本文關(guān)鍵詞：骨髓間充質(zhì)干細胞在染石英塵大鼠體內(nèi)的分布規(guī)律及拮抗肺纖維化作用研究　出處：《首都醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 石英 肺纖維化 BMSCs NIR熒光成像 Micro CT

【摘要】：矽肺是由于在生產(chǎn)過程中長期吸入游離二氧化硅粉塵而引起的以肺組織彌漫性纖維化為主的全身性疾病,是我國發(fā)病最多、對勞動者健康危害最嚴重的職業(yè)病。矽肺基本的病理學(xué)變化為矽結(jié)節(jié)的形成和彌漫性間質(zhì)纖維化,尚無有效的治療方法。骨髓間質(zhì)干細胞(bone marrow mesenchymal stem cells,BMSCs)是最具應(yīng)用前景的細胞與基因治療的理想靶細胞,為矽肺的治療提供了新的思路。本項目利用近紅外(near-infrared,NIR)熒光活體成像系統(tǒng)、微計算機斷層掃描(micro computed tomography,Mirco CT)活體肺組織、病理切片等技術(shù),通過氣管滴注的方式制備大鼠矽肺模型,觀察BMSCs在大鼠體內(nèi)的分布規(guī)律以及BMSCs干預(yù)對大鼠肺纖維化的抑制作用。該研究為進一步探索防治矽肺的有效途徑,提高患者的生活質(zhì)量提供新的思路。本研究旨在:(1)研究BMSCs在大鼠體內(nèi)不同時間點的分布規(guī)律和歸巢作用,從而為后續(xù)實驗確定給藥時間提供實驗依據(jù);(2)研究BMSCs對石英誘導(dǎo)大鼠肺纖維化的拮抗作用及Micro CT對肺纖維化程度和BMSCs拮抗作用的評價效果。第一部分大鼠BMSCs的分離、培養(yǎng)與鑒定目的:利用大鼠全骨髓貼壁法,經(jīng)過分離、培養(yǎng)、鑒定,擴增出大鼠BMSCs。方法:采用全骨髓貼壁法分離培養(yǎng)BMSCs。將沖洗出的骨髓細胞混懸液移至細胞培養(yǎng)皿中,置于37℃、體積分數(shù)為5.00%二氧化碳培養(yǎng)箱內(nèi)。通過換液逐漸去除未貼壁細胞。待細胞生長至80%至90%底面積后開始傳代,對傳至第3代的細胞進行鑒定,包括細胞形態(tài)學(xué)觀察、流式鑒定CD抗原表達情況以及成骨與成脂培養(yǎng)液誘導(dǎo)。結(jié)果:(1)原代BMSCs培養(yǎng),細胞融合度在80%至90%時進行傳代,傳代后的細胞生長速度加快,形態(tài)為梭形或紡錘形,旋渦狀生長;(2)經(jīng)流式鑒定,細胞表面cd90和cd44,陽性率分別為97.1%和99.1%,而cd45和cd11b呈陰性,陽性率分別為2.1%和4.5%,符合bmscs表面標志物的特點;(3)加入成脂、成骨誘導(dǎo)培養(yǎng)液一段時間后,出現(xiàn)脂肪細胞并累積成脂滴,呈串珠狀排列,油紅o染色呈現(xiàn)紅色,茜素紅染色鏡下可見橘紅色鈣化結(jié)節(jié)。第二部分nir熒光成像系統(tǒng)示蹤大鼠尾靜脈注射的bmscs.目的:闡明能否通過nir熒光成像系統(tǒng)在大鼠體內(nèi)觀察到bmscs的分布規(guī)律和歸巢作用,從而為bmscs二次注射的時間點提供實驗依據(jù)。方法:72只成年雌性大鼠(spf級,200-240g)隨機分成4組,每組18只,分別為:對照組、bmscs組、石英+bmscs組和石英+染料組。石英+bmscs組和石英+染料組中的大鼠氣管滴注石英粉塵溶液1ml(50mg/ml);對照組和bmscs組中的大鼠氣管滴入等量生理鹽水。將從雄性大鼠股骨中提取并培養(yǎng)好的第3至5代bmscs用dir(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanineiodide,dir)染料進行標記后,行尾靜脈注射,其中bmscs組和石英+bmscs組尾靜脈注射含有2×106個bmscs的細胞溶液1ml,對照組尾靜脈注射等量生理鹽水,石英+染料組尾靜脈注射dir染料濃度為5μg/ml的溶液1ml。分別在尾靜脈注射后1h、6h、24h、3d、15d和30d在nir系統(tǒng)內(nèi)進行觀察,每個時間點獲取到圖像后,將大鼠處死,取出心、肝、脾、肺和雙腎收集到大皿中,放在系統(tǒng)內(nèi)繼續(xù)觀察分離出來的臟器中熒光信號分布情況,用pcr、免疫熒光等方法進行驗證。結(jié)果:(1)dir染料成功標記bmscs并對bmscs的生存和增殖沒有毒性作用;(2)近紅外熒光成像技術(shù)能夠應(yīng)用于經(jīng)dir染料標記的bmscs在大鼠體內(nèi)分布的監(jiān)測,確定尾靜脈注射后第3天為二次給藥時間點;(3)在石英誘導(dǎo)肺纖維化大鼠體內(nèi),bmscs能夠歸巢到石英損傷的肺組織。第三部分microct對bmscs拮抗石英誘導(dǎo)大鼠肺纖維化的效果評價目的:探討microct能否評價大鼠矽肺纖維化程度并反映bmscs拮抗肺纖維化的效果。方法:wistar雌性大鼠60只,隨機分為對照組、石英組和石英+bmscs組,每組20只。石英組和石英+BMSCs組氣管內(nèi)滴注石英混懸液(50mg/ml);染塵后,石英+BMSCs組尾靜脈注射BMSCs,其它組尾靜脈注射生理鹽水。染塵后不同時間點進行大鼠肺部Micro CT平掃,取肺組織觀察病理改變。結(jié)果:(1)石英組與對照組相比,第7、15、30d,病理分別可見炎細胞浸潤,散在的矽結(jié)節(jié)和矽結(jié)節(jié)的融合;CT分別可見肺門斑塊狀密度增高影,圓形或類圓形細胞結(jié)節(jié)高密度影和片狀高密度影;(2)石英+BMSCs組與石英組相比,第7d,炎性滲出物減少,CT結(jié)果不顯現(xiàn),第15、30d,病理和CT均顯示矽肺纖維化不同程度減輕。結(jié)論:(1)通過建立BMSCs全骨髓貼壁培養(yǎng)法,成功分離、鑒定、純化和擴增BMSCs;(2)利用近紅外熒光活體成像技術(shù)成功示蹤BMSCs在大鼠體內(nèi)的分布情況,發(fā)現(xiàn)BMSCs能夠特異性地遷移到石英損傷的肺組織,確定尾靜脈注射后第3天為二次給藥的時間點;(3)BMSCs對大鼠矽肺纖維化有拮抗作用,Micro CT能夠評價石英誘導(dǎo)的大鼠矽肺纖維化程度并反映BMSCs對肺纖維化的拮抗作用。
[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.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R135.2

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