骨髓间充质干细胞在染石英尘大鼠体内的分布规律及拮抗肺纤维化作用研究

发布时间：2018-01-13 20:14

本文关键词：骨髓间充质干细胞在染石英尘大鼠体内的分布规律及拮抗肺纤维化作用研究　出处：《首都医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：矽肺是由于在生产过程中长期吸入游离二氧化硅粉尘而引起的以肺组织弥漫性纤维化为主的全身性疾病,是我国发病最多、对劳动者健康危害最严重的职业病。矽肺基本的病理学变化为矽结节的形成和弥漫性间质纤维化,尚无有效的治疗方法。骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)是最具应用前景的细胞与基因治疗的理想靶细胞,为矽肺的治疗提供了新的思路。本项目利用近红外(near-infrared,NIR)荧光活体成像系统、微计算机断层扫描(micro computed tomography,Mirco CT)活体肺组织、病理切片等技术,通过气管滴注的方式制备大鼠矽肺模型,观察BMSCs在大鼠体内的分布规律以及BMSCs干预对大鼠肺纤维化的抑制作用。该研究为进一步探索防治矽肺的有效途径,提高患者的生活质量提供新的思路。本研究旨在:(1)研究BMSCs在大鼠体内不同时间点的分布规律和归巢作用,从而为后续实验确定给药时间提供实验依据;(2)研究BMSCs对石英诱导大鼠肺纤维化的拮抗作用及Micro CT对肺纤维化程度和BMSCs拮抗作用的评价效果。第一部分大鼠BMSCs的分离、培养与鉴定目的:利用大鼠全骨髓贴壁法,经过分离、培养、鉴定,扩增出大鼠BMSCs。方法:采用全骨髓贴壁法分离培养BMSCs。将冲洗出的骨髓细胞混悬液移至细胞培养皿中,置于37℃、体积分数为5.00%二氧化碳培养箱内。通过换液逐渐去除未贴壁细胞。待细胞生长至80%至90%底面积后开始传代,对传至第3代的细胞进行鉴定,包括细胞形态学观察、流式鉴定CD抗原表达情况以及成骨与成脂培养液诱导。结果:(1)原代BMSCs培养,细胞融合度在80%至90%时进行传代,传代后的细胞生长速度加快,形态为梭形或纺锤形,旋涡状生长;(2)经流式鉴定,细胞表面cd90和cd44,阳性率分别为97.1%和99.1%,而cd45和cd11b呈阴性,阳性率分别为2.1%和4.5%,符合bmscs表面标志物的特点;(3)加入成脂、成骨诱导培养液一段时间后,出现脂肪细胞并累积成脂滴,呈串珠状排列,油红o染色呈现红色,茜素红染色镜下可见橘红色钙化结节。第二部分nir荧光成像系统示踪大鼠尾静脉注射的bmscs.目的:阐明能否通过nir荧光成像系统在大鼠体内观察到bmscs的分布规律和归巢作用,从而为bmscs二次注射的时间点提供实验依据。方法:72只成年雌性大鼠(spf级,200-240g)随机分成4组,每组18只,分别为:对照组、bmscs组、石英+bmscs组和石英+染料组。石英+bmscs组和石英+染料组中的大鼠气管滴注石英粉尘溶液1ml(50mg/ml);对照组和bmscs组中的大鼠气管滴入等量生理盐水。将从雄性大鼠股骨中提取并培养好的第3至5代bmscs用dir(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanineiodide,dir)染料进行标记后,行尾静脉注射,其中bmscs组和石英+bmscs组尾静脉注射含有2×106个bmscs的细胞溶液1ml,对照组尾静脉注射等量生理盐水,石英+染料组尾静脉注射dir染料浓度为5μg/ml的溶液1ml。分别在尾静脉注射后1h、6h、24h、3d、15d和30d在nir系统内进行观察,每个时间点获取到图像后,将大鼠处死,取出心、肝、脾、肺和双肾收集到大皿中,放在系统内继续观察分离出来的脏器中荧光信号分布情况,用pcr、免疫荧光等方法进行验证。结果:(1)dir染料成功标记bmscs并对bmscs的生存和增殖没有毒性作用;(2)近红外荧光成像技术能够应用于经dir染料标记的bmscs在大鼠体内分布的监测,确定尾静脉注射后第3天为二次给药时间点;(3)在石英诱导肺纤维化大鼠体内,bmscs能够归巢到石英损伤的肺组织。第三部分microct对bmscs拮抗石英诱导大鼠肺纤维化的效果评价目的:探讨microct能否评价大鼠矽肺纤维化程度并反映bmscs拮抗肺纤维化的效果。方法:wistar雌性大鼠60只,随机分为对照组、石英组和石英+bmscs组,每组20只。石英组和石英+BMSCs组气管内滴注石英混悬液(50mg/ml);染尘后,石英+BMSCs组尾静脉注射BMSCs,其它组尾静脉注射生理盐水。染尘后不同时间点进行大鼠肺部Micro CT平扫,取肺组织观察病理改变。结果:(1)石英组与对照组相比,第7、15、30d,病理分别可见炎细胞浸润,散在的矽结节和矽结节的融合;CT分别可见肺门斑块状密度增高影,圆形或类圆形细胞结节高密度影和片状高密度影;(2)石英+BMSCs组与石英组相比,第7d,炎性渗出物减少,CT结果不显现,第15、30d,病理和CT均显示矽肺纤维化不同程度减轻。结论:(1)通过建立BMSCs全骨髓贴壁培养法,成功分离、鉴定、纯化和扩增BMSCs;(2)利用近红外荧光活体成像技术成功示踪BMSCs在大鼠体内的分布情况,发现BMSCs能够特异性地迁移到石英损伤的肺组织,确定尾静脉注射后第3天为二次给药的时间点;(3)BMSCs对大鼠矽肺纤维化有拮抗作用,Micro CT能够评价石英诱导的大鼠矽肺纤维化程度并反映BMSCs对肺纤维化的拮抗作用。
[Abstract]:Silicosis is due to the production process of long-term inhalation of free silica dust in the lung tissue caused by diffuse fibrosis of the systemic disease, is China's highest incidence of occupation disease on the health of workers the most serious hazards. The basic pathological changes of silicosis formation and diffuse interstitial fibrosis nodules, there is no effective methods of treatment. Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) is an ideal target cell and gene therapy are the most promising, provides a new way for the treatment of silicosis. This project using near infrared (near-infrared, NIR) in vivo fluorescence imaging system, micro computer tomography (micro computed tomography, Mirco CT) in lung tissue in vivo, pathological technology, through the model of Silicosis Rats by intratracheal instillation method, observation of BMSCs distribution in rats and BMSCs Inhibitory effect of intervention on pulmonary fibrosis in rats. The study to further explore the effective way of prevention and treatment of silicosis, to provide new ideas to improve patients quality of life. The purpose of this study was to: (1) study of BMSCs in rats at different time points of distribution and homing, and provides the experimental basis for the subsequent experiment time of administration sure; (2) BMSCs on silica induced pulmonary fibrosis in rats and the antagonism of Micro CT to evaluate the degree of pulmonary fibrosis and BMSCs antagonism effect. The first part of BMSCs were isolated, cultured and identified Objective: using rat whole bone marrow adherent method. After the separation, culture, identification, amplified from methods: BMSCs. rats were isolated and cultured BMSCs. will flush out the bone marrow cell suspensions to the cell culture dish by whole bone marrow adherent method. At 37 DEG C, the volume fraction of the incubator is 5% carbon dioxide. Through the gradual removal of liquid exchange The non adherent cells. When the cells grow to 80% to 90% after the start of the bottom area of passage, to identify the spread to the third generation of cells, including cell morphology, flow cytometry and CD antigen expression and osteogenic and adipogenic induction medium. Results: (1) BMSCs were cultured, passaged cells were thawed in 80% to 90%, after passage, cell growth, morphology of spindle shaped or spindle shaped, spiral growth; (2) by flow cytometry, cell surface CD90 and CD44 positive rates were 97.1% and 99.1%, while CD45 and CD11b were negative, the positive rates were 2.1% and 4.5%, in line with BMSCs surface markers characteristic; (3) added into fat, bone induction time after cultured, fat cells and accumulation of lipid droplets, beaded arrangement, oil red O staining showed red alizarin red staining showed orange red nodules of calcification. The second part NIR fluorescence imaging system to trace The intravenous injection of bmscs. Objective: to clarify whether the NIR fluorescent imaging system in rats and observe the BMSCs distribution and homing effect, so as to provide experimental basis for BMSCs two injection time. Methods: 72 adult female rats (SPF, 200-240g) were randomly divided into 4 groups, 18 rats in each group that is: control group, BMSCs group, +bmscs group and quartz quartz + quartz dye group. +bmscs group and group of quartz + dye rat tracheal instillation of silica dust solution 1ml (50mg/ml); the control group and BMSCs group in the rat tracheal instillation of saline. From the femur of male rats extract and cultivate a good third to 5 generation BMSCs with dir (1,1'-dioctadecyl-3,3,3', 3'-tetramethylindotricarbocyanineiodide, DIR) dyes were labeled after tail vein injection, 1ml cell solution in BMSCs group and +bmscs group were injected with quartz containing 2 * 106 bmscs, The control group received intravenous injection of normal saline, quartz + dye group were injected with dir dye concentration 1ml. solution 5 g/ml respectively after intravenous injection of 1H, 6h, 24h, 3D, 15d and 30d were observed in the NIR system, each time point to obtain the image after the rats were killed, taken out the heart, liver, spleen, lung and kidney collected large double dish, put the fluorescence signal distribution, continue to observe isolated organs in the system by PCR, immunofluorescence method was validated. Results: (1) dir dye labeled BMSCs and successfully on the survival and proliferation of BMSCs (no toxicity; 2) near infrared fluorescence imaging technology can be applied to the dir dye labeled BMSCs in monitoring the in vivo distribution of rats, two doses for third days to determine the time point after intravenous injection; (3) in silica induced pulmonary fibrosis in rats in vivo, BMSCs can return to the injury of lung tissue. The third part mi To evaluate the effect of croct on anti BMSCs silica induced pulmonary fibrosis in rats Objective: To investigate whether microCT can evaluate the degree of pulmonary fibrosis in rats and reflect BMSCs antagonistic effect of pulmonary fibrosis. Methods: 60 female Wistar rats were randomly divided into control group, +bmscs group and quartz quartz group, 20 rats in each group. Group of quartz and quartz +BMSCs group intratracheal instillation of silica suspension (50mg/ml); after dust, quartz group +BMSCs intravenous injection of BMSCs, the other group were injected with saline. Rat lung Micro CT scans at different time points after instillation, lung tissue pathological changes were observed. Results: (1) quartz group the control group, and 7,15,30d, respectively. Pathological inflammatory cell infiltration, fusion scattered in silicotic nodules and silicotic nodule CT respectively; hilum plaque density increased, nodules round or oval cells with high density and high density sheet; (2) quartz group +BMSCs Compared with quartz group 7d, inflammatory exudate reduced, the CT result does not appear, 15,30d, pathology and CT showed pulmonary fibrosis severity. Conclusion: (1) through the establishment of BMSCs whole bone marrow adherent culture method, successful separation, identification, purification and amplification of BMSCs; (2) using near infrared in vivo fluorescence imaging technology BMSCs tracer distribution in rats in vivo, we found that BMSCs can selectively migrate to quartz lung tissue damage, two dose time point as determined in third days after intravenous injection; (3) BMSCs had antagonistic effect on rat pulmonary fibrosis induced by Micro, CT can evaluate the quartz the rat pulmonary fibrosis degree and reflect the antagonistic effects of BMSCs on pulmonary fibrosis.

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2

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