脐带间充质干细胞转染HGF基因对肝损伤再生的研究
摘要
目的:
肝脏损伤后的再生和修复始终是肝脏疾病研究中重要的课题,应用干细胞对终末期肝病进行细胞移植治疗或是作为转基因治疗的细胞载体是目前研究的热点之一。但干细胞的来源始终是困扰进一步应用的原因。间充质干细胞是成体干细胞中的一种,分布广泛,具有成体干细胞的一切共有特征,并且目前研究证实其在体外具有很强的免疫抑制能力,其作用对象涉及了NK细胞、T淋巴细胞、B淋巴细胞和树突状细胞等几乎所有的免疫细胞。是目前首选的转基因移植的干细胞。最近几年发现从脐带基质中也可以培育出间充质干细胞(脐带间充质干细胞,UC-MSCs),这为今后间充质干细胞广泛的临床应用提供了一个极为简便充足的来源。但目前关于脐带间充质干细胞的特性的研究还较少,至今为止尚未见到UC-MSCs分化为肝细胞的报道。因此,本研究以UC-MSCs为载体细胞,携带HGF基因到受损的肝脏模型,观察其对肝损伤修复再生的作用。
材料与方法:
1.人脐带间充质干细胞(UC-MSC)的分离和培养(Isolation and culture of humanUC-MSC)
脐带取自经父母授权同意的医院产科的足月产儿,均在取材后24小时内进行处理。首先,在无菌条件下用PBS冲洗去除样本血液,采用组织块培养法和酶消化法提取wharton's jelly的间充质干细胞。试验比较不同浓度的血清,不同的取材方法来取得的干细胞,并进行流式细胞仪检测和染色体核型分析。
2.用慢病毒载体的构建和肝细胞生长因子(HGF)和绿色荧光蛋白(GFP)基因的转染
含人肝细胞生长因子cDNA的构建复合物PUC-SRct/HGF的NotⅠ片段被纯化并被插入到pcDNA3.1(invitrogen)中,继而从pcDNA3.1-HGF构建体中切除肝细胞生长因子的PMEⅠ片段并将其克隆入复制缺陷慢病毒载体pWPI载体,即pWPI-HGF。载体序列的方向和完整性通过测序证实。重组体慢病毒通过磷酸钙转染法瞬时转染入293T细胞中,pWPI和pAPI-HGF的滴定度通过转导的293T细胞的GFP表达量来评估。传代至第三代的MSC(间充质干细胞)在含4μg/mL硫酸鱼精蛋白(Sigma)的MOI 20内进行转导。间充质干细胞的转导效率通过在荧光显微镜(Nikon,Japan)下检测GFP的表达量来评估。用转染后的第三代细胞进行移植。
3.动物模型的建立及间充质干细胞移植治疗
制作SD雄性大鼠CCL4急性肝损伤加部分肝切除模型,进行细胞移植试验。大鼠染毒24小时后行部分肝切除术,切除范围占全肝比例约35%,术前眼底采血以备测肝功。SHAM组施予除肝叶切除以外的类似手术操作作为阴性对照。移植治疗后血清酶学检测肝功能以及免疫组化检测绿色荧光蛋白阳性细胞的数目、形态和分布。western blot检测肝组织中肝细胞生长因子的表达情况。RT-PCR方法检测肝中肝细胞生长因子及相关基因表达。
结论:
1,脐带基质中能够较容易的获得丰富的间充质干细胞,其细胞表面标志物与其他组织来源的间充质干细胞大致相同。该种间充质干细胞能为今后临床间充质干细胞的应用提供一条简便充足的来源;
2,慢病毒作为载体能够简便有效的转染肝细胞生长因子基因到脐带间充质干细胞并获得稳定的表达;
3,用部分肝切除术加腹腔单次注射CCL4的方法能够成功的建立大鼠肝损伤再生的复合型模型。并且该模型建立方法更简单,与临床情况吻合性更好;
4,将转染了肝细胞生长因子基因的脐带间充质干细胞移植到大鼠肝损伤再生复合型模型中,脐带间充质干细胞能够到达受损肝脏并表达肝细胞生长因子基因参与到受体肝损伤的修复和再生过程中,对肝脏的修复和再生过程产生有益的影响,其机理尚需要进一步的研究。
Backgrounds and objective:The regeneration and recovery of liver after cell damage has been a fairly important issue in this field.One of the most promising therapeutic approaches is to use stem cell transplantation,but the resources of the stem cells limit its further application.Mesenchymal stem cell is a type of somatic stem cell and is widely distributed.It demonstrated high ability of immunosuppression and could inhibit the function of NK cell,T lymph cell,B lymph cell and dendrite cell.Therefore,it is the most popular stem cells which can be genetic modified before transplantation. Mesenchymal stem cell can derive from the matrix cell in the umbilical cord(UC-MSCs), which make it even convenient to get.However,there is no report regarding the induction of UC-MSCs into liver cell.The purpose of this study is to observe the therapeutic function of HGF transgenic UC-MSCs in liver damage rat model.
Methods:Mesenchymal stem cell was induced from the matrix cell in the umbilical cord and followed by transfection of HGF expressing lentivirus vector.The rat model was established by partial liver resection plus a single intraperitoneal CCL4 injection.The genetic modified stem cells were transplanted through the hepatic vein.The regeneration and recovery of the liver cells was evaluated by monitoring the following parameters:the liver function by the serum enzyme level,the histopathological change of the hepatic cell by HE staining,the growth of UC-MSCs by immunostaining,and the localization of the UC-MSCs by detecting of HGF expression.
Results:The HGF transgenic mesenchymal stem cell and liver damage rat model were successfully established.After transplantation,the stem cells could be positively detected in the damaged liver cells and these stem cells could locally express HGE Compared with the control group,the treated group showed an improved morphological and functional phenotype based on the serum enzyme level and pathological examination.
Conclusion:The matrix cell in the umbilical cord provides an easy way to obtain mesenchymal stem cell and lentivirus can successfully deliver HGF expression vector into UC-MSCs.Moreover,partial liver resection plus a single intraperitoneal CCL4 injection is a better approach to establish liver damage rat model.And the most important is that UC-MSCs could get to the damaged liver,express HGF and help the regeneration and recovery of the liver cells.The detailed mechanism still remains to be clarified.
肝脏损伤后的再生和修复始终是肝脏疾病研究中重要的课题,应用干细胞对终末期肝病进行细胞移植治疗或是作为转基因治疗的细胞载体是目前研究的热点之一。但干细胞的来源始终是困扰进一步应用的原因。间充质干细胞是成体干细胞中的一种,分布广泛,具有成体干细胞的一切共有特征,并且目前研究证实其在体外具有很强的免疫抑制能力,其作用对象涉及了NK细胞、T淋巴细胞、B淋巴细胞和树突状细胞等几乎所有的免疫细胞。是目前首选的转基因移植的干细胞。最近几年发现从脐带基质中也可以培育出间充质干细胞(脐带间充质干细胞,UC-MSCs),这为今后间充质干细胞广泛的临床应用提供了一个极为简便充足的来源。但目前关于脐带间充质干细胞的特性的研究还较少,至今为止尚未见到UC-MSCs分化为肝细胞的报道。因此,本研究以UC-MSCs为载体细胞,携带HGF基因到受损的肝脏模型,观察其对肝损伤修复再生的作用。
材料与方法:
1.人脐带间充质干细胞(UC-MSC)的分离和培养(Isolation and culture of humanUC-MSC)
脐带取自经父母授权同意的医院产科的足月产儿,均在取材后24小时内进行处理。首先,在无菌条件下用PBS冲洗去除样本血液,采用组织块培养法和酶消化法提取wharton's jelly的间充质干细胞。试验比较不同浓度的血清,不同的取材方法来取得的干细胞,并进行流式细胞仪检测和染色体核型分析。
2.用慢病毒载体的构建和肝细胞生长因子(HGF)和绿色荧光蛋白(GFP)基因的转染
含人肝细胞生长因子cDNA的构建复合物PUC-SRct/HGF的NotⅠ片段被纯化并被插入到pcDNA3.1(invitrogen)中,继而从pcDNA3.1-HGF构建体中切除肝细胞生长因子的PMEⅠ片段并将其克隆入复制缺陷慢病毒载体pWPI载体,即pWPI-HGF。载体序列的方向和完整性通过测序证实。重组体慢病毒通过磷酸钙转染法瞬时转染入293T细胞中,pWPI和pAPI-HGF的滴定度通过转导的293T细胞的GFP表达量来评估。传代至第三代的MSC(间充质干细胞)在含4μg/mL硫酸鱼精蛋白(Sigma)的MOI 20内进行转导。间充质干细胞的转导效率通过在荧光显微镜(Nikon,Japan)下检测GFP的表达量来评估。用转染后的第三代细胞进行移植。
3.动物模型的建立及间充质干细胞移植治疗
制作SD雄性大鼠CCL4急性肝损伤加部分肝切除模型,进行细胞移植试验。大鼠染毒24小时后行部分肝切除术,切除范围占全肝比例约35%,术前眼底采血以备测肝功。SHAM组施予除肝叶切除以外的类似手术操作作为阴性对照。移植治疗后血清酶学检测肝功能以及免疫组化检测绿色荧光蛋白阳性细胞的数目、形态和分布。western blot检测肝组织中肝细胞生长因子的表达情况。RT-PCR方法检测肝中肝细胞生长因子及相关基因表达。
结论:
1,脐带基质中能够较容易的获得丰富的间充质干细胞,其细胞表面标志物与其他组织来源的间充质干细胞大致相同。该种间充质干细胞能为今后临床间充质干细胞的应用提供一条简便充足的来源;
2,慢病毒作为载体能够简便有效的转染肝细胞生长因子基因到脐带间充质干细胞并获得稳定的表达;
3,用部分肝切除术加腹腔单次注射CCL4的方法能够成功的建立大鼠肝损伤再生的复合型模型。并且该模型建立方法更简单,与临床情况吻合性更好;
4,将转染了肝细胞生长因子基因的脐带间充质干细胞移植到大鼠肝损伤再生复合型模型中,脐带间充质干细胞能够到达受损肝脏并表达肝细胞生长因子基因参与到受体肝损伤的修复和再生过程中,对肝脏的修复和再生过程产生有益的影响,其机理尚需要进一步的研究。
Backgrounds and objective:The regeneration and recovery of liver after cell damage has been a fairly important issue in this field.One of the most promising therapeutic approaches is to use stem cell transplantation,but the resources of the stem cells limit its further application.Mesenchymal stem cell is a type of somatic stem cell and is widely distributed.It demonstrated high ability of immunosuppression and could inhibit the function of NK cell,T lymph cell,B lymph cell and dendrite cell.Therefore,it is the most popular stem cells which can be genetic modified before transplantation. Mesenchymal stem cell can derive from the matrix cell in the umbilical cord(UC-MSCs), which make it even convenient to get.However,there is no report regarding the induction of UC-MSCs into liver cell.The purpose of this study is to observe the therapeutic function of HGF transgenic UC-MSCs in liver damage rat model.
Methods:Mesenchymal stem cell was induced from the matrix cell in the umbilical cord and followed by transfection of HGF expressing lentivirus vector.The rat model was established by partial liver resection plus a single intraperitoneal CCL4 injection.The genetic modified stem cells were transplanted through the hepatic vein.The regeneration and recovery of the liver cells was evaluated by monitoring the following parameters:the liver function by the serum enzyme level,the histopathological change of the hepatic cell by HE staining,the growth of UC-MSCs by immunostaining,and the localization of the UC-MSCs by detecting of HGF expression.
Results:The HGF transgenic mesenchymal stem cell and liver damage rat model were successfully established.After transplantation,the stem cells could be positively detected in the damaged liver cells and these stem cells could locally express HGE Compared with the control group,the treated group showed an improved morphological and functional phenotype based on the serum enzyme level and pathological examination.
Conclusion:The matrix cell in the umbilical cord provides an easy way to obtain mesenchymal stem cell and lentivirus can successfully deliver HGF expression vector into UC-MSCs.Moreover,partial liver resection plus a single intraperitoneal CCL4 injection is a better approach to establish liver damage rat model.And the most important is that UC-MSCs could get to the damaged liver,express HGF and help the regeneration and recovery of the liver cells.The detailed mechanism still remains to be clarified.
引文
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