小鼠间充质祖细胞体外优化培养及向神经元样细胞定向诱导分化的实验研究
发布时间：2018-09-19 16:16
【摘要】： 目的:对体外培养C57小鼠密质骨来源的间充质祖细胞(mesenchymal progenitor cells,MPC)的方法进行研究,建立适合小鼠MPC增殖的培养方案;探讨小鼠密质骨来源的MPC体外向神经元样细胞定向诱导分化的实验研究。 方法: 取材方法选择健康C57雌性小鼠,清洁级,3周龄,18±2 g,取股骨、胫骨碎片经Ⅱ型胶原蛋白酶消化后作为MPC来源。 分组取得消化后的骨碎片,选取干细胞培养中经常使用的DMEM/F12培养基、IMDM培养基、α-MEM培养基和TBD公司生产的胎牛血清(FCS)、GIBCO公司生产的胎牛血清,按不同搭配进行培养,随机分为DMEM/F12+10%TBD胎牛血清组、DMEM/F12 +10%GIBCO胎牛血清组、IMDM+10%TBD胎牛血清组、IMDM +10%GIBCO胎牛血清组、α-MEM +10%TBD胎牛血清组、α-MEM +10%GIBCO胎牛血清组,共6组,每组培养6份。 培养方法将小鼠密质骨碎片置入六孔板中,按不同分组加入相应培养液,放入37℃、5% CO2的培养箱内,48 h后首次换液,以后每隔2-3 d换液一次。每天观察细胞生长情况,当生长最好的实验组原代(P0)细胞细胞贴壁超过培养板底面积80%时吸出培养液,用含0.04% EDTA的0.25%胰蛋白酶消化、收集细胞,按固定细胞浓度传入培养瓶。传代后以后每2-3 d换液一次,当生长最好的实验组细胞贴壁超过培养瓶底面积70%-80%时按固定细胞浓度1×103个/cm2传代。 MPC纯度鉴定采用流式细胞术分析,单标法检测第三代(P3)MPC,取FITC标记的兔抗鼠CD29、CD31、CD44、CD90和PE标记的兔抗鼠CD45、CD106作为标记物,平行对照组为加入同型对照抗体的MPC。考虑到实验的需要以及研究生学习阶段时间有限,只选择采用优化方案进行培养的MPC进行纯度鉴定。 MPC的多向分化鉴定对MPC进行向骨细胞、脂肪细胞的定向诱导分化,验证其具有干细胞的多向分化潜能,用茜素红和油红O染色检测诱导分化结果。同样只选择采用优化方案进行培养的MPC进行多向分化能力鉴定。 MPC定向诱导成神经元样细胞的方法取优化方案培养的P4代MPC,用微环境体液(神经元原代培养上清液)进行诱导,取诱导24 h后的MPC作为实验标本。对其进行形态学观察;免疫细胞化学检测神经元特异性标志物神经元特异性烯醇化酶(neuron specific enolase ,NSE)及神经丝蛋白(neurofilament protein,NF)的表达情况,同时进行免疫荧光分析。 结果: C57小鼠间充质祖细胞(MPC)体外优化培养 原代培养5 d后,可见培养器皿底部有细胞贴壁生长,以圆形和扁圆形为主,10 d后贴壁细胞逐渐铺开,形成梭形或多角形,形态饱满,折光性强,细胞间无重叠,有接触抑制现象;传代后细胞形态趋于一致,大部分为梭形,排列紧密,生长旺盛。当某个实验组细胞贴壁超过底面积70%-80%时,对所有细胞进行传代计数,观察3代。结果发现在每一代的细胞计数中,DMEM/F12+10% GIBCO胎牛血清组的细胞数量都超过其他各组,通过统计学检验验证DMEM/F12+10% GIBCO胎牛血清组细胞数量与其余各组相比有显著差异(P㩳0.05)。说明DMEM/F12+10%GIBCO胎牛血清更有利于MPC的体外培养增殖。 MPC纯度鉴定 优化方案培养的MPC表达间质细胞标记CD29、CD44、CD90和CD106,不表达造血细胞标记CD31和CD45。说明得到的是同源性好、纯度高、排除了造血干细胞影响的MPC。 MPC多向分化能力鉴定 在用优化方案培养的MPC定向诱导成骨细胞的实验中,通过茜素红染色发现诱导后细胞外基质中大量钙盐沉积;在定向诱导成脂肪细胞的实验中,通过油红O染色发现细胞中出现的脂滴被染成点状红色,表明我们得到的MPC能向骨细胞、脂肪细胞分化。说明我们得到的是活性好、具有多向分化能力的MPC。 MPC体外向神经元样细胞定向诱导分化 经诱导24 h后,MPC细胞形态发生变化,自胞体有突起长出,各细胞突起长短不一,类似神经元。免疫细胞化学检测结果表明,诱导后MPC的NSE(73.73%±9.88%)及NF(60.26%±7.19%)均阳性表达;免疫荧光检测也证实免疫细胞化学检测的结果。 结论 1.实验证明,用DMEM/F12+10% GIBCO胎牛血清为培养液进行小鼠密质骨来源的MPC体外培养最有利于其数量的扩增,得到的是同源性好、纯度高、增殖活力强、具有多向分化潜能的MPC细胞群。 2.通过微环境体液(神经元原代培养上清液)诱导的方法能够使小鼠密质骨来源的MPC定向分化为神经元样细胞。
[Abstract]:AIM: To study the method of culturing mesenchymal progenitor cells (MPCs) derived from mouse compact bone in vitro and establish a culture scheme suitable for the proliferation of mouse MPCs.
Method:
Methods Healthy C57 female mice, clean grade, 3 weeks old, 18 + 2 g, were selected as MPC source.
Digested bone fragments were obtained by grouping. DMEM/F12 medium, IMDM medium, alpha-MEM medium, fetal bovine serum (FCS) produced by TBD company and fetal bovine serum produced by GIBCO company were selected and cultured in different combinations. They were randomly divided into DMEM/F12+10% TBD fetal bovine serum group, DMEM/F12+10% GIBCO fetal bovine serum group, IM/F12+10% GIBCO fetal bovine serum group. DM+10% TBD fetal bovine serum group, IMDM+10% GIBCO fetal bovine serum group, alpha-MEM+10% TBD fetal bovine serum group, alpha-MEM+10% GIBCO fetal bovine serum group, a total of 6 groups, each group culture 6.
Methods The dense bone fragments of mice were put into six-hole plate, and the corresponding culture medium was added according to different groups. The culture medium was first changed after 48 hours, and then changed every 2-3 days. The culture medium was digested with 0.25% trypsin containing 0.04% EDTA. The cells were collected and passed into the culture flask at fixed cell concentration. After passage, the culture medium was changed every 2-3 days. When the adherence area of the best growing experimental group exceeded 70% -80% of the bottom area of the culture flask, the cells were subcultured at fixed cell concentration of 1 *103 cells per cm 2.
The purity of MPC was determined by flow cytometry. The third generation (P3) MPC was detected by single-label method. The FITC-labeled rabbit anti-mouse CD29, CD31, CD44, CD90 and PE-labeled rabbit anti-mouse CD45 and CD106 were used as markers. The parallel control group was used as MPC with homologous control antibodies. The purity of MPC was determined by the culture.
MPC was differentiated into osteoblasts and adipocytes by directional induction. The differentiation potential of MPC was verified by alizarin red and oil red O staining.
MPC was induced into neuron-like cells by microenvironment humor (supernatant of primary culture of neurons) and 24 hours after induction. Morphological observation and immunocytochemical detection of neuron-specific marker neuron-specific enolase (neuron-specific enolase) were performed. The expression of specific enolase, NSE and neurofilament protein (NF) was analyzed by immunofluorescence.
Result:
In vitro optimization culture of C57 mouse mesenchymal progenitor cells (MPC)
After primary culture for 5 days, the cells adhered to the wall and grew mainly round and oblate. After 10 days, the adherent cells gradually spread out, forming spindle or polygonal shape, full shape, strong refraction, no overlap between cells, and contact inhibition phenomenon. After passage, the cell morphology tended to be consistent, most of them were spindle-shaped, arranged closely, and grew vigorously. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group was higher than that in other groups in each generation. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group and other groups were verified by statistical test. The results showed that DMEM/F12+10% GIBCO fetal bovine serum was more conducive to the proliferation of MPC in vitro.
Purity identification of MPC
MPC cultured in the optimized scheme expressed interstitial cell markers CD29, CD44, CD90 and CD106, but did not express hematopoietic cell markers CD31 and CD45.
MPC identification of multiple differentiation ability
In the experiment of directional induction of osteoblasts by MPC cultured with optimized scheme, a large amount of calcium salt was found in the extracellular matrix after induction by alizarin red staining; in the experiment of directional induction of adipocytes, fat droplets were found to be dotted red by oil red O staining, indicating that the obtained MPC could be directed to osteoblasts, lipids. The differentiation of cells shows that we have obtained MPC. with good activity and multiple differentiation ability.
In vitro differentiation of MPC into neuron like cells in vitro
After 24 hours of induction, the morphology of MPC cells changed, and the processes grew out of the cell body. The length of each cell process was different, which was similar to neurons.
conclusion
1. Experiments showed that DMEM/F12+10% GIBCO fetal bovine serum was the best medium for the expansion of MPC derived from mouse compact bone in vitro. The MPC cells with good homology, high purity, strong proliferative activity and multi-differentiation potential were obtained.
2. Microenvironment bodily fluid (supernatant of primary culture of neurons) can induce mouse compact bone-derived MPCs to differentiate into neuron-like cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2250658
[Abstract]:AIM: To study the method of culturing mesenchymal progenitor cells (MPCs) derived from mouse compact bone in vitro and establish a culture scheme suitable for the proliferation of mouse MPCs.
Method:
Methods Healthy C57 female mice, clean grade, 3 weeks old, 18 + 2 g, were selected as MPC source.
Digested bone fragments were obtained by grouping. DMEM/F12 medium, IMDM medium, alpha-MEM medium, fetal bovine serum (FCS) produced by TBD company and fetal bovine serum produced by GIBCO company were selected and cultured in different combinations. They were randomly divided into DMEM/F12+10% TBD fetal bovine serum group, DMEM/F12+10% GIBCO fetal bovine serum group, IM/F12+10% GIBCO fetal bovine serum group. DM+10% TBD fetal bovine serum group, IMDM+10% GIBCO fetal bovine serum group, alpha-MEM+10% TBD fetal bovine serum group, alpha-MEM+10% GIBCO fetal bovine serum group, a total of 6 groups, each group culture 6.
Methods The dense bone fragments of mice were put into six-hole plate, and the corresponding culture medium was added according to different groups. The culture medium was first changed after 48 hours, and then changed every 2-3 days. The culture medium was digested with 0.25% trypsin containing 0.04% EDTA. The cells were collected and passed into the culture flask at fixed cell concentration. After passage, the culture medium was changed every 2-3 days. When the adherence area of the best growing experimental group exceeded 70% -80% of the bottom area of the culture flask, the cells were subcultured at fixed cell concentration of 1 *103 cells per cm 2.
The purity of MPC was determined by flow cytometry. The third generation (P3) MPC was detected by single-label method. The FITC-labeled rabbit anti-mouse CD29, CD31, CD44, CD90 and PE-labeled rabbit anti-mouse CD45 and CD106 were used as markers. The parallel control group was used as MPC with homologous control antibodies. The purity of MPC was determined by the culture.
MPC was differentiated into osteoblasts and adipocytes by directional induction. The differentiation potential of MPC was verified by alizarin red and oil red O staining.
MPC was induced into neuron-like cells by microenvironment humor (supernatant of primary culture of neurons) and 24 hours after induction. Morphological observation and immunocytochemical detection of neuron-specific marker neuron-specific enolase (neuron-specific enolase) were performed. The expression of specific enolase, NSE and neurofilament protein (NF) was analyzed by immunofluorescence.
Result:
In vitro optimization culture of C57 mouse mesenchymal progenitor cells (MPC)
After primary culture for 5 days, the cells adhered to the wall and grew mainly round and oblate. After 10 days, the adherent cells gradually spread out, forming spindle or polygonal shape, full shape, strong refraction, no overlap between cells, and contact inhibition phenomenon. After passage, the cell morphology tended to be consistent, most of them were spindle-shaped, arranged closely, and grew vigorously. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group was higher than that in other groups in each generation. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group and other groups were verified by statistical test. The results showed that DMEM/F12+10% GIBCO fetal bovine serum was more conducive to the proliferation of MPC in vitro.
Purity identification of MPC
MPC cultured in the optimized scheme expressed interstitial cell markers CD29, CD44, CD90 and CD106, but did not express hematopoietic cell markers CD31 and CD45.
MPC identification of multiple differentiation ability
In the experiment of directional induction of osteoblasts by MPC cultured with optimized scheme, a large amount of calcium salt was found in the extracellular matrix after induction by alizarin red staining; in the experiment of directional induction of adipocytes, fat droplets were found to be dotted red by oil red O staining, indicating that the obtained MPC could be directed to osteoblasts, lipids. The differentiation of cells shows that we have obtained MPC. with good activity and multiple differentiation ability.
In vitro differentiation of MPC into neuron like cells in vitro
After 24 hours of induction, the morphology of MPC cells changed, and the processes grew out of the cell body. The length of each cell process was different, which was similar to neurons.
conclusion
1. Experiments showed that DMEM/F12+10% GIBCO fetal bovine serum was the best medium for the expansion of MPC derived from mouse compact bone in vitro. The MPC cells with good homology, high purity, strong proliferative activity and multi-differentiation potential were obtained.
2. Microenvironment bodily fluid (supernatant of primary culture of neurons) can induce mouse compact bone-derived MPCs to differentiate into neuron-like cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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