低强度脉冲超声波对大鼠脂肪干细胞增殖和成骨分化的影响及成骨机制的初步研究

发布时间：2018-07-16 19:20

【摘要】：目的从SD大鼠脂肪组织中分离得到间质干细胞,为后续研究提供种子细胞。 方法 1.从SD大鼠的腹股沟部位切取脂肪组织,结合酶消化法获得一群细胞。 2.经免疫磁珠系统分选后体外培养,观测其形态学特征及鉴定细胞表面标记。 结果 1.获取的细胞呈现类成纤维细胞样,可在体外稳定扩增传代。 2.获取的细胞表面标志符合目前公认的间质干细胞表型特征。 结论通过机械分离结合酶消化的方法,经分选后成功获得SD大鼠ADSCs,可体外培养,为后续研究提供种子细胞。 目的探讨LIPUS对体外分离培养的大鼠ADSCs增殖及成骨分化的影响。 方法 1.流式细胞仪检测阴性对照组和LIPUS组中1d、2d、3d、4d和5d时ADSCs细胞周期分布,计算增殖指数。 2.CCK-8法检测阴性对照组和LIPUS组1d、3d、5d和7d时细胞计数,比较增殖程度。 3.设置阴性对照组、LIPUS组、成骨诱导组及LIPUS联合成骨诱导组,于LIPUS刺激7d、14d时测定各组细胞碱性磷酸酶(ALP)活性；于LIPUS刺激21d时采用Von kossa染色比较各组钙结节形成。 4. RT-PCR检测阴性对照组和LIPUS组1d、3d、5d和7d时成骨相关基因(包括Runx2、ALP、OC、BSP、Coll Ⅰ)的表达。 5. Western blot检测阴性对照组和LIPUS组7d和14d时成骨相关蛋白(包括Runx2和BSP)的表达。 结果 1. LIPUS组细胞增殖指数4d和5d时较对照组提高。 2. LIPUS组细胞增殖程度3d、5d和7d时较对照组加快。 3. LIPUS组ALP活性7d或14d时较对照组提高,但低于成骨诱导组及LIPUS联合成骨诱导组；成骨诱导组与LIPUS联合成骨诱导组间差异无统计学意义；另外各组细胞ALP活性测定结果在上述2个时间点间组内差异均无统计学意义。LIPUS作用能致ADSCs形成矿化结节,但其矿化结节数量不及成骨诱导组和LIPUS联合成骨诱导组。 4. LIPUS组3d、5d或7d后,Runx2和ALP mRNA的表达增加；5d或7d后,LIPUS组BSP和OCN mRNA也表达增加；没有观察到Coll Ⅰ mRNA的改变。 5. LIPUS作用7d和14d后,Runx2和BSP蛋白表达量均增加,BSP蛋白表达的增加从7d维持到14d。但14d后Runx2蛋白量较7d时稍有下调。 结论LIPUS能加速大鼠ADSCs增殖,促进大鼠ADSCs向成骨细胞分化。 β-catenin shRNA陧病毒载体构建和沉默β-catenin基因效果检测 目的构建慢病毒载体,沉默大鼠脂肪源干细胞β-catenin基因表达,为分析经典的Wnt通路在LIPUS促进大鼠ADSCs成骨分化中的作用提供基础。 方法 1.针对大鼠ADSCs的β-catenin基因设计出3种shRNA打靶序列,分别构建3种pCsilencerTM U6/Neo/GFP/RNAi质粒载体,转染至大鼠ADSCs,筛选出最有效的shRNA打靶序列。 2.构建含有该shRNA序列的慢病毒载体。 3.用慢病毒载体转染大鼠ADSCs。 4.检测慢病毒载体转染后,沉默β-catenin基因的效果。 结果 1.用质粒载体筛选出有效的β-catenin基因shRNA序列。 2.用构建病毒载体转染大鼠ADSCs后,可有效沉默β-catenin基因。 结论一种较为经济的方法,稳定转染,沉默大鼠脂肪源干细胞β-catenin基因。藉此可分析经典的Wnt通路在LIPUS促进大鼠ADSCs成骨分化中的作用。 经典Wnt信号通路在LIPUS促进大鼠ADSCs成骨分化中的作用 目的初步探讨经典的Wnt/β-catenin信号通路在LIPUS促进大鼠ADSCs成骨分化过程中的作用。 方法 1. Real-time PCR检测正常ADSCs组、未转染刺激组、转染刺激组1d、3d、5d和7d时成骨相关基因(包括Runx2、ALP、BSP、Coll Ⅰ)的表达； 2. Western blot检测正常ADSCs组、未转染刺激组、转染刺激组7d和14d时成骨相关蛋白(包括Runx2和BSP)的表达。 结果 1.正常的ADSCs在不接受LIPUS刺激的情况下,所有基因基本呈现一致表达。 2.未转染刺激组3d、5d和7d后Runx2基因的表达出现上调；转染刺激组3d、5d和7d后Runx2基因的表达也同样出现上调,但上调的幅度下降。 3.未转染刺激组3d、5d和7d后出现ALP基因表达的上调；转染刺激组3d、5d和7d后也同样出现较一致的上调,上调的幅度无下降。 4.未转染刺激组5d和7d后出现BSP基因的上调表达；转染刺激组5d和7d后也同样出现上调,但上调的幅度有所下降。 5.而三组之间,Ⅰ型胶原基因的表达始终没有明显的变化。 6.未转染刺激组和转染刺激组在7d和14d后,均出现Runx2蛋白的表达水平增加,但14d后增幅稍低；转染刺激组与未转染刺激组相比,7d时增幅低。 7.未转染刺激组7d和14d后,出现BSP蛋白表达增加；转染刺激组仅14d后,出现BSP蛋白增加。 结论有效沉默大鼠ADSCs的β-catenin基因,阻断经典的Wnt通路后,能部分抑制LIPUS对ADSCs的促成骨分化。经典的Wnt通路在LIPUS促进对体外分离培养的大鼠ADSCs成骨分化中,仅起着部分的作用。
[Abstract]:Objective to isolate mesenchymal stem cells from adipose tissue of SD rats and provide seed cells for subsequent research.
Method
1. the adipose tissue was harvested from the groin area of SD rats, and a group of cells were obtained by enzyme digestion.
2. after immunomagnetic beads sorting, the morphological characteristics and identification of cell surface markers were observed.
Result
1. the cells obtained were similar to fibroblast like cells and could be amplified steadily in vitro.
2. the cell surface markers obtained are in line with the currently recognized phenotype of mesenchymal stem cells.
Conclusion the SD rat ADSCs can be successfully obtained by mechanical isolation and enzyme digestion, and can be cultured in vitro to provide seed cells for subsequent research.
Objective to investigate the effects of LIPUS on proliferation and osteogenic differentiation of rat ADSCs cultured in vitro.
Method
1. flow cytometry was used to detect the cell cycle distribution of 1D, 2D, 3D, 4D and 5D in negative control group and LIPUS group, and the proliferation index was calculated.
2.CCK-8 method was used to detect cell count and proliferation of 1D and 3D, 5D and 7d in negative control group and LIPUS group.
3. the negative control group, LIPUS group, osteogenic induction group and LIPUS combined with osteogenic induction group, LIPUS stimulated 7d and 14d to determine the activity of alkaline phosphatase (ALP) in each group, and LIPUS stimulation 21d used Von Kossa staining to compare the formation of calcium nodules in each group.
4. RT-PCR was used to detect the expression of osteogenic related genes (including Runx2, ALP, OC, BSP, and Coll) in negative control group and LIPUS group 1D, 3D, 5D and 7d.
5. Western blot was used to detect the expression of osteogenic associated proteins (including Runx2 and BSP) in negative control group and LIPUS group at 7d and 14d.
Result
1. the cell proliferation index 4D and 5D in group LIPUS were higher than those in control group.
2. the cell proliferation of LIPUS group was faster than that of control group at 3D, 5D and 7d.
3. LIPUS group ALP activity 7d or 14d higher than the control group, but lower than the osteogenic induction group and LIPUS joint osteogenic induction group, bone induction group and LIPUS joint osteogenic induction group difference is not statistically significant; in addition, the determination of ALP activity in each group of cells in the 2 time points among the differences are not statistically significant.LIPUS action can cause ADSC S formed mineralized nodules, but the number of mineralized nodules was less than that of osteogenic induction group and LIPUS combined osteogenic induction group.
4. after LIPUS, 3D, 5D or 7d, the expression of Runx2 and ALP mRNA increased. 5D or 7d increased the expression of BSP and 7d in LIPUS group, and no change in the expression of No.
After 5. LIPUS acting on 7d and 14d, the expression of Runx2 and BSP increased, and the expression of BSP protein increased from 7d to 14D., but the amount of Runx2 protein after 14d decreased slightly compared with that of 7D.
Conclusion LIPUS can accelerate the proliferation of ADSCs in rats and promote the differentiation of ADSCs into osteoblasts in rats.
Construction of beta -catenin shRNA virus vector and detection of silencing of beta -catenin gene
Objective to construct the lentivirus vector, to silence the expression of beta -catenin gene in rat fat source stem cells, and to provide a basis for the analysis of the role of the classical Wnt pathway in promoting the differentiation of ADSCs osteogenesis in rats by LIPUS.
Method
1. 3 kinds of shRNA targeting sequences were designed for the beta -catenin gene of rat ADSCs, and 3 pCsilencerTM U6/Neo/GFP/RNAi plasmid vectors were constructed respectively, and transfected to rat ADSCs, and the most effective shRNA targeting sequence was screened.
2. a lentivirus carrier containing the shRNA sequence was constructed.
3. transfection of rat ADSCs. with lentivirus vector
4. to detect the effect of silencing the beta -catenin gene after lentiviral vector transfection.
Result
1. the effective shRNA sequence of the beta -catenin gene was screened by plasmid vector.
2. transfection of rat ADSCs with a viral vector can effectively silence the beta -catenin gene.
Conclusion a more economical method to stabilize the transfection and silence the beta -catenin gene of rat adipose derived stem cells can be used to analyze the role of the classical Wnt pathway in LIPUS induced ADSCs osteogenesis in rats.
Role of classical Wnt signaling pathway in LIPUS promoting osteogenic differentiation of ADSCs in rats
Objective to explore the role of the classical Wnt/ beta -catenin signaling pathway in LIPUS promoting ADSCs osteogenesis in rats.
Method
1. Real-time PCR was used to detect the expression of osteogenic related genes (including Runx2, ALP, BSP, Coll I) in normal ADSCs group, untransfected stimulation group, transfection group 1D, 3D, 5D and 7d.
2. Western blot was used to detect the expression of osteogenic associated proteins (including Runx2 and BSP) in normal ADSCs group, untransfected stimulation group and transfection group 7d and 14d.
Result
1. normal ADSCs did not receive LIPUS stimulation, and all genes basically expressed consistent expression.
2. the expression of Runx2 gene in the untransfected group of 3D, 5D and 7d was up-regulated, and the expression of Runx2 gene after 3D, 5D and 7d in the transfection group was also up - regulated, but the range of up - regulation decreased.
3. untransfected stimulation group 3D, 5D and 7d showed up regulation of ALP gene expression, and 3D, 5D and 7d in the transfected group were also up to be up, and the range of up - regulation was not decreased.
4. upregulated expression of BSP gene after 5D and 7d in the untransfected stimulation group. The 5D and 7d in the stimulation group also increased, but the rate of upregulation decreased.
There was no significant change in the expression of type I collagen gene between 5. and three groups.
6. the expression level of Runx2 protein increased after 7d and 14d in the untransfected stimulation group and the transfection group, but the increase was slightly lower after 14d, and the increase of the transfection stimulation group was lower than that of the untransfected group. The increase in 7d was low.
7. the expression of BSP protein was increased after 7d and 14d in the non transfected stimulation group, and the BSP protein increased after transfection in the stimulation group only after 14d.
Conclusion effectively silencing the beta -catenin gene of ADSCs in rats, blocking the classical Wnt pathway, can partially inhibit the LIPUS induced bone differentiation of ADSCs. The classical Wnt pathway only plays a part in LIPUS promoting the bone differentiation of rat ADSCs in vitro, which is isolated and cultured in vitro.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【参考文献】

相关期刊论文 前3条

1 梁伟;苟三怀;齐进;魏立;欧阳跃平;刘岩;;低强度脉冲超声波对体外培养的兔骨髓基质细胞成骨作用的影响[J];第二军医大学学报;2007年06期

2 孔璐;低剂量脉冲式超声波的生物学效应研究概况[J];中国公共卫生;2002年12期

3 张超,梁国穗,张颖恺,胡蕴玉,阮狄克;低强度脉冲超声波刺激对人骨髓基质细胞和骨膜细胞生物学效应的影响[J];中华物理医学与康复杂志;2005年11期

，

本文编号：2127416