【作者】 杨丽华；

【导师】 李肖蓉； 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2008， 硕士

【摘要】 目的:骨髓间充质干细胞(Bone marrow mesenchyme stem cell,BMSCs)来源于中胚层,具有自我更新和多向分化潜能,可以体外分离培养,经一定条件诱导,可以向心肌细胞方向分化。5-氮胞苷(5-azacytidine,5-aza)诱导后,可使得某些基因的肌源性决定部位去甲基化,转录激活,引起细胞向肌源性细胞分化。成纤维细胞生长因子-2(FGF-2)是一种有丝分裂原,具有促进细胞分裂增殖的效应。据报道,此因子还具有心脏保护作用,可以诱导心肌肥大,并且可以提高心肌细胞在缺氧条件下的存活率。超声介导的白蛋白微泡破裂可以促进DNA分子穿透细胞膜和核膜并整合入细胞基因组中。本研究目的:(1)体外克隆FGF-2基因,重组并构建其真核表达载体;(2)探讨BMSCs体外分离培养并诱导其向心肌细胞分化方法;(3)探讨超声介导白蛋白微泡破裂促进FGF-2转移BMSCs的方法,并和脂质体(lipsome2000)介导转移BMSCs相比较。方法:(1)采用全骨髓培养法和Ficoll分离法体外分离培养大鼠BMSCs,观察其生长特性,表面抗原表达等,HE染色观察BMSCs细胞形态,胞核和胞浆,MTT法分别测定诱导和未诱导BMSCs的生长曲线。在第4代加入10gmol/L 5-aza预诱导BMSCs24小时(hr),第7代再一次加入10pμmol/L 5-aza诱导24hr;流式细胞仪技术、免疫荧光及RT-PCR实验对诱导前后的BMSCs进行表型鉴定;(2)分离培养新生大鼠心室肌细胞,观察其体外生长的形态、特性,以及抗原表达等特征:(3)收集体外培养的心室肌细胞,Trizol法提取心室肌细胞总mRNA,RT-PCR克隆扩增大鼠FGF-2基因,与PIRES2-EGFP载体连接,构建重组PIRES2-EGFP-FGF-2真核表达载体;(4)调整超声强度和作用时间,寻找最佳作用条件,在超声介导白蛋白微泡破裂作用下促进FGF-2基因转移BMSCs,24hr后荧光显微镜下观察EGFP表达情况;(5)在一定剂量的lipsome2000作用下,促进FGF-2基因转移BMSCs:(6)台盼蓝染色法测定不同超声强度不同作用时间之后BMSCs的活性。(7)流式细胞仪技术检测超声组和脂质体组转移FGF-2之后报告基因EGFP的表达。结果:(1)由新生大鼠心室肌组织分离培养得到心室肌细胞呈簇样生长并重叠成致密有边界的细胞团,培养10天左右,倒置相差显微镜下可见成团的心室肌细胞出现节律性收缩,收缩时间长达半个月之久。(2)从心室肌细胞中扩增FGF-2基因,构建重组质粒PIRES2-EGFP-FGF-2,测序正确的阳性克隆保种备用。(3)由骨髓分离得到的BMSCs体外培养呈长梭形贴壁生长,部分造血细胞悬浮在培养基中,MTT生长曲线显示,细胞在接种后第2天即进入对数生长期,细胞增殖活跃,倒置相差显微镜下观察细胞向外周伸展,两端有轴状突起,细胞呈长梭形,有双核分裂相的BMSCs多见,胞核较圆,核质丰富,核仁可见。随着细胞生长密度增大,彼此相连;7天可铺满瓶底,生长迅速,可大量繁殖,之后如不传代,则有老化现象。12天左右,BMSCs生长缓慢,进入平台期。(4)运用免疫荧光和流式细胞仪技术检测显示该群细胞高表达CD44(99.3%),CD54(97.7%),CD90(99.4%),CD71(98.2%),CD106(25.8%)表达相对较低,低表达CD45(8.9%),CD31(15.9%)等造血细胞表面标志。BMSCs经传代之后,细胞形态比较均一,生长速度较快,平均3-4d可铺满瓶底,细胞接种密度较大,接触抑制比较明显。第10代BMSCs HE染色,细胞仍呈长梭形,两端有轴状突起,胞浆丰富深染成红色,胞核圆形或椭圆形,呈蓝色,核仁明显。(5)BMSCs经5-aza诱导5天后,倒置相差显微镜下观察,细胞呈集落样生长,体积变大变圆,大小不等,核质丰富深染,比较粗糙,胞核较圆,核仁清楚,多分裂相,增殖能力强,细胞之间互相交联,紧密连接。免疫荧光染色后发现经5-aza诱导后的BMSCs可表达心肌细胞特异性分子肌球蛋白重链(MHC-β)、连接蛋白43(connexin43)、结蛋白(desmin)以及横纹肌肌动蛋白(actinin-α);而部分经诱导的细胞也表达血管内皮细胞特异性分子(Flk-1/VEGFR2/KDR)。经RT-PCR检测,诱导的BMSCs表达心肌细胞早期发育基因NKX2.5和GATA4。(6)FGF-2基因在超声介导白蛋白微泡破裂作用下转移到BMSCs。荧光显微镜下可看到绿色荧光,细胞表达EGFP;lipsome2000介导的FGF-2基因转移大鼠BMSCs,24hr后荧光显微镜下观察,可见到绿色荧光,细胞表达EGFP,FGF-2基因转移BMSCs中。(7)不同超声强度和作用时间对于BMSCs活力影响不同,通过测定,在不超过0.75W/cm~2的超声强度,32sec的作用时间之内,对于细胞都是安全的。0.75W/cm~2为最好转移强度,32sec为最好转移时间。(8)FGF-2转移BMSCs 48hr后进行流式细胞仪技术测定报告基因EGFP的表达,脂质体组报告基因EGFP表达率(49.35a±9.34)%,明显高于超声组(30.42±4.58)%,有统计学差异(P＜0.05);脂质体组转移效率高于超声组。结论:(1)采用组织块培养法,可体外成功分离培养新生Sprague-Dawley(SD)大鼠心室肌细胞,采用免疫荧光方法,可对表面抗原进行鉴定。(2)收集SD大鼠心室肌细胞,Trizol法抽提RNA,依照NCBI Genebank中大鼠FGF-2基因序列,设计适当引物,可克隆出大鼠FGF-2基因,并成功构建了其真核表达载体。(3)一定浓度的5-aza可诱导BMSCs向心肌细胞分化。(4)不同超声强度、不同作用时间对于BMSCs活力影响不同。(5)脂质体和超声介导的白蛋白微泡破裂均可以促进FGF-2转移BMSCs。(6)超声介导白蛋白微泡破裂可促进FGF-2转移BMSCs,为非病毒载体进行基因转移提供了新的方法,但转移效率有待进一步提高。更多还原

【Abstract】 Objective:Bone marrow mesenchymal stem cells(BMSCs)are derived from mesoderm. BMSCs have the ability of differentiation into a variety of tissues.They can be isolated and cultured in vitro,and can differentiate into myocardial cells under some special environments.Induction of 5-azacytidine(5-aza)can cause demethylation of muscle-derived genetic locus,transcription activation and cells differentiation into muscle-derived cells.Fibroblast growth factor-2(FGF-2)is a kind of mitogen.It has the effect of promoting cell division and proliferation.It is reported that FGF-2 can protect heart by inducing myocardial hypertrophy and increasing survival rate of myocardial cells under anoxia condition.Ultrasound-mediated albumin microbubble destruction can promote exogenous gene transfer into genome of eukaryotic cells.In present study,our objectives are as follows:(1)to clone rat FGF-2 gene in vitro and construct its’ eukaryotic expression vector.(2)to investigate the method of fractional cultivation of BMSCs and induce its differentiation into myocardial cells.(3)to investigate transfer of FGF-2 into BMSCs in vitro by ultrasound-mediated albumin microbubble destruction method and compare with the lipsome2000 method.Methods:(1)From Sprague-Dawley(SD)rats’ bone marrow,BMSCs were isolated and cultured in vitro successfully,their growth characteristic and phenotypes were observed. Nucleus,endochylema and cell morphology of BMSCs were observed with HE staining. Growth curves of induced and non-induced BMSCs were detected by MTT method. BMSCs were induced for 24hr by 5-aza(10μmol/L)during primary culture in generation 4 and 7.The phenotypes of BMSCs induced or not were identified by flow cytometry, immunofluorescence and reverse transcription-polymerase chain raction(RT-PCR).(2) Ventricular myocardial cells were isolated and cultured from neonate SD rat,Growth characteristic and phenos were observed.(3)Ventricular myocardial cells were collected, Total mRNA were extracted through Trizol method.The complementary gene encoding rat FGF-2 was amplified by RT-PCR,and then the target gene fragment was inserted into vector PIRES2-EGFP after being digested with EcoR I and BamH I.Eukaryotic expression vectors was constructed at last.(4)Intensity and time of ultrasound were regulated to confirm the best parameters.The plasmid DNA was mixed gently with microbubbles,and then the mixture were transfered into BMSCs by ultrasound-mediated albumin microbubble destruction method.The green fluorescence was observed under inverted phase contrast Microscope after 24hr,It shows that Green Fluorescence Protein(GFP)was expressed by transfered BMSCs.(5)Lipsome2000 could promote FGF-2 transfer into BMSCs at some does.(6)Cell activity was detected by trypan blue staining after different ultrasound intensity and time.(7)Expression of EGFP was detected by flow cytometry after transferring BMSCs with FGF-2 24hr later.Results:(1)BMSCs were derived from SD rat bone marrow,grew adhering to the wall in shuttle shape,part of hematopoietic cells floated in substratum.MTT growth curve indicated that cells came into exponential phase of growth on the second day after inoculation and proliferated actively.Observed under contrast phase microscope,BMSC extended,axle protuberance,in shuttle shape,two or more circular nucleuses,abundant karyoplasms.Cell density increased,connected with each other.After the cells were cultured about seven days,cells-could spread the whole bottom,and expanded rapidly. After the cells were cultured about twelve days,BMSCs grew torpidity and came into platform phase.(2)Immunofluorescence and flow cytometer test indicated that BMSCs highly expressed CD44(99.3%),CD54(97.7%),CD90(99.4%),CD71(98.2%),CD106(25.8%) correspondence lower,and lowly expressed CD45(8.9%),CD31(15.9%).After passage, BMSCs was in uniform shape,grew rapidly and spread the whole bottom in 3 to 4 days. Contact inhibition was obvious because of high cell inoculum density.Cell morphology of 10 generation BMSCs in HE staining is shuttle shape,axle protuberance,abundant cytoplasm and blue circular nucleus.(3)Induced by 5-aza for 5 days,BMSCs size was getting bigger and rounder. Observed under contrast phase microscope,cells were localized in areas with high cell density in different size with abundant cytoplasm and blue circular nucleus.Cells proliferated and connected with each other tightly.Immunofluorescence staining indicated BMSCs induced by 5-aza could express MHC-β,connexin43,desmin and actinin-a.While part of induced cells could also express vascular endothelial cell specific moleculars (Flk-1/VEGFR2/KDR).RT-PCR test indicated that induced BMSCs could express myocardial cell early development gene NKX2.5 and GATA4.(4)Ventrieular myocardial cells which were isolated from neonate rats heart grew in cluster and overlapped into compact cell masses.Cultured for about 10 days,conglobate myocardial cells under contrast phase microscope show rhythmic contraction,which continued for half a month.(5)The FGF-2 gene was amplificated from ventricular myocardial cells of neonate rat by RT-PCR,and then was inserted into eukaryon expression vector PIRES2-EGFP after being digested with EcoR I and BamH I to construct the plasmid vector PIRES2-EGFP-FGF-2.(6)FGF-2 gene was transferred into BMSCs induced by ultrasound-mediated albumin microbubble destruction method.BMSCs could express EGFP observed under fluorescence microscope.FGF-2 gene were transferred into BMSCs mediated by Lipsome2000,after 24hr,BMSCs could express EGFP observed under fluorescence microscope.It showed that FGF-2 has been tansferred into BMSCs.(7)After different intensity and time of ultrasound,BMSCs activity was different;it indicated that it was salty within 0.75W/cm~2,32see extent.(8)Expression of EGFP was detected by flow cytometry after transferring BMSCs with FGF-2 24hr.In liposome group, expression efficiency was(49.35±9.34)%,compared with(30.42±4.58)%in ultrasound group,P＜0.05.It showed that transfection efficiency in liposome group was higher than in ultrasound group. Conclusions:(1)Ventricular myocardial cells were isolated from neonate Sprague-Dawley (SD)rats heart successfully.The antigen of ventricular myocardial cells tested by immunofluorescence.(2)Ventricular myocardial cells were collected;Total mRNA was extracted by Trizol method.The complementary gene encoding rat FGF-2 was amplified by RT-PCR,and then the target gene fragment was inserted into vector PIRES2-EGFE Eukaryotic expression vectors was constructed successfully.(3)BMSCs could differentiate into myocardial cell which were induced by 5-aza.(4)Cell activity could be influenced by different intensity and time of ultrasound.(5)Both lipsome and Ultrasound-mediated albumin microbubble destruction could improve FGF-2 transferring into BMSCs.(6)Ultrasound-mediated albumin microbubble destruction could promote FGF-2 transfer into BMSCs.It is a new method for transferring with non-virus vector,but transfer efficiency should be increased更多还原