摘要
背景:动物实验和初期的临床研究表明干细胞静脉途径移植后,干细胞归巢到缺血心肌组织,通过多种机制改善心功能,从而为心肌梗死的治疗开辟了一条崭新的途径。但干细胞在归巢过程中的大量缺失影响了干细胞归巢的治疗效果。一些研究显示,敲除MMP-2、MT1-MMP或TIMP-2基因使干细胞侵袭能力受损,而沉默的TIMP -1能够增强干细胞迁移能力。在体内外的实验中均证实TIMP-1基因在细胞的迁移过程中起阻碍作用,本试验利用慢病毒载体转染TIMP-1-shRNA基因的骨髓间充质干细胞,来研究沉默TIMP-1基因的骨髓干细胞能否在体内外提高干细胞的侵袭能力,增加干细胞归巢受损心肌的数量,并进一步探讨干细胞治疗缺血性心肌病的机理。
第一部分携带TIMP-1-shRNA基因的慢病毒表达载体的构建
目的:设计合成3对以骨髓间充质干细胞TIMP-1基因不同位点为靶点的短发夹状RNA(shRNA),构建携带沉默TIMP-1基因不同位点的3组慢病毒表达载体质粒,并对经鉴定纯化的载体质粒进行包装、滴度测定。
方法:设计合成3对针对TIMP-1基因的特异性shRNA片段,退火后与经Hpa I和Xho 1双酶切的Pll3.7载体连接,扩增后提取质粒,经双酶切鉴定和测序后,将测序正确的载体质粒和慢病毒包装质粒共转染293T细胞,收集、纯化和浓缩富含慢病毒颗粒的细胞上清液。
结果:双酶切鉴定和测序结果均表明3组重组的RNA干扰慢病毒载体均有一段与我们设计合成的靶向链完全一致。?
结论:成功地合成了3组沉默大鼠TIMP-1基因的寡核甘酸序列,并构建了携带shRNA-TIMP-1基因的慢病毒表达载体质粒,经293T细胞包装生产出高滴度的重组慢病毒。
第二部分使用慢病毒包装系统建立沉默TIMP-1基因的稳转骨髓间充质干细胞
目的:使用慢病毒包装系统转染3组骨髓间充质干细胞株,利用RNA干扰技术沉默TIMP-1基因的不同靶点。在构建好的转染不同TIMP-1-shRNA的3组细胞株中筛选出沉默TIMP-1基因效率最高的细胞株。
方法:使用慢病毒包装系统建立沉默TIMP-1基因不同位点的3组骨髓间充质干细胞株(SH1、SH2、SH3组),同时设立转染空载体组和阴性对照组,观察各组细胞转染前后形态和荧光的表达。再抽提蛋白,应用Western blot技术检测TIMP-1蛋白的表达。RT-PCR技术检测各组细胞的TIMP-1基因的mRNA水平。
结果:慢病毒转染MSCs 5天后,各组均可检测到强荧光出现,转染前后细胞形态无改变。Western blot免疫印迹技术表明在5个实验组中,SH2组TIMP-1蛋白表达最低; RT-PCR技术检测结果表明转染1周后SH2组mRNA水平下降最大。
结论:使用慢病毒包装系统成功建立沉默沉默TIMP-1基因的3组骨髓间充质干细胞株,并在构建好的沉默不同shRNA-TIMP-1基因位点的3组细胞株中成功筛选出沉默TIMP-1基因效率最高的细胞株。
第三部分大鼠骨髓间充质干细胞(MSCs)的分离、培养和心梗模型的建立
目的:从大鼠的骨髓中分离出具有多向分化潜能的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)以及通过开胸结扎前降支建立大鼠缺血性心肌病的动物模型。
目的:建立大鼠骨髓间充质干细胞体外培养方法,为干细胞归巢的实验研究提供细胞材料。
方法:大鼠的骨髓MSCs分离纯化后,流式细胞仪检测细胞表面标记。
结果:体外培养的原代MSCs 10～14d达到融合,细胞周期显示89.43%的细胞处于G0/G1期。超过90%大鼠骨髓间充质干细胞性表达CD29、CD90和CD44;但不表达造血干细胞表面标志CD34。
结论:利用MSCs贴壁特性,在体外条件下成功分离、培养和扩增了大鼠骨髓MSCs,可用于干细胞的归巢研究。
第二节大鼠心肌梗死模型的建立
目的:经开胸结扎大鼠冠状动脉前降支(LAD),建立大鼠心肌梗死的模型。
方法: 24只大鼠开胸结扎大鼠冠状动脉前降支,致前室壁梗死。结扎成功标志为左心室前壁及心尖周围心肌组织颜色晦暗或青紫和运动减弱。建模前后观察心电图变化,建模前和建模4周后行心脏超声、血液动力学检测,4周后行组织学检查。
结果:与对照组相比,心梗(MI)组左室舒张未内径(LVEDd)增大(P<0.05),射血分数(EF)下降(P<0.05);左室收缩压(LVSP)、、左室内压最大上升和下降速率(±dp/dt )显著降低(P<0.01);而左室舒张末压(LVEDP)显著增加(P<0.01)。组织学检测证实MI组前室壁、室间隔形成了梗死灶。
结论:这种模型接近临床病理生理过程,稳定可靠。
第四部分转染TIMP-1-shRNA的MSCs和MSCs体外侵袭能力比较及其机制的研究
目的:检测对比转染慢病毒空载体细胞、骨髓间充质干细胞和转染沉默TIMP-1基因细胞的体外趋化侵袭能力,同时检测细胞培养上清液的明胶酶活性以及TIMP-1和MMP-9的表达,研究影响细胞体外侵袭能力的因素并探讨其机制。方法:使用Costar穿膜小室系统研究转染慢病毒空载体细胞、P2代骨髓间充质
干细胞和转染沉默TIMP-1基因细胞的体外趋化侵袭能力。检测不同细胞株培养上清液中明胶酶的活性及TIMP-1、MMP-9蛋白含量。
第一节大鼠骨髓间充质干细胞(MSCs)的分离、培养及鉴定
结果:与转染慢病毒空载体细胞、P2代骨髓间充质干细胞相比,敲除TIMP-1基因的MSCs株体外侵袭能力提高,细胞培养上清液中明胶酶活性增强,细胞培养上清液TIMP-1含量下降,而MMP-9含量升高(P<0.05)。
结论:敲除MSCs的TIMP-1基因能够增加细胞培养上清液中MMP-9的活性和表达,提高了MSCs的体外侵袭能力。
第五部分大鼠骨髓MSCs静脉输入治疗缺血性心肌病的实验研究
目的:在大鼠的心梗模型平台上,研究输入转染TIMP-1-siRNA的MSCs能否在体内较MSCs、转染慢病毒空载体的MSCs更显著地改善缺血性心肌病的心脏功能,来探讨MSCs和基因联合应用治疗缺血性心肌病的可行性,以及研究干细胞归巢治疗缺血性心肌病的机制。
方法:取1只SD大鼠骨髓,采用细胞差异贴壁筛选法分离纯化、体外扩增。利用慢病毒载体建立转染空载体的MSCs、正常传代的MSCs和转染shRNA-TIMP-1基因的细胞株。再选用SD大鼠(n=71),经开胸结扎大鼠前降支建立心梗模型,建模成功后存活56只大鼠随机分入4组:空白组(A组,n=14)、MSCs组(B组,n=14)、GFP-MSCs组(C组,n=14)和TIMP-1-KD-MSCs组(D组,n=14)。1周后经静脉途径植入细胞悬液。
建模前后和细胞静脉途径移植4周后行心脏超声心动图检测,评估心功能。细胞静脉途径移植3天后,随机选取4只处死,TUNEL检测心肌细胞凋亡率。4周后,对比各组心肌梗死面积,评估梗死区和梗死周边区的有血流灌注的毛细血管的数量;另外,采用ELISA法检测血清TGF-β1在建模前和干细胞移植治疗心梗前后的变化。最后,流式细胞仪检测大鼠骨髓MSCs归巢到心肌组织的数量。
结果:心超示:与建模前对比,梗死心脏的左室舒张末径(LVEDd)增加,收缩能力明显下降。干细胞移植后,B、C、D组的心功能较移植前有改善(P<0.05),D组的心功能改善优于其他3组(P<0.05)。移植的MSCs能防止梗死后的心室扩张,增加心肌收缩能力;TUNEL检测显示:MSCs组和GFP-MSCs组梗死区心肌细胞的凋亡率无显著差异(P>0.05),但低于空白组,高于TIMP-1-KD组(P<0.05)。TIMP-1-KD组、MSCs组和GFP-MSCs组心肌梗死面积与空白组对比明显缩小(P<0.05);TIMP-1-KD组心肌梗死面积又小于MSCs组和GFP-MSCs组。(P<0.05)。与空白组相比,B、C、D组梗死区的毛细血管密度明显增加(P<0.05)。D组的增加比B、C组显著(P<0.05)。血清TGF-β1的水平在梗死后有所增加,在细胞移植后逐渐下降,D组TGF-β1的水平下降最明显(P<0.05)。流式细胞仪检测证实D组MSCs归巢心肌组织的数量显著大于其他三组(P<0.05)。
结论:MSCs能在宿主心肌组织中存活,通过减少梗死后心肌细胞凋亡数量、抑制收缩功能异常而改善左室功能;这种改善可能是MSCs通过心肌再生、血管再生和细胞外基质增生引起的。一些细胞因子在其中起非常重要的作用。敲除TIMP-1增加了MMP-9的活性,促进了MMCs侵袭能力,增加了MMCs归巢缺血心肌的数量,使参与心肌修复的干细胞的绝对数量增加,从而可更加显著改善心脏功能。
沉默MSCs的TIMP-1基因,能够促进MSCs静脉移植后归巢损伤心肌的数量是治疗缺血性心肌病的一种有效的方法。
Background: Animal experiments and early clinical studies have shown that intravenous administration of adult bone marrow-derived Mesenchymal stem cells (MSCs) could home to the ischemic myocardial tissue, and improve cardiac function through a variety of mechanisms, thus intravenously administered MSCs may be a new therapeutic strategy for the treatment of myocardial infarction. However, a loss of a large number of stem cells in the process of homing affected stem cell homing therapeutic effect. The molecular mechanisms, however, that control MSC homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. Matrix metalloproteinases (MMPs) were believed to play a pivotal role in invasion behavior of stem cells. Some studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired MSC invasion, whereas silencing of TIMP-1 enhanced cell migration. Lentiviral vectors provide an efficient means for TIMP-1-shRNA delivery into MSCs. Accordingly, we investigated whether intravenously transplanted TIMP-1-silencing MSCs can improve the invasion and homing capacity in vivo and in vitro, increase the number of homing MSCs, and further explore stem cell therapy for ischemic cardiomyopathy mechanism.
PartⅠConstruction of lentiviral expression vector of TIMP-1-shRNA gene
Objective: Designed short hairpin RNA (shRNA), targeted to silence TIMP-1 gene of bone marrow-derived mesenchymal stem cells, constructed lentiviral expression vector carrying TIMP-1-shRNA gene, then dentified and purified packaged plasmid.
Methods: Designed and synthesized three pairs for the TIMP-1 gene-specific oligonucleotide, annealed and connected with Pll3.7 vector that was digested by Hpa I and Xho1 enzyme. Plasmids were amplified, extracted, identified by double enzyme digestion and then sequenced. Vectors that have the correct sequence and the lentiviral packaging plasmid were co-transfected 293T cells. At last, collect, purify and concentrate the virus particles which are rich in cell supernatant.
Results: Double enzyme digestion and sequencing results showed that three groups of recombinant lentiviral vector of shRNA exactly have a fragment and were consistent with the target chain that were designed and synthesized by our.
Conclusion: Successfully synthesized oligonucleotide sequences to silence the rat TIMP-1 gene, and constructed carrying shRNA-TIMP-1 gene lentiviral expressing vector, then packaged by the 293T cells to produce high titer lentivirus particles.
PartⅡUsing an HIV-based lentiviral vector to transfect Mesenchymal stem cells to knock-out TIMP-1 gene stably
Objective: Using an HIV-based lentiviral vector to transfect Mesenchymal stem cells to knock-out TIMP-1 gene stably in vitro,then selecting out cell which have the highest efficiency of gene silencing in three cell lines
Methods: Three lentiviral expression vector of carrying different shRNA-TIMP-1 gene was transfected into MSCs (SH1, SH2, SH3 group), and set up an empty vector transfection group and negative control group. The morphology of cells was observed in each group before and after cell transfection. After five day culturing, GFP (green) was detected in the fluorescence microscope. Protein was extracted, Western blot was carried out to detect TIMP-1 protein expression levels in each group cells and RT-PCR was carried out to detect TIMP-1 mRNA levels in each group cells,and GAPDH was used as a housekeeping gene.
Results: On the lentivirus-transfected MSCs 5th days, strong fluorescence can be detected in each groups, cell morphology did not change before and after transfection. Western blot analysis showed that SH2 group expressed the lowest TIMP-1 Protein levels; RT-PCR results showed that the largest decline in the levels of TIMP-1 mRNA expression could be detected distinctly in SH2 group.
Conclusion: Lentiviral vector carrying shRNA-TIMP-1 gene could make MSCs stably to knockout TIMP-1 gene.Cell lines of SH2 group has the highest efficiency of gene silencing in three cell lines
.PartⅢ
Section A Culture of Mesenchymal Stem Cells from Rat Bone Marrow
Objective: To establish a method for isolation and cultivation of mesenchymal stem cells (MSCs) from rat bone marrow and to study their phenotypical properties to offerg an experimental foundation for their further differentiation and actual application.
Methods: Marrow was isolated from the long bonesof rat by centrifugation and plated in tissue-culture dishes. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer. Epitope analyses were detected by flow cytometry.
Results: The adherent, fibroblast-like cells were confluent in single layer after plating for 10～14 days. The cell cycle analysis showed that 89.43% of MSCs was in G0/G1 phase. More than 90% of rat bone marrow-derived mesenchymal stem cells expressed CD29, CD90 and CD44, but not CD34 which was hematopoietic stem cell surface marker. Conclusion: Application of MSCs adherent properties in vitro conditions, rat bone marrow MSCs which were successful isolated, cultured and amplificated can be used to study stem cell homing. It is suggesting that the method can provide cell material for cell homing experiment.
Section B A Model of Myocardial Infarction in Rat
Objective: Myocardial infarction (MI) was induced by left anterior descending artery (LAD) ligation.
Methods: MI was induced by ligation of the LAD in 24 rats. Doppler echocardiography was performed to compare myocardial function in two groups of rats. Electrocardiogram (ECG) changes were observed in the operation and postoperative. Pathology was also performed 4 weeks later.
Results: Coronary artery ligation resulted in anterior and lateral infarction of the left ventricular wall. The immediate changes visible post ligation were immediately pallor of the myocardium. LVEDd (the LV end-diastolic dimension) in MI increased highly compare to control group (control versus MI: 5.68±0.41 mm vs 7.35±0.67 mm, p<0.01), and the ejection fraction (EF) decreased (control versus MI: 88.36±3.83% vs 49.36±6.25%, p<0.001) in the MI group. Histological tests confirmed that coronary artery ligation resulted in anterior and lateral infarction of the left ventricular wall.
Conclusion: This rat model of MI is reliable, reproducible, and similar to the human condition.
PartⅣContrast MSCs transfected TIMP-1-shRNA and MSCs in vitro invasive ability and investigate its mechanism in vitro.
Objective: Contrast MSCs transfected lentiviral empty vector, MSCs and MSCs transfected shRNA-TIMP-1 gene chemotactic invasive ability in vitro, while detected the gelatin activity in cell culture supernatant, as well as TIMP-1 and MMP-9 expression, and investigated its mechanism affecting the cells invasive ability in vitro.
Methods: Using Costar trans-membrane chamber system to study MSCs transfected lentiviral empty vector, P2 generation MSCs and MSCs transfected shRNA-TIMP-1 gene the chemotactic invasion ability in vitro.After culturing in serum-free medium in 5% CO2 at 37℃for 24 h,harvested culture supernatants, detected gelatinase activity and TIMP-1, MMP-9 levels in culture supernatants of different cell lines.
Results: Compared with three groups, the invasive ability of MSCs line transfected shRNA-TIMP-1 gene was the highest (P<0.05), gelatin activity of cell culture supernatant also increased, TIMP-1 content of cell culture supernatant decreased, while MMP-9 expression levels increased(P<0.05).
Conclusion: MSCs line transfected shRNA-TIMP-1 gene can increase the MMP-9 activity and expression in cell culture supernatant, and increase invasion ability of MSCs. The improvement of MSCs invasion ability in vitro was associated with knockout TIMP-1 gene.
PartⅤEffect of Intravenously Injected Marrow Mesenchymal Cells in Rat Infarcted Myocardium
Objective: To investigate whether intravenously injected MSCs improved cardiac function in rat myocardial infarction model, and explore whether the combined application of MSCs and shRNA-TIMP-1 gene in the treatment of MI is feasible, as well as investigate stem cell homing mechanism for the treatment of MI.
Methods: A commercially available self-inactivating lentiviral vector has been modified for the delivery of TIMP-1-siRNA into MSCs. MSCs from healthy rat were isolated, cultured and transduced with a lentiviral vector containing either TIMP-1-siRNA and green fluorescent protein (GFP; TIMP-1-siRNA /GFP vector) or GFP alone (control vector).
MI model was induced by occluding the LAD in 71 SD rats (230-385g). After the establishment of MI model, survival 56 rats were randomly divided into 4 groups: control group (A group, n = 14), MSCs group (B group, n = 14), GFP-MSCs group (C group, n = 14), and TIMP-1-KD-MSCs group (D group, n = 14). One week later, intravenously injected MSCs or suspensions were performed.
The echocardiographic studies were performed to measure cardiac function in the preoperative and postoperative 4 weeks. Intravenously injected MSCs three days later, four rats were killed randomly. TUNEL technology was performed to detect myocardial cell apoptosis. Four weeks later, triphenyltetrazolium chloride (TTC) staining was performed to compare infarct sizes (IS) in each group and assess the number of blood perfusion capillary in the infarct zone and infarct border zone. In addition, ELISA assay were performed to measure serum TGF-β1 content before MI and stem cell transplantation preoperative and postoperative for treatment of myocardial infarction. Finally, the number of MSCs homing to myocardial tissue was measured by flow cytometry.
Results: Myocardial infarction was defined by echocardiography as any segmental wall motion abnormality. Left ventricular end-diastolic diameter (LVEDd) increase and contractility decreased significantly after MI (P<0.05). After intravenously injected MSCs, the cardiac function in B, C, D group improved compared with control group (P<0.05), the cardiac function improvement in D group was better than the other three groups (P<0.05). MSCs transplantation can prevent post-infarction ventricular dilatation, increased cardiac contractility.
TUNEL test showed: Apoptotic myocytes of B group and C group were no significantly difference (P>0.05), but lower than the control group, higher than D group (P<0.05). Myocardial infarct size of D group, B group and C group were reduced significantly compared with the control group (P<0.05), Myocardial infarct size in D group was smaller than B group and C groups (P<0.05). Compared with the control group, B, C, D group infarct zone capillary density was significantly increased (P <0.05), the number of blood perfusion capillary of D group in the infarct zone was the most highest in B, C, D group (P <0.05). The serum TGF-β1 levels began to increase in post-infarction and gradually decreased after cells transplantation, the level of TGF-β1 of D group decreased significantly Compared with A,B, C group (P<0.05). The number of MSCs homing to myocardial tissue was measured by flow cytometry confirmed D group was significantly higher than the other three groups (P<0.05).
Conclusion: MSCs may both migrate and differentiate extensively, might improve the cardiac function by attenuating contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells. Some cytokines maybe play an important role in repairing the ischemic myocardium.
TIMP-1 gene knockout increased the MMP-9 activity and promoted the invasive ability of MSCs, and increased the number of MSCs homing to ischemic myocardium, so that the quantitations of MSCs that participating in myocardial repair increased, and the therapeutic benefit was more powerful.
Use of lentiviral vectors for delivery of TIMP-1-shRNA enhanced invasion and homing efficiency of intravenously injected MSCs in rat infarcted myocardium, so the strategies will be an effective approach for the treatment of MI.
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