低強度脈沖超聲波對大鼠脂肪干細胞增殖和成骨分化的影響及成骨機制的初步研究
發(fā)布時間：2018-07-16 19:20
【摘要】：目的從SD大鼠脂肪組織中分離得到間質(zhì)干細胞,為后續(xù)研究提供種子細胞。 方法 1.從SD大鼠的腹股溝部位切取脂肪組織,結(jié)合酶消化法獲得一群細胞。 2.經(jīng)免疫磁珠系統(tǒng)分選后體外培養(yǎng),觀測其形態(tài)學特征及鑒定細胞表面標記。 結(jié)果 1.獲取的細胞呈現(xiàn)類成纖維細胞樣,可在體外穩(wěn)定擴增傳代。 2.獲取的細胞表面標志符合目前公認的間質(zhì)干細胞表型特征。 結(jié)論通過機械分離結(jié)合酶消化的方法,經(jīng)分選后成功獲得SD大鼠ADSCs,可體外培養(yǎng),為后續(xù)研究提供種子細胞。 目的探討LIPUS對體外分離培養(yǎng)的大鼠ADSCs增殖及成骨分化的影響。 方法 1.流式細胞儀檢測陰性對照組和LIPUS組中1d、2d、3d、4d和5d時ADSCs細胞周期分布,計算增殖指數(shù)。 2.CCK-8法檢測陰性對照組和LIPUS組1d、3d、5d和7d時細胞計數(shù),比較增殖程度。 3.設(shè)置陰性對照組、LIPUS組、成骨誘導組及LIPUS聯(lián)合成骨誘導組,于LIPUS刺激7d、14d時測定各組細胞堿性磷酸酶(ALP)活性；于LIPUS刺激21d時采用Von kossa染色比較各組鈣結(jié)節(jié)形成。 4. RT-PCR檢測陰性對照組和LIPUS組1d、3d、5d和7d時成骨相關(guān)基因(包括Runx2、ALP、OC、BSP、Coll Ⅰ)的表達。 5. Western blot檢測陰性對照組和LIPUS組7d和14d時成骨相關(guān)蛋白(包括Runx2和BSP)的表達。 結(jié)果 1. LIPUS組細胞增殖指數(shù)4d和5d時較對照組提高。 2. LIPUS組細胞增殖程度3d、5d和7d時較對照組加快。 3. LIPUS組ALP活性7d或14d時較對照組提高,但低于成骨誘導組及LIPUS聯(lián)合成骨誘導組；成骨誘導組與LIPUS聯(lián)合成骨誘導組間差異無統(tǒng)計學意義；另外各組細胞ALP活性測定結(jié)果在上述2個時間點間組內(nèi)差異均無統(tǒng)計學意義。LIPUS作用能致ADSCs形成礦化結(jié)節(jié),但其礦化結(jié)節(jié)數(shù)量不及成骨誘導組和LIPUS聯(lián)合成骨誘導組。 4. LIPUS組3d、5d或7d后,Runx2和ALP mRNA的表達增加；5d或7d后,LIPUS組BSP和OCN mRNA也表達增加；沒有觀察到Coll Ⅰ mRNA的改變。 5. LIPUS作用7d和14d后,Runx2和BSP蛋白表達量均增加,BSP蛋白表達的增加從7d維持到14d。但14d后Runx2蛋白量較7d時稍有下調(diào)。 結(jié)論LIPUS能加速大鼠ADSCs增殖,促進大鼠ADSCs向成骨細胞分化。 β-catenin shRNA隉病毒載體構(gòu)建和沉默β-catenin基因效果檢測 目的構(gòu)建慢病毒載體,沉默大鼠脂肪源干細胞β-catenin基因表達,為分析經(jīng)典的Wnt通路在LIPUS促進大鼠ADSCs成骨分化中的作用提供基礎(chǔ)。 方法 1.針對大鼠ADSCs的β-catenin基因設(shè)計出3種shRNA打靶序列,分別構(gòu)建3種pCsilencerTM U6/Neo/GFP/RNAi質(zhì)粒載體,轉(zhuǎn)染至大鼠ADSCs,篩選出最有效的shRNA打靶序列。 2.構(gòu)建含有該shRNA序列的慢病毒載體。 3.用慢病毒載體轉(zhuǎn)染大鼠ADSCs。 4.檢測慢病毒載體轉(zhuǎn)染后,沉默β-catenin基因的效果。 結(jié)果 1.用質(zhì)粒載體篩選出有效的β-catenin基因shRNA序列。 2.用構(gòu)建病毒載體轉(zhuǎn)染大鼠ADSCs后,可有效沉默β-catenin基因。 結(jié)論一種較為經(jīng)濟的方法,穩(wěn)定轉(zhuǎn)染,沉默大鼠脂肪源干細胞β-catenin基因。藉此可分析經(jīng)典的Wnt通路在LIPUS促進大鼠ADSCs成骨分化中的作用。 經(jīng)典Wnt信號通路在LIPUS促進大鼠ADSCs成骨分化中的作用 目的初步探討經(jīng)典的Wnt/β-catenin信號通路在LIPUS促進大鼠ADSCs成骨分化過程中的作用。 方法 1. Real-time PCR檢測正常ADSCs組、未轉(zhuǎn)染刺激組、轉(zhuǎn)染刺激組1d、3d、5d和7d時成骨相關(guān)基因(包括Runx2、ALP、BSP、Coll Ⅰ)的表達； 2. Western blot檢測正常ADSCs組、未轉(zhuǎn)染刺激組、轉(zhuǎn)染刺激組7d和14d時成骨相關(guān)蛋白(包括Runx2和BSP)的表達。 結(jié)果 1.正常的ADSCs在不接受LIPUS刺激的情況下,所有基因基本呈現(xiàn)一致表達。 2.未轉(zhuǎn)染刺激組3d、5d和7d后Runx2基因的表達出現(xiàn)上調(diào)；轉(zhuǎn)染刺激組3d、5d和7d后Runx2基因的表達也同樣出現(xiàn)上調(diào),但上調(diào)的幅度下降。 3.未轉(zhuǎn)染刺激組3d、5d和7d后出現(xiàn)ALP基因表達的上調(diào)；轉(zhuǎn)染刺激組3d、5d和7d后也同樣出現(xiàn)較一致的上調(diào),上調(diào)的幅度無下降。 4.未轉(zhuǎn)染刺激組5d和7d后出現(xiàn)BSP基因的上調(diào)表達；轉(zhuǎn)染刺激組5d和7d后也同樣出現(xiàn)上調(diào),但上調(diào)的幅度有所下降。 5.而三組之間,Ⅰ型膠原基因的表達始終沒有明顯的變化。 6.未轉(zhuǎn)染刺激組和轉(zhuǎn)染刺激組在7d和14d后,均出現(xiàn)Runx2蛋白的表達水平增加,但14d后增幅稍低；轉(zhuǎn)染刺激組與未轉(zhuǎn)染刺激組相比,7d時增幅低。 7.未轉(zhuǎn)染刺激組7d和14d后,出現(xiàn)BSP蛋白表達增加；轉(zhuǎn)染刺激組僅14d后,出現(xiàn)BSP蛋白增加。 結(jié)論有效沉默大鼠ADSCs的β-catenin基因,阻斷經(jīng)典的Wnt通路后,能部分抑制LIPUS對ADSCs的促成骨分化。經(jīng)典的Wnt通路在LIPUS促進對體外分離培養(yǎng)的大鼠ADSCs成骨分化中,僅起著部分的作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329
本文編號：2127416
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329
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