本文選題：肺泡 + 上皮細(xì)胞�。� 參考：《復(fù)旦大學(xué)》2009年碩士論文

【摘要】： 研究背景 肺泡Ⅱ型上皮細(xì)胞(Alveolar epithelial typeⅡcells, AECⅡ)是肺泡上皮的祖細(xì)胞,能夠變?yōu)棰裥头闻萆掀ぜ?xì)胞參與肺損傷修復(fù),分泌肺泡表面活性物質(zhì)(Pulmonary surfactant, PS)維持肺泡穩(wěn)定性、防止肺泡萎陷、保持肺的順應(yīng)性,能夠?qū)⒎闻萆掀ろ敹四っ娴拟c、水轉(zhuǎn)運(yùn)至肺間質(zhì),并參加了肺對(duì)各種吸入有害物質(zhì)及微生物的天然免疫等。AECⅡ僅占肺部細(xì)胞的15%,對(duì)肺組織或肺混合細(xì)胞的培養(yǎng)難以明確AECⅡ的具體功能。目前尚無(wú)具有全部AECⅡ功能的細(xì)胞系,AECⅡ的原代培養(yǎng)成為解決這一問題的重要手段。以往研究大多是對(duì)成年大鼠、小鼠和兔AECⅡ的分離培養(yǎng)研究,也有少量新生動(dòng)物細(xì)胞分離培養(yǎng)報(bào)道,但細(xì)胞產(chǎn)量均較低,此外由于小動(dòng)物生長(zhǎng)發(fā)育周期及生理病理過程短,生理及解剖特點(diǎn)也不利于長(zhǎng)期動(dòng)物實(shí)驗(yàn)研究,對(duì)于人新生兒至嬰幼兒期的生理和病理生理學(xué)研究適用性有限。本研究的目的是建立新生豬AECⅡ分離、純化及鑒定方法,為AECⅡ的生物學(xué)特性研究及與AECⅡ有關(guān)的在體動(dòng)物實(shí)驗(yàn)研究奠定基礎(chǔ)。 目的 1、建立新生豬AECⅡ的分離、純化及鑒定方法； 2、觀察AECⅡ的體外生長(zhǎng)變化特點(diǎn),明確其體外實(shí)驗(yàn)的最佳時(shí)間。 方法 1、取體重1000-1300 g足月新生豬肺,經(jīng)氣道灌入0.1%胰酶及不同濃度彈力蛋白酶與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,收集細(xì)胞、計(jì)數(shù)細(xì)胞產(chǎn)量及活力； 2、采用percoll非連續(xù)密度梯度離心及免疫粘附法純化(即panning法)細(xì)胞,計(jì)數(shù)細(xì)胞產(chǎn)量及活力； 3、采用透射電鏡法、堿性磷酸酯酶染色法鑒定AECⅡ； 4、將細(xì)胞以4x105 cells／cm2接種于組織培養(yǎng)板,用DMEM低糖培養(yǎng)基(含10%胎牛血清、100 u／ml青霉素、0.1 g／ml鏈霉素)進(jìn)行培養(yǎng),每24 h換液,觀察細(xì)胞生長(zhǎng)變化情況。 5、在相同的消化條件下：0.1%胰酶、37℃、20 min水浴消化條件下,對(duì)比研究免疫粘附法、percoll非連續(xù)密度梯度離心法純化細(xì)胞后所獲得大鼠、新生豬AECⅡ的產(chǎn)量、活力及純度的差別。 結(jié)果 1、新生豬肺組織細(xì)胞的消化分離：30 u／ml彈力蛋白酶／0.1%胰酶消化肺組織后細(xì)胞產(chǎn)量(5.35±0.54)x106,而20 u／ml彈力蛋白酶／0.1%胰酶消化細(xì)胞產(chǎn)量為(3.16±0.94)x106,40 u／ml彈力蛋白酶／0.1%胰酶消化細(xì)胞產(chǎn)量為(3.09±0.86)x106,0.1%胰酶消化細(xì)胞產(chǎn)量為(2.76±0.65)x10+,30 u／ml彈力蛋白酶／0.1%胰酶消化細(xì)胞產(chǎn)量顯著高于其他3組(P0.01)； 2、新生豬肺AECⅡ的純化：免疫粘附法純化后的細(xì)胞產(chǎn)量為(37.97±27.98)x106,percoll非連續(xù)密度梯度離心法純化細(xì)胞的產(chǎn)量(11.07±10.59)x106,前者明顯高于后者； 3、新生豬肺AECⅡ的培養(yǎng)：原代培養(yǎng)24 h,AECⅡ開始貼壁,細(xì)胞呈圓形、多角形,呈島狀分布。培養(yǎng)至48 h,細(xì)胞形態(tài)一致,為多角形。第3-4天細(xì)胞平展,連接成細(xì)胞單層,胞漿內(nèi)有大量反差明顯的小顆粒,細(xì)胞核明顯。第5-7天,細(xì)胞內(nèi)顆粒逐漸減少,胞漿空泡,細(xì)胞體積增大,邊緣模糊。AECⅡ在原代培養(yǎng)24-96 h處于最佳狀態(tài),此階段適合作體外實(shí)驗(yàn)研究。 4、在相同消化、分離及純化方法條件下,新生豬肺AECⅡ的純化產(chǎn)量明顯高于大鼠AECⅡ純化產(chǎn)量。大鼠AECⅡ的percoll法純化細(xì)胞產(chǎn)量顯著高于免疫黏附法。新生豬AECⅡ的純化,免疫粘附法產(chǎn)量則明顯高于percoll法。純化后大鼠AECⅡ的陽(yáng)性率近90%,而新生豬AECⅡ的陽(yáng)性率僅為70%左右。 結(jié)論 1、新生豬AECⅡ消化分離的最佳消化條件是：30 u／ml彈力蛋白酶／0.1%胰酶聯(lián)合使用,37℃、20 min消化；免疫粘附法純化新生豬AECⅡ優(yōu)于percoll法；AKP染色法是簡(jiǎn)單、實(shí)用的AECⅡ鑒定方法,細(xì)胞染色陽(yáng)性率與電鏡鑒定結(jié)果一致,可用于新生豬AECⅡ的鑒定。 2、AECⅡ體外研究的最佳時(shí)間為原代培養(yǎng)的第24-96 h。 3、在消化條件及純化方法相同的情況下,新生豬肺AECⅡ的純化產(chǎn)量明顯高于大鼠AECⅡ純化產(chǎn)量,AECⅡ純度可達(dá)70%左右,可用于進(jìn)一步的體外實(shí)驗(yàn)研究。 研究背景 內(nèi)皮祖細(xì)胞(Endothelial progenitor cells，EPC)是循環(huán)中骨髓來源的祖細(xì)胞之一，能分化為成熟血管內(nèi)皮細(xì)胞，又稱為血管內(nèi)皮細(xì)胞的前體細(xì)胞。正常生理狀態(tài)下，外周血中EPC數(shù)量很少，缺血、血管損傷等因素可以使外周血中EPC數(shù)量增加并參與了損傷血管的修復(fù)過程。對(duì)急性肺損傷(Acute lung injury,ALI)的研究表明，炎癥反應(yīng)抑制劑、炎癥因子特異性阻斷劑等治療效果并不佳。目前干細(xì)胞治療在組織修復(fù)和重建領(lǐng)域中備受關(guān)注，EPC很可能是極具前景的急性肺損傷治療方法之一。本研究的目的是建立幼豬EPC分離培養(yǎng)方法，為EPC對(duì)急性肺損傷修復(fù)作用機(jī)制研究奠定基礎(chǔ)。 目的 建立幼豬外周血EPC體外分離、純化及鑒定方法，探討其體外培養(yǎng)條件及生長(zhǎng)變化特點(diǎn)。為進(jìn)一步的EPC生物學(xué)特性研究及內(nèi)皮祖細(xì)胞的急性肺損傷修復(fù)研究奠定基礎(chǔ)。 方法 取幼豬外周血10-20 ml，用密度梯度離心法分離出血中單個(gè)核細(xì)胞，貼壁選擇法純化EPC，EGM-2MV培養(yǎng)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)培養(yǎng)細(xì)胞，觀察細(xì)胞生長(zhǎng)變化特點(diǎn)。 結(jié)果 1．使用淋巴細(xì)胞分離液(LTS 1110)，采用密度梯度離心法分離豬外周血單個(gè)核細(xì)胞的方法穩(wěn)定。 2．幼豬外周血單個(gè)核細(xì)胞貼壁選擇法純化后培養(yǎng)觀察：細(xì)胞貼壁較為緩慢，貼壁細(xì)胞大小較為均一、圓形為主。培養(yǎng)7天見較多長(zhǎng)梭形細(xì)胞，形態(tài)相對(duì)單 一、觸角少。長(zhǎng)梭形細(xì)胞可逐漸增多，，培養(yǎng)至3周左右時(shí)見長(zhǎng)梭形細(xì)胞相互連接呈管腔樣結(jié)構(gòu)分布。 3。透射電鏡觀察管腔樣結(jié)構(gòu)分布細(xì)胞：可見細(xì)胞間有細(xì)胞連接。 結(jié)論 使用淋巴細(xì)胞分離液(LTS 1110)，密度梯度離心法分離幼豬外周血單個(gè)核細(xì)胞，貼壁選擇純化的方法，可用于與EPC有關(guān)的進(jìn)一步的實(shí)驗(yàn)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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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本文編號(hào)：1832669