摘要
第一部分Snail真核表达质粒扩增及转染骨髓间充质干细胞的实验研究
目的:研究Snail基因在人骨髓间充质干细胞(MSCs)中的转染和表达,Snail对骨髓基质细胞生物学性状及生长等方面的影响,探讨骨髓基质细胞作为Snail基因受体细胞的可行性。
方法:采用密度梯度离心法、贴壁筛选法并结合传代对成人骨髓MSC进行纯化和体外扩增,观察细胞形态和表面标志物的表达特点,流式细胞仪检测骨髓MSC表面抗原表达,脂质体法将重组真核表达载体(pCAGGSneo-snail-HA)及对照空质粒(pCAGGSneo)转染MSCs,G418筛选稳定表达,绘制转染后细胞的生长曲线,分别于转染后48小时和4周利用免疫荧光染色技术及免疫印迹法检测MSCs报告基因HA及目的基因Snail表达情况。
结果:成人骨髓MSC培养3天后有散在呈针尖状的贴壁细胞,7~10天后形成集落或融合呈纤维状,3周左右细胞生长可汇合至80-90%,间充质样细胞呈长梭形,走向趋向一致,传代后1周左右即可融合,3~5代后基本纯化,细胞形态单一,细胞排列具有典型的旋涡状结构。流式细胞仪检测结果显示:CD34表达阴性及CD29表达阳性;500μg/ml的G418是最适筛选浓度,pCAGGSneo-Snail-HA转染后不同时期的MSCs在蛋白水平均可检测到目的基因snail及报告基因HA的表达;生长曲线示重组真核表达载体(pCAGGSneo-snail-HA)及对照空质粒(pCAGGSneo)转染后的细胞(即MSCs-Sna和MSCs-neo)生长曲线与未转染的同代MSCs相比,倍增时间稍稍延长,一代时间约比未转染的细胞多一天,而MSCs-Sna和MSCs-neo之间倍增时间及一代时间没有明显差异。
结论:分离培养的细胞为间充质干细胞且成分单一,第3、5代细胞很纯且生长旺盛,适用于做转染或以后的研究,pCAGGSneo-snail-HA可以在MSCs内获得瞬时和长期表达,且Snail表达对MSCs的生长速度无明显影响。MSCs可以作为Snail基因的受体细胞,为下一步研究Snail对MSCs迁移趋化、存活抗凋亡及基质金属蛋白酶2分泌等生物学特性的影响奠定基础。
第二部分Snail基因修饰诱导AKT、ERK活化对骨髓间充质干细胞迁移的影响
目的:探讨Snail基因修饰对骨髓间充质干细胞迁移力的影响,及磷脂酰肌醇3激酶( phosphatidylinosital3-kinase , PI-3K )、细胞外信号调节激酶1/2 (extracellular signal-regulated kinase, ERK1/2)及P38丝裂原活化的蛋白激酶p38(mitogen-activated protein kinase,P38 MAPK)等信号通路在Snail转染骨髓间充质干细胞迁移能力改变中的调控作用。
方法:采用免疫荧光细胞化学、Western Blot方法检测Snail基因修饰后骨髓间充质干细胞内PI3K、ERK1/2和P38/MAPK信号通路的表达变化,用跨膜迁移实验(Transwell实验)来评价MSCs的侵袭迁移能力,比较转染Snail质粒和转染对照空质粒的骨髓间充质干细胞的差异,并比较用和不用各信号通路干预剂的差异。
结果:Western blot结果表明, MSCs-Sna细胞P-ERK1/2与总ERK1/2的比值(ERK的磷酸化活性)显著高于MSCs-neo组(0.14±0.04 vs. 0.6±0.09, P<0.05)。同时,MSCs-Sna细胞内p-Akt与总Akt的比值(AKT的磷酸化活性)较MSCs-neo组亦显著性增加(0.20±0.05 vs. 0.75±0.1, P<0.05)。免疫荧光细胞化学染色显示P-ERK1/2、p-Akt在MSCs-neo中均为较弱的绿色荧光,且多分布在细胞胞浆内。而在MSCs-Sna中P-ERK1/2、p-Akt荧光强度明显增强,核内亦存在较多分布。p-P38在MSCs-neo和MSCs-Sna细胞核和胞浆中均呈阴性表达。Transwell体外跨膜侵袭迁移实验显示Snail质粒转染MSCs(MSCs-Sna)较对照空质粒转染MSCs(MSCs-neo)细胞迁移率增加(p<0.05),PI-3K信号通路特异性抑制剂Wortmannin和ERK通路抑制剂PD98059能显著抑制此迁移率的增加(p<0.05)。各浓度P38通路抑制剂SB203580对MSCs-neo和MSCs-Sna的跨膜迁移数量均无明显抑制作用(P>0.05)。
结论: Snail基因修饰可增加骨髓间充质干细胞AKT、ERK1/2磷酸化水平,并促进骨髓间充质干细胞的迁移,PI3K通路的特异性抑制剂Wortmannin和ERK通路抑制剂PD98059可分别抑制Snail修饰诱导的AKT、ERK1/2的磷酸化,降低骨髓间充质干细胞迁移能力;SB203580对Snail的促细胞迁移作用无明显影响。PI3K和ERK信号通路在Snail基因促进骨髓间充质干细胞迁移功能的调控中可能发挥了重要作用。
第三部分Snail基因修饰对骨髓间充质干细胞CXCR4表达水平及向SDF-1趋化能力影响的研究
目的:观察MSCs在Snail基因修饰后细胞膜表面相应特异性受体CXCR4表达水平、及对SDF-1的趋化能力的变化。为探索提高MSCs移植后向受损后局部常高表达SDF-1的器官组织趋化迁移能力的方法提供研究基础。
方法:采用免疫荧光细胞化学染色、荧光标记流式细胞仪技术及RT-PCR技术检测Snail基因修饰后的骨髓间充质干细胞上CXCR4受体的表达水平;采用体外Transwell跨膜趋化实验评价MSCs向SDF-1的趋向迁移能力,观察大小不同浓度的抗CXCR4中和抗体对趋化实验的干预作用。
结果:流式细胞仪荧光检测显示,MSCs-neo和MSCs-Sna细胞表面均有CXCR4受体的表达,其中MSCs-Sna的阳性表达率明显高于MSCs-neo的阳性表达率(64.25%±7.22% vs. 7.45%±0.91%, P<0.05)。免疫荧光细胞化学检测显示CXCR4在MSCs-neo和MSCs-Sna的细胞膜上均有阳性表达,但在MSCs-neo中为较弱的绿色荧光,而在MSCs-Sna中CXCR4荧光强度明显增强。RT-PCR结果显示,以GAPDH mRNA为内参照,在MSCs-neo、MSCs-Sna细胞中都有CXCR4 mRNA的表达,但在MSCs-Sna细胞中的表达显著高于MSCs-neo(0.916±0.102 vs. 0.410±0.050,P<0.05)。体外跨膜趋化迁移实验显示,MSCs-Sna在SDF-1诱导下的细胞迁移量较MSCs-neo显著增加(p<0.05)。阻断性CXCR4单抗(12G5)(中和抗体)加于微孔隔离室的上室后可显著减少SDF-1α诱导的MSCs-Sna趋化运动,且10ug/ml的CXCR4单抗对SDF-1α诱导的细胞趋化运动的抑制作用较强1ug/ml的CXCR4单抗作用更为明显。同时,IgG阴性对照抗体对SDF-1α诱导的MSCs-Sna趋化运动没有抑制效应。
结论:Snail可上调MSCs趋化因子受体CXCR4分子的表达,并可通过这一环节促进骨髓间充质干细胞对相应趋化因子SDF-1的趋化作用,这提示了通过上调Snail的表达而提高MSCs向正调节表达SDF-1的受损组织(例如脑的缺血半暗带等部位)迁移效率的可行性,为提高MSCs的迁移能力及移植效率的研究方向提供了新思路和实验依据,并为以后进一步开展提高MSCs向受损组织器官迁移的在体实验研究提供了实验基础。
第四部分Snail基因修饰对骨髓间充质干细胞骨架结构稳定作用及对IL-4和无血清培养诱导细胞凋亡的保护作用研究
目的:观察Snail对骨髓MSCs在离体无血清培养下细胞骨架的变化及凋亡的影响,并研究Snail对MSCs在含IL-4培养基中发生凋亡的保护作用,为探索提高MSCs移植后存活能力的方法提供研究基础。
方法:MSCs-Sna及MSCs-neo于体外无血清培养基中培养后,β-Actin免疫荧光细胞染色观察细胞骨架,流式细胞仪检测细胞Sub-G1凋亡峰,及Annexin V/PI双标法检测细胞早期凋亡率(Annexin V+/PI- %),并比较snail转染及未转染的MSCs(即MSC-sna及MSC-neo)在以上方面的差异。同样的方法,MSCs-Sna及MSCs-neo在含IL-4(20ng/ml)和不含IL-4的完全培养基中培养24小时后,收集细胞流式细胞仪检测细胞凋亡。
结果:无血清培养24小时后,MSCs-Sna细胞骨架排列保持正常有序分布状态,细胞长极存在较长的应力纤维束,细胞轮廓清晰;而MSCs-neo细胞骨架排列出现紊乱,基本细胞形态尚保持。无血清培养72小时后可见,MSCs-neo不能维持正常形态,轮廓模糊,萎缩成团状,细胞骨架断裂紊乱;与之相比,MSCs-Sna无血清培养72小时后尚能维持基本形态,细胞轮廓尚清晰,细胞骨架依然可见有序排列。无血清培养24小时,MSCs-Sna和MSCs-neo之间比较凋亡率没有显著差异;无血清培养72小时后MSCs-Sna Sub-G1凋亡细胞率和Annexin V+/PI-%早期凋亡率(34.33%±5.51%和12.5%±2.2%)较MSCs-neo凋亡率(52%±8.19%和30%±4.3%)低,且有统计学差异(p<0.05);细胞在含IL-4(20ng/ml)和不含IL-4的完全培养基中培养24小时后,检测annexin-V+PI- %(早期凋亡率),比较MSCs-Sna和MSCs-neo凋亡率的差异:加入IL-4后MSCs-neo的早期凋亡较未加入IL-4时增高达5倍,而对于转染Snail表达载体后的MSCs(即MSCSs-Sna),加入IL-4后MSCs-Sna早期凋亡增高仅达未加入IL-4的2.6倍。
结论:经Snail基因修饰,MSCs骨架结构的稳定性及在体外无血清培养或IL-4等促凋亡因素存在的环境中抗凋亡能力增加。
第五部分Snail基因修饰对骨髓间充质干细胞MMP2表达的影响及其信号传导通路的研究
目的研究Snail基因修饰对骨髓间充质干细胞MMP-2表达的影响,及细胞外信号调节激酶1/2 (extracellular signal-regulated kinase, ERK1/2)和PI-3K/AKT信号通路在Snail转染骨髓间充质干细胞MMP-2表达改变中的调控作用。
方法应用脂质体介导的方法将携带Snail基因的重组载体pCAGGSneo-Snail-HA导入体外培养的骨髓间充质干细胞中,用G418筛选获得阳性细胞。采用RT-PCR检测细胞内MMP-2 mRNA的表达,明胶电泳酶谱分析细胞上清液中基质金属蛋白酶2(MMP-2)的活力,并比较转染Snail质粒和转染对照空质粒的MSCs的差异;阻断实验以不同浓度ERK激酶特异性抑制剂(PD98059)或和PI3K通路特异性抑制剂(Wortmannin)干预后Western-blot检测MSCs细胞ERK、AKT磷酸化水平变化,确定PD98059和Wortmannin分别抑制Snail诱导的MSCs细胞ERK激酶和AKT激酶磷酸化的有效浓度,进而研究其对Snail转染MSCsMMP-2 mRNA水平及培养上清中MMP-2活性的影响。
结果:(1)Western blot分析显示MSCs-Sna细胞p-Akt和p-ERK水平较MSCs-neo均明显增高;Wortmannin和PD98059可分别以剂量依赖性抑制MSCs-Sna细胞p-Akt和p-ERK水平;可有效抑制Snail诱导的MSCs细胞ERK激酶及AKT激酶磷酸化的PD98059和Wortmannin相应浓度分别为50μmol/L和40 nmol/L。(2)以GAPDH mRNA为内参照,RT-PCR结果显示,MSCs-Sna细胞中MMP-2 mRNA的表达显著高于MSCs-neo(0.680±0.104 vs. 1.803±0.301,P<0.05);PD98059(50μmol/L)、Wortmannin(40 nmol/L)明显抑制了Snail修饰诱导的MMP-2 mRNA水平,抑制率分别为33%和30%;而两者联合给予对MSCs-Sna细胞MMP-2基因表达的抑制作用具有协同作用,抑制率为56%,与MSCs-Sna组相比,差异有统计学意义(0.803±0.160 vs. 1.807±0.304, P<0.05)。(3)明胶酶谱实验显示MSCs-Sna有较高水平MMP-2活性,为对照组(MSCs-neo)的1.79±0.22倍(p<0.05);而在50μmol/L PD98059作用后下降至(1.28±0.12)倍(P<0.05);在40 nmol/L Wortmannin作用后下降至(1.24±0.21)倍(P<0.05);PD98059与Wortmannin联合干预后下降至(0.98±0.23)(P<0.05);而SB203580处理对MSCs-Sna细胞MMP-2活力的影响无显著性差异(P>0.05)。
结论Snail能促进MSCs的MMP-2的转录及MMP-2蛋白的表达。MMP-2可能是MSCs的细胞内受转录因子Snail调控作用的耙基因之一,且ERK1/2及PI3K转导通路的激活在此过程中具有调控作用。
Part I Study on the amplification of Snail eukaryotic expressing plasmid and its transfer to bone mesenchymal stem cells
Objective: To investigate the transfer and expression of Snail gene in human bone mesenchymal stem cells(MSCs), and determine its effects on biologic characteristics change of the growth of MSCs, for the purpose of the feasibility study on MSCs used as accepted cells of human Snail gene.
Methods: MSCs were isolated from adult human bone marrow via density gradient centifugation. Light microscopy was used to study the morphologic features and flow cytometry to examine the expression of cell surface antigens. Then the recombinant eukaryotic expression plasmid PCAGGSneo-Snail-HA or the control vector of PCAGGSneo was transferred into the cells by the liposome LipofectamineTM 2000. Then stably transfected cell colonies were obtained by G418 screening. After transfection, the marker gene coding HA and the destination gene coding Snail were observed with the fluorescent microscope and Western-blot. The growth curve of cell proliferation was also obtained based on the observation of the proliferation status of MSCs transfected by the vectors or not.
Results: Bone mesenchymal stem cells in original generation were all in the long shuttle-like shape and adhered to the disk. MSCs of 3rd, 5th generation were well purified and all in the whirlpool-shape. These cultured cells showed immunore activity to CD29 but not CD34.The expression of Snail and HA in MSCs transducted with PCAGGsneo-Snail-HA (MSCS-Sna), compared with the MSCS transducted with the control vector of PCAGGsneo (MSCS-neo), was confirmed by Western-Blot and Immunofluorescence both in 48hr and 4w culture interval after transfection. Growth curve showed a little longer lag in cell doubling time of cells transfected (including MSCs-Sna and MSCs-neo) than not. However there was no significant difference of growth rate and doubling time between MSCs-Sna and MSCs-neo.
Conclusion: Purified MSCs can be obtained via this protocol. Cells of 3rd, 5th generation with high proliferation are fit to gene transfer and the further experiment. The Snail gene is able to provide transient and persistent expression in MSCs,which has no significant influence on the cells’activity. This provides a further foundation for studies of effects of Snail gene expression on the survival, migration and expression of Matrix Metalloproteinase-2 in MSCs.
Part II Up-regulation of Snail expression accelerates MSCs migration in vitro through PI-3K/Akt and ERK/MAPK-dependent pathway
Objective To investigate the advantage for Snail to promote MSCs migration and to test whether PI3K or other signaling pathway is implicated in Snail-mediated MSCs migration.
Methods: Stably transfected cell colonies were obtained by Snail gene transfer with liposome and G418 screening. The role of Snail in MSCs motility was assessed by comparing the migration of MSCS-Sna with that of MSCs-neo in Transwell System. Migration was calculated from the number of cells found to have passed through an 8-um pore size polycarbonate membrane. Levels of PI3K and ERK/MAPK activity were detected by fluorescent microscope and Western-blot. To test further whether PI-3K or MAPK is implicated in Snail-mediated MSCs migration, we next used a variety of inhibitors in our migration assay.
Results: With Western blot technique, the ratio of p-Akt to Akt or of p-ERK1/2 to total ERK1/2 in MSCs-Sna was obviously higher than that in the control group of MSCs-neo (P<0.05). Furthermore, immunofluorescence staining confirmed a considerably higher quantity of p-Akt and p-ERK present in MSCs-Sna than in MSCs-neo and confirmed the Snail-induced increase in translocation of p-Akt or p-ERK1/2 to the nucleus. MSCs-Sna showed more significantly migration than MSCs-neo in the transwell migration system (p<0.05). And suppression of PI-3K activation by the specific PI-3K inhibitor, Wortmannin, or suppression of ERK activation by the specific ERK inhibitor, PD98059 brought on a reduction in Snail-mediated MSCs migration (P<0.05). In contrast, treatment with the P38 Mitogen-activated protein kinase (MAPK) inhibitor, SB203580, did not show any significant effect on the Snail-mediated cell migration (P>0.05).
Conclusion: Our results suggested that Snail expression in MSCs can increase the migration ability of MSCs. Snail-accelerated MSCS migration was mainly associated with PI-3K and ERK/MAPK, but not with P38/MAPK.
Part III Study on effects of Snail gene modification on the CXCR4 expression of human bone mesenchymal stem cells and their capacity of migration to SDF-1 in vitro
Objective: To investigate effects of Snail gene modification on the CXCR4 expression of human MSCs and their capacity of migration to SDF-1 in vitro, for providing the possibility for us to optimize the migration or targeting of transplanted MSCS toward damaged tissues where SDF-1 has been shown to be upregulated as part of the injury response.
Methods: Stably transfected cell colonies were obtained by Snail gene transfer with liposome and G418 screening. FACS analysis and immunofluorescence staining were used to study the expression of CXCR4 on the surface of MSCs-Sna and MSCs-neo. And chemokine receptor CXCR4 mRNA was detected by RT-PCR. Chemotaxis assays were performed, using the Transwell Chamber assay and SDF-1α, a ligand for CXCR4, as a chemoattractive agent in the lower chamber, to evaluate the migratory capacity of MSCs-Sna and MSCs-neo to SDF-1 in vitro. For the blocking assay, CXCR4 bloking antibody(1μg/ml, 10μg/ml) was added into cell culture.
Results: Fluorescence-activated cell sorting(FACS) analysis showed that CXCR4 expression was higher in the Snail transfectants of MSCs(MSCs-Sna) than that in the vector control cells(MSCs-neo) (64.25%±7.22% vs. 7.45%±0.91%, P<0.05). The increase in CXCR4 expression by Snail was further supported by fluorescence confocal microscopy. Furthermore, CXCR4 mRNA was enhanced more in MSCs-Sna than in MSCs-neo, confirmed by RT-PCR(0.916±0.102 vs. 0.410±0.050,P<0.05). Chemotaxis assays showed that SDF-1αstimulated the in vitro migratory activity in MSCs-Sna more than MSCs-neo(p<0.05). Moreover, the SDF-1α-induced migration activity of MSCs-Sna was inhibited in a concentration-dependent manner by a CXCR4-blocking antibody, but not by a control IgG antibody.
Conclusion: Snail enhances CXCR4 expression by MSCs, providing a plausible mechanism for Snail-mediated MSCs transmigration in vitro or metastasis to damaged tissues in vivo where SDF-1 has been shown to be upregulated as part of the injury response.
PartⅣThe advantages for Snail expression to protect against serum-deprivation triggered actin cytoskeleton damage and apoptosis of bone mesenchymal stem cells
Objective: To examine whether Snail gene expression acts in the cytoskeleton stabilization and anti-apoptosis capacity of MSCs, providing the feasibility for transplanted MSCs to survive and maintain subsequently in the local microenvionment of injured tissue.
Methods: Actin cytoskeleton staining by Immunofluorescence was performed to analyze the cytoskeleton of MSCs-Sna. The percentages of sub-G1 apoptotic cells and the annexin-V+PI- apoptotic cells were detected by flow cytometry.
Results: After the cells were cultured in serum-free medium for 24 h, MSCs-Sna maintain the normal distribution ofβ-Actin, and there were a few bundles of stress filaments running the whole length of the cell body; however MSCs-neo displayed the disordered actin cytoskeleton, though the cellular outline still remained. Furtheremore, When cultured in in serum-free medium for 72 h, MSCs-neo even couldn’t maintain the essential cellular shape, associated with damaged, depolymerized and aggregated filaments of the cytoskeletons, compared to the MSCs-Sna, which still appeared normal cellular shape with stable and well organized cytoskeletal network. Both MSCS-Sna and MSCS-neo cultured in conventional medium exhibited a low proportion of apoptotic cells as indicated by the “Sub-G1 peak”and the annexin-V+PI- cell percentage. We failed to detect the significant difference in apoptosis between MSCS-neo and MSCS-Sna, after the cells were cultured in serum-free medium for 1 day. Apoptotic percentages in both MSCS-Sna and MSCS-neo were increased after incubation in serum-free medium (3 days). However these percentages were significantly lower in MSCs-Sna (34.33%±5.51% and 12.5%±2.2%) than MSCs-neo (52%±8.19% and 30%±4.3%)(P<0.05). MSCs-neo or MSCs-Sna were examined following a 24-h cultured in complete medium with or without IL-4(20ng/ml). The apoptotic annexin-V+PI- MSCs-neo percentage was 5-fold enhanced by addition of IL-4. However the annexin-V+PI- percentage of MSCs-Sna cultured in the presence of IL-4 was just 2.6-fold enhanced, compared with controls, cultured in medium without IL-4.
Conclusion: MSCs overexpressing Snail exhibited a greater resistance to the cytoskeleton damage and serum-deprivation or IL-4 triggered-apoptosis of MSCs. These support the idea that Snail may play a role in survival capacity of MSCs.
Part V Snail gene modification stimulates Matrix Metalloproteinase-2 production by human bone mesenchymal stem cells through activation of ERK and PI3K pathways
Objective: To examine whether Snail-induced expression of matix metalloproteinase-2 (MMP-2) is involved in MSCs and investigate the molecular mechanism involved in it.
Methods: After both MSCs-Sna and MSCs-neo were treated with PD98059(0、10、30、50、100μmol/L)or Wortmannin(0、10、20、40、80 nmol/L) for 24h, the levels of phosphorylated Akt(p-Akt) and phosphorylated ERK1/2 (p-ERK1/2) were examined by Western blot to determine the effective concentrations for PD98059 and Wortmanin to inhibit the Snail-induced ERK and PI3K activity of cells. Then we employed these specific inhibitors with respective effective concentrations to incubate MSCs-Sna and MSCs-neo and detect the expression of MMP-2 mRNA and gelatinolytic activity by MSCs-Sna or MSCs-neo with RT-PCR and gelatin zymographic assay.
Results: (1) Increased levels of p-ERKs (phospho-p42 and phospho-p44) and active AKT, but not phosphor-p38, in BMSC-Sna, were confirmed by Western-blots. Snail-induced increased activity of p-Akt and p-ERKs in MSCs-Sna could be inhibited in a concentration-dependent manner by Wortmannin and PD98059. The effective concentrations for PD98059 and Wortmanin to inhibit the Snail-induced ERK and PI3K activity of cells are 50μmol/L and 40nmol/L respectively. (2)&(3) MMP2 mRNA was up-regulated in MSCs-Sna than in MSCs-neo, which is confirmed by RT-PCR(0.680±0.104 vs. 1.803±0.301,P<0.05). The significantly decreased levels in the MMP-2 gelatinalytic activity by BMSC-Sna exposed to PD98059 (PD, 50μmol/L) or Wortmannin (WM, 40 nmol/L), compared to the MSCs-Sna untreated, were detected in zymographic assay (p<0.05). Furthermore, treatment with a combination of PD and WM induced such a decrease more (p<0.05). However MSCs-Sna exposed to the inhibitor of p38 pathway (SB203580) did not show a significant decrease of MMP-2 mRNA expression and of the gelatinolytic activity of the latent 72-kDa form of MMP-2, compared with MSCs-Sna untreated(P>0.05).
Conclusion: Overexpression of Snail can induce expression and secretion of MMP-2 by MSCs through activation of ERK/MAPK and PI3K signaling pathways.
引文
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