体外培养及其诱导分化人胚胰腺巢蛋白和神经元素3阳性细胞的实验

An experimental study on isolation, culture and inductive differentiation of the Nestin and Neurogenin 3 positive cells from human fetal pancreas in vitro

目的:观察从人胚胎胰腺体外分离培养的巢蛋白和神经元素3阳性细胞,分析其基因表达及诱导分化能力,探讨为胰腺组织工程移植提供种子细胞的可能性。方法:实验于2002-01/2004-05在北京大学干细胞中心完成。实验采用标本为北京市海淀妇产医院产科药物引产的人胚胎,引产药物为米索。使用的胚胎经孕妇同意,并签署同意书。选取18例胎龄为12～24周的人胚胰腺,用Ⅴ型胶原酶消化获得贴壁生长的人胚胰腺细胞,观察其原代培养、传代培养、增殖能力及体外诱导分化能力;胰腺干细胞标志巢蛋白和神经元素3阳性细胞及胰岛素表达应用免疫荧光染色及反转录聚合酶链反应检测;细胞总蛋白中胰岛素含量应用化学发光法检测。结果:18例胎龄为12～24周的人胚胰腺全部纳入实验。①人胚胎胰腺组织体外培养和诱导分化:人胚胎胰腺组织消化后可获得胰岛样结构,能贴壁生长,传代20代以上,然后进入凋亡期。汇合的细胞加入诱导分化培养基,24h后即可见大部分细胞聚集成团;周围散在贴壁细胞突起细长,立体感增强。随着诱导时间的延长,细胞增殖缓慢,个别细胞脱落、死亡。细胞团易于脱落漂浮,继而死亡。但仍有部分细胞及细胞团贴壁生长。②巢蛋白、胰岛素、胰高血糖素、广谱角蛋白及角蛋白19含量测定:无论是第2代还是第10代细胞中均无胰岛素、胰高血糖素CK-Pan和CK-19染色阳性的细胞。第2代细胞中,巢蛋白阳性细胞约占总细胞数的20%,个别细胞核神经元素3阳性,多数细胞为两者均阴性。第10代细胞中95%以上为巢蛋白强阳性细胞,约90%细胞神经元素3阳性且阳性部位同时存在于细胞核和细胞质中,并与巢蛋白共表达。但第18代细胞,荧光染色神经元素3染色转阴性(反转录聚合酶链反应可检测到少量mRNA的表达),而巢蛋白阳性无明显变化。③诱导前后巢蛋白和神经元素3阳性细胞及胰岛素表达:诱导前有巢蛋白、胰十二指肠同源盒基因-1、神经元素3的表达,无胰岛素表达。诱导后出现胰岛素表达,胰十二指肠同源盒基因-1表达增高,巢蛋白和神经元素3表达下降。④胰岛素含量测定结果:诱导分化后,巢蛋白和神经元素3阳性细胞的总蛋白质中含有胰岛素量为2.10IU/g总蛋白,作为阳性对照的人胚胎胰腺组织总蛋白中的含量为2.70IU/g总蛋白,而未诱导的细胞的总蛋白质中未检测到胰岛素。结论:从人胚胎胰腺可以体外分离获得巢蛋白和神经元素3阳性人胚胎胰腺干细胞,为未分化细胞,能在体外大量扩增,可长期培养。总蛋白中有胰岛素表达,有分化为胰岛素阳性细胞的潜能,有望为胰腺组织工程移植提供足够的种子细胞。

AIM： To isolate and culture Nestin and Neurogenin 3（Ngn 3） positive human fetal pancreatic cells and analyze the genes expression and the capacity of differentiation to insulin-positive cells in vitro for seeking the possibility of providing seeding cells for pancreatic tissue engineering. METHODS： The experiment was done in the Stem Cell Center of Peking University between January 2002 and May 2004. Human fetuses with Misoprofil induced labor were supported by the Department of Obstetrics, Beijing Haidian Obstetrics and Gynecology hospital. Informed consent was obtained from gravida. Eighteen fetuses at 12-24 weeks were selected to obtain human fetal pancreas. Human fetal pancreatic cells were gained by digestion of collagenase V. Primary culture, passage culture, the capacity of expansion and inductive differentiation in vitro were observed; lmmunofluorescence stain and reverse transcription-polymerase chain reaction （RT-PCR） techniques were used to exam the expression of the markers of pancreatic stem cells and insulin; The content of insulin in total protein was detected by the chemiluminescence technique. RESULTS： Human fetal pancreases from 18 fetuses were all involved in the experiment. ① In vitro culture and differentiation： Human fetal pancreatic tissues could present insulin-like structure after digestion, grow adherently with the passage numbers of over 20, and then enter the apoptotic phase. Cell aggregation was formed in most cells 24 hours after the cells were cultured in inductive differentiation medium. Adherent cells with long and thin processes scattered around with strong stereogenosis. Along with inductive differentiation, cell proliferation became slowly, and individual cells died or shed. Cell assemblies were likely to shed and drift and then die. However, there were still some adherent cells or cell assemblies. ② Measurement of Nestin, insulin, glucagons, broad-spectrum keratin （CK- Pan） and keratin 19 （CK-19）： There were no insulin, glucagons, CK-Pan and CK-19 positive cells either in the 2^nd passage or in the 10^th passage. In the cells of the 2^nd passage, Nestin-positive cells accounted for 20% in all the cells. Some cells showed positive Ngn 3 and nucleus, and most cells showed negative. In the 10^th passage cells, over 95% cells had strong Nestin-positive expression, and about 90% cells had positive Ngn 3 expression in nucleus and cytoplasm. However, in the cells of the 18^th passage, fluorescence staining showed that Ngn 3 presented negative expression （a few mRNA expressions were detected by RT-PCR technique） , and no changes were found in Nestin expression. ③ Expression of Nestin and Ngn3 positive cell and insulin： There were expressions of Nestin, pancreaticoduodenal homeobox gene 1, Ngn 3, but no expression of insulin before differentiation. After differentiation, the expression of insulin appeared, the expression of pancreaticoduodenal homeobox gene 1 increased, and the expressions of Nestin and Ngn 3 decreased. ④ Measurement of insulin： After differentiation, the content of insulin was 2.10 IU/g protein in Nestin and Ngn 3, and 2.70 IU/g in human fetal pancreatic tissue. There was no insulin expression in undifferentiated cells. CONCLUSION： The Nestin and Ngn 3 positive stem cells isolated from human fetal pancreas can be proliferated massively, cultured for long time in vitro and induced to differentiate to insulin-positive cells. They might offer enough seeding cells for pancreatic tissue engineering.