【作者】 张雁敏；

【导师】 邱泽武；

【作者基本信息】 中国人民解放军军事医学科学院 ， 内科学， 2010， 硕士

【摘要】 研究背景百草枯（PQ)是目前广泛使用的一种有机杂环类速效触杀型除草剂，由于肺内存在着胺类物质转运系统,而百草枯和二胺、多胺、及二硫胱胺具有结构上的相似性,百草枯可与胺类物质竞争通过肺泡Ⅱ型细胞的能量依赖性多胺摄取途径而积聚在肺内。因此，肺脏是百草枯中毒的主要靶器官(俗称百草枯肺）。大量口服百草枯后会出现急性中毒反应，表现为肺泡上皮细胞受损，肺泡内出血水肿，炎性细胞浸润，死亡率极高。少量口服百草枯后，早期肺部症状比较轻微或不明显，随着毒性作用时间加长，肺部损伤逐渐由炎性反应过渡到不可逆的肺纤维化（此过程大约需要28天），患者生存质量严重下降，继而进行性呼吸困难，最后导致呼吸衰竭甚至死亡。这种慢性肺纤维化是临床百草枯中毒的常见死亡原因之一。因此，为改善生存状况、降低死亡率，就要积极预防或者减轻肺纤维化的形成.虽然目前[1-4]对百草枯中毒有过很多机制及治疗方面的研究与探索，且获到一些成效，但都未取得突破性进展，仍无有效解毒药物，仍以激素、免疫抑制剂治疗和对症支持治疗为主，中毒者病死率高达50％－60％。所以，寻求PQ中毒引起肺损伤的新的治疗策略，降低PQ中毒病死率具有重要现实意义。目前医学界研究和探讨的重点是如何将干细胞成功应用于各类临床严重疾患的治疗方面，为诸多患者带来佳音。骨髓间充质干细胞(mesenchymal stemcells，MSCs)也称骨髓基质来源干细胞，是存在于骨髓中除造血干细胞以外另一类具有多向分化潜能的干细胞。在一定诱导条件下，这类细胞可定向分化为多种造血以外的组织，特别是中胚层和神经外胚层来源的组织细胞[5]，例如成骨细胞、成软骨细胞、脂肪细胞、腱细胞、肌肉细胞、神经细胞等。因此,我们认为MSCs移植有可能替代百草枯中毒肺部受损细胞，从而改善和恢复肺的通气换气，降低肺纤维化的形成。近年来已将MSCs引入到各种肺损伤的治疗中，包括肺纤维化损伤，但研究多集中在MSCs的移植对博莱霉素、放射性骨髓抑制以及LPS诱导的肺损伤的动物疾病模型的治疗作用方面，而关于MSC移植对PQ中毒所致肺损伤的研究还没有报道。观察骨髓间充质干细胞(MSCs)对大鼠百草枯(PQ)中毒肺损伤的疗效及可能机制。实验方法与结果1.分离、培养和鉴定大鼠骨髓MSC取SPF级Wistar大鼠100-130g,雌雄不拘,全骨髓贴壁分离法培养大鼠骨髓间充质干细胞，至第四代镜下观察细胞贴壁生长，形态统一，呈一致的梭形或长条形。取四代MSCs分别行流式细胞仪细胞表型检测及成骨、成脂诱导分化实验，结果显示四代MSCs均表达CD90,不表达CD45和CD34,即CD34-/CD45-/CD90+，且在一定诱导条件下，成功向成骨、成脂肪诱导转化。这就说明我们用的全骨髓贴壁分离筛选法可以获得足够量、生长旺盛、高纯度、具有多向分化潜能的大鼠骨髓见充质干细胞。2.增强型绿色荧光蛋白标记的骨髓间充质干细胞在百草枯中毒大鼠肺组织中的植入60只SPF级Wistar大鼠,体重200g左右,雌雄不拘，随机分为4组，每组15只:A组，正常对照组；B组，肺损伤模型组(PQ+PBS)，腹腔注射质量分数为20%的百草枯18mg/kg,4h后经尾静脉注射1mlPBS;C组，MSCs治疗组(PQ+MSCs)，腹腔注射质量分数为20%的百草枯18mg/kg,4h后经尾静脉注射MSCs-EGFP，1×106/只(重悬于1mlPBS中);D组，MSCs对照组(MSCs+PBS)，腹腔注射等量的PBS，4h后经尾静脉注射MSCs-EGFP，1×106/只(重悬于1mlPBS中)。取第三代MSCs，加入携带增强型绿色荧光蛋白基因的腺病毒载体Ad5-EGFP，转染成功后,在染毒后4h经尾静脉注射入大鼠体内，分别在给予MSCs后1d、3d、7d随机处死每组大鼠各5只，行肺组织病理切片，荧光倒置显微镜观察骨髓间充质干细胞的迁移。结果显示细胞在转染前后，生长状况良好，形态相同，基本不受病毒影响。在大鼠肺组织中，荧光显微镜下可见MSCs治疗组大鼠在给予MSCs后一天、三天肺组织中有少量绿色荧光标记的细胞，分布散在，无明显规律，而肺损伤组，MSCs对照组，及正常对照组均未发现绿色荧光标记的细胞。染毒后7天均未见到阳性细胞。3.骨髓间充质干细胞对百草枯致大鼠肺损伤的保护作用20只SPF级wistar大鼠，体重200g左右,雌雄不拘，按上述方法随机分为4组,观察各组大鼠的一般情况，染毒后28d处死各组大鼠取相应标本,检测肺系数、肺匀浆中羟脯氨酸（HYP）的含量及血清转化生长因子-β1(TGF-β1)水平,同时行实验目的:肺组织病理学观察。结果显示MSCs治疗组大鼠肺纤维化及实变程度较百草枯中毒模型组轻。与正常对照组比较,各组血清转化生长因子-β1(TGF-β1)，肺系数、肺匀浆中经脯氨酸（HYP）的含量均显著升高(P<0.05)。MSCs治疗组各组指标含量均好于百草枯中毒肺损伤模型组,差异均有统计学意义(P<0.05)。结论：1、全骨髓贴壁分离法可成功分离纯化大鼠骨髓间充质干细胞，并在体外稳定培养。2、携带增强型绿色荧光蛋白基因的腺病毒Ad5-EGFP可以成功转染大鼠骨髓间充质干细胞。3、大鼠尾静脉注入骨髓MSCs后，MSCs可以在体内向百草枯中毒肺损伤肺组织中植入。4、骨髓间充质干细胞的趋化和定植受百草枯中毒所致微环境的影响和制约，肺损伤越重，植入越明显，而移植而来的骨髓间充质干细胞又可以反过来改变PQ中毒肺部微环境，通过拮抗转化生长因子β，有效降低PQ中毒大鼠肺组织中的HYP，进一步明确了MSCs可有效减轻大鼠百草枯中毒所致肺损伤及肺纤维化。更多还原

【Abstract】 Paraquat(PQ) is a quarternary nitrogen herbicide widely used for broadleaf weedcontrol.As lung is rich of oxygen,and alveolus have a amine like matter transferringsysterm,which is similar to PQ,so the primary injuried target organ is the lung(it’s saidParaquat lung). If a great volume of pq solution is taken,it would occur acut PQpoisoning,which will lead to a much high mortality.On the contrary,if a little of PQ istaken ,the early respiration symptom is not evident or be mild,but as the toxicity existpersistently and form into inreversible pulmonary fibrosis gradually,the dyspneaprogressively wiil occur,even being dead one month later.So,to improve living statusand reducing the mortality,we would actively prevent chronical pulmonaryfibrosis.Although a lot of basic and clinical research were carried out,and achieveingsome effect,unfortunately we have not made substantial breakthrough.Now there is noselectively antitoxic agent.The main methods to cure are anti-inflammatory,anti-oxygen and to clear inflammatory factor etc. Except the anti-oxygen medicines,lucocorticosteroid associated with immune depressant cyclophosphamide is alsoapplied,but the effect is not well. The mortality be up to above 60%，so it is a greatimportant to find a new way to reduce the mortality of PQ poisoning.Mesenchymal stem cells,also known as bone marrow derived stem cells,areanother multi-directional differertiation potential stem cells in bone marrow,inaddition to hematopoietic stem cells.In some condiyion,MSCs can be directlytransformed into a variety of tissues except hematopoietic systerm,especially in themesoderm and neuroectodermal tissue cells,for example,bone cells,cartilage cells,fatcells,muscle cells,never cellsn and so on.Therefore,we believe that the transplantationof MSCs may replace damaged cells in the lung of paraquat poisoning in order toimprove and restore the ventilation of lung,reduce the formation of pulmonaryfibrosis.In recent years,MSCs has been introduced to cure a variety of lunginjury,including pulmonary fibrosis injury,but research mostly concentrated in curingthe injury by bleomycin or radiation，and the MSCs transplantation for chronic lunginjury induced by PQ have not reported. ObjectiObjective of the experiment:to explore the possible mechanism and protective effect of MSC on rats withparaquat-induced chronic injury.Methed and Result of the experiment:1、MSCs is culture and identificationSPF Wistar rats were randomly taken,no matter male or female.MSC wereisolated from rat bone marrow and purified by means of adherent way.Themicroscope was applied to observe the changes of the cell and the cells had aspindled，fibroblast appearance in culture.Flow cytometry analysis demonstrated thatphenotypes of MSCs were CD34-/CD45-/CD90+.It is demonstrated that MSCsretained their ability to form ostcoblasts and adipocytes at Passages4 throughdifferentiation assays ,which showing a multipotent plasticity.All the descriptionabove is consistent with MSCs.2、MSCs which were transfected with Ad5-EGFP virus occur in rat lung tissueEighty SPF Wistar rats were randomly divided into four group：PQ plus PBSgroup（n=20），PQ plus MSCs group（n=20），MSCs plus PBS group（n=20），normal group（n=20）.20% paraquat 18mg/Kg was injected peritoneally for poisoningand in the normal group,PBS was used.The third generation MSCs were transfectedwith Ad5-EGFP virus vector. inverted microscope was applied to observe the changesof the cell,then MSCs-EGFP was delivered through the tail vein of rats 4h afterPQ .subgroup of animals were killed at 1d、3d、7d、28d after MSCs and slicepulmonary coefficient and histological to observe the migration of MSCs.The resultshowed that MSCs are growing in a good condition and have the same shape,whichmeans MSCs have no affect of the virus.The positive MSCs can be detected in lungtissue at 1d、3d after PQ which PQ and MSCs were given.The heavier the lung fromthe rat was damaged,the more MSCs were occur.We have not deteced positive cell at7d、28d,and we also have not deteced positive cell in other groups.3、The potential efficacy of transplanted bone marrow derived mesenchymalstem cells in treating and repairing the chronic lung injury is studied.Twenty SPF Wistar rats were randomly divided into four group,the generalsituation in rats from all of group were observated .At 28 days after paraquat injection,5 rats in each group were killed respectively.Serum transforming growthfactor-β1(TGF-β1) and hydroxyproline(HYP) level in the lung homogenate weremeasured,and pulmonary coefficient and histological changes were observed.The lungtissue pathological investigation showed that the pulmonary fibrosis and consolidationin the treatment group were milder than those in model group.In the treatmentgroup,the levels of serum TGF-β1 and lung tissue HYP,pulmonary coefficient wereless than those of model group,and the different was significant(P<0.05).ConclusionConclusion:1、Using adherent screening can successfully separate,purificy and culture MSCsdericed from rat bone marrow in vitro..2、Ad5-EGFP virus can successfully transfect mesenchymal stem cells derived fromrat bone marrow.3、MSCs can engraft in rat with paraquat-induced lung tissue after tail vein injectionof MSCs.4、In one hand,chemotaxis and colonization of mesenchymal stem cells are impactedand constraint by its micro-environment.The heavier the lung from the rat wasdamaged,the more MSCs were occur.In the other hand,mesenchymal stem cells can inturn change the micro-environment of the damaged part. Mesenchymal stem cells caneffectively reduce lung injury and pulmonary fibrosis in rat which is caused byantagonizing TGF-β1 and reducing HYP in rat lung tissue.更多还原