CKLF1-C19多肽对肺纤维化大鼠的治疗作用研究
发布时间：2018-07-26 18:56
【摘要】：目的 1.采用气管给药制造大鼠肺间质纤维化模型；2.观察CKLFl-C19多肽及氢化可的松琥珀酸钠对博莱霉素致肺纤维化大鼠的治疗作用；3.探讨CKLFl-C19多肽及氢化可的松琥珀酸钠对大鼠肺间质纤维化模型肺组织中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、转化生长因子β1(transforming growth factor-β1,TGF-β1)含量的影响；4.对比CKLFl-C19多肽与氢化可的松琥珀酸钠对大鼠肺间质纤维化模型肺组织细胞形态学及肺组织中TNF-α、TGF-β1蛋白含量的影响差异。 方法 健康Wistar大鼠60只,雄性,体重(200±20)g,实验大鼠随机分为4组,即对照组、模型组、CKLFl-C19多肽组及激素组各15只。用10%水合氯醛(3m1.kg-1)腹腔注射麻醉大鼠,将其头和四肢固定后,采用气管给药法制造肺纤维化模型,方法如下：用开口器撬开口腔,拉出舌,在额镜直视下,趁大鼠吸气的瞬间,迅速将与1ml注射器相连的硬脊膜外麻醉导管插入气管内约1～2厘米,见到导管内液面随大鼠呼吸而波动后,向模型组、CKLFl-C19多肽组及激素组的大鼠气管内一次性注入0.4%博莱霉素0.25mL (5mg.kg-1),同时对照组注入生理盐水0.25ml作为阴性对照,注药后立即将大鼠直立并左右旋转,使药液在肺内分布均匀。造模后第二天开始,对照组与模型组每天给予生理盐水(2mL/只)灌胃,CKLFl-C19多肽组给予CKLFl-C19多肽(100μ g)腹腔注射,激素组给予氢化可的松琥珀酸钠(25mg/g)腹腔注射干预。每天观察各组动物的一般情况(体型、精神状态、皮毛、摄食、活动、粪便等)并称重。在给药后的第7、14、28天采用腹主动脉放血法将各组大鼠分别处死5只,打开胸腔后,分离并取出肺组织,观察各组大鼠双肺外观情况后称重,将左肺浸于10%中性甲醛溶液中固定,常规石蜡包埋、切片,HE染色,评价肺泡炎及肺纤维化程度,并采用免疫组化法测定肺组织中TGF-β1的含量。取右肺,用冷生理盐水洗去浮血,在冰浴上剪成碎块,制成10%肺匀浆,3000r.min-1离心10min后取上清液放置于-80℃冰箱中保存,采用酶联免疫吸附法(ELISA法)测定TNF-α的含量。 结果 1.大鼠的一般情况：正常对照组大鼠反应灵敏,体格肥壮,皮毛光亮、致密,摄食正常。模型组大鼠均有不同程度的咳嗽和呼吸困难,摄食减少,体重下降,精神差,活动明显减少,皮毛晦暗,无光泽。CKLF1-C19多肽组前期表现类似模型组,给药7天后有所好转,治疗14天后明显好转,精神状态转好,食欲及活动量较前均有所增加。激素组大鼠表现与CKLF1-C19多肽组无明显差异。 2.肺组织大体病理变化：对照组大鼠双肺外观正常,肺叶饱满,呈粉红色,表面光滑,弹性良好。模型组第7天双肺体积增大,呈暗红色,表面可见散在点状出血；14天双肺体积较前缩小,弹性差,除散在点状出血点外,还可见大小不等的散在灰白色结节；第28天双肺体积较前明显缩小,呈灰白色,弹性差且硬度增加,可见大量灰白色结节。CKLF1-C19多肽组部分肺叶表面光滑,呈暗红色,部分局部见大小不等的灰白色结节,部分边缘有散在出血点,部分肺叶体积缩小,硬度增加。激素组与CKLF1-C19多肽组相似。 3.肺系数的比较：模型组、CKLF1-C19多肽组、激素组第7、14天和28天时肺系数均显著大于对照组(P0.05), CKLF1-C19多肽组、激素第7、14、28时肺系数均小于模型组(P0.05), CKLF1-C19多肽组与激素组无明显差异。 4.病理切片肺泡炎和肺纤维化程度的比较：对照组大鼠肺组织大多数结构清晰,各观察时间点均未出现明显的形态学改变。模型组第7天可见明显炎症改变,以巨噬细胞、淋巴细胞为主的大量炎性细胞浸润,成纤维细胞增多,伴有肺泡隔增宽；第14天时可见大量炎性细胞浸润,肺泡间隔明显增宽,肺泡变形,伴有成纤维细胞、平滑肌细胞增殖及胶原纤维沉积；28天时肺部广泛纤维化,肺泡间隔继续增宽,肺泡腔明显缩小,大部分肺泡消失,细胞成分减少,肺间质被胶原纤维和成纤维细胞替代,纤维成分增多较前更为明显,但肺部炎性细胞浸润有所减轻。CKLF1-C19多肽组和激素组各时间点的肺泡炎程度与模型组比均明显减轻(P0.05),28天时同对照组比较已无统计学意义(P0.05)。肺纤维化程度与与模型组比较也有所减轻,28天时肺纤维化的缓解程度较模型组有所减缓,但较对照组仍较严重。CKLF1-C19多肽组和激素组肺泡炎及纤维化程度在各观察点比较差别不大(P0.05)。 5.肺组织中TNF-α、TGF-β1含量变化：模型组、CKLF1-C19多肽组及激素组肺组织匀浆中TNF-α含量明显高于对照组(P0.05), CKLF1-C19多肽组和激素组低于模型组(P0.05),而CKLF1-C19多肽组和激素组无明显差异；第7到第28天,模型组、CKLF1-C19多肽组及激素组大鼠肺组织TNF-α含量有递减趋势。TGF-β1蛋白在对照组表达弱阳性,仅在少数细支气管黏膜上皮细胞的胞浆中出现,模型对照组TGF-β1的表达在第14天时为最强,随时间推移呈递减趋势,主要位于肺泡巨噬细胞、支气管黏膜上皮细胞、血管内皮细胞及成纤维细胞的胞浆中。CKLF1-C19多肽组和激素组第14、28天肺组织中TGF-β1的表达均低于模型组(P0.05), CKLF1-C19多肽组和激素组的差异无统计学意义。 结论 1. CKLF1-C19多肽和氢化可的松琥珀酸钠对博莱霉素致大鼠肺纤维化模型的肺泡炎和肺纤维化有一定的治疗作用； 2. CKLF1-C19多肽和氢化可的松琥珀酸钠可能部分通过降低TNF-α、TGF-β1的含量抑制博莱霉素致肺部炎症及肺纤维化的发展。 3. CKLF1-C19多肽与氢化可的松琥珀酸钠治疗对博莱霉素致大鼠肺纤维化模型的肺泡炎与肺纤维化的疗效无明显差异。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
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相关期刊论文 前10条
1 张东伟,王继峰,牛建昭,高宝华,赵景民,周光德;盐酸博莱霉素致大鼠肺纤维化动物模型HRCT的评价[J];解放军医学杂志;2004年06期
2 李安;田琼;梁向艳;李超锋;;佤药灯台树对大鼠博莱霉素所致肺损伤早期作用的研究[J];科学技术与工程;2007年01期
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