腺病毒介导的骨形态蛋白-9基因转染脂肪干细胞的实验研究
发布时间：2018-07-18 10:35
【摘要】： 目的: 1.通过对兔脂肪干细胞的分离、培养及鉴定,获得纯化的兔脂肪干细胞(ADSCs);建立一种稳定、高效的兔ADSCs的培养方法。 2.寻求一种BMP-9基因转染靶细胞ADSCs的稳定的实验方法;证实BMP-9基因转染后的ADSCs可向成骨细胞分化,以获得可用于骨组织工程的基因修饰的种子细胞。 方法: 1.ADSCs的分离、培养及鉴定:采用I型胶原酶消化法获取原代ADSCs。扩增和纯化ADSCs后,显微镜下观察兔ADSCs形态,MTT、细胞计数分析ADSCs的体外增殖情况,免疫组化检测ADSCs表面抗原CD29、CD44的表达。 2.BMP-9转染ADSCs的实验:载BMP-9的腺病毒直接感染P3代的ADSCs。荧光显微镜观察转染效果,流式细胞仪测定转染率,RT-PCR检测转染后ADSCs内BMP-9 mRNA的表达。碱性磷酸酶活性、碱性磷酸酶和茜素红钙结节染色检测转染后ADSCs的成骨活性。 结果: 1.ADSCs的分离、培养及鉴定:(1)分离、培养的原代ADSCs细胞形态呈梭形或多角状,类似成纤维细胞,团簇状生长,在10 d左右达到90%融合。(2)经过传代、纯化的ADSCs呈成纤维细胞样,分布均匀、大小一致、细胞紧密排列,漩涡状生长,5 d左右达90%融合,显示出活跃的增殖能力。(3)生长曲线显示:1~3 d为传代培养的滞留期,4~6 d为对数增殖期,7~9 d为生长抑制期,此时细胞生长速度变慢。(4)累计倍增曲线显示:群体倍增时间大约为55 h,P3代细胞增殖速度最快,P8代以后的细胞增殖速度减慢,细胞出现衰老征象。(5)免疫组化染色显示:ADSCs细胞内表面抗原CD29、CD44均阳性表达。 2.BMP-9转染ADSCs的实验:(1)BMP-9基因腺病毒载体可成功转染ADSCs。(2)腺病毒介导的BMP-9基因转染ADSCs后24 h即有荧光表达,并随时间延长荧光强度逐渐增强,转染3d转染效率达到80%以上。(3)转染后ADSCs停滞期略延长,数量轻度下降,倍增时间稍延长,但不影响细胞增殖。(4) RT-PCR检测结果显示,各转染组细胞内hBMP-9的mRNA持续阳性表达,未转染组未见阳性条带。(5)ADSCs转染后,ALP染色及茜素红钙结节染色为阳性,细胞内ALP活性呈增长趋势,转染组显著高于未转染组。 结论: 1.从兔脂肪组织中分离、培养、纯化的ADSCs具有明显的干细胞特征,体外生长旺盛,扩增迅速,长期传代仍能保持稳定的增殖能力,为BMP-9转染ADSCs诱导成骨的实验奠定了基础。 2.腺病毒介导的BMP-9基因可以安全、有效地转染ADSCs,其转染的BMP-9基因可获得较高水平的表达,且具有明显的诱导ADSCs向成骨细胞转化的作用;而携带人全长BMP-9基因的腺病毒转染ADSCs可作为研究BMP-9诱导ADSCs成骨作用实验的可靠方法。
[Abstract]:Objective:
1. through isolation, culture and identification of rabbit adipose derived stem cells, purified rabbit adipose derived stem cells (ADSCs) were obtained, and a stable and efficient method for ADSCs culture was established.
2. seek a stable experimental method of transfection of BMP-9 gene to target cell ADSCs, and confirm that ADSCs transfected by BMP-9 gene can differentiate into osteoblasts to obtain gene modified seed cells that can be used for bone tissue engineering.
Method:
The separation, culture and identification of 1.ADSCs: after I type collagenase digestion was used to obtain the original ADSCs. amplification and purification of ADSCs, the ADSCs morphology of rabbit was observed under microscope, MTT, cell count was used to analyze the proliferation of ADSCs in vitro, and the expression of ADSCs surface antigen CD29 and CD44 was detected by immunohistochemistry.
The transfection of ADSCs by 2.BMP-9: the adenovirus carrying BMP-9 was directly infected with the P3 generation ADSCs. fluorescence microscope to observe the transfection effect. The transfection rate was measured by flow cytometry. The expression of BMP-9 mRNA in ADSCs after the transfection was detected by RT-PCR. The activity of alkaline phosphatase, alkaline phosphatase and alizarin red calcium nodule staining were used to detect the osteogenic activity of ADSCs in the transfected ADSCs.
Result:
1.ADSCs isolation, culture and identification: (1) isolated, cultured primary ADSCs cells have spindle or multi angle shape, similar to fibroblasts, cluster like growth and 90% fusion at about 10 d. (2) after passage, the purified ADSCs is fibroblast like, evenly distributed and uniform in size, cells closely arranged, whirlpool, 5 d to 90% fusion, (3) the growth curve showed that 1~3 D was the stagnation period of subculture, 4~6 D was a logarithmic proliferation period, 7~9 D was a growth inhibition period, and the growth rate of cell slowed. (4) the cumulative doubling curve showed that the multiplication time of the population was about 55 h, the proliferation rate of P3 generation was the fastest, and the proliferation rate slowed down after the P8 generation. The cells showed signs of aging. (5) immunohistochemical staining showed that the surface antigen CD29 and CD44 of ADSCs cells were all positive.
2.BMP-9 transfection of ADSCs: (1) BMP-9 gene adenovirus vector could be transfected successfully with ADSCs. (2) adenovirus mediated BMP-9 gene transfection to ADSCs after transfection to 24 h, and the fluorescence intensity was gradually increased with time, and the transfection efficiency of 3D transfection reached more than 80%. (3) the stagnation period of ADSCs was slightly prolonged, the number of 3D was slightly decreased and the doubling time was delayed slightly after transfection. Long, but did not affect cell proliferation. (4) the results of RT-PCR detection showed that the mRNA of hBMP-9 in the cells of each transfection group was positive and no positive bands were not found in the untransfected group. (5) after ADSCs transfection, ALP staining and alizarin red calcium nodule staining were positive, and the intracellular ALP activity was increasing, and the transfection group was significantly higher than that of the untransfected group.
Conclusion:
1. isolated, cultured and purified ADSCs from rabbit adipose tissue has obvious characteristics of stem cell. It grows exuberant in vitro, expands rapidly, and can still maintain stable proliferative ability for a long time. It lays a foundation for the experiment of BMP-9 transfection of ADSCs to induce osteogenesis.
2. adenovirus mediated BMP-9 gene can be safely and effectively transfected to ADSCs. The transfected BMP-9 gene can obtain a high level of expression, and it can obviously induce the transformation of ADSCs into osteoblasts, and the transfection of ADSCs with the adenovirus carrying human BMP-9 gene can be used as a reliable method for the study of BMP-9 induced ADSCs osteogenesis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2131639
[Abstract]:Objective:
1. through isolation, culture and identification of rabbit adipose derived stem cells, purified rabbit adipose derived stem cells (ADSCs) were obtained, and a stable and efficient method for ADSCs culture was established.
2. seek a stable experimental method of transfection of BMP-9 gene to target cell ADSCs, and confirm that ADSCs transfected by BMP-9 gene can differentiate into osteoblasts to obtain gene modified seed cells that can be used for bone tissue engineering.
Method:
The separation, culture and identification of 1.ADSCs: after I type collagenase digestion was used to obtain the original ADSCs. amplification and purification of ADSCs, the ADSCs morphology of rabbit was observed under microscope, MTT, cell count was used to analyze the proliferation of ADSCs in vitro, and the expression of ADSCs surface antigen CD29 and CD44 was detected by immunohistochemistry.
The transfection of ADSCs by 2.BMP-9: the adenovirus carrying BMP-9 was directly infected with the P3 generation ADSCs. fluorescence microscope to observe the transfection effect. The transfection rate was measured by flow cytometry. The expression of BMP-9 mRNA in ADSCs after the transfection was detected by RT-PCR. The activity of alkaline phosphatase, alkaline phosphatase and alizarin red calcium nodule staining were used to detect the osteogenic activity of ADSCs in the transfected ADSCs.
Result:
1.ADSCs isolation, culture and identification: (1) isolated, cultured primary ADSCs cells have spindle or multi angle shape, similar to fibroblasts, cluster like growth and 90% fusion at about 10 d. (2) after passage, the purified ADSCs is fibroblast like, evenly distributed and uniform in size, cells closely arranged, whirlpool, 5 d to 90% fusion, (3) the growth curve showed that 1~3 D was the stagnation period of subculture, 4~6 D was a logarithmic proliferation period, 7~9 D was a growth inhibition period, and the growth rate of cell slowed. (4) the cumulative doubling curve showed that the multiplication time of the population was about 55 h, the proliferation rate of P3 generation was the fastest, and the proliferation rate slowed down after the P8 generation. The cells showed signs of aging. (5) immunohistochemical staining showed that the surface antigen CD29 and CD44 of ADSCs cells were all positive.
2.BMP-9 transfection of ADSCs: (1) BMP-9 gene adenovirus vector could be transfected successfully with ADSCs. (2) adenovirus mediated BMP-9 gene transfection to ADSCs after transfection to 24 h, and the fluorescence intensity was gradually increased with time, and the transfection efficiency of 3D transfection reached more than 80%. (3) the stagnation period of ADSCs was slightly prolonged, the number of 3D was slightly decreased and the doubling time was delayed slightly after transfection. Long, but did not affect cell proliferation. (4) the results of RT-PCR detection showed that the mRNA of hBMP-9 in the cells of each transfection group was positive and no positive bands were not found in the untransfected group. (5) after ADSCs transfection, ALP staining and alizarin red calcium nodule staining were positive, and the intracellular ALP activity was increasing, and the transfection group was significantly higher than that of the untransfected group.
Conclusion:
1. isolated, cultured and purified ADSCs from rabbit adipose tissue has obvious characteristics of stem cell. It grows exuberant in vitro, expands rapidly, and can still maintain stable proliferative ability for a long time. It lays a foundation for the experiment of BMP-9 transfection of ADSCs to induce osteogenesis.
2. adenovirus mediated BMP-9 gene can be safely and effectively transfected to ADSCs. The transfected BMP-9 gene can obtain a high level of expression, and it can obviously induce the transformation of ADSCs into osteoblasts, and the transfection of ADSCs with the adenovirus carrying human BMP-9 gene can be used as a reliable method for the study of BMP-9 induced ADSCs osteogenesis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【参考文献】
相关期刊论文 前8条
1 王燕,陈光辉,邵建华,田新利;大鼠脂肪组织源性间充质干细胞的分离及向心肌细胞的诱导分化[J];山东大学学报(医学版);2005年07期
2 胡静;戚孟春;韩立赤;李继华;周海孝;;BMP-7基因促进大鼠下颌牵张成骨的研究[J];实用口腔医学杂志;2006年05期
3 谭祖键,李起鸿;BMP及其诱导成骨的分子生物学基础[J];中华骨科杂志;1996年09期
4 石冰,邓典智,王翰章,王大章;腭裂植骨对上颌骨生长发育的影响(动物实验研究)[J];中华口腔医学杂志;1998年03期
5 段智霞;郑启新;郭晓东;白玉;袁泉;陈顺广;;BMP-2活性多肽体外定向诱导大鼠BMSCs向成骨方向分化的实验研究[J];中国矫形外科杂志;2007年21期
6 康焱;廖威明;盛蹼义;;hBMP7瞬时转染对兔骨髓间充质干细胞生物学行为的影响[J];中山大学学报(医学科学版);2006年S1期
7 佘小明;田锟;钱宁;吴曙光;赵菊花;;贵州小型猪腭裂动物实验模型的建立[J];遵义医学院学报;2007年01期
8 徐晓斐;王健;徐海艇;余力;朱昌;张波;;组织工程方法修复羊腭裂骨缺损的初步研究[J];组织工程与重建外科杂志;2008年02期
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