间充质干细胞对内毒素诱导急性肺损伤大鼠肺保护作用及机制研究
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摘要
研究背景及目的急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)主要是由胃酸吸入、感染、创伤、脂肪栓塞等引起,感染是最常见原因。不论何种原因所致急性肺损伤,炎症反应都在其病理改变中起了主要作用。虽然,大量的研究试图阐明急性肺损伤/急性呼吸窘迫综合症的发病机理,但是,至今仍没发现特别有效的治疗手段。目前,急性肺损伤/急性呼吸窘迫综合征的治疗上主要以支持治疗、药物治疗和辅助通气治疗为主,临床上仍然保持着很高的病死率,大约维持在30%～40%,因此迫切需要深入研究其发病机制,寻找更加有效的治疗方法。间充质干细胞(MSC)不仅具有低免疫原性、多向分化潜能、诱导免疫耐受、还具有免疫抑制的作用。由于MSCs具有独特的免疫学特性及良好的可塑性,并且MSCs的利用不涉及伦理争议,因此,到目前为止MSCs已成为细胞治疗和组织工程的最佳候选细胞材料。但是,其治疗机制仍然没有完全清楚。核转录因子KB(NF-κB)是近年研究较多的一个促炎因子,其在炎症发生及调节中起了非常重要的作用。近几年的文献报道,阻断肺上皮细胞的NF-κB信号通路可以减轻内毒素诱导的急性肺损伤大鼠的粒细胞性肺炎症反应和肺水肿程度,是肺炎症疾病的有效治疗方法之一。亲环素A(CyPA)是白细胞趋化因子,其通过与其受体CD147结合激活MAPK及核因子KB(NF-κB)通路从而调节促炎因子的释放。目前未发现间充质干细胞在内毒素诱导急性肺损伤中对NF-κB与CyPA的影响的文献报道。本研究旨在研究间充质干细胞对内毒素诱导急性肺损伤的生物学作用及对NF-κB与CyPA的影响,以期进一步阐明MSCs治疗ALI的机制,为临床发现更有效的治疗方法提供实验线索。
目的观察C57BL/6J小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外培养时,细胞的鉴定、形态及定向诱导分化为成骨细胞的特点。
方法1取C57BL/6J小鼠双侧股骨和胫骨骨髓细胞进行体外全骨髓贴壁筛选法原代培养并传代。2.取原代的MSCs培养至第7代细胞,倒置相差显微镜下观察其生长形态及特征。3.提取第1、3代细胞,胰酶消化后计数,以2×103个/孔接种于96孔板,每孔加入10μlCell Counting Kit-8(CCK-8)溶液,37℃,饱和湿度为5%CO2,培养箱孵育2h后,用酶联免疫检测仪于450nm波长下检测吸光度值。然后在检测后第1、2、3、4、5、6、7天同一时间作相同检测。用吸光度值绘制细胞增殖曲线。4.第5代生长状态良好的C57小鼠BMSCs制成单细胞悬液,以2×103/ml浓度接种于6孔板,待细胞达60%融合时,分别加入成骨诱导培养液及成脂诱导剂进行体外分化培养。5.第5代生长状态良好的C57小鼠BMSCs制成单细胞悬液,收集约2×106个细胞上机进行流式细胞检测。
结果1.原代培养的MSCs在24h后开始贴壁,48h后贴壁的MSCs逐渐伸展为不规则圆形、多角形、梭形、排列不规则。2.成骨诱导后镜下观察钙结节染成橘红色、成脂诱导后脂肪细胞里的脂滴呈红色,其它细胞则不着色。3.MSCs的生长曲线呈S型,其生长周期分别为滞后期,对数期和平台期。4.C57小鼠MSCs分化的流式细胞分析显示：MSCs诱导分化后的细胞表面主要有Sca-1、CD34、CD29和CD44标记分子、缺乏CD117分子。
结论1.密度离心结合贴壁法可以成功培养、分离出C57BL/6J小鼠骨髓间充质干细胞。2.1×1011L-1的细胞密度是较为理想的原代全骨髓接种密度,2×108L-1是MSCs较为理想的传代密度。待MSCs生长到第3-4天时,细胞生长融合至80%-90%时,培养皿中的细胞是最佳的传代时间。3MSCs能于体外培养大量扩增和存活,在增殖多代后仍然可以保持良好的细胞活性和扩增能力,并于一定条件下可以诱导分化为成骨细胞。4MSCs诱导分化后的细胞表面主要有Sca-1、CD34、CD29、CD44标记分子、缺乏CD117分子。
目的首先观察间充质干细胞对内毒素诱导急性肺损伤大鼠的保护作用,然后观察其对大鼠氧化应激及炎症反应的影响。
方法1.90只SD大鼠随机分为5组：A组：正常对照组(n=18)尾静脉注射等量生理盐水；B组：内毒素组(LPS)(n=18)：经尾静脉注射LPS5mg/kg；C组：高剂量干细胞组(BMSCH组)(n=18)：经尾静脉注射LPS5mg/kg+BMSC2×106/ml0.5ml；D组：低剂量干细胞组(BMSCL组)(n=18)：LPS5mg/kg+BMSC1×106/ml0.5ml；干细胞对照组(BMSC组)(n=18)：骨髓间充质干细胞2×106/ml0.5ml。分别于造模后6、24、72小时,处死动物收取标本,每个时间点6只(LPS的24小时组因1只死亡固只处死5只)。2.取右肺上叶用于测定肺湿重/干重,取右肺下叶组织(约0.5cm2)放入多聚甲醛中固定,用于组织形态学观察。取其余肺组织于生理盐水中迅速漂洗干净后,立即放入液氮中冷冻,后转入-70℃冰箱中保存备用,腹主动脉采血立即送血气分析。分别测定肺湿重/干重、肺组织MDA、SOD、MP0、TNF-α和IL-1β。3.统计学分析：采用SPSS13.0软件包处理,计量资料以均数±标准差表示,不同处理组间资料均数比较采用析因设计资料的方差分析。各组间同一时间点之间的比较及组内不同时间点间的比较采用完全随机资料的方差分析(one-way ANOVA法),以P<0.05判断差异有显著性。在进行方差分析前,首先进行Levene方差齐性检验。对方差齐性的资料采用LSD法进行多重比较；对方差不齐的资料采用近似F检验的Welch法进行方差分析,并采用Tambane's T2法进行多重比较。
结果1.病理所见：A、E组肺组织结构清晰,肺泡腔无炎性细胞浸润,肺泡壁薄,间质血管无扩张,支气管粘膜上皮完整。B组肺泡结构受到广泛破坏,肺组织水肿及肺间质明显的增宽,肺泡腔内有浆液性渗出,可见大量红细胞以及中性粒细胞,C组为BMSC高剂量组,大部分肺组织及间质结构较完整,小部分肺泡间隔稍增宽,可见肺间质水肿减轻,有少量红细胞及中性粒细胞浸润。D组为BMSC (?)低剂量组,肺泡结构部分被破坏,肺间质中度的水肿,肺泡腔内有多量渗出的红细胞及炎性细胞浸润。2.W/D：不同处理组间有显著差异(F=57.741,P=0.000),时间因素和分组因素交互效应显著(F=3.129,P=0.004)。B组均明显高于A组；24h和72h两个时间点,C、D组低于B组；Pa02比较：B组明显低于A组,C组三个时间点与B组比较,差异均有统计学意义。3.MDA：不同处理组间有显著差异(F=25.742,P=0.000),时间因素和分组因素无显著交互效应(F=0.690,P=0.699)。三个时间点B组MDA明显高于A组,差异有统计学意义；C、D组明显低于B,差异有统计学意义；A组和E组差异无统计学意义。SOD：不同处理组间有显著差异(F=138.052,P=0.000),时间因素和分组因素无显著交互效应(F=0.910,P=0.513)。三个时间点B组低于A组,差异有统计学意义；C、D组明显高于B,差异有统计学意义；E组和A组差异无统计学意义。MPO：不同处理组间有显著差异(F=52.057,P=0.000),时间因素和分组因素无显著交互效应(F=1.096,P=0.376)。三个时间点B组明显高于A组,差异有统计学意义；C、D组明显低于B,差异有统计学意义；A组和E组差异无统计学意义。TNF-a和IL-1β：IL-1β不同处理组间有显著差异(F=151.807,P=0.000),时间因素和分组因素交互效应显著(F=2.085,P=0.048)。TNF-a不同处理组间有显著差异(F=134.814,P=0.000),时间因素和分组因素无显著交互效应(F=1.394,P=0.213)。TNF-α和IL-1β不同组之间存在显著差异(F=123.507,P<0.001)各组同一时间点的比较：B组较A组显著性升高,P<0.05；C、D组低于B组,差异有统计学意义；E组与A组差异无统计学意义。
结论1.间充质干细胞移植对内毒素诱导急性肺损伤大鼠具有肺保护作用。2.间充质干细胞移植可以降低急性肺损伤大鼠的氧化应激反应和炎症因子的表达。3.SD大鼠对小鼠的间充质干细胞移植无明显的排异反应,无明显的毒副作用。
目的1.观察NF-κB在内毒素诱导急性肺损伤大鼠肺组织的活性变化及间充质干细胞对NF-κB活性的影响,探索NF-κB在内毒素诱导急性肺损伤的发病机理及间充质干细胞治疗机理中的作用。2.观察CyPA在内毒素诱导急性肺损伤大鼠肺组织表达的变化及间充质干细胞对CyPA表达的影响,探索CyPA在内毒素诱导急性肺损伤的发病机理及间充质干细胞治疗机理中的作用。
方法1.间充质干细胞的制备：同第一部分。2.肺组织NF-κB及CyPA的Western blot:a、配制SDS-PSGE电泳胶。b、处理样品：依次取样(上样量为10ug)加入到20ul2×SDS凝胶加样缓冲液中,沸水中煮沸8分钟使蛋白变性,然后10000rpm离心5分钟。c、上样：先将梳子小心的移出,把凝胶固定在电泳装置上,内外槽各加入1×Tris-甘氨酸电泳缓冲液,取样品用加样器向孔内加入样品,加样量通常在1020ul,将电泳装置与电源连接,接通电源凝胶上胶电压为60伏,当染料前沿进入分离胶后(跑平即可),把电压提高到120伏。继续电泳直至溴酚蓝到达分离胶底部上方约0.5cm,然后关闭电源,从电泳装置上卸下玻璃板,用刮勺撬开玻璃板,在凝胶上部一角处切去一角标注加样顺序。d、免疫印迹(Western Blot):(?)将电泳结束后的胶片放入转印缓冲液中,将转印膜也放入转印缓冲液中浸润,进行转膜,膜在正极,胶在负极,分别用滤纸3张/面压住胶和膜。转移时电压100伏,转移时间1小时,电转移时会产热,固应在转移装置的外面加入冰块进行除热,转移结束后,卸下电泳装置,把硝酸纤维素转移膜放入盛有10%脱脂奶粉或10%BSA的封闭液中,封闭2小时或过夜,封闭完毕后用PBST洗膜5次,每次间隔5分钟,加一抗。反应1小时,洗膜5次每次间隔5分钟。(一抗HPS701：200)(一抗HSV11：200),加辣根过氧化物酶标记二抗,按说明书要求的稀释比例加入,反应1小时。洗膜5次每次间隔5分钟。(二抗1：1000),取ECL荧光底物(Pierce公司,货号：37071)A液和B液1各mL,混匀后,将PVDF膜浸泡其中,室温孵育3-5分钟后,用滤纸将PVDF膜表面液体擦干,并将其置于暗室,取医用X光底片覆盖,曝光1分钟后,依次显影、定影即可。3CyPA的RNA提取和Real-PCR:a、抽提RNA。b、反转录：融解反应所需的试剂,上下轻微颠倒混匀,进行短暂离心后放置冰上待用,然后配制RT反应液(所有反应都在冰上进行操作),混匀反应Mix,短暂离心后37-C孵育30小时,反应结束后,85℃灭活处理5min,对所得到的cDNA用灭菌水稀释5倍后做好标记并-20℃保存反转录产物。c、定量PCR实验：通过引物验证预实验确定选取特异性扩增,无引物二聚体、灵敏度高的引物为下游各基因表达量差异分析的使用引物,将All-in-One qPCRMix在室温下融解,轻柔得上下颠倒混匀并进行短暂离心,同时在使用过程中始终保持避光,制备PCR reaction mix(在冰上操作),PCR reaction mix制备后,迅速将稍混匀,并加入96孔板中。4.统计学分析：采用SPSS13.0软件包处理,计量资料以均数±标准差表示,不同处理组间资料均数比较采用析因设计资料的方差分析。各组间同一时间点之间的比较及组内不同时间点间的比较采用完全随机资料的方差分析(one-way ANOVA法),以P<0.05判断差异有显著性。在进行方差分析前,首先进行Levene方差齐性检验。对方差齐性的资料采用LSD法进行多重比较；对方差不齐的资料采用近似F检验的Welch法进行方差分析,并采用Tambane's T2法进行多重比较。
结果1.本研究通过检测核提取物中NF-κBp65的蛋白表达量来决定NF-κB的活性。不同处理组间有显著差异(F=681.495,P=0.000),时间因素和分组因素交互效应显著(F=8.672,P=0.000)。各组NF-κB方差齐性检验：F=1.769,P=0.150,方差齐性。各组之间多重比较选用LSD法。方差分析(ANOVA)结果：F=185.399P=0.000,组间差异有统计学意义。各组同一时间点的比较：三组NF-κB的核内表达量各个时间点之间均有显著性差异(6小时：F=273.165,P<0.001：24小时：F=179.975,P<0.001；72小时：F=254.031,P<0.001=,以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。2.肺组织CyPA蛋白表达(Western blot)水平的变化：不同处理组间有显著差异(F=984.853,P=0.000),时间因素和分组因素交互效应显著(F=16.884,P=0.000)。各组CyPA方差齐性检验：F=1.865,P=0.130,方差齐性。各组之间多重比较选用LSD法。方差分析(ANOVA)结果：F=278.073P=0.000,组间差异有统计学意义。各组同一时间点的方差分析：三组CyPA的表达量各个时间点之间均有显著性差异(6小时：F=278.01,P<0.001：24小时：F=436.923,P<0.001；72小时：F=412.712,P<0.001),以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。3.肺组织CyPA mRNA转录(RT-PCR)水平的变化：不同处理组间有显著差异(F=53.680,P=0.000),时间因素和分组因素无显著交互效应(F=0.015,P=1.000)。各组CyPA方差齐性检验：F=2.199,P=0.046,方差不齐。各组之间多重比较选用Tambane's T2法。方差分析选用近似F检验的Welch法,方差分析结果：F=11.964P=0.000,组间差异有统计学意义。各组同一时间点的两两比较：三组CyPA转录水平各个时间点之间均有显著性差异(6小时：F=18.445,P<0.001：24小时：F=15.117,P<0.001；72小时：F=20.820,P<0.001),以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。
结论1.亲环素A和NF-κB在尾静脉注射内毒素诱导急性肺损伤大鼠的肺组织中表达升高。2.注射内毒素后立即经尾静脉注射小鼠骨髓间充质干细胞显著降低急性肺损伤大鼠的肺组织中的亲环素A和NF-κB的表达。3.亲环素A和NF-κB在内毒素诱导急性肺损伤的发病中起到重要作用,骨髓间充质干细胞可以通过抑制NF-κB的活性和抑制亲环素A的表达而起到治疗作用。
Background and purpose of the research:Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are mainly caused by the gastric acid inhalation, infection, vulnus and fat embolism with infection as the most common reason. And the inflammatory reaction plays an important role in the cute lung injury caused by whatever reason. Although lots of researches aim to expound the pathogeny of the cute lung injury/acute respiratory distress syndrome, especially effective therapy has not been found so far. At present, the therapy of acute lung injury/acute respiratory distress syndrome gives first place to supporting therapy, drug treatment and assisted ventilation therapy, there is still a high case fatality rate in clinic, at about30%-40%, therefore, there are crying needs for the intensive study of the pathogenesis to find out the more effective treatment method. Mesenchymal stem cells (MSC) not only possess the low immunogenicity, multilineage differentiation, immune tolerance of induction but also have the function of immunosuppression. Since the MSCs have the unique immunological characteristics and good plasticity and its application does not relate to the ethical debates, therefore. MSCs has been the best candidate cytologic material of the cell therapy and tissue engineering. However, its treatment mechanism is still not completely clear. The nuclear transcription factorκB(NF-κB)is one proinflammatory factor which receives great many researches and which plays
an important role in the occurrence and regulation of the inflammation, In the last few years, the NF-κB signalling channel of the blocking-up pulmonary epithelial cells can reduce the degrees of granulocytic pneumonia disease and pulmonary edema of the acute lung injury rat caused by the endotoxin induction, it is one of the effective treatment methods of pneumonia disorder disease. Cyclophilin A (CyPA) is the leukocyte chemotactic factor (LCF). It makes use of the combination between it and its receptor to activate the channel of MAPK and its nuclear factorκB (NF-κB) to regulate the release of proinflammatory factors. Nowadays, the literature reports on the impacts of mesenchymal stem cells on NF-κB and CyPA in endotoxin-induction acute lung injury can not be found. The research aims to study the biological function of mesenchymal stem cells in endotoxin-induction acute lung injury and the impacts on NF-κB and CyPA to further expound the treating mechanism MSCs on ALI and to provide the experimental clues for clinic to find more effective treatment method.
Objective To observe the features of the cells in the identification, shape and directed differentiation into osteoblast when mesenchymal stem cells of C57BL/6J small mouse are cultured in vitro.
Methods1.Take bone marrow cells from the bilateral thighbone and shin bone of C57BL/6J small mouse and give primary culture and passage through adherent screening the medulla ossium in vitro.2.Culture the primary MSCs to the7th-generation cell and put them under the phase contrast microscope to observe its growth form and feature.3. Pick up the cells of the1st and3rd generations and count them after trypsinization and then inoculate them into the96-orifice plate at a rate of2×103pieces/orifice and add10ul Cell Counting Kit-8(CCK-8) solution, CO2whose saturated humidity is5%into each orifice and after2-hour incubator incubation at37℃,make use of the thermo labsystems to detect the absorbance value under the450nm wavelength. And then, after this detection, perform the same detection at the same time on the1st,2nd,3rd,4th,5th,6th,7th days. Make use of the absorbance value to draw the cell proliferation curve.4. Take the BMSCs from the5th-generation C57small mouse which is in good condition to make unicellular suspension and inoculate it into the6-orifice plate at the density of2×103/ml and then add the culture solution of bone induction and fat inducer separately for differentiated culture in vitro when the cells fuse by60%.5. Take the BMSCs from the5th C57small mouse which is in good condition to make unicellular suspension and collect about2×106pieces of cells for flow cytometry detection on the device.
Results1. The MSCs of primary culture begin adherence after24hours and the adherent MSCs gradually spread into irregular round, polygon, fusiformis and irregular arrangement after48hours.2. After the bone induction, calcium nodules become jacinth while the lipid droplet inside the adipocytes become red and the other cells are not colored after fat induction.3. The growth curve of the MSCs presents a form of S and the growth cycle includes lag phase, log phase and plateau phase.4. The analysis of the flow cytometry cells differentiated from the MSCs of C57small mouse shows that:there are mainly Sca-1, CD34, CD29and CD44 labeled molecules on the surface of the MSCs after inducted differentiation, with short of CD117molecules.
Conclusion1. The method of density centrifugation and adherence can successfully culture and differentiate the mesenchymal stem cells of the C57BL/6J small mouse. The2.1×1011L-1cell density is much more ideal inoculum density of the medulla ossium of primary generation. While2.1×1011L-1cell density is much more ideal passage density. When MSCs grow for3-4days and the cells grow and fuse by80%-90%, the cells in the culture dish are the best generation transfer time.3. MSCs can greatly amplify and survive through culture in vitro and they can keep the good cytoactive and amplification ability after many generations of multiplication, and can differentiate into osteoblast through induction.4. There are mainly Sca-1, CD34, CD29and CD44labeled molecules on the surface of the MSCs after inducted differentiation, with short of CD117molecules.
Objective Firstly it's to observe the protective function of the mesenchymal stem cells on the endotoxin-induction acute lung injury rat and then it's to observe the influence on the oxidatie stress and inflammatory reaction of the rat.
Methods1. Randomly separate the90rats into5groups:Group A:the normal group (n=18):given the equivalent normal saline through tail vein injection; Group B:the lipopolysaccharide group (LPS)(n=18):given5mg/kg LPS through tail vein injection; Group C:the group of high-dose stem cells(BMSCH group)(n=18):given5mg/kg+BMSC2×106/ml0.5ml LPS through tail vein injection; Group D:the group of low-dose stem cells (BMSCL group):LPS5mg/kg+BMSC1×106/ml0.5ml; the control group of stem cells(BMSC group)(n=18):given2×106/ml0.5ml medulla ossium mesenchymal stem cells. When it's6hours,24hours and72hours after molding, collect the sample from the animal which is put to death,6samples at each time(since one of the24-hour group of LPS dies, so only the other five are put to death).2. Take the superior lobe of right lung for detecting the wet weight/dry weight of the lung while take the tissues of the inferior lobe of right lung (about0.5cm2) and put them into the paraformaldehyde to fix, which are used for observing the histomorphology. And add the rest tissues of the lung into the normal saline for rapid rinsing, and then immediately put them into the liquid nitrogen for freezing and then transfer them to-70℃refrigerator to save for spare use. Take abdominal aortic blood and immediately deliver for blood gas analysis. Determine the wet weight/dry weight of the lung and lung tissues like MDA, SOD, MPO, and TNF-α and IL-1β.3. Statistical analysis:To make use of SPSS13.0software to dispose and the measurement data are shown in the form of mean±standard deviation, the comparison between the means of the data of different disposed groups adopts the way of variance of reason analysis design data.the comparison of the data of same time between different group and the comparison of the data of different time within same group adopt the way of variance analysis of completely-random data(one-way ANOVA) and making use of P<0.05to determine the significance of difference. Homogeneity of variance must be tested Before taking variance analysis.The LSD would be adopted if the data meet Homogeneity of variance. If not, The Welch would be adopted to test the variance analysis,and Tambane's T2would be adopted.
Results1. Pathological findings:the structures of the lung tissues of Group A and Group B are clear, the non-inflammatory cells of pulmonary alveoli chamber infiltrate and the wall of pulmonary alveoli is thin, and the mesenchymal vessels do not expand and the mucous epithelium of bronchus is complete. The structures of pulmonary alveoli of the Group B are destroyed widely and there is lung tissue oedema and obvious broadening of the lung mesenchyme and there is serous fluid exudation from the pulmonary alveoli chamber, a large number of red blood cells and neutrophile granulocytes can be found; Group C is the BMSC high-dose group, and most lung tissues and mesenchymal structures are more complete while the pulmonary alveoli of intervals of small parts slightly broaden, so it can be seen that the lung mesenchyme edema lightens while a few red blood cells and neutrophile granulocyte infiltrate. While Group D is the BMSC high-dose group, whose structures of pulmonary alveoli are partly destroyed and lung mesenchyme suffers moderate edema and there are mickle red blood cells leaking and inflammatory cells infiltrating.2. W/D:The W/D is significantly different between different group(F=57.741, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=3.129, P=0.004). that of Group B is obviously higher than that of Group A; that of Group C and Group D is lower than that of Group B at24h and72h; PaO2comparison:that of Group A is obviously lower than that of Group A, comparing that of Group C at the three points of the time with that of Group B, the differences between them are of statistical meaning.3. MDA:The MDA is significantly different between different group(F=25.742, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.690, P=0.699).MDA of Group B at the three points of the time is obviously higher than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is lower than that of Group B and the differences between them are of statistical meaning; the differences between Group A and Group E are not of statistical meaning. SOD:The SOD is significantly different between different group(F=138.052, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.910, P=0.513).that of Group B at the three points of time is lower than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is obviously higher than that of Group B and the differences between them are of statistical meaning; the differences between Group E and Group A are not of statistical meaning. MPO:The MPO is significantly different between different group(F=52.057, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=1.096, P=0.376). that of Group B at the tree points of time is obviously higher than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is obviously lower than that of Group B and the differences between them are of statistical meaning; the differences between Group A and Group E are not of statistical meaning. TNF-α and IL-1β:TNF-α is significantly different between different group(F=134.814, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=1.394, P=0.213). IL-1β is significantly different between different group(F=151.807, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=2.085, P=0.048).There are significant differences between TNF-α and IL-1β of different groups(F=123.507,P<0.001) by comparing them at the same point of time:compared with Group A, the significance of Group B rises, P<0.05; Group C and Group D are lower than Group B and the differences between them are of statistical meaning; the differences between Group E and Group A are not of statistical meaning.
Conclusion1. Mesenchymal stem cell transplantation has protective function for the lung of the endotoxin-induction acute lung injury rat.2. Mesenchymal stem cell transplantation can reduce the express of the oxidative stress and inflammatory factors.3. There is no apparent rejection of SD rat for the mesenchymal stem cell transplantation of small mouse and there is no significant side effect.
Objective1. Observe the activity changes of NF-κB in lung tissues of endotoxin-induction acute lung injury rat and the influence of mesenchymal stem cells on the activity of NF-κB and explore the functin of NF-κB in the pathogenesis of endotoxin-induction acute lung injury and the function of mesenchymal stem cells in the cure mechanism.2. Observe the activity changes of CyPA in lung tissues of endotoxin-induction acute lung injury rat and the influence of mesenchymal stem cells on the activity of CyPA and explore the functin of CyPA in the pathogenesis of endotoxin-induction acute lung injury rat and the function of mesenchymal stem cells in the cure mechanism.
Methods1. The preparation of mesenchymal stem cells:it is the same as the first part.2. Western blot of the lung tissues NF-κB and CyPA:a.Prepare the SDS-PSGE running gel. b. Sample handling:take samples successively (the loading amount is10ug)and add into20ul2×SDS gel sampling buffer solution and boil for8minutes in the boiling water to make the albuminous degeneration and then perform10000rpm centrifugation for5minutes. c. Loading:firstly move the comb out carefully and fix the gel on the electrophoresis device and add1×Tris-glycine electrophoresis buffer solution into the inside and outside slots, then take samples and make use of sample injector to add the sample into the orifice and the dose of sample is usually at10—20ul; connect the electrophoresis device with the power supply and switch on the power supply gel and the loading voltage is60volts; when the front edge of dye enters into separation gel,raise the voltage to120volts. Continue the electrophoresis until the bromophenol blue reach the position which is about0.5cm away from the bottom of separation gel and then switch off the power supply and demount the glass board from the electrophoresis device, and make use of the spatula to force the glass board open, and then cut off one corner of the top of gel to lable the loading order, d. Western blot:add the film at the end of electrophoresis into the blotting buffer solution and also add the blotting film into the blotting buffer solution for infiltration to transfer the film with the film at the positive pole and the gel at the negative pole, and separately make use of the filter paper whose size is3pieces/side to pin the gel and the film. At the time of transfer, the voltage is100volts and the time of transfer is1hour; at the time of electro-transfer, there will be heat production so it is necessary to add ice to the exterior face of the transfer device to get rid of the heat; after the transfer, dismount the electrophoresis device and put the nitrocellulose transfer film into the10%nonfat dried milk or10%sealing liquid of BSA and seal up for2hours or overnight, and after sealing up, make use of PBST to wash the film for5times at the rate of once every5minutes and add the first antibody. Wash the film for5times at the rate of once every5minutes and in the1-hour reaction.(The first antibody HPS701:200)(The first antibody HSV11:200), and add the horse radish peroxidase and mark it as the second antibody according to the required dilution ratio of the instruction book for1-hour reaction. Wash the film for5times at the rate of once every5minutes.(The second resist1:1000), take lml of ECL fluorogenic substrate (Pierce, article No.37071) A and B separately, and immerse the PVDF film in it after blending and perform room-temperature incubation for3-5minutes, and make use of the filter paper to wipe the liquid on the surface of PVDF film dry and put it in the darkroom and cover it by medical X-ray film and after1minute, development and fixation will be ok in proper order.3. RNA collection of CyPA and Real-PCR:a. Extract RNA. b. Reverse transcription:dissolve the required reagent and blend it by slight up and down perversion and add ice for spare use after short centrifugation and then prepare the RT reaction liquid (all the reaction shall be carried out on the ice); incubate for30hours at37℃after the blending reaction and short centrifugation and perform inactivated processing for5minutes at85℃after the reaction and make use of the sterilization water to dilute the obtained cDNA by5times and mark it and store the reverse transcription products at-20℃. c. Quantitative PCR test:make use of the primer to test and verify the test to be verified and determine the amplification of selecting specificity, non-primer dimer and the primer with high sensitivity and the primer which is used for the variance analysis of gene expression; dissolve the All-in-One qPCR Mix at the room temperature and blend them by slight up and down perversion and perform short centrifugation and at the same time, keep out of the sun during the process of use from beginning to end and prepare PCR reaction mix(operate on the ice) and blend it rapidly after the preparation of PCR reaction mix and add it into the96-orifice plate.4. Statistical analysis:to make use of SPSS13.0software to dispose and the measurement data are shown in the form of mean±standard deviation, the comparison between the means of the data of different disposed groups adopts the way of variance of reason analysis design data.the comparison of the data of same time between different group and the comparison of the data of different time within same group adopt the way of variance analysis of completely-random data(one-way ANOVA) and make use of P <0.05to determine the significance of difference. Homogeneity of variance must be tested Before taking variance analysis.The LSD would be adopted if the data meet Homogeneity of variance. If not, The Welch would be adopted to test the variance analysis,and Tambane's T2would be adopted.
Results1.The research makes use of the protein expression of NF-κB p65in the testing nuclear extractive to determine the activity of NF-κB. NF-κB is significantly different between different group(F=681.495, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=8.672, P=0.000).The test of Homogeneity of variance:F=1.769,P=0.150, Homogeneity of variance was meeted.The multiple comparation adopt LSD.ANOVA result:F=185.399,P=0.000, the difference between the groups is significant. The comparation of the groups at the same time:the expression inside the nuclear of the three groups of NF-κB has a significant difference at the corresponding points of time (6-hour:F=273.165,P<0.001;24-hour:F=179.975, P <0.001;72-hour:F=254.031, P<0.001, with Group B highest followed by Group C and the Group Alowest and the multiple comparison among the groups at the same time also has significant difference, P<0.05).2. The level change of CyPA western blot of lung tissues:CyPA is significantly different between different group(F=984.853, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=16.884, P=0.000).The test of Homogeneity of variance:F=1.865,P=0.130, Homogeneity of variance was meeted.The multiple comparation adopt LSD.ANOVA result:F=278.073,P=0.000, the difference between the groups is significant. the comparison of the groups at the same time: the CyPA expression of the three groups has a significant difference at the points of time (6-hour:F=278.01,P<0.001;24-hour:F=436.923,P<0.001;72-hour: F=412.712,P<0.001), with Group B highest followed by Group C and the Group A lowest and the multiple comparison among the groups at the same time also has significant difference(P<0.05).3. The level change of CyPA mRNA transcription of the lung tissues (RT-PCR):CyPA is significantly different between different group(F=53.680, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.015, P=1.000).The test of Homogeneity of variance:F=2.199,P=0.046, Homogeneity of variance was not meeted.The multiple comparation adopt Tambane's T2and ANONA adopt Welch. ANOVA result:F=11.964P=0.000, the difference between the groups is significant, the multiple comparison of the groups at the same time:the CyPA transcription levels of the three groups have a significant difference at the same time (6-hour: F=18.445,P<0.001;24-hour:F=15.117, P<0.001;72-hour:F=20.820, P<0.001), with Group B highest followed by Group C and the Group A lowest and the multiple comparison among the groups at the corresponding points of time also has significant difference(P<0.05).
Conclusion1. The expression of cyclophilin A and NF-κB rise in the lung tissues of endotoxin-induction acute lung injury rat which is injected in caudal vein.2. Aftering injecting the endotoxin, the injected-in-caudal-veine medulla ossium mesenchymal stem cells of the small mouse immediately reduce the expression of Cyclophilin A and NF-κB in the lung tissues of acute lung injury rat.3. Cyclophilin A and NF-κB play an important role in the morbidity of endotoxin-induction acute lung injury and the medulla ossium mesenchymal stem cells can perform the therapeutic effect by the level of Cyclophilin A.
目的观察C57BL/6J小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外培养时,细胞的鉴定、形态及定向诱导分化为成骨细胞的特点。
方法1取C57BL/6J小鼠双侧股骨和胫骨骨髓细胞进行体外全骨髓贴壁筛选法原代培养并传代。2.取原代的MSCs培养至第7代细胞,倒置相差显微镜下观察其生长形态及特征。3.提取第1、3代细胞,胰酶消化后计数,以2×103个/孔接种于96孔板,每孔加入10μlCell Counting Kit-8(CCK-8)溶液,37℃,饱和湿度为5%CO2,培养箱孵育2h后,用酶联免疫检测仪于450nm波长下检测吸光度值。然后在检测后第1、2、3、4、5、6、7天同一时间作相同检测。用吸光度值绘制细胞增殖曲线。4.第5代生长状态良好的C57小鼠BMSCs制成单细胞悬液,以2×103/ml浓度接种于6孔板,待细胞达60%融合时,分别加入成骨诱导培养液及成脂诱导剂进行体外分化培养。5.第5代生长状态良好的C57小鼠BMSCs制成单细胞悬液,收集约2×106个细胞上机进行流式细胞检测。
结果1.原代培养的MSCs在24h后开始贴壁,48h后贴壁的MSCs逐渐伸展为不规则圆形、多角形、梭形、排列不规则。2.成骨诱导后镜下观察钙结节染成橘红色、成脂诱导后脂肪细胞里的脂滴呈红色,其它细胞则不着色。3.MSCs的生长曲线呈S型,其生长周期分别为滞后期,对数期和平台期。4.C57小鼠MSCs分化的流式细胞分析显示：MSCs诱导分化后的细胞表面主要有Sca-1、CD34、CD29和CD44标记分子、缺乏CD117分子。
结论1.密度离心结合贴壁法可以成功培养、分离出C57BL/6J小鼠骨髓间充质干细胞。2.1×1011L-1的细胞密度是较为理想的原代全骨髓接种密度,2×108L-1是MSCs较为理想的传代密度。待MSCs生长到第3-4天时,细胞生长融合至80%-90%时,培养皿中的细胞是最佳的传代时间。3MSCs能于体外培养大量扩增和存活,在增殖多代后仍然可以保持良好的细胞活性和扩增能力,并于一定条件下可以诱导分化为成骨细胞。4MSCs诱导分化后的细胞表面主要有Sca-1、CD34、CD29、CD44标记分子、缺乏CD117分子。
目的首先观察间充质干细胞对内毒素诱导急性肺损伤大鼠的保护作用,然后观察其对大鼠氧化应激及炎症反应的影响。
方法1.90只SD大鼠随机分为5组：A组：正常对照组(n=18)尾静脉注射等量生理盐水；B组：内毒素组(LPS)(n=18)：经尾静脉注射LPS5mg/kg；C组：高剂量干细胞组(BMSCH组)(n=18)：经尾静脉注射LPS5mg/kg+BMSC2×106/ml0.5ml；D组：低剂量干细胞组(BMSCL组)(n=18)：LPS5mg/kg+BMSC1×106/ml0.5ml；干细胞对照组(BMSC组)(n=18)：骨髓间充质干细胞2×106/ml0.5ml。分别于造模后6、24、72小时,处死动物收取标本,每个时间点6只(LPS的24小时组因1只死亡固只处死5只)。2.取右肺上叶用于测定肺湿重/干重,取右肺下叶组织(约0.5cm2)放入多聚甲醛中固定,用于组织形态学观察。取其余肺组织于生理盐水中迅速漂洗干净后,立即放入液氮中冷冻,后转入-70℃冰箱中保存备用,腹主动脉采血立即送血气分析。分别测定肺湿重/干重、肺组织MDA、SOD、MP0、TNF-α和IL-1β。3.统计学分析：采用SPSS13.0软件包处理,计量资料以均数±标准差表示,不同处理组间资料均数比较采用析因设计资料的方差分析。各组间同一时间点之间的比较及组内不同时间点间的比较采用完全随机资料的方差分析(one-way ANOVA法),以P<0.05判断差异有显著性。在进行方差分析前,首先进行Levene方差齐性检验。对方差齐性的资料采用LSD法进行多重比较；对方差不齐的资料采用近似F检验的Welch法进行方差分析,并采用Tambane's T2法进行多重比较。
结果1.病理所见：A、E组肺组织结构清晰,肺泡腔无炎性细胞浸润,肺泡壁薄,间质血管无扩张,支气管粘膜上皮完整。B组肺泡结构受到广泛破坏,肺组织水肿及肺间质明显的增宽,肺泡腔内有浆液性渗出,可见大量红细胞以及中性粒细胞,C组为BMSC高剂量组,大部分肺组织及间质结构较完整,小部分肺泡间隔稍增宽,可见肺间质水肿减轻,有少量红细胞及中性粒细胞浸润。D组为BMSC (?)低剂量组,肺泡结构部分被破坏,肺间质中度的水肿,肺泡腔内有多量渗出的红细胞及炎性细胞浸润。2.W/D：不同处理组间有显著差异(F=57.741,P=0.000),时间因素和分组因素交互效应显著(F=3.129,P=0.004)。B组均明显高于A组；24h和72h两个时间点,C、D组低于B组；Pa02比较：B组明显低于A组,C组三个时间点与B组比较,差异均有统计学意义。3.MDA：不同处理组间有显著差异(F=25.742,P=0.000),时间因素和分组因素无显著交互效应(F=0.690,P=0.699)。三个时间点B组MDA明显高于A组,差异有统计学意义；C、D组明显低于B,差异有统计学意义；A组和E组差异无统计学意义。SOD：不同处理组间有显著差异(F=138.052,P=0.000),时间因素和分组因素无显著交互效应(F=0.910,P=0.513)。三个时间点B组低于A组,差异有统计学意义；C、D组明显高于B,差异有统计学意义；E组和A组差异无统计学意义。MPO：不同处理组间有显著差异(F=52.057,P=0.000),时间因素和分组因素无显著交互效应(F=1.096,P=0.376)。三个时间点B组明显高于A组,差异有统计学意义；C、D组明显低于B,差异有统计学意义；A组和E组差异无统计学意义。TNF-a和IL-1β：IL-1β不同处理组间有显著差异(F=151.807,P=0.000),时间因素和分组因素交互效应显著(F=2.085,P=0.048)。TNF-a不同处理组间有显著差异(F=134.814,P=0.000),时间因素和分组因素无显著交互效应(F=1.394,P=0.213)。TNF-α和IL-1β不同组之间存在显著差异(F=123.507,P<0.001)各组同一时间点的比较：B组较A组显著性升高,P<0.05；C、D组低于B组,差异有统计学意义；E组与A组差异无统计学意义。
结论1.间充质干细胞移植对内毒素诱导急性肺损伤大鼠具有肺保护作用。2.间充质干细胞移植可以降低急性肺损伤大鼠的氧化应激反应和炎症因子的表达。3.SD大鼠对小鼠的间充质干细胞移植无明显的排异反应,无明显的毒副作用。
目的1.观察NF-κB在内毒素诱导急性肺损伤大鼠肺组织的活性变化及间充质干细胞对NF-κB活性的影响,探索NF-κB在内毒素诱导急性肺损伤的发病机理及间充质干细胞治疗机理中的作用。2.观察CyPA在内毒素诱导急性肺损伤大鼠肺组织表达的变化及间充质干细胞对CyPA表达的影响,探索CyPA在内毒素诱导急性肺损伤的发病机理及间充质干细胞治疗机理中的作用。
方法1.间充质干细胞的制备：同第一部分。2.肺组织NF-κB及CyPA的Western blot:a、配制SDS-PSGE电泳胶。b、处理样品：依次取样(上样量为10ug)加入到20ul2×SDS凝胶加样缓冲液中,沸水中煮沸8分钟使蛋白变性,然后10000rpm离心5分钟。c、上样：先将梳子小心的移出,把凝胶固定在电泳装置上,内外槽各加入1×Tris-甘氨酸电泳缓冲液,取样品用加样器向孔内加入样品,加样量通常在1020ul,将电泳装置与电源连接,接通电源凝胶上胶电压为60伏,当染料前沿进入分离胶后(跑平即可),把电压提高到120伏。继续电泳直至溴酚蓝到达分离胶底部上方约0.5cm,然后关闭电源,从电泳装置上卸下玻璃板,用刮勺撬开玻璃板,在凝胶上部一角处切去一角标注加样顺序。d、免疫印迹(Western Blot):(?)将电泳结束后的胶片放入转印缓冲液中,将转印膜也放入转印缓冲液中浸润,进行转膜,膜在正极,胶在负极,分别用滤纸3张/面压住胶和膜。转移时电压100伏,转移时间1小时,电转移时会产热,固应在转移装置的外面加入冰块进行除热,转移结束后,卸下电泳装置,把硝酸纤维素转移膜放入盛有10%脱脂奶粉或10%BSA的封闭液中,封闭2小时或过夜,封闭完毕后用PBST洗膜5次,每次间隔5分钟,加一抗。反应1小时,洗膜5次每次间隔5分钟。(一抗HPS701：200)(一抗HSV11：200),加辣根过氧化物酶标记二抗,按说明书要求的稀释比例加入,反应1小时。洗膜5次每次间隔5分钟。(二抗1：1000),取ECL荧光底物(Pierce公司,货号：37071)A液和B液1各mL,混匀后,将PVDF膜浸泡其中,室温孵育3-5分钟后,用滤纸将PVDF膜表面液体擦干,并将其置于暗室,取医用X光底片覆盖,曝光1分钟后,依次显影、定影即可。3CyPA的RNA提取和Real-PCR:a、抽提RNA。b、反转录：融解反应所需的试剂,上下轻微颠倒混匀,进行短暂离心后放置冰上待用,然后配制RT反应液(所有反应都在冰上进行操作),混匀反应Mix,短暂离心后37-C孵育30小时,反应结束后,85℃灭活处理5min,对所得到的cDNA用灭菌水稀释5倍后做好标记并-20℃保存反转录产物。c、定量PCR实验：通过引物验证预实验确定选取特异性扩增,无引物二聚体、灵敏度高的引物为下游各基因表达量差异分析的使用引物,将All-in-One qPCRMix在室温下融解,轻柔得上下颠倒混匀并进行短暂离心,同时在使用过程中始终保持避光,制备PCR reaction mix(在冰上操作),PCR reaction mix制备后,迅速将稍混匀,并加入96孔板中。4.统计学分析：采用SPSS13.0软件包处理,计量资料以均数±标准差表示,不同处理组间资料均数比较采用析因设计资料的方差分析。各组间同一时间点之间的比较及组内不同时间点间的比较采用完全随机资料的方差分析(one-way ANOVA法),以P<0.05判断差异有显著性。在进行方差分析前,首先进行Levene方差齐性检验。对方差齐性的资料采用LSD法进行多重比较；对方差不齐的资料采用近似F检验的Welch法进行方差分析,并采用Tambane's T2法进行多重比较。
结果1.本研究通过检测核提取物中NF-κBp65的蛋白表达量来决定NF-κB的活性。不同处理组间有显著差异(F=681.495,P=0.000),时间因素和分组因素交互效应显著(F=8.672,P=0.000)。各组NF-κB方差齐性检验：F=1.769,P=0.150,方差齐性。各组之间多重比较选用LSD法。方差分析(ANOVA)结果：F=185.399P=0.000,组间差异有统计学意义。各组同一时间点的比较：三组NF-κB的核内表达量各个时间点之间均有显著性差异(6小时：F=273.165,P<0.001：24小时：F=179.975,P<0.001；72小时：F=254.031,P<0.001=,以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。2.肺组织CyPA蛋白表达(Western blot)水平的变化：不同处理组间有显著差异(F=984.853,P=0.000),时间因素和分组因素交互效应显著(F=16.884,P=0.000)。各组CyPA方差齐性检验：F=1.865,P=0.130,方差齐性。各组之间多重比较选用LSD法。方差分析(ANOVA)结果：F=278.073P=0.000,组间差异有统计学意义。各组同一时间点的方差分析：三组CyPA的表达量各个时间点之间均有显著性差异(6小时：F=278.01,P<0.001：24小时：F=436.923,P<0.001；72小时：F=412.712,P<0.001),以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。3.肺组织CyPA mRNA转录(RT-PCR)水平的变化：不同处理组间有显著差异(F=53.680,P=0.000),时间因素和分组因素无显著交互效应(F=0.015,P=1.000)。各组CyPA方差齐性检验：F=2.199,P=0.046,方差不齐。各组之间多重比较选用Tambane's T2法。方差分析选用近似F检验的Welch法,方差分析结果：F=11.964P=0.000,组间差异有统计学意义。各组同一时间点的两两比较：三组CyPA转录水平各个时间点之间均有显著性差异(6小时：F=18.445,P<0.001：24小时：F=15.117,P<0.001；72小时：F=20.820,P<0.001),以B组最、高C组其次、A组最低,且同一时间点各组之间两两比较也均有显著性差异(P<0.05)。
结论1.亲环素A和NF-κB在尾静脉注射内毒素诱导急性肺损伤大鼠的肺组织中表达升高。2.注射内毒素后立即经尾静脉注射小鼠骨髓间充质干细胞显著降低急性肺损伤大鼠的肺组织中的亲环素A和NF-κB的表达。3.亲环素A和NF-κB在内毒素诱导急性肺损伤的发病中起到重要作用,骨髓间充质干细胞可以通过抑制NF-κB的活性和抑制亲环素A的表达而起到治疗作用。
Background and purpose of the research:Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are mainly caused by the gastric acid inhalation, infection, vulnus and fat embolism with infection as the most common reason. And the inflammatory reaction plays an important role in the cute lung injury caused by whatever reason. Although lots of researches aim to expound the pathogeny of the cute lung injury/acute respiratory distress syndrome, especially effective therapy has not been found so far. At present, the therapy of acute lung injury/acute respiratory distress syndrome gives first place to supporting therapy, drug treatment and assisted ventilation therapy, there is still a high case fatality rate in clinic, at about30%-40%, therefore, there are crying needs for the intensive study of the pathogenesis to find out the more effective treatment method. Mesenchymal stem cells (MSC) not only possess the low immunogenicity, multilineage differentiation, immune tolerance of induction but also have the function of immunosuppression. Since the MSCs have the unique immunological characteristics and good plasticity and its application does not relate to the ethical debates, therefore. MSCs has been the best candidate cytologic material of the cell therapy and tissue engineering. However, its treatment mechanism is still not completely clear. The nuclear transcription factorκB(NF-κB)is one proinflammatory factor which receives great many researches and which plays
an important role in the occurrence and regulation of the inflammation, In the last few years, the NF-κB signalling channel of the blocking-up pulmonary epithelial cells can reduce the degrees of granulocytic pneumonia disease and pulmonary edema of the acute lung injury rat caused by the endotoxin induction, it is one of the effective treatment methods of pneumonia disorder disease. Cyclophilin A (CyPA) is the leukocyte chemotactic factor (LCF). It makes use of the combination between it and its receptor to activate the channel of MAPK and its nuclear factorκB (NF-κB) to regulate the release of proinflammatory factors. Nowadays, the literature reports on the impacts of mesenchymal stem cells on NF-κB and CyPA in endotoxin-induction acute lung injury can not be found. The research aims to study the biological function of mesenchymal stem cells in endotoxin-induction acute lung injury and the impacts on NF-κB and CyPA to further expound the treating mechanism MSCs on ALI and to provide the experimental clues for clinic to find more effective treatment method.
Objective To observe the features of the cells in the identification, shape and directed differentiation into osteoblast when mesenchymal stem cells of C57BL/6J small mouse are cultured in vitro.
Methods1.Take bone marrow cells from the bilateral thighbone and shin bone of C57BL/6J small mouse and give primary culture and passage through adherent screening the medulla ossium in vitro.2.Culture the primary MSCs to the7th-generation cell and put them under the phase contrast microscope to observe its growth form and feature.3. Pick up the cells of the1st and3rd generations and count them after trypsinization and then inoculate them into the96-orifice plate at a rate of2×103pieces/orifice and add10ul Cell Counting Kit-8(CCK-8) solution, CO2whose saturated humidity is5%into each orifice and after2-hour incubator incubation at37℃,make use of the thermo labsystems to detect the absorbance value under the450nm wavelength. And then, after this detection, perform the same detection at the same time on the1st,2nd,3rd,4th,5th,6th,7th days. Make use of the absorbance value to draw the cell proliferation curve.4. Take the BMSCs from the5th-generation C57small mouse which is in good condition to make unicellular suspension and inoculate it into the6-orifice plate at the density of2×103/ml and then add the culture solution of bone induction and fat inducer separately for differentiated culture in vitro when the cells fuse by60%.5. Take the BMSCs from the5th C57small mouse which is in good condition to make unicellular suspension and collect about2×106pieces of cells for flow cytometry detection on the device.
Results1. The MSCs of primary culture begin adherence after24hours and the adherent MSCs gradually spread into irregular round, polygon, fusiformis and irregular arrangement after48hours.2. After the bone induction, calcium nodules become jacinth while the lipid droplet inside the adipocytes become red and the other cells are not colored after fat induction.3. The growth curve of the MSCs presents a form of S and the growth cycle includes lag phase, log phase and plateau phase.4. The analysis of the flow cytometry cells differentiated from the MSCs of C57small mouse shows that:there are mainly Sca-1, CD34, CD29and CD44 labeled molecules on the surface of the MSCs after inducted differentiation, with short of CD117molecules.
Conclusion1. The method of density centrifugation and adherence can successfully culture and differentiate the mesenchymal stem cells of the C57BL/6J small mouse. The2.1×1011L-1cell density is much more ideal inoculum density of the medulla ossium of primary generation. While2.1×1011L-1cell density is much more ideal passage density. When MSCs grow for3-4days and the cells grow and fuse by80%-90%, the cells in the culture dish are the best generation transfer time.3. MSCs can greatly amplify and survive through culture in vitro and they can keep the good cytoactive and amplification ability after many generations of multiplication, and can differentiate into osteoblast through induction.4. There are mainly Sca-1, CD34, CD29and CD44labeled molecules on the surface of the MSCs after inducted differentiation, with short of CD117molecules.
Objective Firstly it's to observe the protective function of the mesenchymal stem cells on the endotoxin-induction acute lung injury rat and then it's to observe the influence on the oxidatie stress and inflammatory reaction of the rat.
Methods1. Randomly separate the90rats into5groups:Group A:the normal group (n=18):given the equivalent normal saline through tail vein injection; Group B:the lipopolysaccharide group (LPS)(n=18):given5mg/kg LPS through tail vein injection; Group C:the group of high-dose stem cells(BMSCH group)(n=18):given5mg/kg+BMSC2×106/ml0.5ml LPS through tail vein injection; Group D:the group of low-dose stem cells (BMSCL group):LPS5mg/kg+BMSC1×106/ml0.5ml; the control group of stem cells(BMSC group)(n=18):given2×106/ml0.5ml medulla ossium mesenchymal stem cells. When it's6hours,24hours and72hours after molding, collect the sample from the animal which is put to death,6samples at each time(since one of the24-hour group of LPS dies, so only the other five are put to death).2. Take the superior lobe of right lung for detecting the wet weight/dry weight of the lung while take the tissues of the inferior lobe of right lung (about0.5cm2) and put them into the paraformaldehyde to fix, which are used for observing the histomorphology. And add the rest tissues of the lung into the normal saline for rapid rinsing, and then immediately put them into the liquid nitrogen for freezing and then transfer them to-70℃refrigerator to save for spare use. Take abdominal aortic blood and immediately deliver for blood gas analysis. Determine the wet weight/dry weight of the lung and lung tissues like MDA, SOD, MPO, and TNF-α and IL-1β.3. Statistical analysis:To make use of SPSS13.0software to dispose and the measurement data are shown in the form of mean±standard deviation, the comparison between the means of the data of different disposed groups adopts the way of variance of reason analysis design data.the comparison of the data of same time between different group and the comparison of the data of different time within same group adopt the way of variance analysis of completely-random data(one-way ANOVA) and making use of P<0.05to determine the significance of difference. Homogeneity of variance must be tested Before taking variance analysis.The LSD would be adopted if the data meet Homogeneity of variance. If not, The Welch would be adopted to test the variance analysis,and Tambane's T2would be adopted.
Results1. Pathological findings:the structures of the lung tissues of Group A and Group B are clear, the non-inflammatory cells of pulmonary alveoli chamber infiltrate and the wall of pulmonary alveoli is thin, and the mesenchymal vessels do not expand and the mucous epithelium of bronchus is complete. The structures of pulmonary alveoli of the Group B are destroyed widely and there is lung tissue oedema and obvious broadening of the lung mesenchyme and there is serous fluid exudation from the pulmonary alveoli chamber, a large number of red blood cells and neutrophile granulocytes can be found; Group C is the BMSC high-dose group, and most lung tissues and mesenchymal structures are more complete while the pulmonary alveoli of intervals of small parts slightly broaden, so it can be seen that the lung mesenchyme edema lightens while a few red blood cells and neutrophile granulocyte infiltrate. While Group D is the BMSC high-dose group, whose structures of pulmonary alveoli are partly destroyed and lung mesenchyme suffers moderate edema and there are mickle red blood cells leaking and inflammatory cells infiltrating.2. W/D:The W/D is significantly different between different group(F=57.741, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=3.129, P=0.004). that of Group B is obviously higher than that of Group A; that of Group C and Group D is lower than that of Group B at24h and72h; PaO2comparison:that of Group A is obviously lower than that of Group A, comparing that of Group C at the three points of the time with that of Group B, the differences between them are of statistical meaning.3. MDA:The MDA is significantly different between different group(F=25.742, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.690, P=0.699).MDA of Group B at the three points of the time is obviously higher than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is lower than that of Group B and the differences between them are of statistical meaning; the differences between Group A and Group E are not of statistical meaning. SOD:The SOD is significantly different between different group(F=138.052, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.910, P=0.513).that of Group B at the three points of time is lower than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is obviously higher than that of Group B and the differences between them are of statistical meaning; the differences between Group E and Group A are not of statistical meaning. MPO:The MPO is significantly different between different group(F=52.057, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=1.096, P=0.376). that of Group B at the tree points of time is obviously higher than that of Group A and the differences between them are of statistical meaning; that of Group C and Group D is obviously lower than that of Group B and the differences between them are of statistical meaning; the differences between Group A and Group E are not of statistical meaning. TNF-α and IL-1β:TNF-α is significantly different between different group(F=134.814, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=1.394, P=0.213). IL-1β is significantly different between different group(F=151.807, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=2.085, P=0.048).There are significant differences between TNF-α and IL-1β of different groups(F=123.507,P<0.001) by comparing them at the same point of time:compared with Group A, the significance of Group B rises, P<0.05; Group C and Group D are lower than Group B and the differences between them are of statistical meaning; the differences between Group E and Group A are not of statistical meaning.
Conclusion1. Mesenchymal stem cell transplantation has protective function for the lung of the endotoxin-induction acute lung injury rat.2. Mesenchymal stem cell transplantation can reduce the express of the oxidative stress and inflammatory factors.3. There is no apparent rejection of SD rat for the mesenchymal stem cell transplantation of small mouse and there is no significant side effect.
Objective1. Observe the activity changes of NF-κB in lung tissues of endotoxin-induction acute lung injury rat and the influence of mesenchymal stem cells on the activity of NF-κB and explore the functin of NF-κB in the pathogenesis of endotoxin-induction acute lung injury and the function of mesenchymal stem cells in the cure mechanism.2. Observe the activity changes of CyPA in lung tissues of endotoxin-induction acute lung injury rat and the influence of mesenchymal stem cells on the activity of CyPA and explore the functin of CyPA in the pathogenesis of endotoxin-induction acute lung injury rat and the function of mesenchymal stem cells in the cure mechanism.
Methods1. The preparation of mesenchymal stem cells:it is the same as the first part.2. Western blot of the lung tissues NF-κB and CyPA:a.Prepare the SDS-PSGE running gel. b. Sample handling:take samples successively (the loading amount is10ug)and add into20ul2×SDS gel sampling buffer solution and boil for8minutes in the boiling water to make the albuminous degeneration and then perform10000rpm centrifugation for5minutes. c. Loading:firstly move the comb out carefully and fix the gel on the electrophoresis device and add1×Tris-glycine electrophoresis buffer solution into the inside and outside slots, then take samples and make use of sample injector to add the sample into the orifice and the dose of sample is usually at10—20ul; connect the electrophoresis device with the power supply and switch on the power supply gel and the loading voltage is60volts; when the front edge of dye enters into separation gel,raise the voltage to120volts. Continue the electrophoresis until the bromophenol blue reach the position which is about0.5cm away from the bottom of separation gel and then switch off the power supply and demount the glass board from the electrophoresis device, and make use of the spatula to force the glass board open, and then cut off one corner of the top of gel to lable the loading order, d. Western blot:add the film at the end of electrophoresis into the blotting buffer solution and also add the blotting film into the blotting buffer solution for infiltration to transfer the film with the film at the positive pole and the gel at the negative pole, and separately make use of the filter paper whose size is3pieces/side to pin the gel and the film. At the time of transfer, the voltage is100volts and the time of transfer is1hour; at the time of electro-transfer, there will be heat production so it is necessary to add ice to the exterior face of the transfer device to get rid of the heat; after the transfer, dismount the electrophoresis device and put the nitrocellulose transfer film into the10%nonfat dried milk or10%sealing liquid of BSA and seal up for2hours or overnight, and after sealing up, make use of PBST to wash the film for5times at the rate of once every5minutes and add the first antibody. Wash the film for5times at the rate of once every5minutes and in the1-hour reaction.(The first antibody HPS701:200)(The first antibody HSV11:200), and add the horse radish peroxidase and mark it as the second antibody according to the required dilution ratio of the instruction book for1-hour reaction. Wash the film for5times at the rate of once every5minutes.(The second resist1:1000), take lml of ECL fluorogenic substrate (Pierce, article No.37071) A and B separately, and immerse the PVDF film in it after blending and perform room-temperature incubation for3-5minutes, and make use of the filter paper to wipe the liquid on the surface of PVDF film dry and put it in the darkroom and cover it by medical X-ray film and after1minute, development and fixation will be ok in proper order.3. RNA collection of CyPA and Real-PCR:a. Extract RNA. b. Reverse transcription:dissolve the required reagent and blend it by slight up and down perversion and add ice for spare use after short centrifugation and then prepare the RT reaction liquid (all the reaction shall be carried out on the ice); incubate for30hours at37℃after the blending reaction and short centrifugation and perform inactivated processing for5minutes at85℃after the reaction and make use of the sterilization water to dilute the obtained cDNA by5times and mark it and store the reverse transcription products at-20℃. c. Quantitative PCR test:make use of the primer to test and verify the test to be verified and determine the amplification of selecting specificity, non-primer dimer and the primer with high sensitivity and the primer which is used for the variance analysis of gene expression; dissolve the All-in-One qPCR Mix at the room temperature and blend them by slight up and down perversion and perform short centrifugation and at the same time, keep out of the sun during the process of use from beginning to end and prepare PCR reaction mix(operate on the ice) and blend it rapidly after the preparation of PCR reaction mix and add it into the96-orifice plate.4. Statistical analysis:to make use of SPSS13.0software to dispose and the measurement data are shown in the form of mean±standard deviation, the comparison between the means of the data of different disposed groups adopts the way of variance of reason analysis design data.the comparison of the data of same time between different group and the comparison of the data of different time within same group adopt the way of variance analysis of completely-random data(one-way ANOVA) and make use of P <0.05to determine the significance of difference. Homogeneity of variance must be tested Before taking variance analysis.The LSD would be adopted if the data meet Homogeneity of variance. If not, The Welch would be adopted to test the variance analysis,and Tambane's T2would be adopted.
Results1.The research makes use of the protein expression of NF-κB p65in the testing nuclear extractive to determine the activity of NF-κB. NF-κB is significantly different between different group(F=681.495, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=8.672, P=0.000).The test of Homogeneity of variance:F=1.769,P=0.150, Homogeneity of variance was meeted.The multiple comparation adopt LSD.ANOVA result:F=185.399,P=0.000, the difference between the groups is significant. The comparation of the groups at the same time:the expression inside the nuclear of the three groups of NF-κB has a significant difference at the corresponding points of time (6-hour:F=273.165,P<0.001;24-hour:F=179.975, P <0.001;72-hour:F=254.031, P<0.001, with Group B highest followed by Group C and the Group Alowest and the multiple comparison among the groups at the same time also has significant difference, P<0.05).2. The level change of CyPA western blot of lung tissues:CyPA is significantly different between different group(F=984.853, P=0.000), there is significant reciprocation effection between the time factor and group factor (F=16.884, P=0.000).The test of Homogeneity of variance:F=1.865,P=0.130, Homogeneity of variance was meeted.The multiple comparation adopt LSD.ANOVA result:F=278.073,P=0.000, the difference between the groups is significant. the comparison of the groups at the same time: the CyPA expression of the three groups has a significant difference at the points of time (6-hour:F=278.01,P<0.001;24-hour:F=436.923,P<0.001;72-hour: F=412.712,P<0.001), with Group B highest followed by Group C and the Group A lowest and the multiple comparison among the groups at the same time also has significant difference(P<0.05).3. The level change of CyPA mRNA transcription of the lung tissues (RT-PCR):CyPA is significantly different between different group(F=53.680, P=0.000), there isnot significant reciprocation effection between the time factor and group factor (F=0.015, P=1.000).The test of Homogeneity of variance:F=2.199,P=0.046, Homogeneity of variance was not meeted.The multiple comparation adopt Tambane's T2and ANONA adopt Welch. ANOVA result:F=11.964P=0.000, the difference between the groups is significant, the multiple comparison of the groups at the same time:the CyPA transcription levels of the three groups have a significant difference at the same time (6-hour: F=18.445,P<0.001;24-hour:F=15.117, P<0.001;72-hour:F=20.820, P<0.001), with Group B highest followed by Group C and the Group A lowest and the multiple comparison among the groups at the corresponding points of time also has significant difference(P<0.05).
Conclusion1. The expression of cyclophilin A and NF-κB rise in the lung tissues of endotoxin-induction acute lung injury rat which is injected in caudal vein.2. Aftering injecting the endotoxin, the injected-in-caudal-veine medulla ossium mesenchymal stem cells of the small mouse immediately reduce the expression of Cyclophilin A and NF-κB in the lung tissues of acute lung injury rat.3. Cyclophilin A and NF-κB play an important role in the morbidity of endotoxin-induction acute lung injury and the medulla ossium mesenchymal stem cells can perform the therapeutic effect by the level of Cyclophilin A.
引文
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