本文選題：骨髓間充質干細胞 + 肝細胞��； 參考：《吉林大學》2007年碩士論文

【摘要】： 骨髓來源的間充質干細胞(Bone marrow mesenchymal stem cells,BMSCs)是骨髓內具有多種分化潛能的一類干細胞亞群,并且在一定條件下可以向多種不同組織細胞分化。本論文主要用具有脂肪細胞分化潛能的MSCs,進行向肝細胞定向誘導分化,為肝細胞移植提供可靠的種子來源。 目的:分離、純化、培養(yǎng)和鑒定大鼠骨髓間充質干細胞;采用損傷肝組織和血清對MSCs進行定向誘導分化,并確定MSCs體外誘導分化為肝樣細胞的最佳條件。 方法:無菌條件下取周齡大乳鼠四肢骨骨髓,全血培養(yǎng)所得的貼壁細胞用1.077g/mlFICOLL(淋巴細胞分離液)密度梯度離心去除成纖維細胞和殘留血細胞,傳代,通過控制消化時間得到純度較高的骨髓MSC。取第三代細胞(P3),用成脂誘導的方法檢測其分化潛能,用損傷肝組織和血清定向誘導分化MSCs,利用免疫細胞化學和RT-PCR等方法對MSCs及誘導后肝樣細胞進行細胞特異性標志物的鑒定。 結果:1.全血培養(yǎng)的骨髓原代細胞呈細沙樣,換液后,貼壁細胞呈集落樣生長,細胞貼壁后由于密度的不同而表現(xiàn)不同的形態(tài)。當密度較大時,細胞呈梭形,呈旋渦狀,放射狀或平行狀生長。當密度較小時,細胞呈延伸態(tài)紡錘形或三角形。密度梯度離心后細胞均質性明顯提高,從形態(tài)學上看具有MSC的特點。 2.傳代培養(yǎng)的第三代(P3)MSCs經免疫細胞化學檢測CD105呈陽性,提示細胞具有MSC特異性標記。 3.MSCs在20%馬血清、20%馬血清+氫化可的松、DX+IBMX+IS+ID,這些成脂誘導劑均能分化為脂肪細胞,陽性率達到90%以上。油紅O結合蘇木素染色后可見橘紅色的脂肪滴和淺藍色的細胞核。其中DX+IBMX+IS+ID誘導的脂肪細胞脂滴體積最大,誘導分化時間最短,提示雖然三組誘導環(huán)境均可檢測MSCs的分化潛能,但DX+IBMX+IS+ID誘導效果最明顯。 4.損傷肝組織和血清誘導MSCs后,免疫細胞化學染色ALB陽性,糖原染色陽性,RT-PCR可檢測出肝特有基因AFP、ALB的表達�；旌辖MALB及糖原染色陽性率高于單因素組,提示混合組比單因素組能更高效的誘導MSCs向肝細胞分化。 結論:1.本實驗采用全血培養(yǎng)法和密度梯度離心法對大鼠骨髓MSCs進行分離、純化、體外培養(yǎng),同時從細胞形態(tài)和生物學特性進行觀察和檢測,該方法是有效、簡便,可獲得高純度MSCs的可靠方法,MSCs在體外不但可進行大量培養(yǎng)擴增,而且其增殖和傳代能力很強。 2.骨髓MSCs在成脂誘導劑誘導下,可以定向分化為脂肪細胞,陽性率均在90%以上,說明所得到的MSCs具有多向分化潛能。其中DX+IBMX+IS+ID組誘導效果最明顯。 3.損傷肝組織和血清定向誘導具有脂肪細胞分化潛能MSCs(P3),分化為肝樣細胞,并表達肝細胞的特異性標志物,說明損傷肝組織及血清中存在使MSCs分化為肝樣細胞的相關因子或成分。結果提示混合組為MSCs向肝細胞分化提供了更適宜的誘導環(huán)境。
[Abstract]:Bone marrow derived mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) are a type of stem cell subgroup with multiple differentiation potential in bone marrow, and can differentiate into a variety of different tissue cells under certain conditions. This paper mainly uses MSCs with the potential of adipocyte differentiation to induce differentiation to liver cells and be the liver. Cell transplantation provides a reliable source of seed.
Objective: to isolate, purify, culture and identify rat bone marrow mesenchymal stem cells (MSCs), and to induce differentiation of MSCs by using damaged liver tissue and serum, and to determine the best conditions for MSCs to induce differentiation into hepatocyte like cells in vitro.
Methods: under aseptic conditions, the bone marrow of the limbs of the four limbs of the old rats was taken. The adherent cells obtained from the whole blood culture were removed by 1.077g/mlFICOLL (lymphocyte separation) density gradient centrifugation to remove the fibroblasts and residual blood cells. By controlling the digestion time, the third generation cells (P3) with high purity MSC. were obtained by controlling the digestion time, and the method of lipid induction was used. The differentiation potential was detected, the differentiated MSCs was induced by damaged liver tissue and serum, and the specific markers of MSCs and induced hepatocyst were identified by immunocytochemistry and RT-PCR.
Results: 1. the primary cells in the whole blood culture were fine sand samples. After changing the liquid, the adherent cells were colonies, and the cells displayed different forms due to the different density. When the density was large, the cells showed spindle shaped, radial or parallel growth. When the density was small, the cells showed an extended state spindle shape or triangle. After gradient centrifugation, the cell homogeneity was obviously improved, and MSC was characteristic in morphology.
2. the third generation (P3) MSCs of passage culture was positive for CD105 by immunocytochemistry, indicating that the cells had MSC specific markers.
3.MSCs in 20% horse serum, 20% horse serum + hydrocortisone, DX+IBMX+IS+ID, these lipid inducers can differentiate into adipocytes, the positive rate is more than 90%. After the oil red O combined with hematoxylin, the orange red fat drops and the light blue nuclei are visible. Among them, the fat cell lipid droplets induced by DX+IBMX+IS+ID are the largest and induce the differentiation time. The shortest suggested that although the induction potential of the three groups could detect the differentiation potential of MSCs, the induction effect of DX+IBMX+IS+ID was the most obvious.
4. after MSCs injury liver tissue and serum induced, immunocytochemical staining ALB positive, glycogen staining positive, RT-PCR can detect the expression of liver specific gene AFP, ALB expression. The positive rate of ALB and glycogen staining in the mixed group is higher than that of the single factor group, suggesting that the mixed group can induce the differentiation of MSCs to the liver cells more efficiently than the single factor group.
Conclusion: 1. the total blood culture and density gradient centrifugation were used to separate the bone marrow MSCs of rats, to be purified and cultured in vitro, and to observe and detect the morphology and biological characteristics of the cells. This method is effective and simple, and a reliable method for obtaining high purity MSCs can be obtained. MSCs can not only be expanded in vitro, but also increase in vitro. The ability to colonization and passage is very strong.
2. bone marrow MSCs can differentiate into adipocytes under the induction of lipid inducer, and the positive rate is above 90%, indicating that the obtained MSCs has multipotential differentiation potential, of which the DX+IBMX+IS+ID group has the most obvious induction effect.
3. the hepatic tissue and serum were directed to induce the differentiation potential of adipocyte MSCs (P3), differentiate into hepatocyte like cells, and express the specific markers of liver cells, indicating the existence of related factors or components that differentiate MSCs into hepatocyte like cells in the injured liver tissue and serum. The results suggest that the mixed group is more suitable for the differentiation of MSCs to liver cells. Induce the environment.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

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