人孤雌胚胎干细胞诱导分化为肠上皮干细胞的研究
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摘要
背景和目的：
肠上皮损伤包括克隆氏病、溃疡性结肠炎、短肠综合征、放射性肠炎等,可引起消化吸收障碍,严重时甚至危及到患者生命,是当今临床治疗上的一个难题。将具有肠上皮修复潜能的肠上皮干细胞(intestinal epithelial stem cell, IESC)应用于肠上皮损伤的治疗,已成为目前国内外研究的热点之一,但在体外原代培养IESC比较困难,大大限制了其在临床中的应用。人孤雌胚胎干细胞(human parthenogenetic embryonic stem cell, hPESC)由于可避免移植免疫排斥、伦理学争端等问题,有望成为诱导分化为IESC的重要细胞来源。本研究拟探讨hPESC体外培养的最佳条件和体外分阶段诱导hPESC分化为IESC的可行性及条件。
方法：
1、采用胰蛋白酶消化法原代培养人包皮成纤维细胞(human foreskin fibroblast, hFF),倒置显微镜下观察其生长状态,并采用免疫细胞化学染色对其进行鉴定。
2、建立hPESC体外培养的三种不同体系：以MEF为饲养层、以hFF为饲养层和无血清无饲养层体系,观察hPESC在不同体系中的生长状态,采用碱性磷酸酶(alkaline phosphatase, AKP)检测和核型分析研究hPESC的生物学特性,优化hPESC体外培养的最佳条件。
3、体外诱导hPESC向限定性内胚层(definitive endoderm, DE)分化：以ActivinA和Wnt3a作为诱导因子,同时作用于hPESC以提高向DE分化的比例,采用流式细胞术(flow cytometry, FCM)和实时定量PCR方法动态监测DE的标志物CXCR4、ECD、Sox-17和Gsc的表达,确定hPESC向DE分化比例最高的时间点。
4、体外诱导DE向IESC分化：以EGF作为诱导因子,定向诱导DE向肠上皮干细胞(intestinal epithelial stem cell, IESC)分化,采用免疫细胞化学双染色和实时定量PCR方法动态测定ESC标记物Msil和Hesl的表达,确定DE向IESC分化比例最高的时间点。
结果：
1、经原代培养成功获得了hFF,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性。
2、生长在hFF饲养层上的hPESC克隆形态规则、生长状态良好、不易分化,经体外培养25代,hPESC仍能够保持基本生物学特性和正常核型；生长在MEF饲养层上的hPESC克隆形态良好、生长速度稳定、自发分化率低,经体外培养15代,hPESC仍能够保持基本生物学特性；生长在无血清无饲养层体系中的hPESC克隆形态良好、生长速度较慢、分化率极低,经体外培养15代,hPESC仍能够保持基本生物学特性。
3、经过15代的体外培养,比较三种不同培养体系中hPESC克隆的形态、贴壁率、自发分化率,hPESC的扩增速度,动物源性污染,实验的工作量及成本问题,发现hPESC-hFF体系具有无可比拟的优势。
4、hPESC在诱导因子Activin A和Wnt3a共同作用下向DE分化,FCM检测显示：CXCR4和ECD于诱导2天时达到峰值；实时定量PCR结果表明：Sox-17和Gsc的表达于诱导2天时达到峰值,即hPESC向DE分化在诱导2天时比例最高。
5、DE在诱导因子EGF作用下向ESC定向分化,免疫细胞化学双染色显示：Msil定位于胞核,呈蓝紫色,Hesl定位于胞浆,呈红色,Msil和Hesl双阳性细胞的比例在诱导5天时达到峰值；实时定量PCR结果表明：在诱导第5天时Msil及Hesl同步达到峰值,即DE向IESC分化在诱导5天时比例最高。
结论：
1、经胰蛋白酶消化法原代培养成功获得了hFF。
2、在适宜的条件下,hPESC-MEF体系、hPESC-hFF体系和无血清无饲养层hPESC体系均能长期有效地支持hPESC的生长并维持其未分化状态。
3、hPESC在hFF饲养层上能够保持基本生物学特性和正常核型。作为人源性饲养层,hFF能够有效地支持hPESC的生长并维持其未分化状态。经多方面的比较,hPESC-hFF体系具有无可比拟的优势,最适合本研究中hPESC的体外培养。
4、联合应用诱导因子Activin A和Wnt3a可促进hPESC向DE分化；诱导分化2天时,hPESC向DE分化的比例最高,是进一步向IESC诱导分化的理想前体细胞群。
5、诱导因子EGF可促进DE向IESC分化；诱导分化5天时,DE向IESC分化的比例最高,为肠上皮损伤临床开展IESC移植治疗提供了潜在的细胞来源,为小肠上皮结构重建及小肠组织工程研究奠定了基础和并提供了理论依据。
6、通过分阶段诱导分化方法,成功将hPESC诱导分化为IESC。
Background and Objective:
Damage of intestinal epithelium including crohn's disease, ulcerative colitis, short bowel syndrome, radiation enterocolitis, and so on, can cause malabsorption and even endanger the life of patient in severe case. Damage of intestinal epithelium is an intractable problem in clinic. At present, treating damage of intestinal epithelium with intestinal epithelial stem cell(IESC) which owns the potency of repairing the intestinal epithelium becomes a research focus at home and abroad. However, primary culture of IESC in vitro is difficult, which constraints the application of IESC in clinic. Human partheno genetic embryonic stem cell(hPESC) can avoid many problems such as transplant rejection and ethical issues. Therefore, hPESC is expected to become a significant cell source of IESC. In this study optimum culture condition of hPESC in vitro will be studied at first, then the feasibility and condition of inducing hPESC to differentiate into IESC in vitro will be explored.
Methods:
1. Trypsin digestion method was adopted to carry on primary culture of hFF. Then morphology of hFF was observed with inverted microscope, and hFF was identified by immunohistochemistry method.
2.Three different culture systems of hPESC were established as follows:mouse embryonic fibroblast(MEF) as feeder-layer, human foreskin fibroblast(hFF) as feeder-layer and feeder-free system. Morphology of hPESC in three culture systems were observed respectively. Alkaline phosphatase activity and karyotypes analysis were used to detect the biological characteristics of hPESC. Culture condition of hPESC in vitro was optimized.
3. hPESC was induced to differentiate into definitive endoderm(DE) in vitro. Wnt3a and Activin A were adopted simultaneously as inducing factors to promote hPESC to differentiate into DE. Markers of DE including CXCR4、ECD、Sox-17and Gsc, were monitored dynamically with flow cytometry and real-time quantitative PCR, thus the time when the percentage of DE culminated was detected.
4. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Markers of IESC including Msil and Hesl, were monitored dynamically with immunocytochemical double staining and real-time quantitative PCR, thus the differentiated time of IESC was detected in vitro.
Results:
1. hFF was obtained successfully by primary culture. Morphology and characteristic of hFF were verified with inverted microscope and immunocytochemical stain.
2. Morphous of hPESC on hFF feeder-layer was regular and growth state of hPESC was well. hPESC on hFF feeder-layer was not apt to differentiate. After subcultured for25passages in vitro, hPESC still maintained biological characteristics and normal karyotype. Morphous of hPESC on MEF feeder-layer was regular and growth velocity of hPESC was stable. Self-differentiation of hPESC on MEF feeder-layer seldom occurred. After subcultured for15passages in vitro, hPESC still maintained biological characteristics. Morphous of hPESC in feeder-free system was regular and growth velocity of hPESC was slow. Self-differentiation ratio of hPESC in feeder-free system was extremely low. After subcultured for15passages in vitro, hPESC still maintained biological characteristics.
3. After hPESC has been subcultured for15passages in vitro, morphous, adherence ratio, self-differentiate ratio and amplification velocity of hPESC, animal-derived contamination, workload and cost in three culture systems were considered thoroughly. We found that as feeder-layer of hPESC hFF owns a significant advantage.
4. Under the effect of Wnt3a and Activin A, hPESC was induced to differentiate into DE. The results of flow cytometry indicated that expression of CXCR4and ECD culminated on the second day. The results of real-time quantitative PCR displayed that expression of Sox-17and Gsc culminated on the second day. Therefore, the percentage of DE was highest on the second day.
5. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Immunocytochemical double staining displayed that Msil+Hesl+cells culminated on the fifth day. The results of real-time quantitative PCR revealed that the expression of Msil and Hesl culminated synchronously on the fifth day. Therefore, the percentage of IESC was highest on the fifth day.
Conclusions:
1. hFF was obtained successfully by trypsin digestion method.
2. Under the appropriate conditions all of three systems could sustain the growth of hPESC and keep hPESC undifferentiated.
3. hPESC could keep biological characteristics and normal karyotype on hFF feeder-layer. As a kind of human-derived feeder-layer, hFF could sustain the growth of hPESC and keep hPESC undifferentiated. After various comparison, we found that hFF feeder-layer have a significant advantage.
4. Combined application of inducing factors Activin A and Wnt3a can promote hPESC to differentiate into DE; the percentage of DE was highest on the second day. The hPESC induced with Wnt3a and Activin A on the second day is the ideal progenitor cell of IESC.
5. Inducing factor EGF can promote DE to differentiate into IESC. The percentage of IESC was highest on the fifth day. It will provide potential cell source for the IESC transplantation treatment of damage of intestinal epithelium and theoretical basis for reconstruction of intestinal epithelium and intestinal tissue engineering research.
6. hPESC was successfully induced to differentiate into IESC by a phased method.
肠上皮损伤包括克隆氏病、溃疡性结肠炎、短肠综合征、放射性肠炎等,可引起消化吸收障碍,严重时甚至危及到患者生命,是当今临床治疗上的一个难题。将具有肠上皮修复潜能的肠上皮干细胞(intestinal epithelial stem cell, IESC)应用于肠上皮损伤的治疗,已成为目前国内外研究的热点之一,但在体外原代培养IESC比较困难,大大限制了其在临床中的应用。人孤雌胚胎干细胞(human parthenogenetic embryonic stem cell, hPESC)由于可避免移植免疫排斥、伦理学争端等问题,有望成为诱导分化为IESC的重要细胞来源。本研究拟探讨hPESC体外培养的最佳条件和体外分阶段诱导hPESC分化为IESC的可行性及条件。
方法：
1、采用胰蛋白酶消化法原代培养人包皮成纤维细胞(human foreskin fibroblast, hFF),倒置显微镜下观察其生长状态,并采用免疫细胞化学染色对其进行鉴定。
2、建立hPESC体外培养的三种不同体系：以MEF为饲养层、以hFF为饲养层和无血清无饲养层体系,观察hPESC在不同体系中的生长状态,采用碱性磷酸酶(alkaline phosphatase, AKP)检测和核型分析研究hPESC的生物学特性,优化hPESC体外培养的最佳条件。
3、体外诱导hPESC向限定性内胚层(definitive endoderm, DE)分化：以ActivinA和Wnt3a作为诱导因子,同时作用于hPESC以提高向DE分化的比例,采用流式细胞术(flow cytometry, FCM)和实时定量PCR方法动态监测DE的标志物CXCR4、ECD、Sox-17和Gsc的表达,确定hPESC向DE分化比例最高的时间点。
4、体外诱导DE向IESC分化：以EGF作为诱导因子,定向诱导DE向肠上皮干细胞(intestinal epithelial stem cell, IESC)分化,采用免疫细胞化学双染色和实时定量PCR方法动态测定ESC标记物Msil和Hesl的表达,确定DE向IESC分化比例最高的时间点。
结果：
1、经原代培养成功获得了hFF,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性。
2、生长在hFF饲养层上的hPESC克隆形态规则、生长状态良好、不易分化,经体外培养25代,hPESC仍能够保持基本生物学特性和正常核型；生长在MEF饲养层上的hPESC克隆形态良好、生长速度稳定、自发分化率低,经体外培养15代,hPESC仍能够保持基本生物学特性；生长在无血清无饲养层体系中的hPESC克隆形态良好、生长速度较慢、分化率极低,经体外培养15代,hPESC仍能够保持基本生物学特性。
3、经过15代的体外培养,比较三种不同培养体系中hPESC克隆的形态、贴壁率、自发分化率,hPESC的扩增速度,动物源性污染,实验的工作量及成本问题,发现hPESC-hFF体系具有无可比拟的优势。
4、hPESC在诱导因子Activin A和Wnt3a共同作用下向DE分化,FCM检测显示：CXCR4和ECD于诱导2天时达到峰值；实时定量PCR结果表明：Sox-17和Gsc的表达于诱导2天时达到峰值,即hPESC向DE分化在诱导2天时比例最高。
5、DE在诱导因子EGF作用下向ESC定向分化,免疫细胞化学双染色显示：Msil定位于胞核,呈蓝紫色,Hesl定位于胞浆,呈红色,Msil和Hesl双阳性细胞的比例在诱导5天时达到峰值；实时定量PCR结果表明：在诱导第5天时Msil及Hesl同步达到峰值,即DE向IESC分化在诱导5天时比例最高。
结论：
1、经胰蛋白酶消化法原代培养成功获得了hFF。
2、在适宜的条件下,hPESC-MEF体系、hPESC-hFF体系和无血清无饲养层hPESC体系均能长期有效地支持hPESC的生长并维持其未分化状态。
3、hPESC在hFF饲养层上能够保持基本生物学特性和正常核型。作为人源性饲养层,hFF能够有效地支持hPESC的生长并维持其未分化状态。经多方面的比较,hPESC-hFF体系具有无可比拟的优势,最适合本研究中hPESC的体外培养。
4、联合应用诱导因子Activin A和Wnt3a可促进hPESC向DE分化；诱导分化2天时,hPESC向DE分化的比例最高,是进一步向IESC诱导分化的理想前体细胞群。
5、诱导因子EGF可促进DE向IESC分化；诱导分化5天时,DE向IESC分化的比例最高,为肠上皮损伤临床开展IESC移植治疗提供了潜在的细胞来源,为小肠上皮结构重建及小肠组织工程研究奠定了基础和并提供了理论依据。
6、通过分阶段诱导分化方法,成功将hPESC诱导分化为IESC。
Background and Objective:
Damage of intestinal epithelium including crohn's disease, ulcerative colitis, short bowel syndrome, radiation enterocolitis, and so on, can cause malabsorption and even endanger the life of patient in severe case. Damage of intestinal epithelium is an intractable problem in clinic. At present, treating damage of intestinal epithelium with intestinal epithelial stem cell(IESC) which owns the potency of repairing the intestinal epithelium becomes a research focus at home and abroad. However, primary culture of IESC in vitro is difficult, which constraints the application of IESC in clinic. Human partheno genetic embryonic stem cell(hPESC) can avoid many problems such as transplant rejection and ethical issues. Therefore, hPESC is expected to become a significant cell source of IESC. In this study optimum culture condition of hPESC in vitro will be studied at first, then the feasibility and condition of inducing hPESC to differentiate into IESC in vitro will be explored.
Methods:
1. Trypsin digestion method was adopted to carry on primary culture of hFF. Then morphology of hFF was observed with inverted microscope, and hFF was identified by immunohistochemistry method.
2.Three different culture systems of hPESC were established as follows:mouse embryonic fibroblast(MEF) as feeder-layer, human foreskin fibroblast(hFF) as feeder-layer and feeder-free system. Morphology of hPESC in three culture systems were observed respectively. Alkaline phosphatase activity and karyotypes analysis were used to detect the biological characteristics of hPESC. Culture condition of hPESC in vitro was optimized.
3. hPESC was induced to differentiate into definitive endoderm(DE) in vitro. Wnt3a and Activin A were adopted simultaneously as inducing factors to promote hPESC to differentiate into DE. Markers of DE including CXCR4、ECD、Sox-17and Gsc, were monitored dynamically with flow cytometry and real-time quantitative PCR, thus the time when the percentage of DE culminated was detected.
4. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Markers of IESC including Msil and Hesl, were monitored dynamically with immunocytochemical double staining and real-time quantitative PCR, thus the differentiated time of IESC was detected in vitro.
Results:
1. hFF was obtained successfully by primary culture. Morphology and characteristic of hFF were verified with inverted microscope and immunocytochemical stain.
2. Morphous of hPESC on hFF feeder-layer was regular and growth state of hPESC was well. hPESC on hFF feeder-layer was not apt to differentiate. After subcultured for25passages in vitro, hPESC still maintained biological characteristics and normal karyotype. Morphous of hPESC on MEF feeder-layer was regular and growth velocity of hPESC was stable. Self-differentiation of hPESC on MEF feeder-layer seldom occurred. After subcultured for15passages in vitro, hPESC still maintained biological characteristics. Morphous of hPESC in feeder-free system was regular and growth velocity of hPESC was slow. Self-differentiation ratio of hPESC in feeder-free system was extremely low. After subcultured for15passages in vitro, hPESC still maintained biological characteristics.
3. After hPESC has been subcultured for15passages in vitro, morphous, adherence ratio, self-differentiate ratio and amplification velocity of hPESC, animal-derived contamination, workload and cost in three culture systems were considered thoroughly. We found that as feeder-layer of hPESC hFF owns a significant advantage.
4. Under the effect of Wnt3a and Activin A, hPESC was induced to differentiate into DE. The results of flow cytometry indicated that expression of CXCR4and ECD culminated on the second day. The results of real-time quantitative PCR displayed that expression of Sox-17and Gsc culminated on the second day. Therefore, the percentage of DE was highest on the second day.
5. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Immunocytochemical double staining displayed that Msil+Hesl+cells culminated on the fifth day. The results of real-time quantitative PCR revealed that the expression of Msil and Hesl culminated synchronously on the fifth day. Therefore, the percentage of IESC was highest on the fifth day.
Conclusions:
1. hFF was obtained successfully by trypsin digestion method.
2. Under the appropriate conditions all of three systems could sustain the growth of hPESC and keep hPESC undifferentiated.
3. hPESC could keep biological characteristics and normal karyotype on hFF feeder-layer. As a kind of human-derived feeder-layer, hFF could sustain the growth of hPESC and keep hPESC undifferentiated. After various comparison, we found that hFF feeder-layer have a significant advantage.
4. Combined application of inducing factors Activin A and Wnt3a can promote hPESC to differentiate into DE; the percentage of DE was highest on the second day. The hPESC induced with Wnt3a and Activin A on the second day is the ideal progenitor cell of IESC.
5. Inducing factor EGF can promote DE to differentiate into IESC. The percentage of IESC was highest on the fifth day. It will provide potential cell source for the IESC transplantation treatment of damage of intestinal epithelium and theoretical basis for reconstruction of intestinal epithelium and intestinal tissue engineering research.
6. hPESC was successfully induced to differentiate into IESC by a phased method.
引文
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