摘要
目的
促进血管形成是当今国内外治疗缺血性疾病研究的热点之一,而血管内皮细胞增殖、分化可导致毛细血管生成。血管内皮细胞生长因子(VEGF)是一种能刺激内皮细胞增殖,增加内皮通透性的血管源性蛋白。大量研究已证实VEGF是刺激血管形成最关键的生长因子。
骨髓间充质干细胞(mesenchymal stem cells, MSCs)具有自我更新,克隆非造血组织的前体,并且具有分化骨细胞,软骨细胞,骨骼肌细胞,等多种细胞的功能。而且在体内及体外被基因转染的MSCs可以保持稳定性。丝/苏氨酸激酶(serine/threonine kinase Akt),又名为蛋白激酶B(protein kinase B, PKB)是生物信号传递途径中的重要因子,并与磷脂酰肌醇-3-羟基激酶(phophatidylinostitol-3-kinase, PI3K)构成重要的PI3K/Akt信号传导通路,在血管生成过程中伴有重要的作用。曾有研究证实MSCs中过量表达Akt可以抑制细胞凋亡,促进细胞存活,而且少量的Akt就有作用。绿色荧光蛋白(green fluorescent protein, GFP)从能发光的生物水母(Aequorea Victoria)中分离出来现已被广泛应用于示踪活细胞内基因的表达,已经成为细胞与分子生物学研究的一种流行的实验工具。
本试验：1、克隆构建GFP/Akt表达载体及鉴定,MSCs的分离培养及鉴定；2、通过脂质体真核细胞转染法,荧光显微镜下观察GFP/Akt表达载体在鼠MSCs中的表达和定位及其对VEGFmRNA和蛋白表达的影响；3、转染GFP/Akt的MSCs对下肢缺血大鼠血管生成的影响。
材料与方法
一、材料
Wistar大鼠[SCXK(辽)2003-0009],4周-6周、体重150-200g(购自中国医科大学动物试验中心)。
二、方法
1、克隆构建GFP/Akt表达载体及鉴定：
GFP-Akt primer设计F 5'-CGAGGAATTCGATGAACGACGTAGCCATTGT-3'(含EcoRⅠ位点)R 5'-TATCAGGATCCACCTCAGGCTGTGCCACTGG-3'(含BamHI位点)。Akt基因PCR扩增：以pcDNA3-Akt为模板PCR扩增此基因的开放阅读框架,引物上游引入EcoRⅠ酶切位点,下游引入BamHI酶切位点。
反应体系如下：DW 72.5ul,10×pyrobest buffer泳100 u1。
反应条件：94℃3min,94℃50s,60℃lmin,72℃3min,72℃10min, 4℃store,30 cycles。PCR产物用1%琼脂糖凝胶电泳验证,在约1400bp处有明显条带,将其回收。酶切,连接,转化,质粒提取,过夜培养,次日小量提取质粒,同时保存菌种。
pCDNA-Akt片断扩增鉴定：Rt-PCR产物进行1%琼脂糖电泳验证,在约1450bp处为一单一条带,跟目的片段大小位置一致
重组质粒酶切鉴定：取2ul质粒进行双酶切鉴定。经1%琼脂糖电泳验证,6个菌落经鉴定正确,在约4700bp处有GFP载体的酶切片段,1450bp处有Akt的酶切片段。次日大量提取质粒。
浓度检测：根据0D值GFP浓度是0.85ug/ul, GFP-Akt浓度是1.874ug/ul。
2、MSCs分离培养及鉴定：
将Wistar大鼠(4周-6周、体重150-200g),颈椎脱臼处死,75%酒精浸泡1分钟消毒,无菌条件下取出股骨及胫骨,剪去骨两端,用6ml肝素化(100u/ml)PBS (PH7.40)冲洗骨髓腔,将冲洗液缓缓加于Percoll(比重1.074g/ml和1.070g/ml)分离液上梯度离心(500g,20mins)后,吸取界面层细胞,用10mlPBS将其吹打制成细胞悬液洗涤(100g,10min)后弃上清,共两次。用含15%FBS的L-DMEM培养液10ml将获得的细胞吹打制成悬液接种于25cm2塑料培养瓶中,在37℃、5%CO2条件下于孵育箱中培养,3天后半量换液以后每隔2-3d全量换液以弃除悬浮,以后培养和传代按细胞培养常规。
3、脂质体真核细胞转染：
取第二代细胞(Passage 2, P2)1.0-2.0×104传至24孔板上,待细胞长至80%-90%融合,分对照组、转染GFP组和转染GFP-Akt组三组(每组8孔),按Lipofectamine TM2000转染试剂说明方法,次日全部换液;对照组换含15%FBS的L-DMEM,转染GFP组及转染GFP-Akt组换含加入G418(400ug/ul)的15%FBS的L-DMEM,三天换液一次,维持筛选作用。两周后,转染细胞单克隆形成,其间荧光显微镜观察稳定转染效率并照相(取同一视野倒置相差显微镜和荧光显微镜观察转染效率：GFP组转染率为7.64%、GFP-Akt组转染率为6.5%)。
4、实验大鼠分组,双下肢缺血大鼠模型的制备及处理
Wistar大鼠30只,鼠龄8-10周,体重150-250g；动物随机分为3组：基因治疗组,非基因治疗组,对照组。大鼠用1%戊巴比妥钠(30mg/kg)腹腔注射麻醉,自其腹股沟韧带中点到膝关节上做一纵型切口,分别离断双侧的股动脉及其分支。基因治疗组、非基因治疗组、对照组左侧内收肌,腓肠肌取7个点分别共肌注1.0×107 pEGFP-C1/AKT转染的MSCs,1.0×107MSCs, PBS;右侧分别肌注PBS、PBS.生理盐水做对照。
5、动脉照影：
实验动物分别于术后第4周在DSA下肢动脉造影,穿刺腹主动脉推注70%泛影葡胺连续摄片,对照观察双下肢血管分布密度。
6、毛细血管密度的测定：
处死动物分别取其左侧的内收肌和半膜肌标本,以10%的甲醛固定,兔抗人F-Ⅷ及CD34抗体染血管内皮细胞,苏木素复染,中性树胶封片。光镜观察血管内皮细胞呈棕黄色。每个标本随机取10个视野,在高倍镜(400倍)下观察,计算毛细血管密度(毛细血管数／高倍镜)。
7、RT-PCR检测AKTmRNA的表达：
(1)分别取1.0×107细胞用RNA提取试剂Trizol提取总RNA。
紫外分光光度计测样品浓度,琼脂糖凝胶电泳鉴定RNA完整性,用MMLV第一链CDNA合成试剂盒反转录合成CDNA,按说明书操作。
取反转录产物4ul进行PCR反应：95℃预变性2 min94℃变性lmin55℃复性lmin72℃延伸lmin,32个循环72℃延伸lmin,32个循环,72℃延伸10min
内对照p-actin(上游5′-GCCAACCGTGAAAAGATG-3',下游5′-CCAGGAT-AGAGCCACCAAT-3')扩增长度681bp(退火温度57℃,循环30次);VEGF(上游5'-GCCTTGCCTTGCTGCTCTA-3',下游5′-TAACTCAAGCTGCCTCGCC-3')扩增长度505bp(退火温度55℃,循环30次)：Akt(上游5′-GAGGAGCGGGAAGAGTG-3',下游5′-GAGACAGGTGGAAGAAGAGC-3')扩增长度672bp(退火温度54℃,循环30次)。
PCR扩增产物用1.5%琼脂糖凝胶电泳,溴化乙锭染色显色,凝胶图像分析系统分析,以Akt mRNA和VEGFmRNA分别与β-actin mRNA灰度比值作为Akt mRNA及VEGF mRNA半定量指标。
(2)取约100mg组织,Trizole液提取总RNA总RNA提取操作均按说明书进行。紫外分光光度计(A260/A280)检测RNA纯度。
逆转录反应：总RNA 1μl,Oligo dT-Adaptor primer 1μl,70℃5min,水浴2 min,按顺序加入10×RNAPCR缓冲液2μl、10 mmol/L Dntp mixture 2μl、RNA酶抑制剂20 U、反转录酶1 ul等,加去离子水至20μl。30℃10 min,50℃30 min,99℃5 min,5℃5 min,水浴1-2 min。逆转录合成的cDNA在-20℃保存用于PCR,以下操作同上
8、Western blot法：
(1)分别取1.0×107细胞,细胞裂解液裂解细胞,蛋白抽提上样,进行10%SDS-PAGF电泳,半干法电转移法100V恒压电泳1.5h将蛋白转印到PVDF膜上。室温下用TBS阻断1h；5%脱脂奶粉封闭1h后加一抗(工作浓度1：400),37℃,2h,洗膜；加入二抗(生物素化山羊抗兔IgG抗体,工作浓度1：400),37℃,2h,洗膜；DAB显色,照相。Western blot条带图像存入计算机,用Quantity One图像分析软件包进行光密度计算。
(2)剪碎约100mg内收肌组织,机械匀浆,离心10 min。考马斯亮蓝R250染色法测总蛋白质浓度,将各组浓度调到同一水平以下操作同上。
三、统计学处理：
应用SPSS 12.0统计软件包进行数据处理,不同组间分析采用方差分析(ANOVA)检验,有显著意义后用最小有意义差异t检验进行均数间的多重比较,检验水准α=0.05。
结果
一、MSCs的分离、培养及鉴定：
MSCs培养5-7 d长满瓶底,此时,细胞呈平行或漩涡状生长,多次传代融合生长时,有更均匀有序的成纤维细胞样分布。CD2、CD44和CD71免疫细胞化学染色均可见棕黄色颗粒沉积于胞膜,而CD34免疫细胞化学染色后未见棕黄色颗粒沉积于胞膜。
二、重组质粒的鉴定和转染：
pEGFP-C1/Akt重组质粒酶切片段分别显现在4700bp和1450bp处,与扩增的pEGFP-C1和Akt目的片断相符。
三、GFP/AKT组、GFP组转染MSCs后AKTmRNA及蛋白与VEGFmRNA及蛋白的相关性：
GFP/AKT组、GFP组转染MSCs后VEGF蛋白和Akt蛋白表达显著正相关,VEGF mRNA和Akt mRNA表达亦正相关。
四、转染GFP/AKT的MSCs、MSCs对下肢缺血大鼠血管生成的相关性：
移植后第28 d腹主动脉造影显示,基因治疗组结扎股动脉处的远端毛细血管生成较明显,血管成网状,优于非基因治疗组及对照组。
结论
一.MSCs分离、培养成功。
二.GFP/AKT重组质粒的构建、转染成功。
三、转染GFP/AKT的MSCs组AKTmRNA及蛋白与VEGFmRNA及蛋白高于转染GFP的MSCs组。
四、转染GFP/AKT的MSCs组对下肢缺血大鼠的血管生成高于MSCs组。
Intoduction
The duration of exposure to VEGF may be a critical determinant of formation of stable neovasculature, VEGF is a multifunctional cytokine with potent vascular permeabilizing properties that stimulates endothelial cell migration, survival, and growth, and its effects on angiogenesis may be via direct action on endothelial cells and less direct effects involving other cells types. Endothelial cells (ECs) are important for the homeostasis of the vasculature. They control vascular tone by producing vasorelaxing substances and prevent thrombosis by control lingthe coagulant status. They also control vascular permeability and are involved in angiogenes.
Mesenchymal stem cells (MSCs), which have been shown to proliferate extensively in vivo and in vitro and, when cued by the appropriate microenvironment, to differentiate along multiple lineages giving rise to muscle, brain, liver, cartilage, bone, fat, and blood vessels MSCs have also been used in a model of cell transplantation, showing that these cells could differentiate into myogenic cells. Therefore, MSCs have many characteristics that make them useful for cellular therapy.
Akt (protein kinase B) is a Ser/Thr kinase that regulates several cellular processes, including cell cycle progression, transcription, glucose uptake and apoptosis. In particular, Akt plays a critical role in mediating cell survival in response to growth factor stimuli, Akt is an excellent therapeutic gene for preserving MSC viability in the early post-transplant period.
Green fluorescent protein (GFP) absorbs blue or ultraviolet light,and gives out bright green fluorescence.GFP has been expressed in a variety of species,and extentively used in tissue engineering.
1. To construct expression vector containing GFP/Akt gene and Isolation of MSCs from bone marrow and cell culture.
2. MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP was evaluated by fluorescene microscopy.
3. The MSCs was transfeced by GFP/Akt transplantation in a rat model of acute Hind Limb Ischemia possibly inducs angiogenesis.and to investigate the expression of GFP-Akt These results raise the possibility that Akt plays a role in modulating vasculogenesis and angio genesis.
Materials and methods
1. Model of ischemic:
Thirty male Sprague-Dawley rats (Department of laboratory animals of China Medical University.china) 16 weeks old, body weight range:300±50 g were used in these studies. All experimental procedures were approved by the animal care and handling committee of China Medical University and conformed to the Guide for the care and use of laboratory animals as adopted by Chinese Association For Laboratory Animal.They were maintained on a 12-h light/dark cycle in a temperature-controlled (19-21℃) room and were allowed free access to water and standard rat diet until 18-20 h before being killed when food was withdrawn. Before the surgical procedures, the rats were anesthetized with an intraperitoneal injection of a mixture of ketamine 80mg/kg and xylazine 5mg/kg. Severe bilateral hind limb ischemia was surgically created via ligation and division from its proximal origin as a branch of the external iliac artery till the bifurcation into saphenous and popliteal arteries. At the time of surgery. rats were randomized to one of three groups (n=10 per group):
2. Expansion of Bone Marrow MSCs
we humanely killed male Sprague-Dawley rats (150g) and harvested bone marrow by flushing their femoral and tibial cavities with 10 ml of DMEM, then the DMEM including bone marrow was layered over 1.073 g/ml percoll solution (GE Healthcare Bio-Sciences AB) and centrifuged at 400 g and room temperature for 25 min. The mononuclear cells were covered at the interface, then were gathered and resuspended in growth medium (DMEM,10% heat inactivated fetal bovine serum,100 U/ml penicillin G and 100 U/ml streptomycin) (GIBCO), and then were plated on 50 cm2 flasks. The cultures maintained at 37_C in a 5% CO2 incubator. A small number of cells developed visible symmetric colonies by days 5 to 7. Nonadherent hematopoietic cells were removed, and the medium was replaced. When the cultures reached 80% of confluence, cells were recovered by 0.25% trypsin-1 mM EDTA and followed by passages,The adherent, spindle-shaped MSCs population expanded to>5×107 cells within≈3to 4passages were used.
3. Construction of Plasmid DNA
The control plasmids, pEGFPC1-Akt and pEGFPC1 were kindly provided by the Laboratory Center of Cell and Molecular Biology, china medical University.
4. Transfection of MSCs:
MSCs at 70-80% confluence were transfected with pEGFPCl-Akt and pEGFPCl-using commercially available cationic vectors; LipofectamineTM 2000 (Invitrogen) according to manufacturers instructions, Stable transformants were identified in media containing 400 ug/ml of G-418 sulfate (Geneticin; Gibco) 48-72 h after transfection. After three to four weeks of growth in the presence of G-418, clones with the highest levels of pEGFPC1-Akt and pEGFPC1 expression were identified and isolated based on GFP fluorescence using fluorescence microscopy. Cells containing pEGFPC1-Akt were designated MSCs-Akt (experimental cells) and those containing pEGFPC1 were named MSCs-neo (control cells)
5. Transplantation of MSCs
Transplantation was performed 2 h after the hind limb ischemia. The MSCs suspension 1×107 MSCs/100μL transfected pEGFP-C1/Akt(.gene therapy group),MSCs suspension 1×107 MSCs/100μL (therapy group),or PBS(control group) was slowly infused into the adductor and semimembranous muscles at 7 spoints of rats left hind limb,while The rats right hind limb in the control group received an equal volume of PBS
6. Angiographic Analysis
Development of collateral arteries was evaluated by angiography on days 28, the rats were anesthetized with an intraperitoneal injection of a mixture of ketamine 80mg/kg and xylazine 5mg/kg, incision was made in thee abdomen, The abdominal aorta. was exposed, and a 3-F catheter was cannulated with its tip positioned just above the aortic bifurcation in the lower abdominal aorta. Angiography was performed by manual injection within 2 s with 2 ml of contrast medium (Iopamiron 300, SCHERING), The angiographic score was calculated for each film as the ratio of grid, The angiographic score was determined by 2 blinded observers.
7. Evaluation of capillary density
Ischemic tissues from the adductor and semimembranous muscles were obtained 28 days after treatment of left hindlimb ischemia. Frozen sections were stained with anti-factor VIII, followed by incubation with TRIC-conjugated secondary antibody. Five fields from 2 muscle samples of each animal were randomly selected for capillary counts. ischemic tissues of Right hindlimb ischemia as control. Analysis of Capillary Density: Immunohistochemical staining for anti-factor VIII revealed the presence of capillary endothelial cells The capillary/muscle fiber ratio in the skeletal muscle obtained 28 days after hindlimb ischemia was significantly increased in the gene therapy (7.1±0.3) (p<0.01) compared with therapy group (4.2±0.4) and control group (1.3±0.2)
8. Immunodetection of Akt and VEGF protein:
MSCs-Akt, MSCs-neo and MSCs were cultured at a concentration of 5×105 cells/ml in 6-well plates for 24 hours. Cellular proteins (30μg) were separated by 10% SDS-PAGE and immunoblotted with polyclonal rabbit anti- AKT antibody antibodies (Danvers,MA, USA). (1:1000) or phospho- Akt (Ser473) polyclonal antibodies (Danvers,MA, USA) (1:1000)at 4℃overnight, conjugated goat-anti-rabbit secondary antibody were added, followed by ECL visualization of the bands. For rehybridization, After incubation with secondary antibodies, bands were visualized by ECL. The blots were scanned for density assays.
The muscles were homogenized in lysis buffer. Lysates were immunoblotted with anti-phospho antibodies and detected with an enhanced chemiluminescence kit (Amersham).Plasma Akt and VEGF concentration was measured by use of the ELISA kit (R&D Systems).
9. RT-PCR of VEGF and Akt Expression In Vitro and In Vivo
RNeasy kit (Qiagen) was used for isolation of total RNA from the cells or rat adductor and semimembranous muscles samples as per lement.) The primer sequences used in this study included the following:GAPDH (457 bp):forward:5'-GCCAACCGTGAAAAGA—TG-3'; reverse:5'-CCAGGATAGAGCCACCAAT-3'; VEGF(505bp):forward:5'-GCCT—TGCCTTGCTGCTCTA-3'; reverse:5'-TAACTCAAGCTGCCTCGCC-3'; Akt(672bp): forward:5'-GAGGAGCGGGAAGAGTG-3'; reverse:5'-GAGACAGGTGGAAGAAG—AGC-3'.
10. Statistical Analysis
All data were described as mean±SEM. To analyze the data statistically, we performed Student's t test and 1-way ANOVA with post hoc analysis. A value of P<0.05 was considered statistically significant.
Conclusions
1. GFP-Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of mouse
2. Akt mRNA and protein, VEGF mRNA and protein(of gene therapy group markedly increased compared with A therapy group. and control group.
3. gene therapy group has more angiogenic action associated with new vessel formation in the hindlimb ischemia model.compare with control group.
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