HNF4α在胎肝干细胞定向分化修复肝组织损伤中作用机理的研究
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摘要
目的
肝组织损伤是各型肝脏疾病的共同病理基础,严重的肝组织损伤将引起肝功能衰竭;干细胞治疗修复肝组织损伤是目前国内外医学界研究的一个热点,但由于存在体外富集纯化困难、定向分化机制不明等问题而使其应用受到限制。胎肝细胞中富含的具有定向分化功能的干细胞是干细胞治疗研究中的重要细胞来源。本研究拟探讨建立体外扩增大鼠胚胎肝干细胞并抑制其分化的培养条件及方法,并在此基础上,将胎肝干细胞通过门静脉植入肝损伤大鼠体内,观察肝脏修复情况,检测治疗前后HNF4α表达变化,探讨HNF4α在胎肝干细胞促进肝组织损伤修复中的作用。本研究内容共分为两部分:1.体外琼脂克隆培养对胎肝干细胞分化的影响;2.HNF4α在胎肝干细胞促肝组织损伤修复中的作用。探讨这一重要调节过程中分子机理将为临床应用胎肝干细胞移植治疗肝脏疾病奠定重要的基础。
方法
1.无菌条件下利用Ⅳ型胶原酶结合机械消化法制备14d胎龄大鼠胎肝干细胞悬液。将原代分离得到的大鼠胎肝干细胞分成两组,其中一组胚胎肝干细胞接种至Ⅰ型胶原包被的培养板中,常规贴壁培养。另一组细胞则接种至无血清软琼脂培养基中进行悬浮培养;悬浮培养基分为上下两层,底层为含HGF的1.2%琼脂,顶层为无血清的0.6%琼脂,将胎肝干细胞接种至顶层培养基中培养。倒置显微镜下观察两组细胞的生长。培养2周后收集两组细胞,电镜观察两组细胞超微结构的差异,流式细胞仪检测其CD90.1+、CD49F+等干细胞标志物表达特征,碱性磷酸酶染色检测其分化状态的差异,实时定量PCR检测两组细胞甲胎蛋白(AFP)、白蛋白(ALB)的表达差异。
2.为研究HNF4α在胎肝干细胞促肝组织损伤修复中的作用,我们采用四氯化碳制作SD大鼠急性化学性肝损伤模型,造模成功后将肝损伤大鼠随机分为移植组(20只)和对照组(20只),移植组将胎肝干细胞经门静脉移植至大鼠肝脏,对照组注射等容剂量的生理盐水。分别于注射前6h,注射后1,3,5周检测大鼠血清谷氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的表达水平;取病理组织切片HE染色,观察肝组织修复情况;取肝组织切片采用免疫组化及提取部分肝组织蛋白用Western blot方法检测肝脏治疗前后HNF4α表达情况。
结果
1.琼脂克隆培养发现部分胎肝干细胞未能有效增殖并形成克隆的细胞,这部分细胞不能在琼脂培养基中存活,随后发生凋亡崩解。剩余的细胞在2天后开始出现小的由3-5个细胞组成的细胞球,随培养时间的延长,干细胞球不断扩大,球内细胞折光性好,连接紧密,未出现分化表现,从形态上判定为干细胞集落。原代细胞接种后贴壁速度、分裂增生较慢,24 h后相差显微镜下可见细胞贴壁生长,48h后观察到分裂。1周后贴壁细胞体积逐渐增大铺展呈上皮样,形成集落。生长至90%左右时传代后增长速度加快,表现出一些分化的特征。
2.电镜下观察,琼脂克隆培养胎肝干细胞直径约7μm~13μm,表面可见少量短小的微绒毛状突起,整个细胞大部分被卵圆形胞核所占据,细胞质少,核质比例大,内质网、线粒体、核糖体等细胞器不发达,表现为原始幼稚未分化细胞特征,即所谓的肝干细胞。贴壁培养胎肝干细胞直径明显大于琼脂克隆培养,约20~40μm,细胞核浆比例小,细胞质内可见大量内质网、高尔基体、核糖体等发达细胞器,显示出明显的分化特征,与成熟肝细胞相似。通过流式分析发现,悬浮克隆培养的胎肝干细胞球高表达CD90.1、CD49F,明显高于贴壁培养培养的胎肝干细胞。碱性磷酸酶染色,观察细胞表面ALP的表达,结果发现悬浮培养组染色为强阳性,贴壁培养为弱阳性。实时定量PCR检测结果则表明,随着培养时间的增加,贴壁培养组的胎肝干细胞AFP表达明显下降,ALB的表达呈明显上升的趋势(P<0.01)。而悬浮培养组的胎肝干细胞AFP和ALB的表达在培养前后未见明显变化(P>0.05)。
3.四氯化碳诱导肝损伤模型后,各组SD大鼠根据肝病理组织显示不同程度的损伤,经胎肝干细胞移植处理后,对照组大鼠肝功能改善缓慢,ALT,AST明显高于移植组,移植组大鼠损伤的肝组织逐渐修复,肝功能恢复正常。第5周移植组大鼠术后存活率显著高于对照组。肝组织损伤后,HNF4α在肝组织的表达明显升高;随着肝组织的逐渐修复,HNF4α表达逐渐下降。Western blot及免疫组化检测亦证实此结果。
结论
琼脂克隆培养的胎肝干细胞较有血清贴壁培养分化程度低,在琼脂克隆培养条件下能增殖形成具有显著干细胞特征的克隆球。HNF4α在肝脏损伤初期起保护作用。可能系通过促进胎肝干细胞向损坏的肝小叶迁移,定向分化成正常肝细胞并适度增殖,从而替代损伤的肝实质,提高肝损伤后的恢复能力。
Objective
Hepatic injury is the common pathological basis of various types of liver disease. Severe liver damage can develop into liver failure or death within several days. At present, stem cell therapy which is able to dramatically improve the treatment of hepatic injury has become a research hot spot, although it is limited because of the difficulty in the in vitro enrichment and purification of hepatic stem cell and the poor knowledge about mechanism of stem cell differentiation. In this study we investigate the therapeutic effect of fetal hepatocytes which is rich in stem cells with directed differentiation potential on severe liver injury, the advantages of 3D Soft-Agar-Culture-System (SACS) for the culture of mouse embryonic hepatic stem cells was also demonstrated. We showed a satisfactory ability of SACS to maintain the undifferentiated state and pluripotency of the suspended hepatic stem cells for a long time. Then suspension of embryonic hepatic stem cells was transplanted into injured livers of rats via portal vein. The therapeutic effect of hepatic stem cells on CCl4-induced hepatic injury was evaluated. Moreover, we determined the protein expression of HNF4αin rat livers at different time points in order to underline its promotive role on the recovery of acute hepatic injury. This study was divided into two parts: 1. Evaluation of the differentiation-inductive effect of suspension culture in the SACS on embryonic hepatic stem cells. 2. Study on the promotive role of HNF4αon the recovery of acute hepatic injury. Current studies provided a better understanding of the molecular mechanisms of the embryonic hepatic stem cells-based cell therapy which warranted the clinical implication of this novel modality for liver disease in the future.
Methods
1. Suspension of ED 14 Fischer (F) 344 rat embryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly, Group 1 were seeded into type I collagen-coated plates for adherent culture while Group 2 were seeded into soft agar medium for suspension culture. Suspension medium was composed of two parts, the basal layer contained 20% fetal bovine serum, 0.6% agar, and EGF with 20μg/ml final concentration. The upper layer was composed of 0.3% agar into which the hepatic stem cells were seeded. After 2-week culture, the morphology and ultrastructure of cells in both two groups were observed by inverted microscope and transmission electron microscope (TEM). Expressions of CD90.1 and CD49F, two specificstem cell surface markers, were tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase staining was performed to detect stem cell differentiation by immunohistochemistry, and the expressions of AFP and ALB were tested by RT-PCR.
2. In the following experiments, SD rats were randomly divided into transplantation group (n = 20) and control group (n = 20) after CCl4- induced hepatic injury. Hepatic stem cells were transplanted into injured livers of rats in transplantation group via portal vein. Equal volume of saline was transplanted in control group. The expression levels of ALT, AST in the serum and histopathological injury of hepatic tissues were analyzed 6 h before, 1w, 3w and 5w after transplantation. Evaluation of HNF4αprotein expression in rat livers both by immunohistochemistry and western blot was simultaneouly performed.
Results
1. A minor part of cells from suspension culture could not proliferate and form clone ball, and then they experienced apoptosis and disintegrated. However, majority of cells developed into colonies containing more than 3-5 cells after 2days. Shapes of the newly formed colonies were irregular and mulberry-like. Newborn cells budding in these clumps could also be observed. Most of the colonies contained more than 20-25 cells which are well refractive and contacted with each other closely. The morphological study of the colonies in SACS showed that all cells were stem cell-like. Primary cells culturing in adherent cultures condition adhered to bottom wall within 24 h after inoculation and began to divide and proliferate within 48 h. The adhered cells developed into colony-forming units finally. Cells passages significantly accelerated the growth rate of adhered cells and their differentiation into mature hepatocytes.
2. TEM revealed that hepatic stem cells in SACS can maintain an undifferentiated state for a long time interval with a mean diameter of 7μm~13μm and rare microvilli in cell surface. Most of the cells had a relatively large nucleus, higher nucleus-cytoplasm ratio, and less cell organelles such as ribosomes, mitochondria, endoplasmic reticulum. The diameter of adhesive culture cells was significantly greater than suspension culture cells (20~40μm). In addition, ultrastructural feature of these cells showed a mature hepatocyte-like phenotype. Flow cytometry demonstrated that soft agar culture resulted in a larger population of cells expressing the undifferentiated stem cell markers CD49F, CD90.1, compared with a smaller population of the cells which were under adhesive culture conditions. The undifferentiated hepatic stem cells in SACS showed strong ALP staining, while cells in adhesive culture conditions stained weakly for ALP. RT-PCR showed a significantly decreased AFP expression along with a incresed ALB expression in the cells under adhesive culture(P<0.01). There was no significant change before and after culturing in the SACS.
3. Rats in each group suffered different degrees of injury after CCl4-induced hepatic injury. In treatment group, the injured hepatic tissues were markedly repaired after stem cell transplantation and the liver function gradually returned to normal physiological level. By contrast, the liver function of control group was slightly repaired and experienced slow recovery, as evidenced by significant increased ALT and AST in serum. Survival rate of the rats in transplantation group was significantly higher than control group at each time point. Western blot and immunohistochemistry demonstrated that expression of HNF4αnotably increased when liver injured, and then slowly decreased in accordance with the gradual restoration of liver tissue.
Conclusions
Embryonic hepatic stem cells cultured in SACS experienced less differentiation than those were adherently cultured in serum-added culture medium, and could proliferate and form clone ball with a specific stem cell feature. HNF4αplays a protective effect in the early stage of hepatic injury. It was carried out probably through promoting the restoration of acute hepatic injury by promoting the migration to damaged hepatic lobule and facilitating cell differentiation, proliferation of embryonic hepatic stem cell.
肝组织损伤是各型肝脏疾病的共同病理基础,严重的肝组织损伤将引起肝功能衰竭;干细胞治疗修复肝组织损伤是目前国内外医学界研究的一个热点,但由于存在体外富集纯化困难、定向分化机制不明等问题而使其应用受到限制。胎肝细胞中富含的具有定向分化功能的干细胞是干细胞治疗研究中的重要细胞来源。本研究拟探讨建立体外扩增大鼠胚胎肝干细胞并抑制其分化的培养条件及方法,并在此基础上,将胎肝干细胞通过门静脉植入肝损伤大鼠体内,观察肝脏修复情况,检测治疗前后HNF4α表达变化,探讨HNF4α在胎肝干细胞促进肝组织损伤修复中的作用。本研究内容共分为两部分:1.体外琼脂克隆培养对胎肝干细胞分化的影响;2.HNF4α在胎肝干细胞促肝组织损伤修复中的作用。探讨这一重要调节过程中分子机理将为临床应用胎肝干细胞移植治疗肝脏疾病奠定重要的基础。
方法
1.无菌条件下利用Ⅳ型胶原酶结合机械消化法制备14d胎龄大鼠胎肝干细胞悬液。将原代分离得到的大鼠胎肝干细胞分成两组,其中一组胚胎肝干细胞接种至Ⅰ型胶原包被的培养板中,常规贴壁培养。另一组细胞则接种至无血清软琼脂培养基中进行悬浮培养;悬浮培养基分为上下两层,底层为含HGF的1.2%琼脂,顶层为无血清的0.6%琼脂,将胎肝干细胞接种至顶层培养基中培养。倒置显微镜下观察两组细胞的生长。培养2周后收集两组细胞,电镜观察两组细胞超微结构的差异,流式细胞仪检测其CD90.1+、CD49F+等干细胞标志物表达特征,碱性磷酸酶染色检测其分化状态的差异,实时定量PCR检测两组细胞甲胎蛋白(AFP)、白蛋白(ALB)的表达差异。
2.为研究HNF4α在胎肝干细胞促肝组织损伤修复中的作用,我们采用四氯化碳制作SD大鼠急性化学性肝损伤模型,造模成功后将肝损伤大鼠随机分为移植组(20只)和对照组(20只),移植组将胎肝干细胞经门静脉移植至大鼠肝脏,对照组注射等容剂量的生理盐水。分别于注射前6h,注射后1,3,5周检测大鼠血清谷氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的表达水平;取病理组织切片HE染色,观察肝组织修复情况;取肝组织切片采用免疫组化及提取部分肝组织蛋白用Western blot方法检测肝脏治疗前后HNF4α表达情况。
结果
1.琼脂克隆培养发现部分胎肝干细胞未能有效增殖并形成克隆的细胞,这部分细胞不能在琼脂培养基中存活,随后发生凋亡崩解。剩余的细胞在2天后开始出现小的由3-5个细胞组成的细胞球,随培养时间的延长,干细胞球不断扩大,球内细胞折光性好,连接紧密,未出现分化表现,从形态上判定为干细胞集落。原代细胞接种后贴壁速度、分裂增生较慢,24 h后相差显微镜下可见细胞贴壁生长,48h后观察到分裂。1周后贴壁细胞体积逐渐增大铺展呈上皮样,形成集落。生长至90%左右时传代后增长速度加快,表现出一些分化的特征。
2.电镜下观察,琼脂克隆培养胎肝干细胞直径约7μm~13μm,表面可见少量短小的微绒毛状突起,整个细胞大部分被卵圆形胞核所占据,细胞质少,核质比例大,内质网、线粒体、核糖体等细胞器不发达,表现为原始幼稚未分化细胞特征,即所谓的肝干细胞。贴壁培养胎肝干细胞直径明显大于琼脂克隆培养,约20~40μm,细胞核浆比例小,细胞质内可见大量内质网、高尔基体、核糖体等发达细胞器,显示出明显的分化特征,与成熟肝细胞相似。通过流式分析发现,悬浮克隆培养的胎肝干细胞球高表达CD90.1、CD49F,明显高于贴壁培养培养的胎肝干细胞。碱性磷酸酶染色,观察细胞表面ALP的表达,结果发现悬浮培养组染色为强阳性,贴壁培养为弱阳性。实时定量PCR检测结果则表明,随着培养时间的增加,贴壁培养组的胎肝干细胞AFP表达明显下降,ALB的表达呈明显上升的趋势(P<0.01)。而悬浮培养组的胎肝干细胞AFP和ALB的表达在培养前后未见明显变化(P>0.05)。
3.四氯化碳诱导肝损伤模型后,各组SD大鼠根据肝病理组织显示不同程度的损伤,经胎肝干细胞移植处理后,对照组大鼠肝功能改善缓慢,ALT,AST明显高于移植组,移植组大鼠损伤的肝组织逐渐修复,肝功能恢复正常。第5周移植组大鼠术后存活率显著高于对照组。肝组织损伤后,HNF4α在肝组织的表达明显升高;随着肝组织的逐渐修复,HNF4α表达逐渐下降。Western blot及免疫组化检测亦证实此结果。
结论
琼脂克隆培养的胎肝干细胞较有血清贴壁培养分化程度低,在琼脂克隆培养条件下能增殖形成具有显著干细胞特征的克隆球。HNF4α在肝脏损伤初期起保护作用。可能系通过促进胎肝干细胞向损坏的肝小叶迁移,定向分化成正常肝细胞并适度增殖,从而替代损伤的肝实质,提高肝损伤后的恢复能力。
Objective
Hepatic injury is the common pathological basis of various types of liver disease. Severe liver damage can develop into liver failure or death within several days. At present, stem cell therapy which is able to dramatically improve the treatment of hepatic injury has become a research hot spot, although it is limited because of the difficulty in the in vitro enrichment and purification of hepatic stem cell and the poor knowledge about mechanism of stem cell differentiation. In this study we investigate the therapeutic effect of fetal hepatocytes which is rich in stem cells with directed differentiation potential on severe liver injury, the advantages of 3D Soft-Agar-Culture-System (SACS) for the culture of mouse embryonic hepatic stem cells was also demonstrated. We showed a satisfactory ability of SACS to maintain the undifferentiated state and pluripotency of the suspended hepatic stem cells for a long time. Then suspension of embryonic hepatic stem cells was transplanted into injured livers of rats via portal vein. The therapeutic effect of hepatic stem cells on CCl4-induced hepatic injury was evaluated. Moreover, we determined the protein expression of HNF4αin rat livers at different time points in order to underline its promotive role on the recovery of acute hepatic injury. This study was divided into two parts: 1. Evaluation of the differentiation-inductive effect of suspension culture in the SACS on embryonic hepatic stem cells. 2. Study on the promotive role of HNF4αon the recovery of acute hepatic injury. Current studies provided a better understanding of the molecular mechanisms of the embryonic hepatic stem cells-based cell therapy which warranted the clinical implication of this novel modality for liver disease in the future.
Methods
1. Suspension of ED 14 Fischer (F) 344 rat embryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly, Group 1 were seeded into type I collagen-coated plates for adherent culture while Group 2 were seeded into soft agar medium for suspension culture. Suspension medium was composed of two parts, the basal layer contained 20% fetal bovine serum, 0.6% agar, and EGF with 20μg/ml final concentration. The upper layer was composed of 0.3% agar into which the hepatic stem cells were seeded. After 2-week culture, the morphology and ultrastructure of cells in both two groups were observed by inverted microscope and transmission electron microscope (TEM). Expressions of CD90.1 and CD49F, two specificstem cell surface markers, were tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase staining was performed to detect stem cell differentiation by immunohistochemistry, and the expressions of AFP and ALB were tested by RT-PCR.
2. In the following experiments, SD rats were randomly divided into transplantation group (n = 20) and control group (n = 20) after CCl4- induced hepatic injury. Hepatic stem cells were transplanted into injured livers of rats in transplantation group via portal vein. Equal volume of saline was transplanted in control group. The expression levels of ALT, AST in the serum and histopathological injury of hepatic tissues were analyzed 6 h before, 1w, 3w and 5w after transplantation. Evaluation of HNF4αprotein expression in rat livers both by immunohistochemistry and western blot was simultaneouly performed.
Results
1. A minor part of cells from suspension culture could not proliferate and form clone ball, and then they experienced apoptosis and disintegrated. However, majority of cells developed into colonies containing more than 3-5 cells after 2days. Shapes of the newly formed colonies were irregular and mulberry-like. Newborn cells budding in these clumps could also be observed. Most of the colonies contained more than 20-25 cells which are well refractive and contacted with each other closely. The morphological study of the colonies in SACS showed that all cells were stem cell-like. Primary cells culturing in adherent cultures condition adhered to bottom wall within 24 h after inoculation and began to divide and proliferate within 48 h. The adhered cells developed into colony-forming units finally. Cells passages significantly accelerated the growth rate of adhered cells and their differentiation into mature hepatocytes.
2. TEM revealed that hepatic stem cells in SACS can maintain an undifferentiated state for a long time interval with a mean diameter of 7μm~13μm and rare microvilli in cell surface. Most of the cells had a relatively large nucleus, higher nucleus-cytoplasm ratio, and less cell organelles such as ribosomes, mitochondria, endoplasmic reticulum. The diameter of adhesive culture cells was significantly greater than suspension culture cells (20~40μm). In addition, ultrastructural feature of these cells showed a mature hepatocyte-like phenotype. Flow cytometry demonstrated that soft agar culture resulted in a larger population of cells expressing the undifferentiated stem cell markers CD49F, CD90.1, compared with a smaller population of the cells which were under adhesive culture conditions. The undifferentiated hepatic stem cells in SACS showed strong ALP staining, while cells in adhesive culture conditions stained weakly for ALP. RT-PCR showed a significantly decreased AFP expression along with a incresed ALB expression in the cells under adhesive culture(P<0.01). There was no significant change before and after culturing in the SACS.
3. Rats in each group suffered different degrees of injury after CCl4-induced hepatic injury. In treatment group, the injured hepatic tissues were markedly repaired after stem cell transplantation and the liver function gradually returned to normal physiological level. By contrast, the liver function of control group was slightly repaired and experienced slow recovery, as evidenced by significant increased ALT and AST in serum. Survival rate of the rats in transplantation group was significantly higher than control group at each time point. Western blot and immunohistochemistry demonstrated that expression of HNF4αnotably increased when liver injured, and then slowly decreased in accordance with the gradual restoration of liver tissue.
Conclusions
Embryonic hepatic stem cells cultured in SACS experienced less differentiation than those were adherently cultured in serum-added culture medium, and could proliferate and form clone ball with a specific stem cell feature. HNF4αplays a protective effect in the early stage of hepatic injury. It was carried out probably through promoting the restoration of acute hepatic injury by promoting the migration to damaged hepatic lobule and facilitating cell differentiation, proliferation of embryonic hepatic stem cell.
引文
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