【摘要】：間充質(zhì)干細(xì)胞是干細(xì)胞家族的重要成員之一,近年來由于它具有自我更新復(fù)制、免疫調(diào)控、多向分化等特性,有關(guān)間充質(zhì)干細(xì)胞的研究也越來越廣泛和深入。間充質(zhì)干細(xì)胞最早在骨髓中發(fā)現(xiàn),隨后發(fā)現(xiàn)脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺等組織以及羊水、臍帶血中也存在間充質(zhì)干細(xì)胞。但是,目前的研究主要集中在骨髓、臍帶血、脂肪等來源的間充質(zhì)干細(xì)胞上,有關(guān)肝臟來源的間充質(zhì)干細(xì)胞的研究很少。迄今為止,還沒有關(guān)于牛胎肝來源的間充質(zhì)干細(xì)胞方面的研究報(bào)道。本研究旨在建立牛胎肝間充質(zhì)干細(xì)胞分離培養(yǎng)體系,探索其增殖、分化等生物學(xué)特性,結(jié)果如下： 1.采用Percoll密度梯度離心法分離約2-4月齡魯西黃牛胎牛肝臟間充質(zhì)干細(xì)胞,在體外培養(yǎng)至26代。體外培養(yǎng)的胎牛肝臟間允質(zhì)干細(xì)胞生長旺盛,具有典型的成纖維形態(tài),多數(shù)呈長梭狀或不規(guī)則三角形,混有少數(shù)不規(guī)則或多邊形細(xì)胞,細(xì)胞長滿時(shí)呈現(xiàn)漩渦狀或火焰狀走勢(shì)。鏡下觀察細(xì)胞胞質(zhì)豐滿,立體感較強(qiáng)。 2.不同代次的胎牛肝臟間充質(zhì)干細(xì)胞的生長曲線都呈典型的“S”形,細(xì)胞經(jīng)歷了潛伏期、對(duì)數(shù)生長期和停滯期,符合細(xì)胞在體外生長的一般規(guī)律。但是隨著傳代次數(shù)的增加,細(xì)胞的增殖分裂能力逐漸下降。 3.分別檢測(cè)P3,P7,P11,P22胎牛肝臟間充質(zhì)干細(xì)胞的克隆形成率,分別為：43.38±8.54%,37.00±2.45%,32.92±2.50%,15.83±5.69%。 4.采用流式細(xì)胞儀分析P3,P7,P11,P22胎牛肝臟間充質(zhì)干細(xì)胞的細(xì)胞周期,結(jié)果顯示,Go/G1期的細(xì)胞隨代次增加逐漸增多,S期的細(xì)胞呈逐漸減少的趨勢(shì)。 5.核型分析表明,體外培養(yǎng)的胎牛肝臟間充質(zhì)干細(xì)胞染色體數(shù)目2n=60,未見異常。二倍體率為93.6%,表明所培養(yǎng)的細(xì)胞均為穩(wěn)定的二倍體細(xì)胞,在體外遺傳性能穩(wěn)定。 6.共凍存細(xì)胞163支,凍存前后的細(xì)胞活率都在90%左右。復(fù)蘇后細(xì)胞存活率略有下降,其主要原因是細(xì)胞在冷凍和復(fù)蘇過程中遭受機(jī)械和化學(xué)損傷所致。 7.采用免疫熒光和RT-PCR鑒定體外培養(yǎng)的P3,P7,P11,P22胎牛肝臟間充質(zhì)干細(xì)胞是否具有間充質(zhì)干細(xì)胞特有的細(xì)胞標(biāo)志。免疫熒光顯示不同代次細(xì)胞均呈CD44,CD29陽性,各個(gè)代次間相對(duì)熒光強(qiáng)度無明顯差別。RT-PCR檢測(cè)結(jié)果表明,不同代次的細(xì)胞均為CD44, CD29, CD73, CD90, CD 106, CD 166陽性,CD34, CD45, BLA-DR陰性,灰度分析顯示,各個(gè)基因不同代次的表達(dá)差異不明顯,證明體外培養(yǎng)的細(xì)胞持續(xù)穩(wěn)定表達(dá)間充質(zhì)干細(xì)胞的特異性標(biāo)志。 8.采用免疫熒光技術(shù)檢測(cè)P3,P7,P11,P22胎牛肝臟間充質(zhì)干細(xì)胞中組蛋白乙�；稽c(diǎn)H4K12乙�；降淖兓�,并利用熒光分析軟件分析不同代次相對(duì)熒光強(qiáng)度的變化。結(jié)果顯示,隨著傳代增加,細(xì)胞的組蛋白乙�；阶兓伙@著。表明細(xì)胞H4K12位點(diǎn)乙�；降木S持可能與間充質(zhì)干細(xì)胞的特異性標(biāo)志表達(dá)的維持之間存在定的關(guān)聯(lián)。 9.牛肝臟間充質(zhì)干細(xì)胞經(jīng)地塞米松、β-甘油磷酸鈉和抗壞血酸成骨誘導(dǎo)后,茜素紅染色觀察到紅色鈣結(jié)節(jié),RT-PCR檢測(cè)Osteopontin和Collagen type I誘導(dǎo)后表達(dá)陽性,誘導(dǎo)前為陰性。牛肝臟間充質(zhì)干細(xì)胞經(jīng)地塞米松、IBMX、胰島素和吲哚美辛誘導(dǎo)后部分細(xì)胞轉(zhuǎn)變?yōu)楸馄�、肥大、含有大量脂滴的脂肪�?xì)胞,用油紅O染色觀察到紅色的脂滴,RT-PCR檢測(cè)LPL和PPAR-y誘導(dǎo)后陽性。表明牛肝臟間充質(zhì)干細(xì)胞具有向中胚層細(xì)胞分化的能力。 10.分別采用有機(jī)誘變劑BHA, DMSO,β-巰基乙醇的兩步誘導(dǎo)法,和N2與bFGF聯(lián)合誘導(dǎo)法對(duì)牛肝臟間充質(zhì)干細(xì)胞進(jìn)行向神經(jīng)細(xì)胞的誘導(dǎo)。誘導(dǎo)后細(xì)胞可見明顯的胞體收縮,突起伸出及末端分支等變化。RT-PCR和免疫熒光檢測(cè)到誘導(dǎo)后神經(jīng)元標(biāo)志MAP-2的表達(dá),表明了牛肝臟間充質(zhì)干細(xì)胞具有一定的向外胚層細(xì)胞分化的能力。 實(shí)驗(yàn)證明,魯西黃牛胎牛肝臟間充質(zhì)干細(xì)胞經(jīng)過體外多次傳代后,仍然保持正常的生長增殖特性和遺傳穩(wěn)定性,及間充質(zhì)干細(xì)胞的特異標(biāo)志,還具有向成骨細(xì)胞、成脂細(xì)胞和神經(jīng)元細(xì)胞分化的能力,證明了牛胎肝間充質(zhì)干細(xì)胞的多能性�？傊�,本實(shí)驗(yàn)建立了牛胎肝間充質(zhì)干細(xì)胞分離培養(yǎng)及鑒定的技術(shù)平臺(tái),并且驗(yàn)證了它們的多向分化能力,對(duì)動(dòng)物種質(zhì)資源的保存利用具有重要意義,并且最終為干細(xì)胞在臨床上的應(yīng)用提供實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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