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

  更多相關(guān)文章： 心肌干細(xì)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(xì)胞(cardiac stem cells,CSCs)是心梗后細(xì)胞再生修復(fù)療法的熱點(diǎn)種子細(xì)胞。低能激光照射(low-level laster irradiation,LLLI)已被證實(shí)能夠影響多種體外培養(yǎng)細(xì)胞的生長狀況及細(xì)胞因子的分泌,但目前尚缺乏LLLI對(duì)心肌干細(xì)胞影響的研究,本實(shí)驗(yàn)旨在探討不同能量強(qiáng)度低能激光照射對(duì)體外培養(yǎng)的豬心肌干細(xì)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(shù)經(jīng)右側(cè)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(xì)胞,以心肌干細(xì)胞培養(yǎng)液進(jìn)行培養(yǎng),視細(xì)胞生長情況進(jìn)行傳代。選取分選純化后的第3代細(xì)胞行流式檢測鑒定細(xì)胞表型:c-kit、CD34、CD45、CD31、CD90、Lineage;行Western Blot檢測心肌早期特異性轉(zhuǎn)錄因子Nkx2.5和GATA-4的表達(dá);誘導(dǎo)劑誘導(dǎo)后行免疫熒光檢測CX43、c Tn T、α-SMA、v WF,以觀察心肌干細(xì)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(qiáng)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(xì)胞毒性分析;MTT呈色法繪制心肌干細(xì)胞增殖曲線;酶聯(lián)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(jié)果:1.傳代分選后細(xì)胞流式細(xì)胞儀鑒定結(jié)果顯示,干細(xì)胞因子受體c-kit表達(dá)陽性,而成纖維細(xì)胞標(biāo)志CD90、造血干細(xì)胞標(biāo)志CD34、血源性標(biāo)志CD45和Lineage以及內(nèi)皮標(biāo)志CD31表達(dá)陰性。Western Blot檢測結(jié)果顯示,心肌早期特異性轉(zhuǎn)錄因子Nkx2.5和GATA-4結(jié)合蛋白表達(dá)陽性。2.傳代分選后的細(xì)胞分別經(jīng)5-氮雜胞苷、rh-PDGF-BB以及b FGF三種誘導(dǎo)劑誘導(dǎo)后,可分化成熟至表達(dá)心肌細(xì)胞特異的CX43和c Tn T蛋白,平滑肌細(xì)胞獨(dú)有的a-SMA蛋白,以及內(nèi)皮細(xì)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(duì)體外培養(yǎng)的心肌干細(xì)胞沒有明顯的細(xì)胞毒性。4.低能激光照射對(duì)CSCs的增殖速率有促進(jìn)作用,最佳能量密度可能為0.75J/cm2;LLLI能夠促進(jìn)心肌干細(xì)胞分泌IGF-1及angiopoietin-2,最適能量密度可能是3J/cm2。結(jié)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(xì)胞,并且具有多向分化潛能,能在體外實(shí)現(xiàn)穩(wěn)定擴(kuò)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(xì)胞的預(yù)處理。3.低能激光照射能夠提高體外培養(yǎng)的心肌干細(xì)胞的增殖速率,并且能夠促進(jìn)心肌干細(xì)胞的分泌細(xì)胞活性因子,效果與照射劑量有關(guān)。因此,LLLI可能作為心肌干細(xì)胞的一種有效的預(yù)處理手段用于心肌梗死的干細(xì)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.22

【共引文獻(xiàn)】

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