硼对非洲雏鸵鸟肺纤维化的影响
发布时间：2018-08-29 19:18
【摘要】：硼是动物机体生长发育的必须微量元素,在机体中参与了多项生理活动。硼的主要吸收形式是无机盐,无机硼酸盐主要通过胃肠道、呼吸道和破损皮肤吸收。呼吸系统与外界相通因而很容易受到外来刺激物的损伤。雏鸵鸟的肺组织尚未发育完善,易受到外界病原攻击,是呼吸系统疾病的易感动物,雏鸵鸟有着高的患病率和死亡率。目前关于硼对机体诸多生理功能的研究已相继报道,尤其是硼对骨、脑和免疫器官等的作用,含硼化合物对癌症的治疗等,而有关硼对呼吸系统的作用尚未报道。为了探明雏鸵鸟呼吸系统对硼的耐受程度,本研究以肺为研究对象,通过饮水添加不同浓度的硼酸,探明硼对非洲雏鸵鸟肺纤维化的影响。刚出壳的1 d鸵鸟,随机分为6组,分别在饮水中添加浓度为0 mg/L(A组)、40 mg/L(B组)、80 mg/L(C组)、160 mg/L(D组)、320 mg/L(E组)、640 mg/L(F组)的硼酸,饲养至90 d取肺组织进行石蜡切片,HE和MASSON染色,SOD、MDA、GSH测定,TUNEL技术和实时荧光定量PCR等方法,探讨不同浓度饮水硼对非洲雏鸵鸟肺纤维化的影响。具体研究内容如下:1硼对雏鸵鸟肺组织形态结构的影响通过HE染色对雏鸵鸟肺组织形态结构变化作初步评估,并对肺组织病理变化进行标准量化分析。结果显示,与对照组相较,硼剂量低于160 mg/L时,肺组织结构清晰,发育良好,硼剂量高于320 mg/L时,肺组织形态结构出现病变,异嗜性粒细胞显著增多,血管内有淋巴细胞,出现炎症反应,细胞间质增厚,疑似肺纤维化,细胞核有碎裂现象,疑似细胞凋亡。2硼对雏鸵鸟血清抗氧化能力的影响使用SOD、MDA和GSH试剂盒测定雏鸵鸟血清的SOD活性,GSH和MDA含量。结果显示:与对照组相比,血清SOD活性在硼剂量低于160 mg/L时升高,且在硼剂量为80 mg/L时有显著差异性(P0.05),在硼剂量高于320 mg/L时降低,且在硼剂量为640 mg/L时有显著差异性(P0.05);血清GSH含量在硼剂量低于160mg/L时升高,且在硼剂量为80 mg/L时差异显著(P0.05),在硼剂量高于320 mg/L时显著降低(P0.05);血清MDA含量在硼剂量低于160 mg/L时降低,但差异不显著(P0.05),在硼剂量高于320 mg/L时升高,且在硼剂量为640 mg/L时有显著差异(P0.05)。综上所述,硼剂量低于160 mg/L时,可提高雏鸵鸟血清SOD活性和GSH含量,降低血清MDA含量,提高机体抗氧化能力;硼剂量高于320 mg/L时,可降低鸵鸟血清SOD活性和GSH含量,提高血清MDA含量,降低机体抗氧化能力。3硼对雏鸵鸟肺组织炎症反应的影响通过HE染色结果可知,硼剂量高于320 mg/L时雏鸵鸟肺组织异嗜性粒细胞增多,有炎症反应。为验证该结果,本试验通过荧光定量PCR技术对肺组织IL-1β、IL-6、IL-10 m RNA表达水平进行相对定量。与对照组相比,IL-1βm RNA表达水平在硼剂量为40-80 mg/L时降低,但差异不显著,在160 mg/L时显著升高(P0.05),在320-640 mg/L时升高极显著(P0.01);IL-6 m RNA表达水平在硼剂量为40-80mg/L时降低,但是降低不显著,在160 mg/L时升高,且升高不显著,在320-640 mg/L时升高极显著(P0.01);IL-10 m RNA表达水平一直在升高,在40 mg/L时变化不显著,在80-160 mg/L时显著升高(P0.05),在320-640 mg/L时升高极显著(P0.01)。由此可见硼剂量低于160 mg/L时可抑制细胞炎症反应;当硼剂量高于320 mg/L时,IL-1β、IL-6、IL-10 m RNA表达水平均显著升高(P0.01),在促炎因子IL-1β、IL-6引起炎症反应时,抗炎因子IL-10也随着增多以抑制过度炎症反应,这是高剂量硼酸引起炎症反应的结果。由此得出硼剂量为40-160 mg/L时可抑制炎症反应,硼剂量为320-640 mg/L时可促进炎症反应。4硼对雏鸵鸟肺组织细胞凋亡的影响已知HE染色在高倍显微镜(1000×)下观察到,硼剂量高于320 mg/L时,肺组织细胞核有碎裂现象,疑似细胞凋亡。为应证该结论,采用TUNEL技术,荧光显微镜成像发现,硼剂量低于160 mg/L时显著抑制细胞凋亡,硼剂量高于320 mg/L时促进细胞凋亡。运用荧光定量PCR技术对caspase-3 m RNA表达水平进行相对定量测定,与对照组相比,硼剂量低于160 mg/L时caspase-3 m RNA表达水平显著降低(P0.05),硼剂量高于320 mg/L时caspase-3 m RNA表达水平显著增加(P0.05)。5硼对雏鸵鸟肺纤维化的影响利用MASSON染色标记胶原纤维,并进行IOD值统计分析。结果发现,硼剂量高于320 mg/L时肺组织胶原纤维明显增加。本试验以细胞外基质和上皮间质转化为肺纤维化的直接评定指标,为探明该现象,本试验通过荧光定量PCR技术对ECM、E-Cadherin、a-SMA和Vimentin m RNA表达水平进行相对定量。随着硼剂量的增加,ECM、a-SMA和Vimentin m RNA表达水平呈上升趋势,E-Cadherin m RNA表达水平呈下降趋势。通过以上指标验证,得出硼剂量低于160 mg/L时对肺纤维化的作用不明显(P0.05),硼剂量高于320 mg/L时能显著促进肺纤维化(P0.05)的结论。综合以上试验结果,得出硼剂量低于160 mg/L时可提高血清抗氧化能力,抑制肺组织发生炎症反应和细胞凋亡,进而降低肺纤维化的发生;硼剂量高于320 mg/L时可损伤血清抗氧化能力,促进肺组织炎性损伤和细胞凋亡,并增加肺纤维化的发生发展。
[Abstract]:Boron is an essential trace element for the growth and development of animals and participates in many physiological activities in the body. The main absorption form of boron is inorganic salts. Inorganic borates are mainly absorbed through the gastrointestinal tract, respiratory tract and damaged skin. Respiratory system is connected with the outside world and therefore vulnerable to foreign stimuli. The lung tissue of ostrich chicks has not yet been found. Being well-developed and vulnerable to external pathogens, ostrich is a susceptible animal to respiratory diseases. Young ostrich has a high morbidity and mortality rate. At present, studies on the physiological functions of boron have been reported, especially the effects of boron bone, brain and immune organs, boron-containing compounds on cancer treatment, etc., while boron on the respiratory system. The effects of Boron on pulmonary fibrosis in African ostrich chicks were studied by adding boric acid to drinking water. The newly-hatched ostriches were randomly divided into 6 groups: 0 mg/L (group A) and 40 mg/L (group B). 80 mg/L (group C), 160 mg/L (group D), 320 mg/L (group E), 640 mg/L (group F) of boric acid were fed to 90 days for paraffin section, HE and MASSON staining, SOD, MDA, GSH determination, TUNEL and real-time fluorescence quantitative PCR to investigate the effects of different concentrations of Boron on pulmonary fibrosis in African ostrich chicks. The results showed that the lung tissue structure was clear and well developed when boron dosage was lower than 160 mg/L, and when boron dosage was higher than 320 mg/L, the lung tissue structure was out of shape. Now pathological changes, heterophilic granulocytes increased significantly, lymphocytes in the blood vessels, inflammation, thickening of the interstitial cells, suspected pulmonary fibrosis, nuclear fragmentation, suspected apoptosis. 2 boron on the serum antioxidant capacity of ostrich chicks using SOD, MDA and GSH kits to determine the serum SOD activity, GSH and MDA content. Compared with the control group, the serum SOD activity increased when the boron dose was lower than 160 mg/L, and there was a significant difference when the boron dose was 80 mg/L (P 0.05), decreased when the boron dose was higher than 320 mg/L, and there was a significant difference when the boron dose was 640 mg/L (P 0.05); the serum GSH content increased when the boron dose was lower than 160 mg/L, and the boron dose was 80 mg/L. There was a significant difference (P 0.05), when the boron dose was higher than 320 mg/L, it decreased significantly (P 0.05); the serum MDA content was lower when the boron dose was lower than 160 mg/L, but the difference was not significant (P 0.05), when the boron dose was higher than 320 mg/L, and when the boron dose was 640 mg/L, there was a significant difference (P 0.05). When boron dosage was higher than 320 mg/L, SOD activity and GSH content in serum of ostrich could be decreased, MDA content in serum could be increased and antioxidant capacity could be decreased. The effect of Boron on inflammatory reaction of lung tissue of ostrich was observed by HE staining. In order to verify the results, the expression levels of IL-1 beta, IL-6 and IL-10 m RNA in ostrich lung tissues were quantified by fluorescence quantitative PCR. Compared with the control group, the expression levels of IL-1 beta m RNA in ostrich lung tissues decreased at boron dosage of 40-80 mg/L, but the difference was not significant. The expression levels increased significantly at 160 mg/L. (P 0.05), at 320-640 mg/L increased significantly (P 0.01); IL-6 m RNA expression level decreased at boron dosage of 40-80 mg/L, but the decrease was not significant, at 160 mg/L increased, and the increase was not significant, at 320-640 mg/L increased extremely significantly (P 0.01); IL-10 m RNA expression level has been increasing, at 40 mg/L did not change significantly, at 80-160 mg/L significantly. The levels of IL-1 beta, IL-6 and IL-10 m RNA were significantly increased when the dose of boron was lower than 160 mg/L (P 0.05). The levels of IL-10, IL-1 beta and IL-6 were inhibited when the dose of boron was higher than 320 mg/L (P 0.01). Excessive inflammation is the result of inflammation induced by high dose of boric acid. It is concluded that boron dose is 40-160 mg/L and 320-640 mg/L can inhibit inflammation. The effect of Boron on apoptosis of lung tissue in ostrich chicks is known to be observed under high power microscopy (1000 x). Boron dose is higher than 320 mg/L. To confirm this conclusion, TUNEL and fluorescence microscopy showed that boron dosage below 160 mg/L significantly inhibited cell apoptosis, and boron dosage above 320 mg/L promoted cell apoptosis. The expression of Caspase-3 m RNA was quantitatively determined by fluorescence quantitative PCR. Compared with the control group, the expression level of Caspase-3 m RNA was significantly decreased (P 0.05) when boron dosage was lower than 160 mg/L, and significantly increased (P 0.05) when boron dosage was higher than 320 mg/L. The effect of boron pulmonary fibrosis in ostrich chicks was studied by MASSON staining and IOD value was statistically analyzed. The expression levels of ECM, E-Cadherin, a-SMA and Vimentin m RNA were quantified by fluorescence quantitative PCR. With the increase of boron dosage, the expression levels of ECM, a-SMA and Vimentin m RNA were measured. The expression of M RNA increased and the expression of E-Cadherin m RNA decreased. The results showed that the effect of boron dosage below 160 mg/L on pulmonary fibrosis was not obvious (P 0.05), and that boron dosage above 320 mg/L could significantly promote pulmonary fibrosis (P 0.05). When boron dosage is higher than 320 mg/L, the serum antioxidant capacity can be damaged, the inflammatory injury and cell apoptosis of lung tissue can be promoted, and the occurrence and development of pulmonary fibrosis can be increased.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.39
本文编号：2212160
[Abstract]:Boron is an essential trace element for the growth and development of animals and participates in many physiological activities in the body. The main absorption form of boron is inorganic salts. Inorganic borates are mainly absorbed through the gastrointestinal tract, respiratory tract and damaged skin. Respiratory system is connected with the outside world and therefore vulnerable to foreign stimuli. The lung tissue of ostrich chicks has not yet been found. Being well-developed and vulnerable to external pathogens, ostrich is a susceptible animal to respiratory diseases. Young ostrich has a high morbidity and mortality rate. At present, studies on the physiological functions of boron have been reported, especially the effects of boron bone, brain and immune organs, boron-containing compounds on cancer treatment, etc., while boron on the respiratory system. The effects of Boron on pulmonary fibrosis in African ostrich chicks were studied by adding boric acid to drinking water. The newly-hatched ostriches were randomly divided into 6 groups: 0 mg/L (group A) and 40 mg/L (group B). 80 mg/L (group C), 160 mg/L (group D), 320 mg/L (group E), 640 mg/L (group F) of boric acid were fed to 90 days for paraffin section, HE and MASSON staining, SOD, MDA, GSH determination, TUNEL and real-time fluorescence quantitative PCR to investigate the effects of different concentrations of Boron on pulmonary fibrosis in African ostrich chicks. The results showed that the lung tissue structure was clear and well developed when boron dosage was lower than 160 mg/L, and when boron dosage was higher than 320 mg/L, the lung tissue structure was out of shape. Now pathological changes, heterophilic granulocytes increased significantly, lymphocytes in the blood vessels, inflammation, thickening of the interstitial cells, suspected pulmonary fibrosis, nuclear fragmentation, suspected apoptosis. 2 boron on the serum antioxidant capacity of ostrich chicks using SOD, MDA and GSH kits to determine the serum SOD activity, GSH and MDA content. Compared with the control group, the serum SOD activity increased when the boron dose was lower than 160 mg/L, and there was a significant difference when the boron dose was 80 mg/L (P 0.05), decreased when the boron dose was higher than 320 mg/L, and there was a significant difference when the boron dose was 640 mg/L (P 0.05); the serum GSH content increased when the boron dose was lower than 160 mg/L, and the boron dose was 80 mg/L. There was a significant difference (P 0.05), when the boron dose was higher than 320 mg/L, it decreased significantly (P 0.05); the serum MDA content was lower when the boron dose was lower than 160 mg/L, but the difference was not significant (P 0.05), when the boron dose was higher than 320 mg/L, and when the boron dose was 640 mg/L, there was a significant difference (P 0.05). When boron dosage was higher than 320 mg/L, SOD activity and GSH content in serum of ostrich could be decreased, MDA content in serum could be increased and antioxidant capacity could be decreased. The effect of Boron on inflammatory reaction of lung tissue of ostrich was observed by HE staining. In order to verify the results, the expression levels of IL-1 beta, IL-6 and IL-10 m RNA in ostrich lung tissues were quantified by fluorescence quantitative PCR. Compared with the control group, the expression levels of IL-1 beta m RNA in ostrich lung tissues decreased at boron dosage of 40-80 mg/L, but the difference was not significant. The expression levels increased significantly at 160 mg/L. (P 0.05), at 320-640 mg/L increased significantly (P 0.01); IL-6 m RNA expression level decreased at boron dosage of 40-80 mg/L, but the decrease was not significant, at 160 mg/L increased, and the increase was not significant, at 320-640 mg/L increased extremely significantly (P 0.01); IL-10 m RNA expression level has been increasing, at 40 mg/L did not change significantly, at 80-160 mg/L significantly. The levels of IL-1 beta, IL-6 and IL-10 m RNA were significantly increased when the dose of boron was lower than 160 mg/L (P 0.05). The levels of IL-10, IL-1 beta and IL-6 were inhibited when the dose of boron was higher than 320 mg/L (P 0.01). Excessive inflammation is the result of inflammation induced by high dose of boric acid. It is concluded that boron dose is 40-160 mg/L and 320-640 mg/L can inhibit inflammation. The effect of Boron on apoptosis of lung tissue in ostrich chicks is known to be observed under high power microscopy (1000 x). Boron dose is higher than 320 mg/L. To confirm this conclusion, TUNEL and fluorescence microscopy showed that boron dosage below 160 mg/L significantly inhibited cell apoptosis, and boron dosage above 320 mg/L promoted cell apoptosis. The expression of Caspase-3 m RNA was quantitatively determined by fluorescence quantitative PCR. Compared with the control group, the expression level of Caspase-3 m RNA was significantly decreased (P 0.05) when boron dosage was lower than 160 mg/L, and significantly increased (P 0.05) when boron dosage was higher than 320 mg/L. The effect of boron pulmonary fibrosis in ostrich chicks was studied by MASSON staining and IOD value was statistically analyzed. The expression levels of ECM, E-Cadherin, a-SMA and Vimentin m RNA were quantified by fluorescence quantitative PCR. With the increase of boron dosage, the expression levels of ECM, a-SMA and Vimentin m RNA were measured. The expression of M RNA increased and the expression of E-Cadherin m RNA decreased. The results showed that the effect of boron dosage below 160 mg/L on pulmonary fibrosis was not obvious (P 0.05), and that boron dosage above 320 mg/L could significantly promote pulmonary fibrosis (P 0.05). When boron dosage is higher than 320 mg/L, the serum antioxidant capacity can be damaged, the inflammatory injury and cell apoptosis of lung tissue can be promoted, and the occurrence and development of pulmonary fibrosis can be increased.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.39
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