本文選題：共聚焦激光顯微內(nèi)鏡 + 上皮細(xì)胞屏障��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：背景與目的 目前非甾體抗炎藥(Non-Steroid Anti-Inflammatory Drugs, NSAIDs)被廣泛應(yīng)用于臨床,但在發(fā)揮治療作用的同時(shí),也可導(dǎo)致上消化道及腸道黏膜不同程度的損傷。60%～70%的NSAIDs相關(guān)腸病患者無特征性的癥狀,嚴(yán)重的并發(fā)癥如消化道大出血、腸道狹窄、腸穿孔等少見,但一旦發(fā)生,卻是致命性的。在NSAIDs相關(guān)胃黏膜損傷中,環(huán)氧合酶起著關(guān)鍵性作用。但研究認(rèn)為環(huán)氧合酶的抑制在NSAIDs相關(guān)腸黏膜損傷過程中并不起主要作用。目前,NSAIDs目關(guān)腸黏膜損傷機(jī)制尚無定論,較為認(rèn)可的是“三次打擊假說”。假說提出腸黏膜上皮屏障的完整性在NSAIDs相關(guān)腸病中至關(guān)重要。腸上皮屏障由上皮細(xì)胞及細(xì)胞間緊密連接構(gòu)成。上皮細(xì)胞再生的速度為脫落速度的一半,這就決定了細(xì)胞脫落后間隙的存在。脫落細(xì)胞間隙與腸黏膜屏障之間有密切的關(guān)系。已有研究證實(shí)脫落細(xì)胞間隙密度可預(yù)測(cè)炎癥性腸病(Inflammatory bowel disease, IBD)的嚴(yán)重程度及其與復(fù)發(fā)之間的關(guān)系。本研究應(yīng)用共聚焦激光顯微內(nèi)鏡(Confocal laser endomicroscopy,CLE),觀察吲哚美辛相關(guān)小腸上皮屏障損傷的脫落細(xì)胞間隙密度,并分析其發(fā)生機(jī)制,同時(shí)研究替普瑞酮和雷貝拉唑?qū)ι掀て琳系谋Ｗo(hù)機(jī)制。 材料及方法 第一部分：NSAIDs相關(guān)小腸黏膜損傷及其藥物干預(yù)的形態(tài)學(xué)研究 雄性Wistar大鼠,按體重完全隨機(jī)分為八組,每組6只,分別為健康對(duì)照組、模型組(采用吲哚美辛造模)、替普瑞酮預(yù)防組、雷貝拉唑預(yù)防組、治療對(duì)照組、替普瑞酮治療組、雷貝拉唑治療組,以及替普瑞酮與雷貝拉唑聯(lián)合治療組(聯(lián)合治療組)。各組模型制備成功后水合氯醛腹腔注射麻醉成功后,沿十二指腸向下約10cm,取上段空腸,沿腸系膜對(duì)側(cè)縱行剖開腸管,切口長(zhǎng)約1-2cm,注意保護(hù)腸管的血流。暴露要觀察的黏膜后心內(nèi)注射熒光素鈉注射液1min后用觀察。將黏膜平鋪開,將optiscan FIVE1探頭垂直,輕柔放置于黏膜表面,觀察時(shí)間控制在20min內(nèi)。選擇圖像質(zhì)量佳的5個(gè)圖像,計(jì)數(shù)1000個(gè)上皮細(xì)胞中脫落細(xì)胞間隙數(shù)。觀察結(jié)束后取小腸上段組織塊長(zhǎng)約1cm,沖洗掉表面血跡及粘液,立即10%中性甲醛固定12h后,沿腸系膜對(duì)側(cè)剖開腸管,大頭針固定于薄板上,盡量展平腸管,繼續(xù)放入中性甲醛中固定12h。固定后的組織脫水后,蠟塊包埋,切片厚度4um,蘇木素-伊紅(Hematoxylin and Eosin, HE)染色后觀察。取空腸上段組織塊,掃描電子顯微鏡(Scanning electron microscopy, SEM)下觀察黏膜表面超微結(jié)構(gòu)。所有數(shù)據(jù)應(yīng)用SPSS13.0統(tǒng)計(jì)軟件分析。數(shù)據(jù)以x±s表示,多樣本間兩兩比較檢驗(yàn)方差齊性,兩兩組間采用LSD-t檢驗(yàn),方差不齊時(shí)兩兩組間比較采用Hamhane's T2檢驗(yàn)。P0.05為差異有統(tǒng)計(jì)學(xué)意義。 第二部分：NSAIDs相關(guān)小腸黏膜損傷及其藥物干預(yù)的分子機(jī)制研究 雄性Wistar大鼠,按體重完全隨機(jī)分為八組,每組6只,分別為健康對(duì)照組、模型組(采用吲哚美辛造模)、替普瑞酮預(yù)防組、雷貝拉唑預(yù)防組、治療對(duì)照組、替普瑞酮治療組、雷貝拉唑治療組,以及替普瑞酮與雷貝拉唑聯(lián)合治療組(聯(lián)合治療組)。各組模型制備成功后水合氯醛腹腔注射麻醉成功后,心內(nèi)穿刺抽取血,離心后取上清,按照酶聯(lián)免疫吸附試驗(yàn)(Enzyme linked immunosorbent assay, ELISA)試劑盒指示測(cè)定血清腫瘤壞死因子-a (Tumor Necrosis Factor-α, TNF-α)濃度；沿十二指腸向下約10cm,取小腸上段組織,剝?nèi)○つ犹崛〉鞍?用蛋白免疫印跡(Western Blotting, WB)方法測(cè)定組織中caspase-3及核因子-κB(nuclear factor-kappa B, NF-κB)表達(dá)及組織中閉合蛋白(occludin)、閉鎖小帶蛋白1(Zonula occludens protein1, ZO-1)的表達(dá)。應(yīng)用SPSS13.0統(tǒng)計(jì)軟件分析數(shù)據(jù)。數(shù)據(jù)以x±s表示,多樣本間比較檢驗(yàn)方差齊性,兩兩組間采用LSD-t檢驗(yàn),方差不齊時(shí)兩兩組間比較采用Hamhane'sT2檢驗(yàn)。P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 第一部分： 1.CLE脫落細(xì)胞間隙 脫落細(xì)胞間隙呈強(qiáng)熒光,較吸收細(xì)胞略小,部分可見射流樣征象。替普瑞酮預(yù)防組和雷貝拉唑預(yù)防組空腸組織的脫落細(xì)胞間隙密度分別為(57.43±24.55)/1000和(59.80±21.14)/1000,均低于模型組的(110.93±50.58)/1000,差異均有統(tǒng)計(jì)學(xué)意義(t=53.50、54.13,P均0.01)。聯(lián)合治療組的脫落細(xì)胞間隙密度為(40.53±15.39)／1000,低于治療對(duì)照組的(93.80±40.65)/1000,差異有統(tǒng)計(jì)學(xué)意義(t=44.27,P0.01)。 2.HE病理學(xué)及SEM脫落細(xì)胞間隙 組織固定后HE病理組織學(xué)觀察,亦可見脫落或正在脫落的細(xì)胞,但脫落細(xì)胞間隙很少能觀察到。掃描電鏡下脫落細(xì)胞間隙清晰可見,呈空洞樣,周圍可見小腸上皮吸收細(xì)胞,與共聚焦激光顯微內(nèi)鏡下表現(xiàn)類似。 第二部分： 1.血清TNF-α濃度 替普瑞酮預(yù)防組和雷貝拉唑預(yù)防組的TNF-α水平分別為(25.80±8.97)和(22.74±7.15) ng/L,均低于模型組的(44.48±7.42) ng/L,差異均有統(tǒng)計(jì)學(xué)意義(t=18.68、21.74,P均0.01)。聯(lián)合治療組TNF-α水平為(13.66±4.98)ng/L,低于治療對(duì)照組的(24.67±6.70)ng/L,差異有統(tǒng)計(jì)學(xué)意義(t=9.02,P0.01)。 2. caspase-3及NF-κB含量 替普瑞酮預(yù)防組和雷貝拉唑預(yù)防組的caspase-3水平分別為1.47±0.35和1.58±0.34,NF-κB水平分別為1.27±0.14和1.21±0.10,與模型組(2.44±0.45和1.69±0.13)相比,差異均有統(tǒng)計(jì)學(xué)意義(t=0.97、0.86、0.42、0.48,P均0.01)。聯(lián)合治療組的caspase-3和NF-κB水平分別為0.66±0.06和0.44±0.21,低于治療對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(t=0.34、0.56,P均0.01)。 3. occludin及ZO-1表達(dá) 替普瑞酮預(yù)防組和雷貝拉唑預(yù)防組的occludin水平分別為0.69±0.16和0.74±0.11,ZO-1水平分別為0.81±0.08和0.84±0.12,與模型組(0.45±0.22和0.64±0.07)相比,差異均有統(tǒng)計(jì)學(xué)意義(t=0.24、0.29、0.17、0.21,P均0.01)。聯(lián)合治療組的occludin和ZO-1水平分別為2.50±0.46和1.76±0.18,高于治療對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(t=1.50、0.76,P均0.01)。 結(jié)論 1.共聚焦激光顯微內(nèi)鏡可以清晰地觀察腸上皮脫落細(xì)胞間隙并進(jìn)行密度測(cè)定,脫落細(xì)胞間隙密度可作為預(yù)測(cè)炎癥反應(yīng)程度及上皮屏障功能以及評(píng)價(jià)藥物療效的有價(jià)值指標(biāo)。 2.TNF-α增加通過NF-κB-caspase-3途徑導(dǎo)致緊密連接蛋白o(hù)ccludin和ZO-1表達(dá)的減少是導(dǎo)致吲哚美辛相關(guān)小腸黏膜損傷過程中細(xì)胞脫落加速的機(jī)制之一。 3.替普瑞酮與雷貝拉唑可以抑制TNF-α增加,通過NF-KB-caspase-3途徑,改善細(xì)胞緊密連接蛋白的表達(dá),改善腸上皮屏障的損傷,從而達(dá)到預(yù)防和治療吲哚美辛相關(guān)小腸黏膜損傷的作用。 研究意義 本研究證實(shí)共聚焦激光顯微內(nèi)鏡可以清晰得觀察腸上皮脫落細(xì)胞間隙并進(jìn)行脫落細(xì)胞間隙密度的測(cè)定,從而客觀得評(píng)價(jià)吲哚美辛相關(guān)小腸黏膜的損傷,將來有望將其運(yùn)用于臨床,用于腸道炎癥嚴(yán)重程度的評(píng)價(jià),成為一種在體檢測(cè)腸上皮細(xì)胞屏障完整性的新方法及評(píng)價(jià)藥物療效的新指標(biāo)。雷貝拉唑和替普瑞酮可以抑制TNF-α增加,通過調(diào)節(jié)NF-κB-caspase-3途徑,改善細(xì)胞緊密連接蛋白的表達(dá),改善腸上皮屏障的損傷,從而達(dá)到預(yù)防和治療吲哚美辛相關(guān)小腸黏膜損傷的作用。這為兩種藥物在NSAIDs所致小腸黏膜損傷治療的臨床應(yīng)用提供動(dòng)物實(shí)驗(yàn)佐證。
[Abstract]:Background and purpose
At present, Non-Steroid Anti-Inflammatory Drugs (NSAIDs) is widely used in clinical practice, but it can also lead to a different degree of damage to the upper digestive tract and intestinal mucosa in.60% to 70% of NSAIDs related bowel disease patients with no characteristic symptoms, serious complications such as massive hemorrhage of digestive tract, and intestinal stenosis. Intestinal perforation is rare, but once it occurs, it is fatal. Cyclooxygenase plays a key role in NSAIDs related gastric mucosal injury. However, the inhibition of cyclooxygenase is not a major role in NSAIDs related intestinal mucosal damage. The mechanism of NSAIDs mesenteric mucous membrane damage is not conclusive, and "three" The hypothesis suggests that the integrity of the intestinal mucosal epithelial barrier is essential in NSAIDs related bowel disease. The intestinal epithelial barrier is composed of close connections between epithelial cells and cells. The rate of epithelial cell regeneration is half of the rate of exfoliation, which determines the existence of the clearance of the cells after the exfoliation of the cells. There has been a close relationship. Studies have shown that exfoliative cell space density can predict the severity of Inflammatory bowel disease (IBD) and its relationship with recurrence. This study used confocal laser endoscopy (Confocal laser endomicroscopy, CLE) to observe the damage of indomethacin related intestinal epithelial barrier injury. The gap density was analyzed and the protective mechanism of tebuprazone and rabeprazole against epithelial barrier was investigated.
Materials and methods
Part one: morphological study of NSAIDs related intestinal mucosal injury and drug intervention
Male Wistar rats were divided into eight groups according to the total weight of body weight, with 6 rats in each group, including the healthy control group, the model group (using indomethacin model), the tipriprazole prevention group, the riprazole prevention group, the treatment control group, the tipriprone treatment group, the riprazol treatment group, and the combined treatment group of tepreone and riprazole and riprazole (combined treatment group). After successful intraperitoneal injection of chloral chloral injection after the successful preparation of the group model, the upper jejunum was taken along the duodenum, the upper jejunum was taken along the duodenum, the intestinal canal was opened along the mesenteric side, the incision was about 1-2cm, and the blood flow of the intestinal tube was protected. After the observation of the intracardiac injection of Fluorescein Sodium Injection 1min, the mucosa was observed. The mucous membrane was paved open and optiscan The FIVE1 probe is vertical and gently placed on the surface of the mucous membrane, and the observation time is controlled within the 20min. 5 images with good image quality are selected and the number of exfoliated cells in the 1000 epithelial cells is counted. After observation, the upper segment of the small intestine is about 1cm long, and the surface blood and mucus are flushed out. After the 10% neutral formaldehyde is fixed for 12h, the mesenteric side section is on the side of the mesentery. Open the intestine, the pin was fixed to the plate, and the intestine was flattened as far as possible. After the tissue dehydration was fixed in the neutral formaldehyde for 12h. fixation, the wax block was embedded, the thickness of the slice was 4um, and the hematoxylin eosin (Hematoxylin and Eosin, HE) was observed. The upper segment of the jejunum was taken and the scanning electron microscope (Scanning electron microscopy, SEM) was taken down. The ultrastructure of the mucosal surface was examined. All data were analyzed by SPSS13.0 software. The data were expressed in X + s. The 22 test variances were homogeneous in the diversity, and the 22 groups were tested by LSD-t test. The difference between the 22 groups using the Hamhane's T2 test.P0.05 was statistically significant.
The second part: NSAIDs related intestinal mucosal injury and the molecular mechanism of drug intervention.
Male Wistar rats were divided into eight groups according to the total weight of body weight, with 6 rats in each group, including the healthy control group, the model group (using indomethacin model), the tipriprazole prevention group, the riprazole prevention group, the treatment control group, the tipriprone treatment group, the riprazol treatment group, and the combined treatment group of tepreone and riprazole and riprazole (combined treatment group). After successful intraperitoneal injection of chloral chloral injection after the successful preparation of the group model, the blood was extracted from the heart, and then the supernatant was taken after centrifugation. The concentration of serum TNF -a (Tumor Necrosis Factor- alpha, TNF- alpha) was determined according to the enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), and the concentration of the serum tumor necrosis factor -a (Tumor Necrosis Factor- a, TNF- alpha) was measured along the duodenum, and down about 10cm, The expression of Caspase-3 and nuclear factor kappa B (nuclear factor-kappa B, NF- kappa B) in tissue and the expression of closed protein (occludin) in tissue were measured by Western Blotting (WB). Analysis data. The data were expressed in X + s, and the variance was homogeneous. The 22 groups were tested by LSD-t test. When the variance was not homogeneous, the difference between the 22 groups using the Hamhane'sT2 test.P0.05 was statistically significant.
Result
Part one:
1.CLE abscission cell space
The exfoliated cell space showed strong fluorescence, slightly smaller than the absorbable cells, and some jet samples were visible. The interspace density of the exfoliated cells in the jejunum tissue of the preventative and riprazole prevention groups was (57.43 + 24.55) /1000 and (59.80 + 21.14) /1000 respectively, which were lower than that of the model group (110.93 + 50.58) /1000, and the difference was statistically significant (t=53.50,54.13 P 0.01). The interspace density of the exfoliated cells in the combined treatment group was (40.53 + 15.39) / 1000, which was lower than that of the treatment control group (93.80 + 40.65) /1000, and the difference was statistically significant (t=44.27, P0.01).
2.HE pathology and SEM exfoliative cell space
After tissue fixation, the HE histopathological observation was also observed, and the exfoliated or falling cells were also visible, but the exfoliated cell space was rarely observed. The exfoliated cell gap was clearly visible under scanning electron microscope, and the epithelial absorption cells were visible around the small intestinal epithelium, similar to the confocal laser microendoscopy.
The second part:
1. serum TNF- alpha concentration
The TNF- alpha levels of the preventonone prevention group and the riprazole prevention group were (25.80 + 8.97) and (22.74 + 7.15) ng/L respectively, which were lower than those of the model group (44.48 + 7.42) ng/L, and the difference was statistically significant (t=18.68,21.74, P 0.01). The level of TNF- alpha in the combined treatment group was (13.66 + 4.98) ng/L, which was lower than that of the treatment control group (24.67 + 6.70) ng/L. T=9.02, P0.01.
2. caspase-3 and NF- kappa B content
The caspase-3 levels of the preventonone prevention group and the riprazole prevention group were 1.47 + 0.35 and 1.58 + 0.34 respectively, and the levels of NF- kappa B were 1.27 + 0.14 and 1.21 + 0.10 respectively. Compared with the model group (2.44 + 0.45 and 1.69 + 0.13), the difference was statistically significant (t=0.97,0.86,0.42,0.48, P all 0.01). The level of Caspase-3 and NF- kappa B in the combined treatment group was 1.58, respectively. 6 + 0.06 and 0.44 + 0.21, lower than the treatment group, the difference was statistically significant (t=0.34,0.56, P all 0.01).
3. occludin and ZO-1 expression
The occludin levels of the preventonone prevention group and the riprazole prevention group were 0.69 + 0.16 and 0.74 + 0.11 respectively, and the levels of ZO-1 were 0.81 + 0.08 and 0.84 + 0.12 respectively. Compared with the model group (0.45 + 0.22 and 0.64 + 0.07), the differences were statistically significant (t=0.24,0.29,0.17,0.21, P were all 0.01). The occludin and ZO-1 levels in the combined treatment group were respectively 0.74 6 and 1.76 + 0.18 were higher than those in the treatment group, and the differences were statistically significant (t=1.50,0.76, P 0.01).
conclusion
1. confocal laser endoscopy can clearly observe the intercellular space of the intestinal epithelium and determine the density of the cells. The gap density of the exfoliated cells can be used as a valuable indicator to predict the degree of inflammation and the function of epithelial barrier and to evaluate the efficacy of the drug.
The increase of 2.TNF- alpha through the NF- kappa B-caspase-3 pathway leads to the decrease in the expression of closely connexin occludin and ZO-1, which is one of the mechanisms of cell exfoliation acceleration during indomethacin related intestinal mucosal injury.
3. tepreone and rabezole can inhibit the increase of TNF- alpha, improve the expression of close connexin by NF-KB-caspase-3 pathway, improve the damage of intestinal epithelial barrier, and thus prevent and treat indomethacin related intestinal mucosal injury.
research meaning
This study confirms that confocal laser endoscopy can clearly observe the interspace of exfoliative cells in the intestinal epithelium and determine the gap density of the exfoliated cells, thus objectively evaluating the damage of the indomethacin related small intestinal mucosa. It is expected to be used in the future for clinical evaluation of the severity of intestinal inflammation and become a kind of intestinal test. A new method for the integrity of the skin cell barrier and a new index for the evaluation of the efficacy of the drug. Rabeprazole and teprirone can inhibit the increase of TNF- alpha, improve the expression of close connexin by regulating the NF- kappa B-caspase-3 pathway, improve the damage of the intestinal epithelial barrier, and thus achieve the prevention and treatment of indomethacin related intestinal mucosal injury. This provides an animal experimental evidence for the clinical application of two drugs in the treatment of small intestinal mucosal injury caused by NSAIDs.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

【共引文獻(xiàn)】

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