体外诱导人脐带间充质干细胞分化为胰岛样细胞研究

发布时间：2018-06-16 02:48

本文选题：人脐带 + MSC_S　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCS)进行分离培养和体外传代扩增,并分离大鼠的胰岛细胞,利用大鼠胰岛细胞的微环境,通过共培养体系诱导hUC-MSCS向胰岛样细胞初步转化,探索hUC-MSCS在体外胰岛细胞的微环境中向胰岛样细胞的分化潜能及功能变化趋势。方法: 1无菌条件下取足月剖宫产胎儿脐带(获得家属知情同意),自两根脐动脉间沿血管长轴剪开脐带被膜,去掉血管,采用组织块贴壁培养法从人脐带Wharton’s Jelly中分离培养MSCS,倒置显微镜下观察其形态变化;传代扩增后经流式细胞仪鉴定CD34抗体、CD45抗体、CD14抗体、CD29抗体、CD44抗体及CD105抗体的表达; 2采用胶原酶原位灌注消化法和滤网过滤法分离大鼠胰岛细胞,特异性的双硫腙(Dithizon,DTZ)染色进行鉴定; 3选取状态良好的第3代或第4代hUC-MSCS,以1×105/ml接种于6孔Transwell板的底层(共培养组)和普通6孔培养板(单纯培养组),倒置显微镜下观察细胞贴壁后,将分离获得的大鼠胰岛细胞团以50个/孔接种于Transwell共培养板的insert中,通过共培养体系对hUC-MSCS进行诱导分化,倒置显微镜下观察诱导过程中hUC-MSCS的形态变化;免疫细胞化学法鉴定诱导后hUC-MSCS的胰岛细胞早期标志即胰十二指肠同源框-1基因(Pancreatic Duodenal Homeobox-1, PDX-1)的表达;放免法定量检测单纯培养组和共培养组细胞的胰岛素及C肽分泌水平,观察两组细胞的胰岛素分泌功能变化趋势及对葡萄糖刺激实验的反应性。结果: 1利用组织块贴壁培养法从人脐带Wharton’s Jelly中分离出的贴壁细胞,倒置显微镜下观察,7天左右可见组织块周围出现贴壁生长的短棒状或梭形样细胞,14天左右细胞形态变为均一的长梭形,20天左右达70-80%融合,可进行传代扩增培养,传代后细胞形态类似成纤维细胞,呈平行排列生长或旋涡状生长;细胞传代培养至第3代,流式细胞学鉴定细胞表面标志,结果高表达间充质细胞的相关表面标志(CD29、CD44、CD105),几乎不表达与造血细胞相关的细胞表面标志(CD34、CD45、CD14);细胞传代培养至第10代,仍然稳定保持着MSCS的特性。 2每只体重为250-300g的正常SD大鼠,其胰腺组织经胶原酶原位灌注消化及滤网过滤分离后可获得300个左右的胰岛细胞团,DTZ染色呈棕红色; 3倒置显微镜下观察,共培养组hUC-MSCS诱导至第3天时,细胞周边突起缩短,形态逐渐变为椭圆形,诱导至第7天时,可见散在的半悬浮生长的胰岛样细胞团,双硫腙染色呈棕红色,诱导至第10天左右,胰岛样细胞团体积变小,形态变得松散不规则,诱导至第14天时,胰岛样细胞团数量减少,体积进一步缩小。单纯培养组的hUC-MSCS在培养过程中形态无明显变化,仍呈长梭形生长; 4共培养组诱导至第3天和第7天时,胰岛样细胞经免疫细胞化学染色鉴定,胰岛细胞早期标志PDX-1表达阳性,诱导至第10天和第14天的胰岛样细胞,PDX-1阳性细胞罕见。单纯培养组PDX-1表达阴性; 5采用放免法在共培养组的培养上清中检测到较高水平的胰岛素及C肽分泌,单纯培养组检测到少量的胰岛素,几乎检测不到C肽。经SPSS13.0统计软件分析,共培养组与单纯培养组相比,细胞培养上清中胰岛素分泌水平在不同的培养时间均有统计学差异(p0.01)。共培养组:第3天和第14天的细胞培养上清中胰岛素的分泌量无明显统计学差异(p0.05),但与第7天和第10天相比均有统计学差异(p0.05),第7天的细胞培养上清中胰岛素的分泌水平最高,第10天的分泌量仅次于第7天,但均明显高于第3天和第14天;C肽的检测结果与胰岛素一致。共培养组胰岛样细胞的胰岛素及C肽的分泌水平随体外诱导培养时间的延长,均呈现先递增后递减的趋势; 6选取共培养组和单纯培养组培养至第7天的细胞进行葡萄糖刺激胰岛素释放实验,共培养组的细胞在含葡萄糖1000mg/dl的无血清L-DMEM培养基中检测到胰岛素的分泌量为19.6025±5.9516μIU/ml,在含葡萄糖4500mg/dl的无血清H-DMEM中检测到胰岛素的分泌量为27.6617±7.14825μIU/ml,单纯培养组的细胞在含葡萄糖1000mg/dl的无血清L-DMEM培养基中胰岛素的分泌量为5.8000±1.006μIU/ml,在含葡萄糖4500mg/dl的无血清H-DMEM中检测到胰岛素的分泌量为6.2100±1.1746μIU/ml。共培养组的胰岛样细胞,在高糖和低糖培养基中胰岛素的分泌水平具有明显的统计学差异(p0.01),胰岛素的分泌量随葡萄糖浓度的增加明显增加,胰岛样细胞的葡萄糖刺激指数为1.4372±0.1390。单纯培养组细胞的胰岛素分泌对葡萄糖的刺激变化不大(p0.05)。结论: 人脐带Wharton’s Jelly中分离出的MSCS,采用Transwell共培养体系,在体外胰岛细胞的微环境中具有向胰岛样细胞分化的潜能,其胰岛素分泌功能随着体外诱导培养时间的延长呈现先递增后递减的趋势。
[Abstract]:Objective: to isolate and culture human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCS), and to isolate rat islet cells, and to use the microenvironment of rat islet cells to induce the initial transformation of hUC-MSCS into the islet like cells by co culture system, and to explore the expression of hUC-MSCS in the islet islets in vitro. The differentiation potential and functional change trend of islet like cells in the microenvironment.
Method:
1 under aseptic conditions, the fetal umbilical cord was taken from the fetal umbilical cord for full term cesarean section (obtaining the informed consent of the family). The umbilical cord was removed from the vascular long axis and removed from the umbilical artery between two umbilical arteries. The tissue block adherence culture was used to isolate and culture the MSCS from the Wharton 's Jelly of the human umbilical cord, and the morphological changes were observed under the inverted microscope; the passage was amplified by the flow cytometry. The expression of CD34 antibody, CD45 antibody, CD14 antibody, CD29 antibody, CD44 antibody and CD105 antibody.
2 the rat pancreatic islet cells were isolated by collagenase perfusion in situ and screen filtration, and identified by Dithizon (DTZ) staining.
3 the third or fourth generation of hUC-MSCS, which were in good condition, were inoculated at the bottom of the 6 hole Transwell plate (co culture group) and the ordinary 6 Hole culture plate (simple culture group). After the inverted microscope observation cells were adhered to the cell wall, the isolated rat islet cell groups were inoculated into the insert of the Transwell co culture plate with 50 / holes, through co culture. HUC-MSCS was induced and differentiated, and the morphological changes of hUC-MSCS during induction were observed under the inverted microscope; the expression of the early pancreatic islet cell marker of hUC-MSCS was identified by immunocytochemical method, namely, the expression of Pancreatic Duodenal Homeobox-1 (PDX-1) gene (Pancreatic Duodenal Homeobox-1, PDX-1), and the radioimmunoassay was used to determine the simple culture group and co culture. Insulin secretion and C peptide secretion level in the maintenance group were observed, and the changes of insulin secretion function and the responsiveness to glucose stimulation in the two groups were observed.
Result:
1 the parietal cells isolated from human umbilical cord Wharton 's Jelly were cultured with tissue block wall culture method. The short rod like or shuttle like cells were found around the tissue around 7 days, and the cell morphology changed into a homogenous long shuttle shape around 14 days, and 70-80% fusion around 20 days. After generation, cell morphology resembles fibroblasts, which are parallel growth or vortexed growth; cells are cultured to third generations, flow cytometry is used to identify cell surface markers, and the result is high expression of related surface markers of mesenchymal cells (CD29, CD44, CD105), almost no expression of cell surface markers associated with hematopoietic cells (CD34, CD45, CD14); cells Subculture to the tenth generation remained stable and maintained the characteristics of MSCS.
2 of the normal SD rats with a body weight of 250-300g, about 300 of the pancreatic islet cells were obtained by collagenase in situ perfusion digestion and filter screen filtration, and DTZ staining was brown red.
3 inverted microscope was observed. When the co culture group hUC-MSCS was induced to third days, the peripheral protuberances of the cells were shortened and the morphology became elliptical. The islet like cell clusters scattered in the semi suspension were seen at seventh days. The dithizone staining was brown red, induced to the left right of tenth days, and the size of the islet like cell group became smaller and the shape became loose and irregular. On the other hand, the number of islet like cell groups decreased and the volume reduced further at fourteenth days. The morphology of hUC-MSCS in the simple culture group was not obviously changed during the culture process, and it was still long spindle shaped.
4 the 4 co culture groups were induced at third days and seventh days. The islet like cells were identified by immunocytochemical staining. The early sign of the islet cells was positive, and the islet like cells were induced to tenth days and fourteenth days, and the PDX-1 positive cells were rare. The expression of PDX-1 in the simple culture group was negative.
5 the high level of insulin and C peptide secreted in the culture supernatant of the co culture group were detected by radioimmunoassay. A small amount of insulin was detected in the simple culture group and almost no C peptide was detected. The insulin secretion level in the culture supernatant of the co culture group was compared with that of the simple culture group. The level of insulin secretion in the cell culture supernatant was in different culture time. There were statistical differences (P0.01). There was no significant difference in the secretion of insulin in the third days and fourteenth days of cell culture supernatant (P0.05), but there were statistically significant differences compared with seventh days and tenth days (P0.05). The secretion level of insulin in the seventh day cell culture supernatant was the highest, and the tenth day secretion was only inferior to seventh days. The results were significantly higher than that of third days and fourteenth days. The results of C peptide were consistent with insulin. The secretion level of insulin and C peptide in the islet like cells in the co culture group increased with the increase of the induced culture time in vitro.
6 the cells in the co culture group and the simple culture group were cultured to seventh days for glucose stimulation of insulin release. The cells in the co culture group detected the insulin secretion in the serum free L-DMEM medium containing glucose 1000mg/dl 19.6025 + 5.9516 mu IU/ml, and the insulin was detected in the serum free H-DMEM containing glucose 4500mg/dl. The secretion of insulin was 27.6617 + 7.14825 IU/ml. The secretion of insulin in the serum-free L-DMEM medium containing glucose 1000mg/dl was 5.8000 + 1.006 IU/ml in the simple culture group, and the insulin secretion in the serum free H-DMEM containing glucose 4500mg/dl was 6.2100 + 1.1746 mu IU/ml. co culture group of islet like cells. The insulin secretion level in the sugar and low sugar medium had significant statistical difference (P0.01). The secretion of insulin increased significantly with the increase of glucose concentration. The glucose stimulation index of islet like cells was 1.4372 + 0.1390., and the insulin secretion of the cells in the simple culture group had little change on the glucose stimulation (P0.05).
Conclusion:
The separation of MSCS from human umbilical cord Wharton 's Jelly, using Transwell co culture system, has the potential to differentiate into islet like cells in the microenvironment of islet cells in vitro, and its insulin secretion function increases first and then decreases with the prolongation of the induced culture time in vitro.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】