骨髓间充质干细胞体外向胰岛样细胞诱导分化的实验研究

发布时间：2018-06-24 14:18

本文选题：骨髓 + 间充质干细胞　；参考：《四川大学》2007年博士论文

【摘要】： 背景与目的骨髓间充质干细胞(BMSCs)是存在于骨髓中的一种具有自我更新和多向分化潜能的细胞。研究表明骨髓间充质干细胞可以向内胚层、中胚层和外胚层组织细胞分化，如肝细胞、心肌细胞、肌细胞、神经细胞、脂肪细胞和胰岛素分泌细胞等。本研究旨在体外分离培养大鼠BMSCs，观察BMSCs的生物学特性，并采用流式细胞检测技术鉴定BMSCs。为进一步观察体外BMSCs向胰岛素分泌细胞分化奠定基础。材料与方法Wister大鼠麻醉致死后，无菌条件下取出胫骨和股骨用L-DMEM培养基从骨腔一端冲洗骨髓腔，冲出骨髓，轻柔充分吹打成单细胞悬液，离心洗涤后，所得细胞按1×10~8/mL密度接种于25cm~2塑料培养瓶中，37℃，5％CO_2，饱和湿度下培养48小时，首次更换培养液，弃去未贴壁细胞，此后每2-3天换液1次，观察细胞形态变化。待细胞生长良好，80％融合后，传代培养。取第3或4代细胞做流式细胞检测，检测获得的贴壁细胞表面标记抗原CD29、CD90和CD45表达情况。结果在最初培养的48h内，可见大量圆形细胞，悬浮生长。48h后可见部分细胞呈贴壁生长。至第5天时，随着换液次数的增加，圆形悬浮细胞逐渐减少，贴壁细胞明显增多，呈聚集样生长，细胞形态呈梭形和多角形。约10-14天后细胞铺满瓶底，呈鱼丛状或漩涡状聚集，细胞形态呈梭形。流式细胞方法检测大鼠BMSCs的表面标记抗原。结果显示：第三代大鼠BMSCs的特异性表面标志物：CD29阳性，阳性率为91．9％；CD45阴性，阳性率为6．9％；CD29/CD45阳性率为7．4％；CD90阳性，阳性率为51．3％；CD90/CD45阳性率为3．6％。结论本实验通过贴壁筛选法和全骨髓培养法成功分离培养了大鼠骨髓间充质干细胞。目的探讨联合应用高糖和GLP-1、活化素A、尼克酰胺和β细胞调节素刺激体外能否诱导人骨髓间充质干细胞向胰岛样细胞分化。材料与方法体外培养人骨髓间充质干细胞，待细胞80％融合时，用0．1％胰蛋白酶消化传代，按2×10~5/ml接种于96孔板。观察细胞生长情况。采用以下诱导刺激方案：先给予23mmol/L高葡萄糖刺激约20天，接着再将刺激后细胞分为7组，分别给予不同的刺激条件：A组：23mmol/L高葡萄糖培养基刺激；B组：L—DMEM培养基中加GLP-1，终浓度为5nmol/L；C组：L—DMEM培养基中加活化素A，终浓度为10ng/ml；D组：L—DMEM培养基中加尼克酰胺，终浓度为10mmol/L；E组：L—DMEM培养基中加GLP-1+活化素A+尼克酰胺+BTC刺激因子协同作用；F组：L—DMEM培养基中加β细胞调节素，终浓度为10ng/ml；G组为普通L—DMEM培养基，设为对照组。在不同的刺激条件下，培养细胞16天，每2～3天更换细胞培养液1次，，待细胞铺满瓶底，80％融合时传代，制备细胞爬片。用放射免疫法检测细胞培养基中的胰岛素水平。RT-PCR检测各刺激组细胞胰岛素mRNA的表达水平。免疫细胞化学方法检测各刺激组细胞胰岛素、胰高血糖素、生长抑素和巢蛋白的表达。检测诱导分化后细胞对高葡萄糖刺激的胰岛素释放实验。将诱导分化后细胞按3×10~5/孔密度接种于24孔培养板中，37℃培养过夜。第二天，收集旧的培养基后，PBS洗涤细胞3次，新鲜L-DMEM1ml，培养1小时，再给予23mmol/L高葡萄糖培养基刺激细胞2小时，分别于0分、30分、1h、2h收集培养基，放射免疫法检测胰岛素分泌水平。胰蛋白酶消化、收集细胞后，将收集的细胞加入酸酒精中，-20℃过夜，次日用细胞超声破碎仪破碎细胞，取上清检测胰岛素浓度。考马斯亮兰法测定细胞内总蛋白浓度。结果1．诱导分化前hBMSCs呈贴壁生长，细胞透明，呈梭形或多边形，鱼丛状或漩涡状聚集生长。未给予任何刺激前人骨髓间充质干细胞培养基中(细胞密度为1．0×10~6/m1)胰岛素水平为2．2867±0．2665μU/ml。放射免疫法检测普通L-DMEM培养基中胰岛素水平为2．3650±0．4296μU/ml(空白对照，本底值)。该两组间的T检验，P值为0．359＞0．05，两组间胰岛素水平无显著性差异。2．给予23mmol/L高葡萄糖刺激刺激20天，再将上述步骤得到的细胞分为不同的7个刺激组，16天后收集细胞培养基，检测胰岛素分泌水平，发现23mmot/L高糖组、GLP-1组、活化素A组、尼克酰胺组、协同作用组、BTC组和普通L-DMEM组胰岛素分泌水平与刺激前hBMSCs细胞培养基(胰岛素分泌量为2．2867±0．2665μU/10~6 cells和/ml)相比，分别增加了3．13、3．00、2．35、2．94、2．84、3．22和2．32倍。协同作用并没有显著增加胰岛素分泌量。3．RT-PCR检测胰岛素mRNA水平发现：GLP-1组和活化素A组可见清晰的胰岛素PCR产物目的片段。协同作用组和BTC组分别在900bp和300bp位置见到特异性条带，而且900bp片段较300bp片段清晰。900bp的未知片段经测序后，发现其与人胰岛素原基因相比较有较多的突变，故未能证实该未知片段的性质。尼克酰胺组隐约可见300bp胰岛素目的片段，但条带亮度太低。高糖组和普通L-DMEM组均未见到胰岛素目的片段。4．免疫细胞化学证实除了高糖+普通L-DMEM培养基外，各刺激组均有胰岛素表达；各刺激组均未表达胰高血糖素；除协同作用组有生长抑素的弱阳性表达外，其它各刺激组均未表达生长抑素；尼克酰胺组和协同作用组可见到巢蛋白的弱阳性表达，其余各刺激组均未表达巢蛋白。5．高糖刺激胰岛素释放实验结果示，经联合高糖和GLP-1、活化素A、BTC、尼克酰胺和共同刺激诱导分化后，BMSCs对高葡萄糖刺激有反应，能相应增加胰岛素的分泌量；6．细胞内胰岛素浓度以活化素A组、BTC组和高糖+普通L-DMEM组最高，其它各刺激组细胞内胰岛素浓度较低。结论本实验应用高糖和GLP-1、活化素A、尼克酰胺、BTC等刺激因子分别或联合于体外诱导人骨髓间充质干细胞向胰岛样细胞分化，经放射免疫方法、RT-PCR、免疫细胞化学等方法证实，高糖、GLP-1、活化素A、尼克酰胺和BTC均可以在体外诱导人BMSCs分化为胰岛素分泌细胞，但胰岛素表达量或产生量仍较低。本实验未发现各刺激条件间有较强的协同作用。
[Abstract]:Background and objective bone marrow mesenchymal stem cells (BMSCs) is a cell with potential for self renewal and multidifferentiation in bone marrow. Studies show that bone marrow mesenchymal stem cells can differentiate into the cells of the endoderm, mesoderm and ectoderm, such as hepatocytes, cardiomyocytes, muscle cells, nerve cells, adipocytes and insulin secretion. The aim of this study was to isolate and culture rat BMSCs in vitro, to observe the biological characteristics of BMSCs, and to identify BMSCs. as the basis for further observation of the differentiation of BMSCs to insulin secreting cells in vitro. Materials and methods Wister rats were killed and the L-DMEM culture medium was removed from the tibia and femur without bacteria. The bone marrow cavity was flushed from one end of the bone cavity, and the bone marrow was washed out into a single cell suspension. After centrifugation, the cells were inoculated in the 25cm~2 plastic culture bottle at 1 x 10~8/mL density. The cells were cultured at 37, 5%CO_2, under saturated humidity for 48 hours. The culture solution was replaced for the first time, and the unadhered cells were abandoned for the first time, and then 1 times every 2-3 days were changed to observe the cell morphologic changes. After the cell growth was good, after 80% fusion, the third or 4 generation cells were used for flow cytometry to detect the expression of the surface labeled antigen CD29, CD90 and CD45 of the adherent cells. With the increase of the changing times, the circular suspension cells gradually decreased and the adherent cells increased obviously. The cell morphology showed spindle shape and polygon. After about 10-14 days, the cells covered the bottom of the bottle and gathered in the shape of fish bushes or whirlpools. The cell morphology was shuttle shaped. The flow cytometry was used to detect the surface labeled antigen of BMSCs in rats. The results showed that third generations. The specific surface markers of BMSCs in rats: CD29 positive, positive rate 91.9%, CD45 negative, positive rate 6.9%, CD29/CD45 positive rate 7.4%, CD90 positive, positive rate 51.3%, CD90/CD45 positive rate of 3.6%.
Conclusion rat bone marrow mesenchymal stem cells were successfully isolated and cultured by adherence screening and whole bone marrow culture.
Objective to investigate whether the combination of high glucose and GLP-1, activin A, nicotinamide and beta cell regulator stimulate the differentiation of human bone marrow mesenchymal stem cells into islet like cells in vitro.
Materials and methods human bone marrow mesenchymal stem cells were cultured in vitro. When the cell 80% was fused, 0.1% trypsin was used to digest the passage and inoculated to 96 orifice plates according to 2 x 10~5/ml. The following induction stimulation scheme was used to stimulate 23mmol/L Hyperglucose for about 20 days, and then the cells were divided into 7 groups after the stimulation, respectively. Different stimulation conditions: group A: 23mmol/L high glucose medium stimulation; group B: GLP-1 in L DMEM medium, final concentration is 5nmol/L; C group: L DMEM culture medium adding activin A, the final concentration is 10ng/ml. A+ NK +BTC stimulator synergistic action; group F: L DMEM medium with beta cytokine and final concentration of 10ng/ml; G group is a common L DMEM medium, set as control group. Under different stimulation conditions, cultured cells for 16 days, replace cell culture 1 times every 2~3 days, PVE cell bottom, 80% fusion generation, preparation Cell crawling. The level of insulin mRNA expression in the cells of each stimulation group was detected by radioimmunoassay.RT-PCR. The expression of insulin, glucagon, somatostatin and nestin in each stimulation group was detected by immunocytochemistry. After the induced differentiation, the cells were inoculated into the 24 hole culture plate at 3 x 10~5/ pore density. After second days, second days, after collecting the old medium, the PBS washing cells were 3 times, fresh L-DMEM1ml, and cultured for 1 hours, and then given the 23mmol/L high glucose medium for 2 hours, 0, 30, 1H, 2h, respectively. After trypsin digestion and collection of cells, the cells collected were added to the acid alcohol, -20 C for the night, the cells were broken by cell ultrasonic breakers on the next day, and the concentration of insulin was detected by the supernatant.
Results 1. before differentiation, hBMSCs was adhered to wall growth, cells were transparent, spindle or polygon, fish plexiform or whirlpool like growth. No stimulation was given to the previous medium of bone marrow mesenchymal stem cells (the cell density of 1 * 10~6/m1) was 2.2867 + 0.2665 micron U/ml. radioimmunoassay in ordinary L-DMEM medium. The insulin level was 2.3650 + 0.4296 U/ml (blank control, background value). The T test between the two groups was 0.359 > 0.05. There was no significant difference in insulin between the two groups. The 23mmol/L Hyperglucose stimulation was given by.2. for 20 days. Then the cells obtained from the above steps were divided into 7 different stimulation groups, and the cell culture medium was collected after 16 days. The level of insulin secretion was detected, and the insulin secretion of 23mmot/L high glucose group, GLP-1 group, activin A group, NK amide group, CO action group, BTC group and common L-DMEM group was compared with that of hBMSCs cell culture medium before stimulation (insulin secretion was 2.2867 + 0.2665 U/10~6 cells and /ml), respectively. .84,3.22 and 2.32 times. The synergistic effect did not significantly increase insulin secretion.3.RT-PCR detection of insulin mRNA levels: the GLP-1 group and the activin A group showed clear insulin PCR product target fragments. The synergistic group and the BTC group saw the specific bands in the 900bp and 300bp positions respectively, and the 900bp fragment was clearer than the 300bp fragment. The unknown fragment of the.900bp was sequenced, and it was found that there were more mutations than the human proinsulin gene, so the nature of the unknown fragment was not confirmed. The niacin group had a vaguely visible 300bp insulin fragment, but the band brightness was too low. Neither the high sugar group nor the common L-DMEM group had seen the.4. immunocytochemistry of the insulin target fragment. It was confirmed that all the stimulation groups had insulin expression except high sugar + ordinary L-DMEM medium, and all the stimulation groups did not express glucagon. Except for the weak positive expression of somatostatin in the synergistic group, the other groups did not express somatostatin, and the other groups showed the weak positive expression of nestin in the group of nicotinamide and the other group. All the stimulation groups did not express the results of insulin release from nestin.5. high glucose stimulated by insulin release. After induced differentiation by combined high glucose and GLP-1, activin A, BTC, niacin and co stimulation, BMSCs responded to Hyperglucose stimulation and could increase the secretion of insulin; 6. the intracellular insulin concentration was in the activin A group, the BTC group and the high glucose +. The normal L-DMEM group was the highest, and the other groups had lower insulin concentration.
Conclusion high sugar and GLP-1, activin A, nicotinamide, BTC and other stimulating factors are used to induce human mesenchymal stem cells to differentiate into islet like cells respectively in vitro. It is confirmed by radioimmunoassay, RT-PCR, immunocytochemistry and other methods that high sugar, GLP-1, activin A, nicotinamide and BTC can all induce human BMSCs in vitro. Differentiation into insulin secreting cells, but the amount of insulin production or production is still low.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【引证文献】