胎盘源性间充质干细胞及血管内皮生长因子对缺血性皮瓣促愈作用的基础研究
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摘要
本研究目的是探索人胎盘源间充质干细胞(HPMSC_S)能否向创伤修复过程中起关键作用的创伤修复细胞---血管内皮细胞分化;并构建PIRE2-EGFP-VEGF双顺反子真核质粒表达载体,并运用脂质体转染法将该真核质粒表达载体转染至HPMSC_S内;通过腹腔内注射途径探索HPMSC_S能否携带目的基因向创伤局部归巢扩充,HPMSC_S本身对创伤修复的促进作用及其作用机制,以及它是否与VEGF在创伤修复过程中有协同作用,探索HPMSC_S及VEGF对超比例皮瓣的促愈作用。
本实验首先通过密度梯度离心法从废弃的胎盘组织中分离、培养了间充质干细胞,并通过流式分析仪对其表面标志加以分析,结果表明该细胞与骨髓源间充质干细胞的表面标志基本相同;并绘制了生长曲线,结果表明该细胞符合干细胞基本特征;为了鉴定HPMSC_S的多向分化能力,我们进行了成脂肪、成骨诱导鉴定,结果表明,该细胞完全具有多向分化潜能。
然后将鉴定后的HPMSC_S加入内皮细胞诱导分化培养体系中,向内皮细胞诱导分化。研究结果显示,HPMSC_S在培养基中持续培养后,形态由“类成纤维细胞样”转变为类似内皮细胞的“铺路石样结构”。成熟内皮细胞特异的表面标志vWF在未诱导的HPMSC_S中不表达,但在这种内皮样细胞却为阳性表达。流式细胞仪检测显示,内皮细胞表面标志CD31、CD34的表达量较未诱导前均有所增加, Dil-Ac-LDL摄取实验发现,诱导后的内皮样细胞具有摄取Dil-Ac-LDL的能力。以上说明本实验的诱导方案能够诱导HPMSC_S出现内皮细胞表型,且随着诱导时间的延长其表型特征也愈加明显,提示HPMSC_S在体外特定条件下具有定向分化为内皮细胞的潜能。
为了研究HPMSC_S能否充当VEGF基因的转运载体,本实验首先构建了PIRE2-EGFP-VEGF双顺反子真核质粒表达载体,并采用脂质体转染法,用Lipofectin脂质体成功将真核表达载体PIRES2-EGFP-VEGF_(165)转入HPMSC_S中,经绿色荧光显微镜下观察,可见EGFP表达的绿色荧光蛋白,提示EGFP已成功转入靶细胞。证明脂质体介导的PIRES2-EGFP-VEGF_(165)转染HPMSC_S是可行的。转染细胞经G418筛选后,出现数量较多的细胞克隆,而非转染组细胞100%死亡,提示VEGF基因转染HPMSC_S的效果和G418筛选的效果都较好。通过VEGF的免疫印迹分析(Western-blot技术)发现有较强VEGF表达,而空载体转染组则表达较弱。通过MTT法检测VEGF的生物学活性发现:转染目的基因的HPMSC_S所表达的VEGF生物学活性较空载体组及对照组明显提高,而空载组要高于对照组,这些结论提示:一方面VEGF_(165)在HPMSC_S中有较强表达并被正确修饰,HPMSC_S可以作为基因治疗的载体细胞;另一方面提示HPMSC_S也可能具有内分泌VEGF的功能,但作用较弱。为了防止转染外源性基因的HPMSC_S出现基因突变,失去多向分化潜能,我们又对转染外源基因的HPMSC_S进行鉴了多向诱导分化定,结果表明,转染后的HPMSC_S仍旧保持干细胞特性。
最后,本实验通过将标记BrdU的携带目的基因pIRES2-EGFP-VEGF_(165)的HPMSC_S注射于大鼠腹腔内,通过皮瓣抗BrdU免疫组化观察了HPMSC_S的迁徙归巢特性,发现实验组皮瓣远端缺血区域内可见被BrdU标记的HPMSC_S广泛分布,且多分布于表皮层及真皮层下方的新生血管周围,说明HPMSC_S具有向创伤缺血部位表皮及真皮下方归巢扩充的特性,并有可能向内皮细胞分化,促进血管新生。通过皮瓣免疫组化染色及微血管计数发现,携带目的基因及空载体的HPMSC_S组以及单纯应用VEGF组皮瓣真皮下方及表皮层内可见大量黄染颗粒,说明皮瓣真皮下方及表皮层内有大量内皮细胞及微血管形成,但空载体组数量少于携带目的基因组及VEGF组,携带目的基因组黄染颗粒细胞数目最多,这就说明VEGF与HPMSC_S联合作用促血管形成作用大于单纯HPMSC_S或VEGF作用,说明VEGF与HPMSC_S有协同作用,VEGF可以促进HPMSC_S向内皮细胞分化、增殖,促进血管新生,HPMSC_S很可能通过内分泌或旁分泌促血管形成因子来间接促进血管新生。HE染色组织病理学检查也证实,携带目的基因pIRES2-EGFP-VEGF_(165)的HPMSC_S的皮瓣下方细胞成分及新生血管均最多,而携带空载体的HPMSC_S皮瓣下方细胞成分丰富,但血管数量少于携带目的基因组及VEGF组,这也证实,HPMSC_S具有向修复细胞表型分化的特点,但其促血管形成作用低于VEGF与HPMSC_S联合作用及单纯应用VEGF。皮瓣成活率检测表明:携带目的基因的HPMSC_S促进皮瓣成率最高,其次为VEGF组,再次为空载组,三者均较对照组有明显促进皮瓣成活的作用。
Tissue coloboma after trauma or tumor operation usually requires the use of the skin flap transplanting to restore. Over the years, even though designs and surgical techniques of the skin flap have improved continuously, and have tried to adopt a number of materia medica to improve the survival of skin flap, but this problem that because of the hypoxia-ischemia in remote tissue of the skin flap and venous reflux impeded after the skin flap transplanting often leading to the dead of the transplanting skip flap tissue has always plagued clinicians. The hot issue on injury and reconstruction of surgery field research is on how to improve the survival rate of over-length skin flap and expand clinical application of random skin flap. Many scholars found through research that there are two keys to solve this problem: (1) after the skin flap transplanting, repair in trauma process should be started as soon as possible, mobilize repair in trauma factor fully, speed up the repair in trauma of the skin flap transplanting section, and promote "bonding" between skin flap with the recipient site, a skin flap with the surrounding tissue(;2) speeding up reconstruction of the remote skin flap ischemia section blood circulation, resolve remote skin flap hypoxia-ischemia status. The two combined actions can promote the survival of super-ratio skin flap effectively.
VEGF (Vascular endothelial growth factor) is a secreted glycoprotein, the proliferation is strong, and is easy to reach the target cells, and has the promoting effect on migration, proliferation, growth of vascular endothelial cell; and can also promote proliferation, migration of the fibroblast; and stimulates the partial reconstruction cell gathering in trauma, has a strong role in promoting repair in trauma, synthesizes and secretes extracellular matrix, has strong promoting repair in trauma. It is only known cytokines on the purpose of treatment of ischemia- hypoxia through direct effects on vascular endothelial cell surface receptors and rapid formation of new blood vessels, in the short term reconstruction blood circulation of the ischemia section. Although VEGF has an efficient ability to promote angiogenesis, but it has very short half-life and is difficult to play persistent effect with expensive price, so it is difficult to be promoted in practical applications. Therefore, looking for a suitable VEGF sustained-release system or VEGF persistent effect system is in urgent need of solution currently.
In recent years, it was discovered that stem cells has important applications capacity in the field of repair in trauma. MSCs (mesenchymal stem cells) and ADSCS (adipose-derived stem cells) could be mobilized, homed and expanded to the injury point to participate in repair in trauma. the specific capacity of MSCS and ADSCS is that they can move directly to injury tissue can be confirmed in the repair in skin trauma, it is proved that MSCS has been involved in the whole process of repair in trauma, MSCS and ADSCS can home to the injury point or differentiate into phenotype of injury cells, and it also can promote the regeneration of the tissue endogenous cell by creating the enhancement of micro-environment. But at present, the fact whether the placenta-derived stem cells in repair in trauma process can home to the trauma section and play a catalytic role in repair in trauma is not reported now. Moreover, stem cells can also act as vector cells of gene therapy, transport gene to target section, exert therapeutic effects. Such as MSC etch adult stem cells can be used as gene therapy vector of multi-diseases of the central nervous system; MSC, rADSC, EPC etch can be used as therapy vector for VEGF, FGF etch cytokines to promote the establishment of collateral circulation and angiogenesis. But at present there is very rare report about HPMSC_S as gene therapy vector
In order to explore whether HPMSC_S can home to the trauma section and can promote repair in trauma, as well as whether it can act as a vector cell of VEGF cells to deliver VEGF to trauma section, exert persistent effect of VEGF, we carried out an experiment as follows:
1. To obtain HPMSCs from the placenta, this experiment separated mononuclear cells from the placenta perfusate using density gradient centrifugation, cultivated adherence, further selected independent clones to cultivate separately, purified HPMSC_S cloning from adherent cells. We've detected its general biological characteristics, and differentiated into fat, bone to identify its multidirectional differentiation potentiality through directed induction.
2 In order to explore whether HPMSCs can differentiate to repair in trauma cells phenotype, we carried out the experiment on "human placenta mesenchymal stem cells (HPMSCs) inducing in vitro to differentiate into vascular endothelial ", by optimizing the endothelial nutrient fluid, after the directed inducing HPMSCs to cultivate 3 d passage, cell morphology changed ,when passage cell grew to 80% ~ 90% confluence, expressed analogy endothelial "cobblestone"-like structure known as "endothelial-like cells", back cell body inducing 7 d or 14 d retracted gradually, enhanced a sense of three-dimension. Specific surface mark vWF of mature endothelial cell did not express in not induced HPMSC_S, but such endothelial-like cells were positive expression. Flow cytom -etry showed that the expression of CD31 was 22.39%, but it had increased compared to not induced HPMSCs; but CD34 was the same to not induced, endothelial-like cells did not express after induction. Dil-Ac-LDL uptake experiments revealed that endothelial-like cells after induction had the ability to uptake Dil-Ac-LDL. In summary, the current induced scheme can induce HPMSCs to appear endothelial cell phenotype, and its phenotypic characteristics are more obvious as the extension of the induction time, HPMSCs has the capacity to direct and different -iate into endothelial cells in vitro specific conditions.
3 In order to prove that HPMSC_S has the function of gene therapy vector cell, the experiment established a pIRES2-EGFP-VEGF_(165) eukaryotic expression vector, and transferred pIRES2-EGFP-VEGF_(165) into HPMSC_S by liposome method successfully, and identified that HPMSC s with gene still has stem cell characteristics by multidirection induced differentiation..
4 In order to explore whether HPMSC_S with pIRES2-EGFP-VEGF_(165) can home to repair in trauma and promote blood circulation in ischemic section, we carried out an vitro experimental study on〝basic research on HPMSC_S of pIRES2-EGFP-VEGF_(165) eukaryotic expression vector impact on the survival of ischemic skin flap〞. Through research we confirmed that HPMSC_S with gene has characteristics to home to repair in trauma section, and differentiate to repair in trauma cell and promote blood circulation of ischemic section, improve the survival of the ultra proportion skin flap.
Sum up fully, this study confirms by experiment that the placenta-derived stem cells has the characteristic to differentiate to repair in trauma cell phenotype , and it can be used as vector cells of the gene therapy to deliver target genes to trauma section, play a role in promoting repair in trauma , and maintain its multi-directional differentiation potentiality; VEGF can express vector by constructed eukaryote, and transfer into HPMSCS, transport it to its target section through the vector cells , and play sufficient effect. This study further improves the theory of the stem cells family to promote repair in trauma, and also provides a new way and methods for VEGF sufficient effect.
本实验首先通过密度梯度离心法从废弃的胎盘组织中分离、培养了间充质干细胞,并通过流式分析仪对其表面标志加以分析,结果表明该细胞与骨髓源间充质干细胞的表面标志基本相同;并绘制了生长曲线,结果表明该细胞符合干细胞基本特征;为了鉴定HPMSC_S的多向分化能力,我们进行了成脂肪、成骨诱导鉴定,结果表明,该细胞完全具有多向分化潜能。
然后将鉴定后的HPMSC_S加入内皮细胞诱导分化培养体系中,向内皮细胞诱导分化。研究结果显示,HPMSC_S在培养基中持续培养后,形态由“类成纤维细胞样”转变为类似内皮细胞的“铺路石样结构”。成熟内皮细胞特异的表面标志vWF在未诱导的HPMSC_S中不表达,但在这种内皮样细胞却为阳性表达。流式细胞仪检测显示,内皮细胞表面标志CD31、CD34的表达量较未诱导前均有所增加, Dil-Ac-LDL摄取实验发现,诱导后的内皮样细胞具有摄取Dil-Ac-LDL的能力。以上说明本实验的诱导方案能够诱导HPMSC_S出现内皮细胞表型,且随着诱导时间的延长其表型特征也愈加明显,提示HPMSC_S在体外特定条件下具有定向分化为内皮细胞的潜能。
为了研究HPMSC_S能否充当VEGF基因的转运载体,本实验首先构建了PIRE2-EGFP-VEGF双顺反子真核质粒表达载体,并采用脂质体转染法,用Lipofectin脂质体成功将真核表达载体PIRES2-EGFP-VEGF_(165)转入HPMSC_S中,经绿色荧光显微镜下观察,可见EGFP表达的绿色荧光蛋白,提示EGFP已成功转入靶细胞。证明脂质体介导的PIRES2-EGFP-VEGF_(165)转染HPMSC_S是可行的。转染细胞经G418筛选后,出现数量较多的细胞克隆,而非转染组细胞100%死亡,提示VEGF基因转染HPMSC_S的效果和G418筛选的效果都较好。通过VEGF的免疫印迹分析(Western-blot技术)发现有较强VEGF表达,而空载体转染组则表达较弱。通过MTT法检测VEGF的生物学活性发现:转染目的基因的HPMSC_S所表达的VEGF生物学活性较空载体组及对照组明显提高,而空载组要高于对照组,这些结论提示:一方面VEGF_(165)在HPMSC_S中有较强表达并被正确修饰,HPMSC_S可以作为基因治疗的载体细胞;另一方面提示HPMSC_S也可能具有内分泌VEGF的功能,但作用较弱。为了防止转染外源性基因的HPMSC_S出现基因突变,失去多向分化潜能,我们又对转染外源基因的HPMSC_S进行鉴了多向诱导分化定,结果表明,转染后的HPMSC_S仍旧保持干细胞特性。
最后,本实验通过将标记BrdU的携带目的基因pIRES2-EGFP-VEGF_(165)的HPMSC_S注射于大鼠腹腔内,通过皮瓣抗BrdU免疫组化观察了HPMSC_S的迁徙归巢特性,发现实验组皮瓣远端缺血区域内可见被BrdU标记的HPMSC_S广泛分布,且多分布于表皮层及真皮层下方的新生血管周围,说明HPMSC_S具有向创伤缺血部位表皮及真皮下方归巢扩充的特性,并有可能向内皮细胞分化,促进血管新生。通过皮瓣免疫组化染色及微血管计数发现,携带目的基因及空载体的HPMSC_S组以及单纯应用VEGF组皮瓣真皮下方及表皮层内可见大量黄染颗粒,说明皮瓣真皮下方及表皮层内有大量内皮细胞及微血管形成,但空载体组数量少于携带目的基因组及VEGF组,携带目的基因组黄染颗粒细胞数目最多,这就说明VEGF与HPMSC_S联合作用促血管形成作用大于单纯HPMSC_S或VEGF作用,说明VEGF与HPMSC_S有协同作用,VEGF可以促进HPMSC_S向内皮细胞分化、增殖,促进血管新生,HPMSC_S很可能通过内分泌或旁分泌促血管形成因子来间接促进血管新生。HE染色组织病理学检查也证实,携带目的基因pIRES2-EGFP-VEGF_(165)的HPMSC_S的皮瓣下方细胞成分及新生血管均最多,而携带空载体的HPMSC_S皮瓣下方细胞成分丰富,但血管数量少于携带目的基因组及VEGF组,这也证实,HPMSC_S具有向修复细胞表型分化的特点,但其促血管形成作用低于VEGF与HPMSC_S联合作用及单纯应用VEGF。皮瓣成活率检测表明:携带目的基因的HPMSC_S促进皮瓣成率最高,其次为VEGF组,再次为空载组,三者均较对照组有明显促进皮瓣成活的作用。
Tissue coloboma after trauma or tumor operation usually requires the use of the skin flap transplanting to restore. Over the years, even though designs and surgical techniques of the skin flap have improved continuously, and have tried to adopt a number of materia medica to improve the survival of skin flap, but this problem that because of the hypoxia-ischemia in remote tissue of the skin flap and venous reflux impeded after the skin flap transplanting often leading to the dead of the transplanting skip flap tissue has always plagued clinicians. The hot issue on injury and reconstruction of surgery field research is on how to improve the survival rate of over-length skin flap and expand clinical application of random skin flap. Many scholars found through research that there are two keys to solve this problem: (1) after the skin flap transplanting, repair in trauma process should be started as soon as possible, mobilize repair in trauma factor fully, speed up the repair in trauma of the skin flap transplanting section, and promote "bonding" between skin flap with the recipient site, a skin flap with the surrounding tissue(;2) speeding up reconstruction of the remote skin flap ischemia section blood circulation, resolve remote skin flap hypoxia-ischemia status. The two combined actions can promote the survival of super-ratio skin flap effectively.
VEGF (Vascular endothelial growth factor) is a secreted glycoprotein, the proliferation is strong, and is easy to reach the target cells, and has the promoting effect on migration, proliferation, growth of vascular endothelial cell; and can also promote proliferation, migration of the fibroblast; and stimulates the partial reconstruction cell gathering in trauma, has a strong role in promoting repair in trauma, synthesizes and secretes extracellular matrix, has strong promoting repair in trauma. It is only known cytokines on the purpose of treatment of ischemia- hypoxia through direct effects on vascular endothelial cell surface receptors and rapid formation of new blood vessels, in the short term reconstruction blood circulation of the ischemia section. Although VEGF has an efficient ability to promote angiogenesis, but it has very short half-life and is difficult to play persistent effect with expensive price, so it is difficult to be promoted in practical applications. Therefore, looking for a suitable VEGF sustained-release system or VEGF persistent effect system is in urgent need of solution currently.
In recent years, it was discovered that stem cells has important applications capacity in the field of repair in trauma. MSCs (mesenchymal stem cells) and ADSCS (adipose-derived stem cells) could be mobilized, homed and expanded to the injury point to participate in repair in trauma. the specific capacity of MSCS and ADSCS is that they can move directly to injury tissue can be confirmed in the repair in skin trauma, it is proved that MSCS has been involved in the whole process of repair in trauma, MSCS and ADSCS can home to the injury point or differentiate into phenotype of injury cells, and it also can promote the regeneration of the tissue endogenous cell by creating the enhancement of micro-environment. But at present, the fact whether the placenta-derived stem cells in repair in trauma process can home to the trauma section and play a catalytic role in repair in trauma is not reported now. Moreover, stem cells can also act as vector cells of gene therapy, transport gene to target section, exert therapeutic effects. Such as MSC etch adult stem cells can be used as gene therapy vector of multi-diseases of the central nervous system; MSC, rADSC, EPC etch can be used as therapy vector for VEGF, FGF etch cytokines to promote the establishment of collateral circulation and angiogenesis. But at present there is very rare report about HPMSC_S as gene therapy vector
In order to explore whether HPMSC_S can home to the trauma section and can promote repair in trauma, as well as whether it can act as a vector cell of VEGF cells to deliver VEGF to trauma section, exert persistent effect of VEGF, we carried out an experiment as follows:
1. To obtain HPMSCs from the placenta, this experiment separated mononuclear cells from the placenta perfusate using density gradient centrifugation, cultivated adherence, further selected independent clones to cultivate separately, purified HPMSC_S cloning from adherent cells. We've detected its general biological characteristics, and differentiated into fat, bone to identify its multidirectional differentiation potentiality through directed induction.
2 In order to explore whether HPMSCs can differentiate to repair in trauma cells phenotype, we carried out the experiment on "human placenta mesenchymal stem cells (HPMSCs) inducing in vitro to differentiate into vascular endothelial ", by optimizing the endothelial nutrient fluid, after the directed inducing HPMSCs to cultivate 3 d passage, cell morphology changed ,when passage cell grew to 80% ~ 90% confluence, expressed analogy endothelial "cobblestone"-like structure known as "endothelial-like cells", back cell body inducing 7 d or 14 d retracted gradually, enhanced a sense of three-dimension. Specific surface mark vWF of mature endothelial cell did not express in not induced HPMSC_S, but such endothelial-like cells were positive expression. Flow cytom -etry showed that the expression of CD31 was 22.39%, but it had increased compared to not induced HPMSCs; but CD34 was the same to not induced, endothelial-like cells did not express after induction. Dil-Ac-LDL uptake experiments revealed that endothelial-like cells after induction had the ability to uptake Dil-Ac-LDL. In summary, the current induced scheme can induce HPMSCs to appear endothelial cell phenotype, and its phenotypic characteristics are more obvious as the extension of the induction time, HPMSCs has the capacity to direct and different -iate into endothelial cells in vitro specific conditions.
3 In order to prove that HPMSC_S has the function of gene therapy vector cell, the experiment established a pIRES2-EGFP-VEGF_(165) eukaryotic expression vector, and transferred pIRES2-EGFP-VEGF_(165) into HPMSC_S by liposome method successfully, and identified that HPMSC s with gene still has stem cell characteristics by multidirection induced differentiation..
4 In order to explore whether HPMSC_S with pIRES2-EGFP-VEGF_(165) can home to repair in trauma and promote blood circulation in ischemic section, we carried out an vitro experimental study on〝basic research on HPMSC_S of pIRES2-EGFP-VEGF_(165) eukaryotic expression vector impact on the survival of ischemic skin flap〞. Through research we confirmed that HPMSC_S with gene has characteristics to home to repair in trauma section, and differentiate to repair in trauma cell and promote blood circulation of ischemic section, improve the survival of the ultra proportion skin flap.
Sum up fully, this study confirms by experiment that the placenta-derived stem cells has the characteristic to differentiate to repair in trauma cell phenotype , and it can be used as vector cells of the gene therapy to deliver target genes to trauma section, play a role in promoting repair in trauma , and maintain its multi-directional differentiation potentiality; VEGF can express vector by constructed eukaryote, and transfer into HPMSCS, transport it to its target section through the vector cells , and play sufficient effect. This study further improves the theory of the stem cells family to promote repair in trauma, and also provides a new way and methods for VEGF sufficient effect.
引文
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