牛胎肝间充质干细胞的分离培养及生物学特性研究

发布时间：2018-09-18 08:31

【摘要】：间充质干细胞是干细胞家族的重要成员之一,近年来由于它具有自我更新复制、免疫调控、多向分化等特性,有关间充质干细胞的研究也越来越广泛和深入。间充质干细胞最早在骨髓中发现,随后发现脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺等组织以及羊水、脐带血中也存在间充质干细胞。但是,目前的研究主要集中在骨髓、脐带血、脂肪等来源的间充质干细胞上,有关肝脏来源的间充质干细胞的研究很少。迄今为止,还没有关于牛胎肝来源的间充质干细胞方面的研究报道。本研究旨在建立牛胎肝间充质干细胞分离培养体系,探索其增殖、分化等生物学特性,结果如下： 1.采用Percoll密度梯度离心法分离约2-4月龄鲁西黄牛胎牛肝脏间充质干细胞,在体外培养至26代。体外培养的胎牛肝脏间允质干细胞生长旺盛,具有典型的成纤维形态,多数呈长梭状或不规则三角形,混有少数不规则或多边形细胞,细胞长满时呈现漩涡状或火焰状走势。镜下观察细胞胞质丰满,立体感较强。 2.不同代次的胎牛肝脏间充质干细胞的生长曲线都呈典型的“S”形,细胞经历了潜伏期、对数生长期和停滞期,符合细胞在体外生长的一般规律。但是随着传代次数的增加,细胞的增殖分裂能力逐渐下降。 3.分别检测P3,P7,P11,P22胎牛肝脏间充质干细胞的克隆形成率,分别为：43.38±8.54%,37.00±2.45%,32.92±2.50%,15.83±5.69%。 4.采用流式细胞仪分析P3,P7,P11,P22胎牛肝脏间充质干细胞的细胞周期,结果显示,Go/G1期的细胞随代次增加逐渐增多,S期的细胞呈逐渐减少的趋势。 5.核型分析表明,体外培养的胎牛肝脏间充质干细胞染色体数目2n=60,未见异常。二倍体率为93.6%,表明所培养的细胞均为稳定的二倍体细胞,在体外遗传性能稳定。 6.共冻存细胞163支,冻存前后的细胞活率都在90%左右。复苏后细胞存活率略有下降,其主要原因是细胞在冷冻和复苏过程中遭受机械和化学损伤所致。 7.采用免疫荧光和RT-PCR鉴定体外培养的P3,P7,P11,P22胎牛肝脏间充质干细胞是否具有间充质干细胞特有的细胞标志。免疫荧光显示不同代次细胞均呈CD44,CD29阳性,各个代次间相对荧光强度无明显差别。RT-PCR检测结果表明,不同代次的细胞均为CD44, CD29, CD73, CD90, CD 106, CD 166阳性,CD34, CD45, BLA-DR阴性,灰度分析显示,各个基因不同代次的表达差异不明显,证明体外培养的细胞持续稳定表达间充质干细胞的特异性标志。 8.采用免疫荧光技术检测P3,P7,P11,P22胎牛肝脏间充质干细胞中组蛋白乙酰化位点H4K12乙酰化水平的变化,并利用荧光分析软件分析不同代次相对荧光强度的变化。结果显示,随着传代增加,细胞的组蛋白乙酰化水平变化不显著。表明细胞H4K12位点乙酰化水平的维持可能与间充质干细胞的特异性标志表达的维持之间存在定的关联。 9.牛肝脏间充质干细胞经地塞米松、β-甘油磷酸钠和抗坏血酸成骨诱导后,茜素红染色观察到红色钙结节,RT-PCR检测Osteopontin和Collagen type I诱导后表达阳性,诱导前为阴性。牛肝脏间充质干细胞经地塞米松、IBMX、胰岛素和吲哚美辛诱导后部分细胞转变为扁平、肥大、含有大量脂滴的脂肪细胞,用油红O染色观察到红色的脂滴,RT-PCR检测LPL和PPAR-y诱导后阳性。表明牛肝脏间充质干细胞具有向中胚层细胞分化的能力。 10.分别采用有机诱变剂BHA, DMSO,β-巯基乙醇的两步诱导法,和N2与bFGF联合诱导法对牛肝脏间充质干细胞进行向神经细胞的诱导。诱导后细胞可见明显的胞体收缩,突起伸出及末端分支等变化。RT-PCR和免疫荧光检测到诱导后神经元标志MAP-2的表达,表明了牛肝脏间充质干细胞具有一定的向外胚层细胞分化的能力。实验证明,鲁西黄牛胎牛肝脏间充质干细胞经过体外多次传代后,仍然保持正常的生长增殖特性和遗传稳定性,及间充质干细胞的特异标志,还具有向成骨细胞、成脂细胞和神经元细胞分化的能力,证明了牛胎肝间充质干细胞的多能性。总之,本实验建立了牛胎肝间充质干细胞分离培养及鉴定的技术平台,并且验证了它们的多向分化能力,对动物种质资源的保存利用具有重要意义,并且最终为干细胞在临床上的应用提供实验依据。
[Abstract]:Mesenchymal stem cells (MSCs) are one of the most important members of the stem cell family. In recent years, due to their self-renewal, replication, immune regulation and multidirectional differentiation, the research on MSCs has become more and more extensive and in-depth. MSCs were first found in bone marrow, and then found in fat, synovium, bone, muscle, lung, liver, pancreas and so on. Mesenchymal stem cells are also found in tissues, amniotic fluid and umbilical cord blood. However, the current research focuses on bone marrow, umbilical cord blood, fat and other sources of mesenchymal stem cells, liver-derived mesenchymal stem cells are rarely studied. So far, there are no reports on bovine fetal liver-derived mesenchymal stem cells. The aim of this study is to establish a bovine fetal liver mesenchymal stem cell isolation and culture system, and explore its proliferation, differentiation and other biological characteristics.
1. Mesenchymal stem cells from Luxi cattle liver were isolated by Percoll density gradient centrifugation and cultured for 26 generations in vitro. Mesenchymal stem cells from fetal bovine liver in vitro grew vigorously with typical fibroblast morphology, most of them were spindle-shaped or irregular triangle, a few irregular or polygonal cells were mixed, and the cells were long. When it was full, it showed a whirlpool or flame like trend. Under the microscope, the cytoplasm of the cells was full and the stereoscopic feeling was strong.
2. The growth curves of different passages of fetal bovine liver mesenchymal stem cells were all typical "S" shaped. The cells experienced latency, logarithmic growth and stagnation, which accorded with the general rule of cell growth in vitro.
3. The cloning rates of P3, P7, P11 and P22 fetal bovine hepatic mesenchymal stem cells were 43.38 (+ 8.54%), 37.00 (+ 2.45%), 32.92 (+ 2.50%) and 15.83 (+ 5.69%) respectively.
4. The cell cycle of P3, P7, P11, P22 fetal bovine liver mesenchymal stem cells was analyzed by flow cytometry. The results showed that the number of Go/G1 phase cells increased with the passage increasing, and the number of S phase cells decreased gradually.
5. Karyotype analysis showed that the chromosome number of fetal bovine liver mesenchymal stem cells cultured in vitro was 2n=60, and there was no abnormality. The diploid rate was 93.6%, indicating that the cultured cells were stable diploid cells and had stable heredity in vitro.
6. There were 163 cryopreserved cells, and the cell viability was about 90% before and after cryopreservation. The cell viability decreased slightly after resuscitation, which was mainly due to mechanical and chemical damage during cryopreservation and resuscitation.
7. Immunofluorescence and RT-PCR were used to identify whether the cultured bovine liver mesenchymal stem cells of P3, P7, P11 and P22 had specific markers of mesenchymal stem cells. All of them were positive for CD44, CD29, CD73, CD90, CD 106, CD 166, negative for CD34, CD45 and BLA-DR. Gray scale analysis showed that there was no significant difference in the expression of each gene in different generations, which proved that the cells cultured in vitro continuously and steadily expressed the specific markers of mesenchymal stem cells.
8. Immunofluorescence technique was used to detect the acetylation of histone H4K12 in P3, P7, P11, P22 fetal bovine hepatic mesenchymal stem cells, and fluorescence analysis software was used to analyze the changes of relative fluorescence intensity in different passages. The maintenance of acetylation at 4K12 locus may be related to the maintenance of the expression of specific markers of mesenchymal stem cells.
9. Bovine liver mesenchymal stem cells were induced by dexamethasone, beta-glycerophosphate and ascorbic acid. Alizarin red staining showed red calcium nodules. RT-PCR detected that Osteopontin and Collagen type I were positive after induction and negative before induction. Bovine liver mesenchymal stem cells were induced by dexamethasone, IBMX, insulin and indomethacin. Some cells were transformed into flat, hypertrophic adipocytes containing a large number of lipid droplets. Red lipid droplets were observed by oil red O staining, and positive results were detected by RT-PCR after induction of LPL and PPAR-y.
10. Bovine hepatic mesenchymal stem cells were induced into neural cells by two-step induction with organic mutagens BHA, DMSO, beta-mercaptoethanol and N2 and bFGF respectively. After induction, obvious changes of somatic contraction, protrusion and terminal branching were observed. MAP, a neuronal marker, was detected by RT-PCR and immunofluorescence. The expression of -2 indicates that bovine liver mesenchymal stem cells have the ability to differentiate into ectoderm cells.
The results showed that the mesenchymal stem cells from the fetal bovine liver of Luxi yellow cattle still maintained normal growth and proliferation characteristics, genetic stability, and special markers of mesenchymal stem cells, and also had the ability to differentiate into osteoblasts, adipocytes and neurons, which proved the pluripotency of bovine fetal liver mesenchymal stem cells. In conclusion, this study establishes a technical platform for isolation, culture and identification of bovine fetal liver mesenchymal stem cells, and verifies their multi-differentiation ability, which is of great significance for the conservation and utilization of Animal Germplasm resources, and ultimately provides experimental basis for the clinical application of stem cells.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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