幼猪肺泡上皮和血管内皮祖细胞分离培养技术探讨

发布时间：2018-05-02 06:08

本文选题：肺泡 + 上皮细胞　；参考：《复旦大学》2009年硕士论文

【摘要】： 研究背景肺泡Ⅱ型上皮细胞(Alveolar epithelial typeⅡcells, AECⅡ)是肺泡上皮的祖细胞,能够变为Ⅰ型肺泡上皮细胞参与肺损伤修复,分泌肺泡表面活性物质(Pulmonary surfactant, PS)维持肺泡稳定性、防止肺泡萎陷、保持肺的顺应性,能够将肺泡上皮顶端膜面的钠、水转运至肺间质,并参加了肺对各种吸入有害物质及微生物的天然免疫等。AECⅡ仅占肺部细胞的15%,对肺组织或肺混合细胞的培养难以明确AECⅡ的具体功能。目前尚无具有全部AECⅡ功能的细胞系,AECⅡ的原代培养成为解决这一问题的重要手段。以往研究大多是对成年大鼠、小鼠和兔AECⅡ的分离培养研究,也有少量新生动物细胞分离培养报道,但细胞产量均较低,此外由于小动物生长发育周期及生理病理过程短,生理及解剖特点也不利于长期动物实验研究,对于人新生儿至婴幼儿期的生理和病理生理学研究适用性有限。本研究的目的是建立新生猪AECⅡ分离、纯化及鉴定方法,为AECⅡ的生物学特性研究及与AECⅡ有关的在体动物实验研究奠定基础。目的 1、建立新生猪AECⅡ的分离、纯化及鉴定方法； 2、观察AECⅡ的体外生长变化特点,明确其体外实验的最佳时间。方法 1、取体重1000-1300 g足月新生猪肺,经气道灌入0.1%胰酶及不同浓度弹力蛋白酶与0.1%胰酶的混合酶溶液(40 u／ml elatase／0.1% trypsin、30 u／mlelatase/0.1% trypsin、20 u/ml elatase/0.1% trypsin) 37℃、20 min水浴消化,收集细胞、计数细胞产量及活力； 2、采用percoll非连续密度梯度离心及免疫粘附法纯化(即panning法)细胞,计数细胞产量及活力； 3、采用透射电镜法、碱性磷酸酯酶染色法鉴定AECⅡ； 4、将细胞以4x105 cells／cm2接种于组织培养板,用DMEM低糖培养基(含10%胎牛血清、100 u／ml青霉素、0.1 g／ml链霉素)进行培养,每24 h换液,观察细胞生长变化情况。 5、在相同的消化条件下：0.1%胰酶、37℃、20 min水浴消化条件下,对比研究免疫粘附法、percoll非连续密度梯度离心法纯化细胞后所获得大鼠、新生猪AECⅡ的产量、活力及纯度的差别。结果 1、新生猪肺组织细胞的消化分离：30 u／ml弹力蛋白酶／0.1%胰酶消化肺组织后细胞产量(5.35±0.54)x106,而20 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.16±0.94)x106,40 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.09±0.86)x106,0.1%胰酶消化细胞产量为(2.76±0.65)x10+,30 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量显著高于其他3组(P0.01)； 2、新生猪肺AECⅡ的纯化：免疫粘附法纯化后的细胞产量为(37.97±27.98)x106,percoll非连续密度梯度离心法纯化细胞的产量(11.07±10.59)x106,前者明显高于后者； 3、新生猪肺AECⅡ的培养：原代培养24 h,AECⅡ开始贴壁,细胞呈圆形、多角形,呈岛状分布。培养至48 h,细胞形态一致,为多角形。第3-4天细胞平展,连接成细胞单层,胞浆内有大量反差明显的小颗粒,细胞核明显。第5-7天,细胞内颗粒逐渐减少,胞浆空泡,细胞体积增大,边缘模糊。AECⅡ在原代培养24-96 h处于最佳状态,此阶段适合作体外实验研究。 4、在相同消化、分离及纯化方法条件下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量。大鼠AECⅡ的percoll法纯化细胞产量显著高于免疫黏附法。新生猪AECⅡ的纯化,免疫粘附法产量则明显高于percoll法。纯化后大鼠AECⅡ的阳性率近90%,而新生猪AECⅡ的阳性率仅为70%左右。结论 1、新生猪AECⅡ消化分离的最佳消化条件是：30 u／ml弹力蛋白酶／0.1%胰酶联合使用,37℃、20 min消化；免疫粘附法纯化新生猪AECⅡ优于percoll法；AKP染色法是简单、实用的AECⅡ鉴定方法,细胞染色阳性率与电镜鉴定结果一致,可用于新生猪AECⅡ的鉴定。 2、AECⅡ体外研究的最佳时间为原代培养的第24-96 h。 3、在消化条件及纯化方法相同的情况下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量,AECⅡ纯度可达70%左右,可用于进一步的体外实验研究。研究背景内皮祖细胞(Endothelial progenitor cells，EPC)是循环中骨髓来源的祖细胞之一，能分化为成熟血管内皮细胞，又称为血管内皮细胞的前体细胞。正常生理状态下，外周血中EPC数量很少，缺血、血管损伤等因素可以使外周血中EPC数量增加并参与了损伤血管的修复过程。对急性肺损伤(Acute lung injury,ALI)的研究表明，炎症反应抑制剂、炎症因子特异性阻断剂等治疗效果并不佳。目前干细胞治疗在组织修复和重建领域中备受关注，EPC很可能是极具前景的急性肺损伤治疗方法之一。本研究的目的是建立幼猪EPC分离培养方法，为EPC对急性肺损伤修复作用机制研究奠定基础。目的建立幼猪外周血EPC体外分离、纯化及鉴定方法，探讨其体外培养条件及生长变化特点。为进一步的EPC生物学特性研究及内皮祖细胞的急性肺损伤修复研究奠定基础。方法取幼猪外周血10-20 ml，用密度梯度离心法分离出血中单个核细胞，贴壁选择法纯化EPC，EGM-2MV培养基(含5％FBS，Hydrocortisone 0．4μl/ml，hFGF-B4μl／ml，VEGF 1 ttl／ml，IGF-11μl／ml，Ascorbic acidμl／ml，hEGF 1μl/ml，GA-10001μl/tl／m1)培养细胞，观察细胞生长变化特点。结果 1．使用淋巴细胞分离液(LTS 1110)，采用密度梯度离心法分离猪外周血单个核细胞的方法稳定。 2．幼猪外周血单个核细胞贴壁选择法纯化后培养观察：细胞贴壁较为缓慢，贴壁细胞大小较为均一、圆形为主。培养7天见较多长梭形细胞，形态相对单一、触角少。长梭形细胞可逐渐增多，，培养至3周左右时见长梭形细胞相互连接呈管腔样结构分布。 3。透射电镜观察管腔样结构分布细胞：可见细胞间有细胞连接。结论使用淋巴细胞分离液(LTS 1110)，密度梯度离心法分离幼猪外周血单个核细胞，贴壁选择纯化的方法，可用于与EPC有关的进一步的实验研究中。
[Abstract]:Research background
The alveolar epithelial cells (Alveolar epithelial type II cells, AEC II) are the progenitor cells of the alveolar epithelium, which can be transformed into type I alveolar epithelial cells to participate in the repair of lung injury, and to secrete the alveolar surfactant (Pulmonary surfactant, PS) to maintain the stability of the alveoli, prevent the alveoli from collapsing, maintain the lung compliance, and the apex of the alveolar epithelium. The membrane surface sodium, water transport to the interstitial lung, and participate in the lung to various inhalation of harmful substances and the natural immunity of microorganism,.AEC II only accounts for 15% of the lung cells. It is difficult to define the specific function of AEC II in the culture of lung tissue or lung mixed cells. At present, there is no cell line with all AEC II function. The primary culture of AEC II is the solution to this The important means of one problem. Most of the previous studies have been on the isolation and culture of adult rats, mice and rabbit AEC II. There are also a few reports of cell isolation and culture of newborn animals, but the yield of cells is low. In addition, the growth cycle and physiological and pathological process of small animals are short, and the characteristics of physiology and anatomy are not conducive to long-term animal research. The purpose of this study is to establish a new method of isolation, purification and identification of new pig AEC II, and to lay the foundation for the study of biological characteristics of AEC II and the study of AEC II related in vivo animal experiments.
objective
1, establish a new pig AEC II separation, purification and identification methods.
2, observe the growth and change characteristics of AEC II in vitro, and determine the best time for its in vitro experiment.
Method
1, the newborn pigs' lung of 1000-1300 g of weight was taken, 0.1% pancreatin and mixed enzyme solution of different concentration of elastase and 0.1% trypsin (40 U / ml elatase / 0.1% trypsin, 30 U / mlelatase/0.1% trypsin, 20 u/ml elatase/0.1% trypsin) were collected through the airway, and 20 min water bath was digested and the cells were collected to count the cell output and vitality.
2, Percoll discontinuous density gradient centrifugation and immune adherence method were used to purify (panning) cells to count cell production and viability.
3, AEC II was identified by transmission electron microscopy and alkaline phosphatase staining.
4, the cells were inoculated with 4x105 cells / cm2 on tissue culture plate and cultured with DMEM low sugar medium (including 10% fetal bovine serum, 100 U / ml penicillin and 0.1 g / ml streptomycin), and the changes of cell growth were observed every 24 h.
5, under the same digestion conditions: 0.1% pancreatin, 37 C and 20 min water bath, the difference in the yield, vitality and purity of AEC II of neonatal pigs was compared with the immune adherence method and the purification of cells by Percoll discontinuous density gradient centrifugation.
Result
1, the digestion and isolation of newborn pig lung tissue cells: the output of 30 U / ml elastase / 0.1% pancreatin digested lung tissue was (5.35 + 0.54) x106, while 20 U / ml elastase / 0.1% trypsin digestion cell production was (3.16 + 0.94) x106,40 U / ml elastase / 0.1% pancreatin / 0.1% pancreatin digestible cells (3.09 + 0.86) x106,0.1% pancreatin digestion Cell yield was (2.76 + 0.65) x10+, and 30 U / ml elastase / 0.1% trypsin digestion cell yield was significantly higher than that of the other 3 groups (P0.01).
2, the purification of AEC II in newborn pig lung: the yield of purified cells after immunization was (37.97 + 27.98) x106, and the yield of purified cells by Percoll discontinuous density gradient centrifugation was (11.07 + 10.59) x106, the former was significantly higher than that of the latter.
3, the culture of newborn pig lung AEC II: primary culture 24 h, AEC II began to adhere to the wall, cells were round, polygonal, island shaped distribution. Culture to 48 h, the shape of the cells is the same polygon. On the 3-4 day, the cells were flat, connected to cell monolayers, there were a large number of small particles in the cytoplasm, and the nuclei were obvious. On day 5-7, the particles gradually decreased, cells in cell gradually decreased, cells in the 5-7 day, cells gradually decreased, the particles gradually decreased in cell, day 5-7 day, cells gradually decreased, cells in cell gradually decreased on day 5-7, cell particles gradually, cell particles gradually decreased in cells on day 5-7, cell particles gradually, cells within cells on day 5-7, cell particles gradually decreased, cell particles gradually decreased in cell, day on day 5-7, cell particles gradually, cell particles gradually decreased in cell, on day 5-7, cell particles gradually, cell particles gradually decreased in cell The cytoplasm vacuoles, cell volume increased and edge blurred.AEC II was at the best state in primary culture 24-96 H. This stage is suitable for in vitro experimental research.
4, under the same digestion, separation and purification methods, the purified yield of AEC II in newborn pig lung was significantly higher than that of AEC II. The purified cell production of AEC II in rats was significantly higher than that of immune adhesion. The purification of AEC II in newborn pigs was higher than that of the Percoll method. The positive rate of AEC II in the purified rat was nearly 9. 0%, and the positive rate of new pig AEC II was only about 70%.
conclusion
1, the best digestion conditions for new pig AEC II digestion were: 30 U / ml elastase / 0.1% pancreatin combined use, 37 C and 20 min digestion; immuno adhesion method was superior to Percoll method for purification of newborn pig AEC II; AKP staining method was a simple and practical identification method of AEC II, and the positive rate of cell staining was consistent with that of electron microscope identification, and could be used in newborn pigs. Identification of AEC II.
2, the best time to study AEC II in vitro was 24-96 h. in primary culture.
3, under the same digestion conditions and the same purification methods, the purified yield of AEC II in newborn pig lung was significantly higher than that of AEC II purified from rat, and the purity of AEC II was about 70%, which could be used for further study in vitro.
Research background
Endothelial progenitor cells (EPC) is one of the progenitor cells of the circulating bone marrow, which can differentiate into mature vascular endothelial cells, also known as the precursor cells of vascular endothelial cells. Under normal physiological state, the number of EPC in peripheral blood is small, ischemia, and vascular damage can increase the number of EPC in peripheral blood and participate in the process. The study of the repair of damaged vessels. The study of Acute lung injury (ALI) shows that the effects of the inflammatory reaction inhibitors and inflammatory factor specific blockers are not good. At present, stem cell therapy has attracted much attention in the field of tissue repair and reconstruction. EPC is likely to be one of the most promising methods for the treatment of acute lung injury. The aim of the study is to establish a method for isolation and culture of EPC from young pigs, and lay a foundation for the study of EPC on the mechanism of acute lung injury repair.
objective
The methods of isolation, purification and identification of EPC in vitro from the peripheral blood of young pigs were established to explore the culture conditions and the characteristics of their growth in vitro, which lay the foundation for further research on the biological characteristics of EPC and the study of the repair of acute lung injury of endothelial progenitor cells.
Method
The peripheral blood of the young pigs was 10-20 ml, and the mononuclear cells were separated by density gradient centrifugation. The EPC, EGM-2MV medium (including 5%FBS, Hydrocortisone 0.4, l/ml, hFGF-B4 Mu L / ml, VEGF 1 TTL / ml) were cultured and cultured cells to observe the cell growth and change. Characteristics.
Result
1. using lymphocyte separation liquid (LTS 1110), the method of separating peripheral blood mononuclear cells from pigs with density gradient centrifugation is stable.
The adherent selection method of peripheral blood mononuclear cells of 2. young pigs showed that the cell adherence was relatively slow, the size of the adherent cells was more uniform and the circle was the main. The cells were more spindle cells for 7 days, and the form was relatively single.
Long spindle cells can be gradually increased. When cultured for about 3 weeks, the spindle cells are interconnected with lumen like structure.
3. the distribution of cells in the lumen like structure was observed by transmission electron microscope.
conclusion
Using the lymphocyte separation solution (LTS 1110), the density gradient centrifugation method is used to separate the peripheral blood mononuclear cells of young pigs, and the method of adherent selection and purification can be used in the further experimental study related to EPC.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】