(jiàn)明顯出血、充血，光鏡下可見(jiàn)肺組織為典型的ARDS病理學(xué)表現(xiàn)；D組大鼠肺組織肉眼觀察仍可見(jiàn)腫脹及充血、出血，但程度較OA組輕，光鏡下可見(jiàn)肺組織仍有與OA組相同的病理改變，但受損面積明顯減��；S組大鼠肺組織肉眼觀與顯微鏡觀察結(jié)果與OA組類似。而C組濕干重比為4.174±0.311，與其他各組比較差異有統(tǒng)計(jì)學(xué)意義（均p0.001），D組濕干重比為5.855±0.200，與OA組及S組比較差異有統(tǒng)計(jì)學(xué)意義（p0.05），OA組濕干重比為6.527±0.239，S組濕干重比為6.664±0.324，OA組及S組比較差異無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。 4.免疫組化法檢測(cè)肺組織PBEF、ICAM-1、VCAM-1蛋白相對(duì)表達(dá)量 免疫組化檢測(cè)可在所有ARDS大鼠肺組織的支氣管粘膜上皮、血管內(nèi)皮、肺泡壁及肺泡水腫液中發(fā)現(xiàn)PBEF蛋白表達(dá)。與C組比較，OA組、S組PBEF、ICAM-1、VCAM-1蛋白相對(duì)表達(dá)量明顯升高，差異有統(tǒng)計(jì)學(xué)意義（均p0.001），而D組各指標(biāo)蛋白相對(duì)表達(dá)量較OA組及S組有所減少，但較C組高，差異有統(tǒng)計(jì)學(xué)意義（均p0.05）。 5.肺泡灌洗液中炎癥因子TNF-α及IL-1β含量 C組大鼠炎癥因子含量均較其他組低，兩者分別與其他各組比較差異有統(tǒng)計(jì)學(xué)意義（p0.05或p0.001），OA組與S組BALF中炎癥因子含量最多，但OA組及S組比較兩者差異均無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。D組各炎癥因子含量較OA組及S組低，差異均有統(tǒng)計(jì)學(xué)意義（p0.05） 6.RT-PCR法半定量及RT-qPCR法定量檢測(cè)肺組織PBEF、TNF-α、IL-1β、ICAM-1及VCAM-1mRNA表達(dá)水平 RT-PCR法半定量檢測(cè)各指標(biāo)結(jié)果顯示，C組各指標(biāo)mRNA相對(duì)表達(dá)量最低，較其他各組差異有統(tǒng)計(jì)學(xué)意義（p0.001或p0.05），D組各指標(biāo)mRNA相對(duì)表達(dá)量較OA組及S組低，差異具有統(tǒng)計(jì)學(xué)意義（p0.05或p0.001），OA組及S組各指標(biāo)mRNA相對(duì)表達(dá)量最高，但兩者相比差異沒(méi)有統(tǒng)計(jì)學(xué)意義（p0.05），RT-qPCR法檢測(cè)結(jié)果與RT-PCR法檢測(cè)結(jié)果所得出結(jié)果類似。 7.Western Blot法半定量檢測(cè)肺組織中NF-κB p65蛋白表達(dá)水平 Western Blot法半定量檢測(cè)各指標(biāo)結(jié)果顯示，C組NF-κB p65相對(duì)表達(dá)量最低，較其他各組差異有統(tǒng)計(jì)學(xué)意義（均p0.001），D組相對(duì)表達(dá)量較OA組及S組低，差異具有統(tǒng)計(jì)學(xué)意義（p0.05或p0.001），OA組及S組相對(duì)表達(dá)量最高，但兩者相比差異沒(méi)有統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論： 1、PBEF能促進(jìn)ARDS大鼠肺組織炎癥因子TNF-α及IL-1β的表達(dá)和生成； 2、PBEF能促進(jìn)ARDS大鼠肺組織粘附分子ICAM-1及VCAM-1的表達(dá)； 3、PBEF能促進(jìn)ARDS大鼠肺組織NF-κB的激活和表達(dá)。 因此， PBEF可能通過(guò)激活炎癥反應(yīng)，促進(jìn)炎癥因子和粘附分子表達(dá)等途徑造成肺組織的炎性損傷以及炎性細(xì)胞的浸潤(rùn)和遷移等，在ARDS發(fā)生發(fā)展中起重要作用。
[Abstract]:Background : ARDS is common disease and multiple diseases in ICU . Various factors such as pulmonary infection , sepsis , severe acute pancreatitis , trauma , transfusion , etc . can lead to ARDS . With the development of medicine , the incidence and mortality rate of ARDS is much lower than before , but the form is still not optimistic . Therefore , the pathogenesis and treatment of ARDS have become the focus of many scholars , and the existing research also shows that the development mechanism of ARDS is very complex , involving many signal pathways and cytokines .

It is suggested that PBEF may be a potential biomarker for ARDS , and PBEF may be a potential biomarker for ARDS , and PBEF may be a new target for the study of ARDS and ARDS .

Objective : To establish a rat ARDS model by intravenous injection of oleic acid tail vein , and observe the effect of pre - B cell clone enhancement factor on pathological score , wet dry weight ratio and arterial oxygen partial pressure in ARDS rats .
Effect of TNF - 偽 and IL - 1尾 in alveolar lavage fluid of ARDS rats
The effects of TNF - 偽 , IL - 1尾 mRNA and ICAM - 1 , VCAM - 1 mRNA and protein expression and the expression of NF - 魏B in the lung tissues of ARDS rats were studied .
In order to further study the role of preB cell clone enhancement factor in ARDS and to provide a new idea and basis for further research on the pathogenesis and pathological process of ARDS and the treatment measures , the role of the former B cell clone enhancement factor in ARDS was tried to confirm the role of the former B cell clone enhancement factor in ARDS .

Methods : Forty adult healthy male SD rats were randomly divided into two groups : blank control group ( group C ) , model group ( OA group ) , drug intervention group ( group D ) , vehicle control group ( S group ) , OA group , group D and S group .
The expression of TNF - 偽 , IL - 1尾 and ICAM - 1 and VCAM - 1 mRNA were detected by RT - PCR and RT - qPCR .

Results :

1 . Behavior and appearance observation

In group C , the general condition of the rats was good , more active , no hair was erected , the lip and skin were pink , and there was no difficulty in breathing ;
There was significant difference between OA group and group S group ( p < 0.05 ) . The difference between OA group and group S was significant ( p < 0.05 ) .

2 . Arterial blood oxygen partial pressure

The arterial oxygen partial pressure in group C was ( 103.2 鹵 11.2 ) mmHg , the difference was statistically significant compared with other groups ( p < 0.05 ) . The arterial oxygen partial pressure in OA group was ( 59.6 鹵 6.7 ) mmHg , and the arterial oxygen partial pressure in group S was ( 59.8 鹵 11.0 ) mmHg , and there was no significant difference between OA group and S group ( p < 0.05 ) .

3 . Lung tissue pathology and wet dry weight ratio

In group C , the lung tissues were pink without obvious congestion , hemorrhage and swelling , and the normal lung tissue structure was observed under the light microscope .
The lung tissues of OA group showed dark red , swelling , obvious hemorrhage , congestion , and lung tissue was typical ARDS pathological manifestation under light microscope .
In group D , swelling and congestion and bleeding were observed in the lung tissues of the rats , but the degree of bleeding was lower than that in OA group , but the lung tissue still had the same pathological changes as OA group , but the damaged area was obviously reduced ;
Compared with OA group , the wet dry weight ratio of group C was 5.855 鹵 0.200 , and the wet dry weight ratio in group D was 6.527 鹵 0.239 , and the wet dry weight ratio in group S was 6.664 鹵 0.324 , and there was no significant difference between the OA group and S group ( p < 0.05 ) .

4 . The expression of PBEF , ICAM - 1 and VCAM - 1 in lung tissues was detected by immunohistochemistry .

Compared with group C , the relative expression of PBEF , ICAM - 1 and VCAM - 1 in OA group and S group was significantly higher than that of OA group and S group ( P < 0.01 ) .

5 . TNF - 偽 and IL - 1尾 in alveolar lavage fluid

In group C , the inflammatory factors were lower in group C than in other groups ( p < 0.05 or p < 0.001 ) . There was no statistical difference between OA group and S group ( p < 0.05 ) . The inflammatory factors in group D were lower than those in group OA and S group ( p < 0.05 ) .

6.RT - PCR and RT - qPCR were used to detect the levels of PBEF , TNF - 偽 , IL - 1尾 , ICAM - 1 and VCAM - 1 mRNA in lung tissues .

RT-PCR娉曞崐瀹氶噺媯

本文編號(hào)：2062420