本文關(guān)鍵詞：肋軟骨膜、肋軟骨和骨髓來源間充質(zhì)干細(xì)胞的比較　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肋軟骨膜 肋軟骨 骨髓 干細(xì)胞 分離培養(yǎng) 鑒定

【摘要】：由于間充質(zhì)干細(xì)胞（Mesenchymal stem cells, MSCs）來源的多樣性、體外誘導(dǎo)的多向分化性，沒有特定的表面表記等特點(diǎn)，對(duì)其起源有不同的觀點(diǎn)。學(xué)者們多認(rèn)為MSCs是存在于組織和器官中的成體干細(xì)胞經(jīng)體外培養(yǎng)擴(kuò)增而成。但Orkin等提出：體內(nèi)也可能不存在MSCs，只是在體外培養(yǎng)分離到的細(xì)胞時(shí)，這些細(xì)胞被激發(fā)轉(zhuǎn)變或者基因重編，成為具有干細(xì)胞特性的細(xì)胞。成熟的細(xì)胞能否轉(zhuǎn)化成干細(xì)胞呢？已有報(bào)道從軟骨中分離到干細(xì)胞，但體內(nèi)僅生長區(qū)外的軟骨組織中無干細(xì)胞，關(guān)節(jié)軟骨也有干細(xì)胞。從軟骨中分離到的干細(xì)胞是來自軟骨中的干細(xì)胞還是由軟骨細(xì)胞轉(zhuǎn)化而成的呢？本實(shí)驗(yàn)擬分別從兔非生長區(qū)肋軟骨膜、肋軟骨和骨髓中分離、培養(yǎng)、擴(kuò)增獲得干細(xì)胞，觀察軟骨細(xì)胞能否轉(zhuǎn)化為干細(xì)胞？幾種來源干細(xì)胞的差異？有助于認(rèn)識(shí)成體干細(xì)胞體外分離獲取的機(jī)制，以及干細(xì)胞臨床應(yīng)用研究的基礎(chǔ)理論。 第一部分兔肋軟骨膜、肋軟骨和骨髓來源干細(xì)胞的分離培養(yǎng) 1材料與方法 取3-4周新西蘭大白兔的肋軟骨（避開肋生長板區(qū)），解剖顯微鏡下剝離軟骨膜，肋軟骨和肋軟骨膜均經(jīng)酶消化法分別獲取肋軟骨和肋軟骨膜細(xì)胞；另取股骨、脛骨的骨髓細(xì)胞。三種來源的細(xì)胞均以貼壁法分離、培養(yǎng)、擴(kuò)增，分別獲得3種來源的干細(xì)胞。觀察3者各代細(xì)胞的形態(tài)和生長。 取三種來源的第3代細(xì)胞，以1×10~4個(gè)細(xì)胞接種于24孔培養(yǎng)板中培養(yǎng)，每天隨機(jī)各取3個(gè)培養(yǎng)孔，共10天，計(jì)數(shù)細(xì)胞數(shù)，繪制生長曲線。 2結(jié)果 從肋軟骨膜中分離的原代細(xì)胞接種12h后開始貼壁，5-6d細(xì)胞融合，聚集成團(tuán)，以圓形和多邊形為主，從第2代開始細(xì)胞形態(tài)均一，以梭形為主，呈漩渦狀排列。目前已傳至15代。從肋軟骨中分離的原代細(xì)胞6h后開始貼壁，5-6天細(xì)胞融合，以鋪路石形態(tài)為主，第2代和第3代細(xì)胞逐漸轉(zhuǎn)變成梭形；但第5代出現(xiàn)細(xì)胞老化，只傳至第6代。原代骨髓細(xì)胞培養(yǎng)4-5h后開始貼壁，10d左右細(xì)胞融合。融合時(shí)細(xì)胞可見梭形，第2代以后細(xì)胞形態(tài)以梭形為主，呈明顯漩渦狀排列，已傳10代，生長良好。 三種來源細(xì)胞第3代細(xì)胞的生長曲線表明：貼壁后生長潛伏適應(yīng)期為1-3d；3d后即進(jìn)入對(duì)數(shù)生長期；6d后進(jìn)入生長平臺(tái)期。 第二部分肋軟骨膜、肋軟骨和骨髓來源干細(xì)胞的比較 1材料與方法 1.1三種來源的每一代細(xì)胞均以免疫組化染色方法檢測(cè)每一代細(xì)胞CD29、CD90、CD34和Ⅱ型膠原的表達(dá)。 1.2取三種來源的第3代干細(xì)胞分別進(jìn)行體外成脂誘導(dǎo)14天、成骨誘導(dǎo)21天；油紅O和硝酸銀染色法檢測(cè)比較三種干細(xì)胞成脂、成骨的分化能力。 2結(jié)果 2.1免疫細(xì)胞化學(xué)染色發(fā)現(xiàn)原代及第1代肋軟骨膜和肋軟骨細(xì)胞均不表達(dá)CD29、CD90、 CD34，從第2代細(xì)胞開始表達(dá)CD29、CD90，仍不表達(dá)CD34。原代骨髓細(xì)胞可表達(dá)CD29、CD90和CD34，不表達(dá)Ⅱ型膠原；隨后只表達(dá)CD29和CD90。三種來源的第3代細(xì)胞均只強(qiáng)表達(dá)CD29、CD90，其均一性都達(dá)到99％以上。作為軟骨細(xì)胞標(biāo)記物之一的Ⅱ型膠原，僅原代及第1代肋軟骨細(xì)胞表達(dá)，從第2代細(xì)胞不再表達(dá)。 2.2三種來源的第3代干細(xì)胞經(jīng)成脂誘導(dǎo)14天后,肋軟骨膜和骨髓來源的干細(xì)胞形狀多由梭形轉(zhuǎn)變?yōu)閳A形，肋軟骨來源的干細(xì)胞則逐漸轉(zhuǎn)變?yōu)殚L梭形，細(xì)胞內(nèi)均可見油紅O染色的紅色小脂滴。成骨誘導(dǎo)21天后，肋軟骨膜和肋軟骨來源的干細(xì)胞呈復(fù)層生長，而骨髓來源的干細(xì)胞成單層生長。經(jīng)過硝酸銀染色后，黑色鈣化小結(jié)節(jié)分散于肋軟骨膜和肋軟骨來源的干細(xì)胞表面；骨髓來源的干細(xì)胞表面的骨結(jié)節(jié)則聚集成團(tuán)。 全文結(jié)論 1.用酶消化法，，成功的分離、培養(yǎng)和擴(kuò)增了兔肋軟骨膜和肋軟骨來源的干細(xì)胞。用貼壁法提取了骨髓細(xì)胞。體外多次傳代后，肋軟骨膜和骨髓來源的干細(xì)胞比肋軟骨來源的干細(xì)胞具有更強(qiáng)的生長增殖能力。 2.免疫組化檢測(cè)顯示肋軟骨膜和肋軟骨細(xì)胞從第2代開始均表達(dá)MSCs的表型，至第三代強(qiáng)表達(dá)。表明這兩種細(xì)胞第3代具有MSCs的表型特征。 3.肋軟骨膜和肋軟骨來源的第3代干細(xì)胞在體外經(jīng)誘導(dǎo)后具有與BMSCs一致的成脂和成骨能力，證明這兩種來源的細(xì)胞具有干細(xì)胞的多向分化特征。 4.原代肋軟骨細(xì)胞表達(dá)軟骨細(xì)胞的標(biāo)記物Ⅱ型膠原，直至第2代細(xì)胞不再表達(dá)，但開始表達(dá)CD29和CD90。這表明：成熟的肋軟骨細(xì)胞在體外培養(yǎng)也可去分化成為干細(xì)胞。 5.盡管肋軟骨膜和肋軟骨來源的干細(xì)胞均具有與BMSCs相同的免疫表型和成脂、成骨誘導(dǎo)分化能力，但肋軟骨膜來源干細(xì)胞的生長明顯優(yōu)于肋軟骨來源去分化后轉(zhuǎn)化成的干細(xì)胞。因此，從軟骨中分離獲取干細(xì)胞時(shí)，應(yīng)盡量保留軟骨膜。
[Abstract]:The mesenchymal stem cells (Mesenchymal stem cells, MSCs) of the various sources, differentiation in vitro, no specific surface token etc, have different opinions about its origin. Scholars believe that MSCs is present in the tissues and organs in the in vitro amplification of adult stem cells but Orkin suggested: in vivo may not have MSCs, only isolated cells, these cells are stimulated or gene transformation become rewoven with the characteristics of stem cells. Cells of mature cells can be transformed into stem cells? Reported isolated stem cells from the cartilage, but no cartilage cells only the growth area of tissue, articular cartilage also has stem cells. Isolated from cartilage stem cells from cartilage stem cells or transformed from cartilage cells? This study respectively from the non rabbit Long area rib cartilage, costal cartilage and bone marrow were isolated and cultured, amplified stem cells, observe chondrocytes can be transformed into stem cells? The difference of several sources of stem cells? To know the mechanism to the separation of somatic stem cells in vitro, and the basic theory of clinical application of stem cells.
Part 1 the isolation and culture of rabbit costal cartilage membrane, costal cartilage and bone marrow stem cells
1 materials and methods
Take 3-4 weeks in New Zealand rabbits (avoid rib rib cartilage growth plate area), dissected cartilage membrane under the microscope, costal cartilage and costal cartilage membrane were confirmed by enzyme digestion were used to obtain costal cartilage and costal cartilage cell membrane; the other femur, tibia bone marrow cells. Three cells are adherent method isolation, culture, amplification, obtained 3 kinds of stem cells were observed. The morphology and growth of the 3 generations of cells.
The third generation cells from three sources were inoculated in 24 pore culture plates with 1 * 10~4 cells, and 3 culturing holes were collected every day for 10 days. The cell number was counted, and the growth curve was drawn.
2 Results
Isolated from rib perichondrium in primary cells inoculated with 12h adhered to the wall, 5-6d cell fusion, gathered together, in round and polygon, from the beginning of the second generation cells were homogeneous, spindle and whirlpool arrangement. At present has been passaged for 15 generations. Separated from the rib cartilage in primary cells 6h after the start of adherent cell fusion in 5-6 days, cobblestone appearance, the second and third generation cells gradually changed into spindle; but the fifth generation cell aging, only to the sixth generation. The primary cultured bone marrow cells after 4-5h adhered to the wall, about 10d cell fusion. Fusion cells can see the spindle after second generations, the cells were mainly spindle shaped, obvious whorls, has 10 generations, the growth is good.
The growth curves of the third generation cells from three sources showed that the incubation latency after 1-3D was 3D, then entered the logarithmic growth phase, and 6D entered the growth plateau stage.
Comparison of second parts of costal cartilage membrane, costal cartilage and bone marrow stem cells
1 materials and methods
1.1 the expression of CD29, CD90, CD34 and type II collagen in each generation of cells in each generation of every generation of three sources was detected by immunohistochemical staining.
1.2, three generation of third generation stem cells were induced in vitro for 14 days, and 21 days for osteogenic induction. Oil red O and silver nitrate staining were used to compare the adipogenic and osteogenic differentiation ability of three kinds of stem cells.
2 Results
2.1 immunocytochemical staining indicated that the primary and the 1 generation of rib cartilage and costal cartilage cells expressed CD29, CD90, CD34, CD29, from the second generation cells began to express CD90, still not CD34. expression in primary bone marrow cells can express CD29, CD90 and CD34, no expression of type II collagen; then only the expression of CD29 and CD90. three sources of the third generation cells were only the strong expression of CD29, CD90, the uniformity is above 99%. As a type II collagen of cartilage cell markers, only the primary and the expression of 1 generation cartilage cells, from the second generation cells no longer expressed.
2.2 the three sources of the third generation of stem cells into fat stem cells after 14 days of induction, the shape of costal perichondrium and bone marrow derived from the spindle into circular rib cartilage derived stem cells is gradually transformed into fusiform cells were observed in the oil red O staining small red lipid droplets into bone. After 21 days of induction, costal perichondrium and cartilage derived stem cells were stratified growth, and bone marrow stem cells into monolayer growth. After silver staining, black calcified nodules scattered in rib cartilage and costal cartilage derived stem cells; bone marrow stem cells on the surface of bone nodules are gathered into the group.
Full text conclusion
1. by enzyme digestion, successful separation, cultivation and expansion of stem cells of rabbit rib perichondrium and cartilage derived by adherent method. The extraction of bone marrow cells in vitro. After several passages, the soft rib periosteum and bone marrow derived stem cells is stronger than cartilage derived stem cell growth and proliferation.
2. immunohistochemical staining showed that the MSCs phenotype was expressed in costal cartilage and costal chondrocytes from the second generation, and it was strongly expressed in the third generation. It indicated that the third generation of these two cells had the phenotypic characteristics of MSCs.
The third generation stem cells derived from the 3. costal soft periosteum and costal cartilage, after induction, have the same lipid and osteogenic ability with BMSCs, which indicates that the two sources have the characteristics of multi-directional differentiation of stem cells.
4. primary costal chondrocytes expressed collagen type II collagen until the second generation cells no longer expressed, but began to express CD29 and CD90.. This indicates that mature rib chondrocytes can also be differentiated into stem cells in vitro.
Although the 5. rib perichondrium and cartilage derived stem cells have the same immunophenotype and BMSCs and adipogenic differentiation ability of bone induction, but rib perichondrium derived stem cell growth was significantly better than that of costal cartilage to differentiation into the source of stem cells. Therefore, to isolate stem cells from the cartilage. Should try to retain the cartilage membrane.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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7 記者王俊鳴;科學(xué)家說用干細(xì)胞可修補(bǔ)腦部損傷[N];科技日?qǐng)?bào);2002年

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9 章靜波;干細(xì)胞——生命科學(xué)的一個(gè)主攻熱點(diǎn)[N];光明日?qǐng)?bào);2000年

10 通訊員 王其玲 記者 羅堅(jiān)梅;干細(xì)胞，燃起人類絕處逢生新希望[N];杭州日?qǐng)?bào);2005年

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