腸道缺血再灌注損傷對(duì)骨髓間充質(zhì)干細(xì)胞旁分泌作用的影響
發(fā)布時(shí)間：2018-12-11 19:18
【摘要】：腸道的缺血再灌注損傷在臨床當(dāng)中日益得到重視。目前認(rèn)為,在氧自由基、中性粒細(xì)胞、炎癥因子、補(bǔ)體等多種因素的的共同作用下,腸道成為在缺血再灌注損傷中極易受到傷害的器官。嚴(yán)重的腸道的損傷可導(dǎo)致導(dǎo)致全身炎癥反應(yīng)綜合征(MIRS)和多器官功能不全綜合征(MODS)。而對(duì)于腸道缺血再灌注損傷的治療,目前仍然缺乏有效的治療手段。 骨髓來(lái)源的間充質(zhì)干細(xì)胞(bone marrow-derived mesenchymal stem cells,BM-MSCs)是一種存在于骨髓內(nèi)的具有多向分化潛能的成體干細(xì)胞。由于BM-MSCs具有易于獲得、多向分化、高度增殖的特點(diǎn),成為近年來(lái)器官損傷修復(fù)的重要手段。在動(dòng)物試驗(yàn)已經(jīng)證實(shí)同種異體BM-MSCs移植可定植于胃腸道,并參與腸道損傷的修復(fù)。對(duì)于BM-MSCs參與受損組織修復(fù)的具體的機(jī)制,目前認(rèn)為有直接分化和旁分泌兩種方式,但仍缺乏進(jìn)一步的研究證實(shí)。本研究試圖模擬腸道缺血再灌注損傷的病理生理環(huán)境,觀察腸道的缺血再灌注刺激對(duì)BM-MSCs旁分泌的影響,以證實(shí)BM-MSCs在腸道修復(fù)中的旁分泌作用。 研究目的 1、完成Wistar雄性大鼠的骨髓間充質(zhì)干細(xì)胞分離、培養(yǎng)、鑒定、染色工作。 2、探討并制備腸道缺血再灌注大鼠模型,證實(shí)其腸道損傷及功能障礙,為后續(xù)的治療和檢測(cè)奠定基礎(chǔ)。 3、觀察腸道的缺血再灌注損傷刺激對(duì)BM-MSCs旁分泌作用的影響,以證實(shí)BM-MSCs在腸道修復(fù)中的旁分泌作用。 研究方法 1、分離、培養(yǎng)、鑒定、染色Wistar雄性大鼠的骨髓間充質(zhì)干細(xì)胞。 2、用腸系膜上動(dòng)脈夾閉的方式制備大鼠腸道缺血再灌注模型,并根據(jù)夾閉時(shí)間的不同分為20分鐘組,30分鐘組,40分鐘組和50分鐘組,并檢測(cè)損傷后腸道的LPS和腸道損傷指數(shù),以尋找最合適的夾閉時(shí)間。 3、分別提取腸道缺血再灌注模型大鼠、正常大鼠的腸上皮粘膜提取物,與BM-MSCs共同培養(yǎng); 4、分別在培養(yǎng)的第0天、第1天、第3天和第5天留取條件培養(yǎng)基,ELISA檢測(cè)不同細(xì)胞條件培養(yǎng)基以及普通培養(yǎng)基中TGF-α、VEGF、FGF、IGF-1的含量。 結(jié)果 1、全骨髓培養(yǎng)法得到的貼壁細(xì)胞完全符合骨髓間充質(zhì)干細(xì)胞的特性,經(jīng)鑒定為BM-MSCs; 2、在制備大鼠腸道缺血再灌注模型的過(guò)程中,夾閉40分鐘組和夾閉50分鐘組死亡率過(guò)高,而夾閉20分鐘腸道損傷程度不能達(dá)標(biāo),只有夾閉30分鐘既可以控制死亡率又能夠保證損傷達(dá)標(biāo); 3、四種生長(zhǎng)因子在含有腸上皮提取物的條件培養(yǎng)基中的含量均明顯高于普通培養(yǎng)基。其中含有缺血再灌注腸上皮提取物的培養(yǎng)基TGF-α、VEGF、FGF的含量明顯高于含有正常腸上皮提取物的培養(yǎng)基。VEGF含量的高峰時(shí)間出現(xiàn)在第三天,而FGF、IGF-1含量的高峰時(shí)間出現(xiàn)在第五天,TGF-α含量在第1-5天未見(jiàn)到明顯差異。 結(jié)論 1、全骨髓培養(yǎng)法是簡(jiǎn)便、可靠的BM-MSCs培養(yǎng)方法,其獲得BM-MSCs穩(wěn)定可靠; 2、在制備大鼠腸道缺血再灌注模型的過(guò)程中,腸系膜上動(dòng)脈夾閉的時(shí)間控制在30分鐘左右是滿足實(shí)驗(yàn)需要的方法。 3、BM-MSCs可以旁分泌TGF-α、VEGF、FGF、IGF-1等生長(zhǎng)因子;而在缺血再灌注損傷的微環(huán)境下,BM-MSCs的分泌功能得到增強(qiáng),TGF-α、VEGF兩種生長(zhǎng)因子的峰值出現(xiàn)在第3天,而FGF、IGF-1兩種生長(zhǎng)因子的水平呈時(shí)間依賴性。
[Abstract]:The intestinal ischemia-reperfusion injury is becoming more and more important in the clinic. It is believed that the intestinal tract is the most vulnerable organ of the ischemia-reperfusion injury under the co-action of various factors such as oxygen free radical, neutrophil, inflammation factor, complement and the like. Severe intestinal injury can lead to systemic inflammatory response syndrome (MIRS) and multiple organ dysfunction syndrome (MODS). For the treatment of intestinal ischemia-reperfusion injury, there is still a lack of effective means of treatment. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a kind of body with multi-directional differentiation potential in the bone marrow. Because BM-MSCs have the characteristics of easy to obtain, multi-directional differentiation and high proliferation, it becomes an important hand in organ damage repair in recent years. Section. Allogeneic BM-MSCs transplantation has been demonstrated in animal testing to be colonized in the gastrointestinal tract and participate in the repair of intestinal injury Complex. For BM-MSCs involved in the repair of damaged tissue, there are two ways of direct differentiation and paracrine, but there is still a lack of further study. Objective: To study the pathological and physiological environment of intestinal ischemia-reperfusion injury, and to observe the effect of ischemia-reperfusion on the secretion of BM-MSCs on the side of BM-MSCs in order to confirm the paracrine secretion of BM-MSCs in the intestinal repair. for use. Objective: To study the isolation, culture and identification of bone marrow mesenchymal stem cells in Wistar male rats. and to study and prepare the rat model of intestinal ischemia-reperfusion, to confirm the intestinal injury and dysfunction and to be a follow-up treatment. and the effect of the injury of the intestinal ischemia-reperfusion injury on the paracrine function of BM-MSCs was observed to confirm that BM-MSCs were in the intestinal tract. repair paracrine effect. Study method 1, isolation, culture, identification, staining of Wistar rats Bone marrow mesenchymal stem cells were prepared in male rats. The rat intestinal ischemia-reperfusion model was prepared by means of the superior mesenteric artery occlusion, and the rats were divided into 20 minutes, 30 minutes, 40 minutes and 50 minutes according to the time of the occlusion, and the LPS and the intestinal loss in the intestinal tract after injury were detected. the injury index was used to find the most suitable clipping time. 3, the intestinal epithelium of the normal rats was adhered to the rat of the intestinal ischemia-reperfusion model, respectively. membrane extract, co-cultured with BM-MSCs; 4, respectively at day 0 of culture on day 1, day 3 and day 5, conditioned medium was taken, and the culture medium of different cells and TGF-in the normal medium were detected by ELISA. in that case of a man, The content of VEGF, FGF, IGF-1 was 1. The adherent cells obtained by the method of full bone marrow culture were in full compliance. The characteristics of bone marrow mesenchymal stem cells were identified as BM-MSCs; 2. In the preparation of rat intestinal ischemia-reperfusion model in that course of the procedure, the mortality rate of the 40-minute group and the clamp-close 50-minute group was too high, while the degree of intestinal injury of the clip was not up to the standard for 20 minutes, only 30 minutes of the clamp can control the death rate and ensure the damage to the standard; and 3, the four growth factors are The content of the condition culture medium with the intestinal epithelial extract is obviously higher than that of the normal culture medium, wherein the culture medium TGF-1, VE containing the ischemia-reperfusion intestinal epithelial extract The content of GF and FGF was significantly higher than that of the medium containing the normal intestinal epithelium extract. The peak time of the content of VEGF appeared on the third day, while the peak time of the content of FGF and IGF-1 appear in that fifth day, the TGF-1 content did not see a significant difference in day 1-5. conclusion 1, the full bone marrow culture method was simple and the method for culturing the BM-MSCs obtained by the method has the advantages that the BM-MSCs are stable and reliable, and 2, after the rat intestinal ischemia-reperfusion model is prepared, In the course, the time control of the superior mesenteric artery occlusion is about 30 minutes to meet the experimental requirements. 3, BM-MSCs can secrete the growth factors such as TGF-1, VEGF, FGF, IGF-1, and the secretion function of BM-MSCs under the microenvironment of ischemia-reperfusion injury. the growth factors of the two growth factors of the enhanced, TGF-1 and VEGF are enhanced,
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：2373086
[Abstract]:The intestinal ischemia-reperfusion injury is becoming more and more important in the clinic. It is believed that the intestinal tract is the most vulnerable organ of the ischemia-reperfusion injury under the co-action of various factors such as oxygen free radical, neutrophil, inflammation factor, complement and the like. Severe intestinal injury can lead to systemic inflammatory response syndrome (MIRS) and multiple organ dysfunction syndrome (MODS). For the treatment of intestinal ischemia-reperfusion injury, there is still a lack of effective means of treatment. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a kind of body with multi-directional differentiation potential in the bone marrow. Because BM-MSCs have the characteristics of easy to obtain, multi-directional differentiation and high proliferation, it becomes an important hand in organ damage repair in recent years. Section. Allogeneic BM-MSCs transplantation has been demonstrated in animal testing to be colonized in the gastrointestinal tract and participate in the repair of intestinal injury Complex. For BM-MSCs involved in the repair of damaged tissue, there are two ways of direct differentiation and paracrine, but there is still a lack of further study. Objective: To study the pathological and physiological environment of intestinal ischemia-reperfusion injury, and to observe the effect of ischemia-reperfusion on the secretion of BM-MSCs on the side of BM-MSCs in order to confirm the paracrine secretion of BM-MSCs in the intestinal repair. for use. Objective: To study the isolation, culture and identification of bone marrow mesenchymal stem cells in Wistar male rats. and to study and prepare the rat model of intestinal ischemia-reperfusion, to confirm the intestinal injury and dysfunction and to be a follow-up treatment. and the effect of the injury of the intestinal ischemia-reperfusion injury on the paracrine function of BM-MSCs was observed to confirm that BM-MSCs were in the intestinal tract. repair paracrine effect. Study method 1, isolation, culture, identification, staining of Wistar rats Bone marrow mesenchymal stem cells were prepared in male rats. The rat intestinal ischemia-reperfusion model was prepared by means of the superior mesenteric artery occlusion, and the rats were divided into 20 minutes, 30 minutes, 40 minutes and 50 minutes according to the time of the occlusion, and the LPS and the intestinal loss in the intestinal tract after injury were detected. the injury index was used to find the most suitable clipping time. 3, the intestinal epithelium of the normal rats was adhered to the rat of the intestinal ischemia-reperfusion model, respectively. membrane extract, co-cultured with BM-MSCs; 4, respectively at day 0 of culture on day 1, day 3 and day 5, conditioned medium was taken, and the culture medium of different cells and TGF-in the normal medium were detected by ELISA. in that case of a man, The content of VEGF, FGF, IGF-1 was 1. The adherent cells obtained by the method of full bone marrow culture were in full compliance. The characteristics of bone marrow mesenchymal stem cells were identified as BM-MSCs; 2. In the preparation of rat intestinal ischemia-reperfusion model in that course of the procedure, the mortality rate of the 40-minute group and the clamp-close 50-minute group was too high, while the degree of intestinal injury of the clip was not up to the standard for 20 minutes, only 30 minutes of the clamp can control the death rate and ensure the damage to the standard; and 3, the four growth factors are The content of the condition culture medium with the intestinal epithelial extract is obviously higher than that of the normal culture medium, wherein the culture medium TGF-1, VE containing the ischemia-reperfusion intestinal epithelial extract The content of GF and FGF was significantly higher than that of the medium containing the normal intestinal epithelium extract. The peak time of the content of VEGF appeared on the third day, while the peak time of the content of FGF and IGF-1 appear in that fifth day, the TGF-1 content did not see a significant difference in day 1-5. conclusion 1, the full bone marrow culture method was simple and the method for culturing the BM-MSCs obtained by the method has the advantages that the BM-MSCs are stable and reliable, and 2, after the rat intestinal ischemia-reperfusion model is prepared, In the course, the time control of the superior mesenteric artery occlusion is about 30 minutes to meet the experimental requirements. 3, BM-MSCs can secrete the growth factors such as TGF-1, VEGF, FGF, IGF-1, and the secretion function of BM-MSCs under the microenvironment of ischemia-reperfusion injury. the growth factors of the two growth factors of the enhanced, TGF-1 and VEGF are enhanced,
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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