本文關(guān)鍵詞： 骨髓基質(zhì)細(xì)胞 成骨潛能 細(xì)胞培養(yǎng) 生物學(xué)特性　出處：《山西醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:骨創(chuàng)傷后的再生能力提示有能夠自我復(fù)制并能多向分化的干細(xì)胞存在。一般認(rèn)為,干細(xì)胞存在于骨、骨膜、骨髓及其他由中胚層發(fā)育來的組織中,在出生后仍然有少量干細(xì)胞繼續(xù)存在,在機(jī)體損傷時參與組織的修復(fù)。組織工程的基礎(chǔ)是有功能的細(xì)胞,骨組織工程需要大量的成骨細(xì)胞。十多年來,國內(nèi)外學(xué)者和臨床工作者對各種組織的細(xì)胞成骨能力進(jìn)行了大量的研究,提出了定向性骨祖細(xì)胞、誘導(dǎo)性骨祖細(xì)胞和基質(zhì)干細(xì)胞的概念。目前,在研究中使用的作為種子細(xì)胞的成骨細(xì)胞來源有以下幾種:骨、軟骨、骨外膜、骨髓和骨外組織。越來越多的證據(jù)表明,骨髓基質(zhì)干細(xì)胞(BMSCs)有多分化潛能,移植人體內(nèi)后可分化為骨、軟骨、肌肉、脂肪、血管內(nèi)皮、肝臟、神經(jīng)等多種細(xì)胞。能夠作為組織工程中不同細(xì)胞的來源;在體內(nèi)正常循環(huán)中能夠分布于多種組織和器官,可以用于細(xì)胞和基因治療。而且取材、分離培養(yǎng)、擴(kuò)增以及導(dǎo)入外源基因也相對方便。BMSC。通過抽取自體骨髓得到,對病人造成創(chuàng)傷較少,且無免疫排斥反應(yīng)。因此,BMSCs在實際應(yīng)用時將具有潛在的優(yōu)勢。木試驗?zāi)康氖墙⒁环N簡單有效的骨髓基質(zhì)細(xì)胞的體外培養(yǎng)及成骨誘導(dǎo)方法,并探討在體外培養(yǎng)中其增殖和分化的相互關(guān)系,總結(jié)骨髓基質(zhì)細(xì)胞體外培養(yǎng)的生物學(xué)規(guī)律,為將來在體外分離、培養(yǎng)、擴(kuò)增出大量的有成骨活性的細(xì)胞以滿足再造骨組織的需要提供了美好的前景。 方法:沖洗法獲取Weistar大鼠骨髓,用貼壁法分離提純骨髓基質(zhì)細(xì)胞(Bone MarrowStromal Cells BMSCs),傳至第3代細(xì)胞后分組:1.分別用普通培養(yǎng)基和誘導(dǎo)培養(yǎng)基(DMEM加入地米松10~(-7)mmol/L,β-甘油磷酸鈉10mmol/L,維生素C50mg/L)培養(yǎng)10天,每3天換液1次。繪制各自細(xì)胞生長曲線。2.分別用普通培養(yǎng)基和誘導(dǎo)培養(yǎng)基培養(yǎng)15天,每3天換液1次,于第4、7、10、13、16天,檢測兩組的堿性磷酸酶表達(dá)量。3.以5×10~4/ml密度接種于6孔培養(yǎng)板各1板,各孔放置載玻片,分別加入基礎(chǔ)培養(yǎng)液和誘導(dǎo)培養(yǎng)液培養(yǎng),每3天換液,20天后分別取出各自細(xì)胞爬片行Von kossa染色。 結(jié)果:1.普通培養(yǎng)基中細(xì)胞的生長速度明顯快于誘導(dǎo)培養(yǎng)基中的細(xì)胞。2.細(xì)胞堿性磷酸酶表達(dá)量兩組有顯著性差異(P＜0.05),普通培養(yǎng)基組明顯低于誘導(dǎo)組。3.對照組細(xì)胞Vonkossa染色陽性,試驗組為陰性。 結(jié)論:1.貼壁篩選法是一種簡單有效的骨髓基質(zhì)細(xì)胞的分離純化方法。2.骨髓基質(zhì)細(xì)胞在體外可誘導(dǎo)分化為成骨細(xì)胞,可作為骨組織工程中種子細(xì)胞的來源。3.骨髓基質(zhì)細(xì)胞的增殖和分化存在相互抑制的關(guān)系。
[Abstract]:Objective: the ability of regeneration after bone trauma suggests the existence of stem cells capable of self-replicating and multidifferentiation. Stem cells are generally thought to exist in bone, periosteum, bone marrow and other tissues developed from mesoderm. After birth, a small number of stem cells continue to exist, participate in the repair of tissue when the body is damaged. Tissue engineering is based on functional cells, bone tissue engineering requires a large number of osteoblasts for more than a decade. Scholars and clinical workers at home and abroad have done a lot of research on the osteogenic ability of various tissues and put forward the concepts of directional bone progenitor cells induced bone progenitor cells and stromal stem cells. The following sources of osteoblasts are used as seed cells in the study: bone, cartilage, periosteum, bone marrow and extraosseous tissue. Bone marrow stromal cells (BMSCs) can differentiate into bone, cartilage, muscle, fat, vascular endothelium and liver after transplantation. Nerve and other cells. Can be used as a source of different cells in tissue engineering; In the normal circulation in the body can be distributed in a variety of tissues and organs, can be used in cell and gene therapy. Amplification and introduction of foreign genes is also relatively convenient .BMSC.Through extraction of autologous bone marrow, the trauma to patients is less and there is no immune rejection. BMSCs will have potential advantages in practical application. The purpose of wood test is to establish a simple and effective method of bone marrow stromal cell culture and osteogenesis induction in vitro. The relationship between proliferation and differentiation of bone marrow stromal cells in vitro was discussed, and the biological rules of bone marrow stromal cells culture in vitro were summarized in order to isolate and culture bone marrow stromal cells in vitro. The amplification of a large number of osteogenic cells to meet the needs of bone tissue reconstruction provides a bright prospect. Methods: bone MarrowStromal Cells BMSCs of Weistar rats were isolated and purified by adherent method. The cells were transferred to the third passage and divided into two groups: 1. DMEM and DMEM were added to DMEM and 10 mmol / L 尾 -glycerophosphate (10 mmol / L, 10 mmol / L), respectively. Vitamin C 50 mg / L) was cultured for 10 days and changed solution once every 3 days. The cell growth curve was drawn. The cells were cultured in normal medium and induction medium for 15 days and once every 3 days. The expression of alkaline phosphatase (ALP) in the two groups was detected for 16 days. At the density of 5 脳 10 ~ (4) / ml, the two groups were inoculated with 6 hole culture plates (1 plate each), and the slides were placed in each hole. The cells were cultured in basic culture medium and induction medium respectively. After 20 days of culture, the cells were removed for Von kossa staining. Results 1. The growth rate of cells in the ordinary medium was significantly faster than that in the induction medium. There was a significant difference in the expression of alkaline phosphatase between the two groups (P < 0.05). The normal medium group was significantly lower than the induction group. 3. The Vonkossa staining was positive in the control group and negative in the experimental group. Conclusion the adherent screening method is a simple and effective method for the isolation and purification of bone marrow stromal cells. Bone marrow stromal cells can be induced to differentiate into osteoblasts in vitro. It can be used as a source of seed cells in bone tissue engineering. 3. The proliferation and differentiation of bone marrow stromal cells can be inhibited by each other.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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