反义整合素真核表达载体转染及恒磁场干预对动脉平滑肌细胞生物行为的影响
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摘要
【研究背景】经皮腔内冠状动脉成形术(PTCA)及支架植入术应用于临床以来，术后20％～30％的再狭窄(RS)率一直制约其远期疗效，因此也成为心血管领域研究的热点和难点问题。随着细胞生物学和分子生物学的飞速进展，细胞外基质(ECM)受到广泛关注和研究。ECM如纤维粘连蛋白(FN)、胶原(Col)和层粘连蛋白(LN)，通过其相应的细胞膜粘附分子整合素受体，激活细胞内多个信号转导途径，调节细胞的粘附、迁移、增殖、分化以及基因表达等多方面生物行为，参与RS形成和发展。因此我们设想，通过阻断ECM－细胞－整合素受体构成的细胞与基质的粘附系统内部之间的联系，并进而阻断其受体后信号转导途径，就可达到阻断平滑肌细胞过度增殖、迁移和基质大量合成这三个关键的环节，可望成为抑制再狭窄的新策略。但是ECM中各种成分如何对平滑肌细胞进行调节，整合素受体各亚基的表达水平及介导产生怎样的效应，均不十分清楚。
【目的】本实验旨在探讨ECM中两种重要成分纤维粘连蛋白(FN)和层粘连蛋白(LN)及其整合素受体α_5β_1对平滑肌细胞生物功能的调节，并进一步采用反义基因转染和物理因素恒磁场干预，观察对细胞-ECM之间相互作用及信号转导的作用，为RS治疗提出新的靶点。
【方法】对人脐动脉平滑肌细胞的原代培养方法进行改进。建立体外FN和LN可溶性和不容性两种物理状态，以模拟体内的病理情况。采用~3H-TdR掺入和MTT法检测细胞增殖能力，~3H-羟脯氨酸检测细胞合成胶原能力，进行细胞粘附实验、Transwell细胞迁移实验检测细胞粘附和迁移能力。流式细胞仪检测细胞周期。单标记和双标记间接免疫荧光染色检测细胞整合素α_5和β_1、FN及细胞骨架蛋白α-SM-Actin的表达和分布。构建整合
刃四旱区大学俗士学世公文7
素反义真核表达载体AS心和AS仙转染平滑肌细胞，或用不同磁感应强
度的恒磁场干预细胞，透射电镜检测干预后细胞超微结构变化，流式细胞
仪检测细胞周期变化，厂乍stem Blot检测细胞表达整合素蛋白和磷酸化FA K
水平，DOt BIOt检测转染前后细胞整合素tRNA水平，用 FltO－3／AM标iC
细胞内时＼ 激光扫描共聚焦显微镜枝术检测细胞内游离比”浓度变化。
【结果】
一、人动脉平滑肌细胞原代培养方法的改良
小动脉平滑肌细胞培养可以采用动脉环直接接种的方法进行培养，接
种后培养瓶翻转前时间适当延长至5－6h，可以使组织块更好贴壁以及去除
成纤维细胞的污染。加入较高浓度bFGF和EGF，使长出时间提前5－7天，
而且模拟了体内局部病理环境。加入NaHCO；无细胞毒性。鉴定平滑肌细
胞时应选取原代培养的前3代，免疫细胞化学SABC法染色及间接免疫荧
光染色证实细胞表达a－SM－Actin，分别呈黄褐色丝状及丝状红色荧光分布
于胞浆内，透射电镜可见典型的平滑肌细胞肌丝形成的密斑和密体及吞饮
小泡。
二、不同物理状态的FN和LN对细胞生物行为的影响
卜 不同物理状态的FN和LN对细胞生物行为的作用不同。用20、40、60、
80、100 pg．／mL五种浓度65 FN和 LN，友现随浓度逐渐增加，iFN组fro胞
吸光度值、‘H＊dR掺入量、细胞粘附百分数、迁移细胞数均显著增加，iLN
则轻度促进增殖和粘附，而抑制细胞迁移，并呈浓度依赖性。sFN和sLN
对 hUASMCS的增殖、粘附呈浓度依赖性抑制，而 SLN则促进细胞迁移。
二 分别力。、抗整合素。；和pl亚基的抗体PIh年NCIO、＊＊＊mP短肽、
酪氨酸激酶抑制剂Gel。istein和钙通道阻断剂Nicaxdipine千预rN和几N介
导的细胞生物行为，友现对细胞增殖和粘附的抑制强度是Genistein＞
GRGDSP短肽＞P4C一PI D6＞Nicardipine。对细胞迁移的子 制强度是
GRGDSP短肽＞P4C10＞G＞flistsifl＞PID6＞NICCdlplflfl。
3．浓度为 60～100卜u／mL的 iFN和 sLN，显著促进 hUASMCs的胶原合成
能力，iFN较 sLN作用更强。但低浓度 20、40pg／mL与对照组无明显差别。
而 SFN、iLN则抑制细胞合成胶原。
4．iFN使细胞表达整合素 15和A 增加，并使其形成微。］、簇状粘着斑，o
sFN组整合素a。和p；及 Actin呈弥散分布，没有形成微小簇状粘着斑。
三、转染反义整合素l。和pl真核表达载体对平滑肌细胞生物行为的改变
l．pECE－a。和pECE｝为模板，进行修饰聚合酶链反应（mPCR），得到两
I’Is伽ie OfO汕ov地cular砌eme风WU 2002
剪曰旱巨大攀俗士学位论文 吕
端分别带有ECORI和HIOdlll酶
The study aimed to investigate the roles of two important ECM protein fibronectin, laminin and their receptor integrin alpha5betal in regulating the VSMCs biological behaviors. And using gene blockade by antisense integrin vector transfection or physically intervening by
Institute of Cardiovascular disease FMMU 2002
static magnetic field, we studied its effects on interaction between VSMCs and ECM and intracellular signal transduction, and provide a new therapeutic target for RS. [ METHODS]
It was successfully improved in primary culture method of human umbilical artery smooth muscle cells(hUASMCs). The coated fibronectin(FN) or laminin(LN) were used as insoluble FN(iFN) or insoluble LN(iLN), while those added to the medium directly were used as soluble FN and LN(sFN and sLN). Under these two status, DNA and collagen synthesis of hLASMCs were determined by H-thymidine and H梡roline incorporation. Total viable cells was detected by MTT assay. Adhesion and Transwell migration assay were also conducted to test the ability of adhesion to FN and LN and migration on them. The expression and distribution of integrin alphaS.hetal and alpha-SM-actin were determined by single or double條abelled indirect immunofluore.scent staining. We further constructed the antisense eukaryotir vectors of integrin alpha5 and betal subunits and transfected hUASMCs, or intervened hLASMCs with static magnetic field(SMF) with different intensity. Transmission electron microscopy showed the ultrastructure of hUASMCs after transfection or exposure to SMF. The expression of integrin alphaS and betal and phosphorylation of focal adhesion kinese(FAK) of hUASMCs were determined by Western Blotting. Dot Boltting were performed to detect the mRNA level of integrin alphaS and betal. The cell cycle were analyzed by flow cytometry alter pmpidium iodide(PI) staining. Intracellular free calcium ion were loaded with fluorescent probe Fluo?/AM and detected by laser scanning confocal microscopy.
[ RESULTS]
1. Modification of primary culture method of human umbilical artery smooth muscle cells.
The umbilical artery were cut into 3?mm artery rings and vertically attached in the culture flask. Do not turn over the flask to soak the artery rings in medium until 5-6 h later, which was longer than usual for better attachment and get ride of the flbroblasts contamination. The primary culture time were shortened by adding higher doses of bFGF and EGF than what reported. There was no rytotoxicity detected when NaHCOi was used. Identification of SMCs had better been done within the first three passages. With the above methods, we got the pure hUASMCs that were confirmed by immunocyto-chemistry and immunofluorescence staining of alpha桽M梐ctin. Transmission electron microscopy demonstrated the typical characteristics o( "dense patch" or "dense body" .
2. Effects of FN and LN in different physical status on die biological behavior of hUASMCs and mechanism.
Institute of Cardiovascular disease FMMU 2002
12
1). Different physical status of FN and LN showed adverse roles in mediating the biological behavior of hUASMCs. In presence of iFN at 20,40,60,80 and 100 iig/mL, the absorbance value, adhesion percentage, migratory cell numbers were increased remarkably in a concentration-dependent manner. iLN also increased the cell adhesion and proliferation, but inhibit migration and collagen syntgesis at the same concentrations. In contrast, sFN and sLN displayed a concentration?dependent inhibition of cell adhesion and proliferation except that sLN could increase the migration and collagen synthesis of hUASMCs slightly. 2). hUASMCs were pretreated with the blocking antibody against integrin alpha5 P1D6 and betal P4C10. GRGDSP. tyrnsine kinase inhibitor genistein and calcium channel blocker nicardipine to explore the mechanism of FN?and LN-mediated effects. The strongest inhibition of adhesion and proliferation were genistein, then came to or P1D6 and nicardipine. While towards migration. GRGDSP, P4C10 was the first two powerful inhibitors
【目的】本实验旨在探讨ECM中两种重要成分纤维粘连蛋白(FN)和层粘连蛋白(LN)及其整合素受体α_5β_1对平滑肌细胞生物功能的调节，并进一步采用反义基因转染和物理因素恒磁场干预，观察对细胞-ECM之间相互作用及信号转导的作用，为RS治疗提出新的靶点。
【方法】对人脐动脉平滑肌细胞的原代培养方法进行改进。建立体外FN和LN可溶性和不容性两种物理状态，以模拟体内的病理情况。采用~3H-TdR掺入和MTT法检测细胞增殖能力，~3H-羟脯氨酸检测细胞合成胶原能力，进行细胞粘附实验、Transwell细胞迁移实验检测细胞粘附和迁移能力。流式细胞仪检测细胞周期。单标记和双标记间接免疫荧光染色检测细胞整合素α_5和β_1、FN及细胞骨架蛋白α-SM-Actin的表达和分布。构建整合
刃四旱区大学俗士学世公文7
素反义真核表达载体AS心和AS仙转染平滑肌细胞，或用不同磁感应强
度的恒磁场干预细胞，透射电镜检测干预后细胞超微结构变化，流式细胞
仪检测细胞周期变化，厂乍stem Blot检测细胞表达整合素蛋白和磷酸化FA K
水平，DOt BIOt检测转染前后细胞整合素tRNA水平，用 FltO－3／AM标iC
细胞内时＼ 激光扫描共聚焦显微镜枝术检测细胞内游离比”浓度变化。
【结果】
一、人动脉平滑肌细胞原代培养方法的改良
小动脉平滑肌细胞培养可以采用动脉环直接接种的方法进行培养，接
种后培养瓶翻转前时间适当延长至5－6h，可以使组织块更好贴壁以及去除
成纤维细胞的污染。加入较高浓度bFGF和EGF，使长出时间提前5－7天，
而且模拟了体内局部病理环境。加入NaHCO；无细胞毒性。鉴定平滑肌细
胞时应选取原代培养的前3代，免疫细胞化学SABC法染色及间接免疫荧
光染色证实细胞表达a－SM－Actin，分别呈黄褐色丝状及丝状红色荧光分布
于胞浆内，透射电镜可见典型的平滑肌细胞肌丝形成的密斑和密体及吞饮
小泡。
二、不同物理状态的FN和LN对细胞生物行为的影响
卜 不同物理状态的FN和LN对细胞生物行为的作用不同。用20、40、60、
80、100 pg．／mL五种浓度65 FN和 LN，友现随浓度逐渐增加，iFN组fro胞
吸光度值、‘H＊dR掺入量、细胞粘附百分数、迁移细胞数均显著增加，iLN
则轻度促进增殖和粘附，而抑制细胞迁移，并呈浓度依赖性。sFN和sLN
对 hUASMCS的增殖、粘附呈浓度依赖性抑制，而 SLN则促进细胞迁移。
二 分别力。、抗整合素。；和pl亚基的抗体PIh年NCIO、＊＊＊mP短肽、
酪氨酸激酶抑制剂Gel。istein和钙通道阻断剂Nicaxdipine千预rN和几N介
导的细胞生物行为，友现对细胞增殖和粘附的抑制强度是Genistein＞
GRGDSP短肽＞P4C一PI D6＞Nicardipine。对细胞迁移的子 制强度是
GRGDSP短肽＞P4C10＞G＞flistsifl＞PID6＞NICCdlplflfl。
3．浓度为 60～100卜u／mL的 iFN和 sLN，显著促进 hUASMCs的胶原合成
能力，iFN较 sLN作用更强。但低浓度 20、40pg／mL与对照组无明显差别。
而 SFN、iLN则抑制细胞合成胶原。
4．iFN使细胞表达整合素 15和A 增加，并使其形成微。］、簇状粘着斑，o
sFN组整合素a。和p；及 Actin呈弥散分布，没有形成微小簇状粘着斑。
三、转染反义整合素l。和pl真核表达载体对平滑肌细胞生物行为的改变
l．pECE－a。和pECE｝为模板，进行修饰聚合酶链反应（mPCR），得到两
I’Is伽ie OfO汕ov地cular砌eme风WU 2002
剪曰旱巨大攀俗士学位论文 吕
端分别带有ECORI和HIOdlll酶
The study aimed to investigate the roles of two important ECM protein fibronectin, laminin and their receptor integrin alpha5betal in regulating the VSMCs biological behaviors. And using gene blockade by antisense integrin vector transfection or physically intervening by
Institute of Cardiovascular disease FMMU 2002
static magnetic field, we studied its effects on interaction between VSMCs and ECM and intracellular signal transduction, and provide a new therapeutic target for RS. [ METHODS]
It was successfully improved in primary culture method of human umbilical artery smooth muscle cells(hUASMCs). The coated fibronectin(FN) or laminin(LN) were used as insoluble FN(iFN) or insoluble LN(iLN), while those added to the medium directly were used as soluble FN and LN(sFN and sLN). Under these two status, DNA and collagen synthesis of hLASMCs were determined by H-thymidine and H梡roline incorporation. Total viable cells was detected by MTT assay. Adhesion and Transwell migration assay were also conducted to test the ability of adhesion to FN and LN and migration on them. The expression and distribution of integrin alphaS.hetal and alpha-SM-actin were determined by single or double條abelled indirect immunofluore.scent staining. We further constructed the antisense eukaryotir vectors of integrin alpha5 and betal subunits and transfected hUASMCs, or intervened hLASMCs with static magnetic field(SMF) with different intensity. Transmission electron microscopy showed the ultrastructure of hUASMCs after transfection or exposure to SMF. The expression of integrin alphaS and betal and phosphorylation of focal adhesion kinese(FAK) of hUASMCs were determined by Western Blotting. Dot Boltting were performed to detect the mRNA level of integrin alphaS and betal. The cell cycle were analyzed by flow cytometry alter pmpidium iodide(PI) staining. Intracellular free calcium ion were loaded with fluorescent probe Fluo?/AM and detected by laser scanning confocal microscopy.
[ RESULTS]
1. Modification of primary culture method of human umbilical artery smooth muscle cells.
The umbilical artery were cut into 3?mm artery rings and vertically attached in the culture flask. Do not turn over the flask to soak the artery rings in medium until 5-6 h later, which was longer than usual for better attachment and get ride of the flbroblasts contamination. The primary culture time were shortened by adding higher doses of bFGF and EGF than what reported. There was no rytotoxicity detected when NaHCOi was used. Identification of SMCs had better been done within the first three passages. With the above methods, we got the pure hUASMCs that were confirmed by immunocyto-chemistry and immunofluorescence staining of alpha桽M梐ctin. Transmission electron microscopy demonstrated the typical characteristics o( "dense patch" or "dense body" .
2. Effects of FN and LN in different physical status on die biological behavior of hUASMCs and mechanism.
Institute of Cardiovascular disease FMMU 2002
12
1). Different physical status of FN and LN showed adverse roles in mediating the biological behavior of hUASMCs. In presence of iFN at 20,40,60,80 and 100 iig/mL, the absorbance value, adhesion percentage, migratory cell numbers were increased remarkably in a concentration-dependent manner. iLN also increased the cell adhesion and proliferation, but inhibit migration and collagen syntgesis at the same concentrations. In contrast, sFN and sLN displayed a concentration?dependent inhibition of cell adhesion and proliferation except that sLN could increase the migration and collagen synthesis of hUASMCs slightly. 2). hUASMCs were pretreated with the blocking antibody against integrin alpha5 P1D6 and betal P4C10. GRGDSP. tyrnsine kinase inhibitor genistein and calcium channel blocker nicardipine to explore the mechanism of FN?and LN-mediated effects. The strongest inhibition of adhesion and proliferation were genistein, then came to or P1D6 and nicardipine. While towards migration. GRGDSP, P4C10 was the first two powerful inhibitors
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