肋软骨膜、肋软骨和骨髓来源间充质干细胞的比较

发布时间：2018-01-06 12:20

本文关键词：肋软骨膜、肋软骨和骨髓来源间充质干细胞的比较　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：由于间充质干细胞（Mesenchymal stem cells, MSCs）来源的多样性、体外诱导的多向分化性，没有特定的表面表记等特点，对其起源有不同的观点。学者们多认为MSCs是存在于组织和器官中的成体干细胞经体外培养扩增而成。但Orkin等提出：体内也可能不存在MSCs，只是在体外培养分离到的细胞时，这些细胞被激发转变或者基因重编，成为具有干细胞特性的细胞。成熟的细胞能否转化成干细胞呢？已有报道从软骨中分离到干细胞，但体内仅生长区外的软骨组织中无干细胞，关节软骨也有干细胞。从软骨中分离到的干细胞是来自软骨中的干细胞还是由软骨细胞转化而成的呢？本实验拟分别从兔非生长区肋软骨膜、肋软骨和骨髓中分离、培养、扩增获得干细胞，观察软骨细胞能否转化为干细胞？几种来源干细胞的差异？有助于认识成体干细胞体外分离获取的机制，以及干细胞临床应用研究的基础理论。第一部分兔肋软骨膜、肋软骨和骨髓来源干细胞的分离培养 1材料与方法取3-4周新西兰大白兔的肋软骨（避开肋生长板区），解剖显微镜下剥离软骨膜，肋软骨和肋软骨膜均经酶消化法分别获取肋软骨和肋软骨膜细胞；另取股骨、胫骨的骨髓细胞。三种来源的细胞均以贴壁法分离、培养、扩增，分别获得3种来源的干细胞。观察3者各代细胞的形态和生长。取三种来源的第3代细胞，以1×10~4个细胞接种于24孔培养板中培养，每天随机各取3个培养孔，共10天，计数细胞数，绘制生长曲线。 2结果从肋软骨膜中分离的原代细胞接种12h后开始贴壁，5-6d细胞融合，聚集成团，以圆形和多边形为主，从第2代开始细胞形态均一，以梭形为主，呈漩涡状排列。目前已传至15代。从肋软骨中分离的原代细胞6h后开始贴壁，5-6天细胞融合，以铺路石形态为主，第2代和第3代细胞逐渐转变成梭形；但第5代出现细胞老化，只传至第6代。原代骨髓细胞培养4-5h后开始贴壁，10d左右细胞融合。融合时细胞可见梭形，第2代以后细胞形态以梭形为主，呈明显漩涡状排列，已传10代，生长良好。三种来源细胞第3代细胞的生长曲线表明：贴壁后生长潜伏适应期为1-3d；3d后即进入对数生长期；6d后进入生长平台期。第二部分肋软骨膜、肋软骨和骨髓来源干细胞的比较 1材料与方法 1.1三种来源的每一代细胞均以免疫组化染色方法检测每一代细胞CD29、CD90、CD34和Ⅱ型胶原的表达。 1.2取三种来源的第3代干细胞分别进行体外成脂诱导14天、成骨诱导21天；油红O和硝酸银染色法检测比较三种干细胞成脂、成骨的分化能力。 2结果 2.1免疫细胞化学染色发现原代及第1代肋软骨膜和肋软骨细胞均不表达CD29、CD90、 CD34，从第2代细胞开始表达CD29、CD90，仍不表达CD34。原代骨髓细胞可表达CD29、CD90和CD34，不表达Ⅱ型胶原；随后只表达CD29和CD90。三种来源的第3代细胞均只强表达CD29、CD90，其均一性都达到99％以上。作为软骨细胞标记物之一的Ⅱ型胶原，仅原代及第1代肋软骨细胞表达，从第2代细胞不再表达。 2.2三种来源的第3代干细胞经成脂诱导14天后,肋软骨膜和骨髓来源的干细胞形状多由梭形转变为圆形，肋软骨来源的干细胞则逐渐转变为长梭形，细胞内均可见油红O染色的红色小脂滴。成骨诱导21天后，肋软骨膜和肋软骨来源的干细胞呈复层生长，而骨髓来源的干细胞成单层生长。经过硝酸银染色后，黑色钙化小结节分散于肋软骨膜和肋软骨来源的干细胞表面；骨髓来源的干细胞表面的骨结节则聚集成团。全文结论 1.用酶消化法，，成功的分离、培养和扩增了兔肋软骨膜和肋软骨来源的干细胞。用贴壁法提取了骨髓细胞。体外多次传代后，肋软骨膜和骨髓来源的干细胞比肋软骨来源的干细胞具有更强的生长增殖能力。 2.免疫组化检测显示肋软骨膜和肋软骨细胞从第2代开始均表达MSCs的表型，至第三代强表达。表明这两种细胞第3代具有MSCs的表型特征。 3.肋软骨膜和肋软骨来源的第3代干细胞在体外经诱导后具有与BMSCs一致的成脂和成骨能力，证明这两种来源的细胞具有干细胞的多向分化特征。 4.原代肋软骨细胞表达软骨细胞的标记物Ⅱ型胶原，直至第2代细胞不再表达，但开始表达CD29和CD90。这表明：成熟的肋软骨细胞在体外培养也可去分化成为干细胞。 5.尽管肋软骨膜和肋软骨来源的干细胞均具有与BMSCs相同的免疫表型和成脂、成骨诱导分化能力，但肋软骨膜来源干细胞的生长明显优于肋软骨来源去分化后转化成的干细胞。因此，从软骨中分离获取干细胞时，应尽量保留软骨膜。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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