【摘要】：目的 1.采用氣管給藥制造大鼠肺間質(zhì)纖維化模型；2.觀(guān)察CKLFl-C19多肽及氫化可的松琥珀酸鈉對(duì)博萊霉素致肺纖維化大鼠的治療作用；3.探討CKLFl-C19多肽及氫化可的松琥珀酸鈉對(duì)大鼠肺間質(zhì)纖維化模型肺組織中腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)、轉(zhuǎn)化生長(zhǎng)因子β1(transforming growth factor-β1,TGF-β1)含量的影響；4.對(duì)比CKLFl-C19多肽與氫化可的松琥珀酸鈉對(duì)大鼠肺間質(zhì)纖維化模型肺組織細(xì)胞形態(tài)學(xué)及肺組織中TNF-α、TGF-β1蛋白含量的影響差異。 方法 健康Wistar大鼠60只,雄性,體重(200±20)g,實(shí)驗(yàn)大鼠隨機(jī)分為4組,即對(duì)照組、模型組、CKLFl-C19多肽組及激素組各15只。用10%水合氯醛(3m1.kg-1)腹腔注射麻醉大鼠,將其頭和四肢固定后,采用氣管給藥法制造肺纖維化模型,方法如下：用開(kāi)口器撬開(kāi)口腔,拉出舌,在額鏡直視下,趁大鼠吸氣的瞬間,迅速將與1ml注射器相連的硬脊膜外麻醉導(dǎo)管插入氣管內(nèi)約1～2厘米,見(jiàn)到導(dǎo)管內(nèi)液面隨大鼠呼吸而波動(dòng)后,向模型組、CKLFl-C19多肽組及激素組的大鼠氣管內(nèi)一次性注入0.4%博萊霉素0.25mL (5mg.kg-1),同時(shí)對(duì)照組注入生理鹽水0.25ml作為陰性對(duì)照,注藥后立即將大鼠直立并左右旋轉(zhuǎn),使藥液在肺內(nèi)分布均勻。造模后第二天開(kāi)始,對(duì)照組與模型組每天給予生理鹽水(2mL/只)灌胃,CKLFl-C19多肽組給予CKLFl-C19多肽(100μ g)腹腔注射,激素組給予氫化可的松琥珀酸鈉(25mg/g)腹腔注射干預(yù)。每天觀(guān)察各組動(dòng)物的一般情況(體型、精神狀態(tài)、皮毛、攝食、活動(dòng)、糞便等)并稱(chēng)重。在給藥后的第7、14、28天采用腹主動(dòng)脈放血法將各組大鼠分別處死5只,打開(kāi)胸腔后,分離并取出肺組織,觀(guān)察各組大鼠雙肺外觀(guān)情況后稱(chēng)重,將左肺浸于10%中性甲醛溶液中固定,常規(guī)石蠟包埋、切片,HE染色,評(píng)價(jià)肺泡炎及肺纖維化程度,并采用免疫組化法測(cè)定肺組織中TGF-β1的含量。取右肺,用冷生理鹽水洗去浮血,在冰浴上剪成碎塊,制成10%肺勻漿,3000r.min-1離心10min后取上清液放置于-80℃冰箱中保存,采用酶聯(lián)免疫吸附法(ELISA法)測(cè)定TNF-α的含量。 結(jié)果 1.大鼠的一般情況：正常對(duì)照組大鼠反應(yīng)靈敏,體格肥壯,皮毛光亮、致密,攝食正常。模型組大鼠均有不同程度的咳嗽和呼吸困難,攝食減少,體重下降,精神差,活動(dòng)明顯減少,皮毛晦暗,無(wú)光澤。CKLF1-C19多肽組前期表現(xiàn)類(lèi)似模型組,給藥7天后有所好轉(zhuǎn),治療14天后明顯好轉(zhuǎn),精神狀態(tài)轉(zhuǎn)好,食欲及活動(dòng)量較前均有所增加。激素組大鼠表現(xiàn)與CKLF1-C19多肽組無(wú)明顯差異。 2.肺組織大體病理變化：對(duì)照組大鼠雙肺外觀(guān)正常,肺葉飽滿(mǎn),呈粉紅色,表面光滑,彈性良好。模型組第7天雙肺體積增大,呈暗紅色,表面可見(jiàn)散在點(diǎn)狀出血；14天雙肺體積較前縮小,彈性差,除散在點(diǎn)狀出血點(diǎn)外,還可見(jiàn)大小不等的散在灰白色結(jié)節(jié)；第28天雙肺體積較前明顯縮小,呈灰白色,彈性差且硬度增加,可見(jiàn)大量灰白色結(jié)節(jié)。CKLF1-C19多肽組部分肺葉表面光滑,呈暗紅色,部分局部見(jiàn)大小不等的灰白色結(jié)節(jié),部分邊緣有散在出血點(diǎn),部分肺葉體積縮小,硬度增加。激素組與CKLF1-C19多肽組相似。 3.肺系數(shù)的比較：模型組、CKLF1-C19多肽組、激素組第7、14天和28天時(shí)肺系數(shù)均顯著大于對(duì)照組(P0.05), CKLF1-C19多肽組、激素第7、14、28時(shí)肺系數(shù)均小于模型組(P0.05), CKLF1-C19多肽組與激素組無(wú)明顯差異。 4.病理切片肺泡炎和肺纖維化程度的比較：對(duì)照組大鼠肺組織大多數(shù)結(jié)構(gòu)清晰,各觀(guān)察時(shí)間點(diǎn)均未出現(xiàn)明顯的形態(tài)學(xué)改變。模型組第7天可見(jiàn)明顯炎癥改變,以巨噬細(xì)胞、淋巴細(xì)胞為主的大量炎性細(xì)胞浸潤(rùn),成纖維細(xì)胞增多,伴有肺泡隔增寬；第14天時(shí)可見(jiàn)大量炎性細(xì)胞浸潤(rùn),肺泡間隔明顯增寬,肺泡變形,伴有成纖維細(xì)胞、平滑肌細(xì)胞增殖及膠原纖維沉積；28天時(shí)肺部廣泛纖維化,肺泡間隔繼續(xù)增寬,肺泡腔明顯縮小,大部分肺泡消失,細(xì)胞成分減少,肺間質(zhì)被膠原纖維和成纖維細(xì)胞替代,纖維成分增多較前更為明顯,但肺部炎性細(xì)胞浸潤(rùn)有所減輕。CKLF1-C19多肽組和激素組各時(shí)間點(diǎn)的肺泡炎程度與模型組比均明顯減輕(P0.05),28天時(shí)同對(duì)照組比較已無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。肺纖維化程度與與模型組比較也有所減輕,28天時(shí)肺纖維化的緩解程度較模型組有所減緩,但較對(duì)照組仍較嚴(yán)重。CKLF1-C19多肽組和激素組肺泡炎及纖維化程度在各觀(guān)察點(diǎn)比較差別不大(P0.05)。 5.肺組織中TNF-α、TGF-β1含量變化：模型組、CKLF1-C19多肽組及激素組肺組織勻漿中TNF-α含量明顯高于對(duì)照組(P0.05), CKLF1-C19多肽組和激素組低于模型組(P0.05),而CKLF1-C19多肽組和激素組無(wú)明顯差異；第7到第28天,模型組、CKLF1-C19多肽組及激素組大鼠肺組織TNF-α含量有遞減趨勢(shì)。TGF-β1蛋白在對(duì)照組表達(dá)弱陽(yáng)性,僅在少數(shù)細(xì)支氣管黏膜上皮細(xì)胞的胞漿中出現(xiàn),模型對(duì)照組TGF-β1的表達(dá)在第14天時(shí)為最強(qiáng),隨時(shí)間推移呈遞減趨勢(shì),主要位于肺泡巨噬細(xì)胞、支氣管黏膜上皮細(xì)胞、血管內(nèi)皮細(xì)胞及成纖維細(xì)胞的胞漿中。CKLF1-C19多肽組和激素組第14、28天肺組織中TGF-β1的表達(dá)均低于模型組(P0.05), CKLF1-C19多肽組和激素組的差異無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論 1. CKLF1-C19多肽和氫化可的松琥珀酸鈉對(duì)博萊霉素致大鼠肺纖維化模型的肺泡炎和肺纖維化有一定的治療作用； 2. CKLF1-C19多肽和氫化可的松琥珀酸鈉可能部分通過(guò)降低TNF-α、TGF-β1的含量抑制博萊霉素致肺部炎癥及肺纖維化的發(fā)展。 3. CKLF1-C19多肽與氫化可的松琥珀酸鈉治療對(duì)博萊霉素致大鼠肺纖維化模型的肺泡炎與肺纖維化的療效無(wú)明顯差異。
[Abstract]:objective
1. the rat pulmonary fibrosis model was made by tracheal administration; 2. the therapeutic effect of CKLFl-C19 polypeptide and hydrocortisone sodium on bleomycin induced pulmonary fibrosis in rats; 3. to explore the tumor necrosis factor alpha (tumor necrosis) in the lung tissue of rat pulmonary fibrosis model with CKLFl-C19 polypeptide and hydrocortisone sodium succinate. Factor- alpha, TNF- alpha), the effect of transforming growth factor beta 1 (transforming growth factor- beta 1, TGF- beta 1), and 4. to compare the effects of CKLFl-C19 polypeptide and hydrocortisone sodium on the cell morphology of lung tissue in rat pulmonary fibrosis model and the difference of the content of TNF- A and TGF- beta 1 egg white in the lung tissue.
Method
60 healthy Wistar rats, male and body weight (200 + 20) g, were randomly divided into 4 groups, namely the control group, the model group, the CKLFl-C19 polypeptide group and the hormone group 15. The rats were intraperitoneally injected with 10% chloral chloral (3m1.kg-1) and the head and extremities were fixed and the lung fibrotic model was made by the gas tube method. The methods were as follows: prying open with an opening device. The mouth, pull out the tongue, under the forehead mirror directly under the eye, while the rat inhaled at the moment, quickly the 1ml syringe attached to the spinal epidural anesthesia catheter inserted into the trachea about 1~2 centimeters, see the catheter liquid level with the rat breathing and fluctuate, the model group, the CKLFl-C19 polypeptide group and the hormone group of rats endotracheal injection of bleomycin 0.25mL (5m G.kg-1), at the same time, the control group was injected with normal saline 0.25ml as negative control. Immediately after the injection, the rats were erect and rotated right and left, and the liquid was distributed evenly in the lungs. After second days of modeling, the control group and the model group were given the saline (2mL/ only) every day, and the CKLFl-C19 polypeptide group gave CKLFl-C19 polypeptide (100 mu g) intraperitoneal injection, hormone group. Intraperitoneal injection of hydrocortisone sodium (25mg/g) was given by intraperitoneal injection. The general condition of each group (body, mental state, fur, feeding, activity, feces, etc.) was observed every day and weighed. 5 rats were killed by abdominal aorta blood release on day 7,14,28 after the administration. After opening the thoracic cavity, the lung tissue was separated and taken out and observed in each group. Group rats were weighed after double lung appearance. Left lung was impregnated in 10% neutral Formaldehyde Solution. Paraffin embedding, slicing, staining, HE staining, evaluating the degree of pulmonary alveolitis and pulmonary fibrosis, and using immunohistochemical method to determine the content of TGF- beta 1 in the lung tissue. The right lung was taken, the cold physiological salt water was washed to float the blood, and the pieces were cut into pieces on the ice bath and made into 10% lung homogenates. 3000r.min-1 after centrifugation 10min, the supernatant was stored in the refrigerator at -80 C, and the content of TNF- alpha was determined by enzyme linked immunosorbent assay (ELISA).
Result
The general situation of the 1. rats: the rats in the normal control group were sensitive to the reaction, the body was fat, the fur was bright, and the feeding was normal. The rats in the model group had different degrees of cough and breathing difficulties, the loss of food intake, the loss of body weight, the decrease of the activity, the obscure fur and the lustrous.CKLF1-C19 polypeptide group, which was similar to the model group in the early period of 7 days. After 14 days the treatment was better, the mental state was better, the appetite and the amount of activity increased. The performance of the hormone group was not significantly different from the CKLF1-C19 polypeptide group.
2. the gross pathological changes of lung tissue: the double lung appearance of the control group was normal, the lung lobes were full, the surface was pink, the surface was smooth and the elasticity was good. In the model group, the volume of two lungs increased in seventh days, the surface was dark red, the surface could be seen scattered on the dot like bleeding; the volume of double lungs was narrower, and the elasticity was poor, except for the point like bleeding on the 14 day of the 14 day. Gray and white nodules; twenty-eighth days the volume of double lung is narrower than before, it is gray white, the elasticity is poor and the hardness increases. It can be seen that a large number of gray white nodules.CKLF1-C19 polypeptide group is smooth, dark red, some local gray nodules with different size, partial edge of the bleeding point, part of the lung volume narrowing, hardness increase. The vegetarian group was similar to the CKLF1-C19 polypeptide group.
3. comparison of lung coefficient: lung coefficient of model group, CKLF1-C19 polypeptide group, 7,14 day and 28 day of hormone group were significantly greater than that of control group (P0.05), CKLF1-C19 polypeptide group, lung coefficient at 7,14,28 of hormone were less than that of model group (P0.05), and there was no significant difference between CKLF1-C19 polypeptide group and hormone group.
4. comparison of pulmonary alveolitis and pulmonary fibrosis in pathological section: most of the structure of lung tissue in the control group was clear, and no obvious morphological changes were found at all time points. In the model group, obvious inflammatory changes were observed on the seventh day of the model group, a large number of inflammatory cells were infiltrated with macrophages and lymphocytes, fibroblasts increased, and alveolar septum was associated with the alveolar septum. In Fourteenth days, a large number of inflammatory cells were infiltrated, alveolar septum widened obviously, alveolar deformability, fibroblasts, smooth muscle cell proliferation and collagen fibrous deposition, pulmonary fibrosis, alveolar septum widening, alveolar cavity narrowing, large partial alveoli disappeared, cell components reduced, and interstitial lung collagen fibers at 28 days With fibroblast replacement, the increase of fiber composition was more obvious than before, but the alveolitis degree of.CKLF1-C19 polypeptide group and hormone group decreased significantly (P0.05), and there was no statistical meaning (P0.05) compared with the control group at 28 days. The degree of pulmonary fibrosis was compared with that of the model group. The degree of pulmonary fibrosis relieved at 28 days was lower than that in the model group, but there was little difference between the control group and the severe.CKLF1-C19 polypeptide group and the hormone group. The degree of pulmonary alveolitis and fibrosis was not very different at the observation points (P0.05).
5. changes in the content of TNF- alpha and TGF- beta 1 in the lung tissue: the content of TNF- a in the lung tissue of the model group, the CKLF1-C19 polypeptide group and the hormone group was significantly higher than that of the control group (P0.05), the CKLF1-C19 polypeptide group and the hormone group were lower than the model group (P0.05), but there was no significant difference between the CKLF1-C19 polypeptide group and the hormone group; the seventh to twenty-eighth days, the model group, the CKLF1-C19 polypeptide group and the excitation. The TNF- alpha content in lung tissue of the rats in the vegetarian group decreased gradually and the expression of.TGF- beta 1 protein was weakly positive in the control group, only in the cytoplasm of a few bronchiolobronchial epithelial cells. The expression of TGF- beta 1 was the strongest in the model control group at fourteenth days, and decreased with the time lapse, mainly in alveolar macrophages, bronchial epithelial cells, and blood. The expression of TGF- beta 1 in the cytoplasm of endothelial cells and fibroblasts in the cytoplasm of.CKLF1-C19 and hormone group 14,28 day was lower than that in the model group (P0.05), and there was no significant difference in the difference between the CKLF1-C19 polypeptide group and the hormone group.
conclusion
1. CKLF1-C19 peptide and hydrocortisone sodium succinate have certain therapeutic effects on alveolar inflammation and pulmonary fibrosis in bleomycin induced pulmonary fibrosis in rats.
2. CKLF1-C19 polypeptide and hydrocortisone sodium succinate may partly inhibit the development of bleomycin induced pulmonary inflammation and pulmonary fibrosis by reducing the content of TNF- alpha and TGF- beta 1.
3. CKLF1-C19 peptide and hydrocortisone sodium succinate showed no significant difference in alveolar inflammation and pulmonary fibrosis in bleomycin induced pulmonary fibrosis in rats.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R563.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張東偉,王繼峰,牛建昭,高寶華,趙景民,周光德;鹽酸博萊霉素致大鼠肺纖維化動(dòng)物模型HRCT的評(píng)價(jià)[J];解放軍醫(yī)學(xué)雜志;2004年06期

2 李安;田瓊;梁向艷;李超鋒;;佤藥燈臺(tái)樹(shù)對(duì)大鼠博萊霉素所致肺損傷早期作用的研究[J];科學(xué)技術(shù)與工程;2007年01期

3 徐國(guó)萍;徐t煷，

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