低能激光照射对猪心肌干细胞增殖及旁分泌作用的影响

发布时间：2018-01-15 08:01

本文关键词：低能激光照射对猪心肌干细胞增殖及旁分泌作用的影响　出处：《天津医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:心肌干细胞(cardiac stem cells,CSCs)是心梗后细胞再生修复疗法的热点种子细胞。低能激光照射(low-level laster irradiation,LLLI)已被证实能够影响多种体外培养细胞的生长状况及细胞因子的分泌,但目前尚缺乏LLLI对心肌干细胞影响的研究,本实验旨在探讨不同能量强度低能激光照射对体外培养的猪心肌干细胞增殖效率及旁分泌作用的影响。方法:选取成年巴马小型猪,雌性,体重25-30kg。通过心脏外科手术经右侧小切口钳取右心耳组织,用组织块培养法获取原代心肌干细胞,以心肌干细胞培养液进行培养,视细胞生长情况进行传代。选取分选纯化后的第3代细胞行流式检测鉴定细胞表型:c-kit、CD34、CD45、CD31、CD90、Lineage;行Western Blot检测心肌早期特异性转录因子Nkx2.5和GATA-4的表达;诱导剂诱导后行免疫荧光检测CX43、c Tn T、α-SMA、v WF,以观察心肌干细胞的多向分化潜能;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)的低强度激光(660nm)照射,检测上清液中的乳酸脱氢酶(lactic dehydrogenase,LDH)含量以行细胞毒性分析;MTT呈色法绘制心肌干细胞增殖曲线;酶联免疫吸附法(ELISA)测定上清液中胰岛素样生长因子-1(Insulin like Growth Factor-l,IGF-1)和促血管生成素-2(angiopoietin-2)的含量。结果:1.传代分选后细胞流式细胞仪鉴定结果显示,干细胞因子受体c-kit表达阳性,而成纤维细胞标志CD90、造血干细胞标志CD34、血源性标志CD45和Lineage以及内皮标志CD31表达阴性。Western Blot检测结果显示,心肌早期特异性转录因子Nkx2.5和GATA-4结合蛋白表达阳性。2.传代分选后的细胞分别经5-氮杂胞苷、rh-PDGF-BB以及b FGF三种诱导剂诱导后,可分化成熟至表达心肌细胞特异的CX43和c Tn T蛋白,平滑肌细胞独有的a-SMA蛋白,以及内皮细胞的v WF因子。3.波长660nm的低能激光在0.50-5.00 J/cm2能量密度下照射,对体外培养的心肌干细胞没有明显的细胞毒性。4.低能激光照射对CSCs的增殖速率有促进作用,最佳能量密度可能为0.75J/cm2;LLLI能够促进心肌干细胞分泌IGF-1及angiopoietin-2,最适能量密度可能是3J/cm2。结论:1.自成年小型猪右心耳组织成功分离获得c-kit+、CD31-、CD34-、CD45-、CD90-、Lineage-的心肌干细胞,并且具有多向分化潜能,能在体外实现稳定扩增。2.波长660nm的低能激光照射剂量为0.50-5.00 J/cm2,可安全用于体外培养心肌干细胞的预处理。3.低能激光照射能够提高体外培养的心肌干细胞的增殖速率,并且能够促进心肌干细胞的分泌细胞活性因子,效果与照射剂量有关。因此,LLLI可能作为心肌干细胞的一种有效的预处理手段用于心肌梗死的干细胞再生疗法。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.22

【共引文献】