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  本文關鍵詞：低能激光照射對豬心肌干細胞增殖及旁分泌作用的影響　出處：《天津醫(yī)科大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 心肌干細胞 低能激光照射 增殖 胰島素樣生長因子-1 促血管生成素-2

【摘要】：目的:心肌干細胞(cardiac stem cells,CSCs)是心梗后細胞再生修復療法的熱點種子細胞。低能激光照射(low-level laster irradiation,LLLI)已被證實能夠影響多種體外培養(yǎng)細胞的生長狀況及細胞因子的分泌,但目前尚缺乏LLLI對心肌干細胞影響的研究,本實驗旨在探討不同能量強度低能激光照射對體外培養(yǎng)的豬心肌干細胞增殖效率及旁分泌作用的影響。方法:選取成年巴馬小型豬,雌性,體重25-30kg。通過心臟外科手術經右側小切口鉗取右心耳組織,用組織塊培養(yǎng)法獲取原代心肌干細胞,以心肌干細胞培養(yǎng)液進行培養(yǎng),視細胞生長情況進行傳代。選取分選純化后的第3代細胞行流式檢測鑒定細胞表型:c-kit、CD34、CD45、CD31、CD90、Lineage;行Western Blot檢測心肌早期特異性轉錄因子Nkx2.5和GATA-4的表達;誘導劑誘導后行免疫熒光檢測CX43、c Tn T、α-SMA、v WF,以觀察心肌干細胞的多向分化潛能;并以不同能量密度(0 J/cm2,0.50 J/cm2,0.75 J/cm2,1.00 J/cm2,3.00 J/cm2,5.00J/cm2)的低強度激光(660nm)照射,檢測上清液中的乳酸脫氫酶(lactic dehydrogenase,LDH)含量以行細胞毒性分析;MTT呈色法繪制心肌干細胞增殖曲線;酶聯免疫吸附法(ELISA)測定上清液中胰島素樣生長因子-1(Insulin like Growth Factor-l,IGF-1)和促血管生成素-2(angiopoietin-2)的含量。結果:1.傳代分選后細胞流式細胞儀鑒定結果顯示,干細胞因子受體c-kit表達陽性,而成纖維細胞標志CD90、造血干細胞標志CD34、血源性標志CD45和Lineage以及內皮標志CD31表達陰性。Western Blot檢測結果顯示,心肌早期特異性轉錄因子Nkx2.5和GATA-4結合蛋白表達陽性。2.傳代分選后的細胞分別經5-氮雜胞苷、rh-PDGF-BB以及b FGF三種誘導劑誘導后,可分化成熟至表達心肌細胞特異的CX43和c Tn T蛋白,平滑肌細胞獨有的a-SMA蛋白,以及內皮細胞的v WF因子。3.波長660nm的低能激光在0.50-5.00 J/cm2能量密度下照射,對體外培養(yǎng)的心肌干細胞沒有明顯的細胞毒性。4.低能激光照射對CSCs的增殖速率有促進作用,最佳能量密度可能為0.75J/cm2;LLLI能夠促進心肌干細胞分泌IGF-1及angiopoietin-2,最適能量密度可能是3J/cm2。結論:1.自成年小型豬右心耳組織成功分離獲得c-kit+、CD31-、CD34-、CD45-、CD90-、Lineage-的心肌干細胞,并且具有多向分化潛能,能在體外實現穩(wěn)定擴增。2.波長660nm的低能激光照射劑量為0.50-5.00 J/cm2,可安全用于體外培養(yǎng)心肌干細胞的預處理。3.低能激光照射能夠提高體外培養(yǎng)的心肌干細胞的增殖速率,并且能夠促進心肌干細胞的分泌細胞活性因子,效果與照射劑量有關。因此,LLLI可能作為心肌干細胞的一種有效的預處理手段用于心肌梗死的干細胞再生療法。
[Abstract]:Objective: cardiac stem cells (cardiac stem cells, CSCs) is after myocardial cell regeneration therapy hot seed cells. Low energy laser irradiation (low-level laster, irradiation, LLLI) has been shown to influence a variety of in vitro growth and secretion of cytokines in cells, but there is still a lack of research on myocardial stem cells LLLI, this study aimed to investigate the effects of different energy intensity of low energy laser irradiation of stem cell proliferation efficiency and paracrine effects on cultured porcine myocardium. Methods: adult Bama miniature pig, female, weight 25-30kg. by heart surgery through right small incision forceps right atrial tissue obtained by stem cells in primary myocardial tissue in the culture medium containing cardiac stem cells, according to the growth of cells were selected. The purified cells of the third generation line flow type cell phenotype detection and identification: C-kit, CD34, CD45, CD31, CD90, Lineage; Western Blot expression for early detection of myocardial specific transcription factors Nkx2.5 and GATA-4 inducers; immunofluorescence was performed on CX43 C, Tn T, V WF, a -SMA, to observe the myocardial stem cell differentiation; and with different energy density (0 J/cm2,0.50 J/cm2,0.75 J/cm2,1.00 J/cm2,3.00 J/cm2,5.00J/cm2) low intensity laser irradiation (660nm), lactate dehydrogenase assay in the supernatant (lactic dehydrogenase, LDH) content for cytotoxicity analysis; MTT color rendering method of cardiac stem cell proliferation curve; enzyme linked immunosorbent assay (ELISA) were measured in the supernatants of insulin-like growth factor -1 (Insulin like Growth Factor-l, IGF-1) and angiopoietin -2 (angiopoietin-2) content. Results: the 1. passage cells after sorting the results of flow cytometry showed that stem cell factor receptor c-kit expression, and fiber Dimensional cell marker CD90, hematopoietic stem cell markers CD34, CD45 and Lineage as well as signs of blood borne endothelial marker CD31 negative.Western Blot test results showed that the cardiac specific transcription factor Nkx2.5 and GATA-4 binding protein positive expression of.2. were sorted cells were treated with 5- azacytidine, rh-PDGF-BB B and FGF three inducer after differentiation to mature expression of cardiomyocyte specific CX43 C and Tn T protein, a-SMA protein unique to smooth muscle cells, and endothelial cells of V WF factor.3. 660nm wavelength in the low-energy laser energy density 0.50-5.00 J/cm2 irradiation on cultured myocardial stem cell proliferation rate and cytotoxicity of.4. without obvious low power laser irradiation promoted CSCs, optimal energy density may be 0.75J/cm2; LLLI can promote myocardial stem cell secretion of IGF-1 and angiopoietin-2, the optimal energy density can be Conclusion: 3J/cm2. can be from 1. adult mini pigs right atrial tissue obtained from c-kit+, CD31-, CD34-, CD45-, CD90-, Lineage-, cardiac stem cells, and have the potential of multi-directional differentiation, in the low-energy laser irradiation dose in vitro to achieve stable amplification of.2. 660nm 0.50-5.00 J/cm2 wavelength, can be safely used in pre cardiac stem cells.3. low energy laser irradiation can increase the proliferation rate of stem cells in vitro cultured myocardial cultured in vitro, and can promote cardiac stem cells secreting cell active factor, the effect related to the dose of radiation. Therefore, LLLI may act as a cardiac stem cells is an effective pretreatment method for regeneration of stem cell therapy for myocardial infarction.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R542.22

【共引文獻】

相關期刊論文 前2條

1 崔曉軍;譚玉珍;王海杰;;促進移植干細胞存活、分化和修復壞死心肌的策略[J];基礎醫(yī)學與臨床;2009年01期

2 國海東;張貴燾;王海杰;王新艷;譚玉珍;;PI3-K/Akt信號通路對骨髓源性心肌干細胞分化的調控作用[J];解剖學報;2008年04期

相關博士學位論文 前4條

1 劉東寧;大鼠骨髓間充質干細胞向視網膜神經細胞誘導分化和體內移植研究[D];第三軍醫(yī)大學;2007年

2 崔曉軍;自聚肽納米纖維對骨髓源性心肌干細胞存活、分化和修復壞死心肌的作用[D];復旦大學;2009年

3 國海東;纖維蛋白膠和自聚肽納米纖維對骨髓源性心肌干細胞存活、分化和改善心功能的作用及其機制[D];復旦大學;2010年

4 孫敬和;心肌樣微環(huán)境下黃芪甲苷聯合5-氮胞苷誘導MSCs分化為心肌樣細胞[D];廣州中醫(yī)藥大學;2012年

相關碩士學位論文 前2條

1 李詠雪;生理性缺血訓練對冠心病患者循環(huán)血管內皮祖細胞的影響[D];南京醫(yī)科大學;2012年

2 艾旗;阿托伐他汀聯合人臍血單個核細胞靜脈移植對急性心梗心肌組織血管再生影響實驗研究[D];中南大學;2012年

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