脂肪组织分泌物诱导脂肪干细胞成脂相关研究
发布时间：2018-07-24 13:14
【摘要】：临床上因创伤、肿瘤术后、感染及先天缺陷的患者，需要大量的脂肪组织来修复软组织缺损。然而，常用的自体脂肪移植有很多的局限。脂肪组织工程是应用干细胞对软组织进行重建的一门新兴技术，干细胞和成脂因子在其中起着关键的作用。脂肪干细胞由于其来源广泛、含量丰富、取材方便、具有多向方分化潜能，已广泛应用于组织工程研究。脂肪组织不仅仅是能量存储的器官，并且能分泌大量细胞因子。我们在脂肪组织块原代法培养脂肪细胞时发现，脂肪干细胞向脂肪细胞方向分化。脂肪组织分泌物可能含有，能诱导脂肪干细胞脂向分化的成脂因子。现在研究比较成熟的体外成脂诱导因子包括：胰岛素、吲哚美辛等，对生理性成脂因子的研究较少。本课题采用组织块原代培养法获取脂肪干细胞，探索脂肪组织分泌物与成脂间的关系，及脂肪组织分泌物中可能的成脂因子。对生理性成脂因子的研究将为脂肪组织工程应用于临床奠定基础。 方法： 1、脂肪组织块原代培养法分离培养大鼠脂肪干细胞，采用倒置显微镜观察细胞的形态和生长状况。将原代细胞培养传代，纯化至第四代后，分别用成脂、成骨、成神经化学诱导剂进行诱导培养，然后进行油红O染色、茜素红染色及荧光免疫实验。 2、脂肪组织块贴壁法收集前三天培养液作为ATE，对第四代生长状态良好的大鼠脂肪干细胞，进行诱导。而后进行油红O染色实验。 3、用含有FBS和无FBS的培养基对脂肪组织块进行培养并收集ATE，分别对大鼠脂肪干细胞进行成脂诱导，进行油红O染色实验。 4、收集无FBS培养基获取的ATE，分为100μg/ml、250μg/ml、500μg/ml组分，对第四代大鼠脂肪干细胞进行诱导，分别观察各诱导组成脂情况，进行油红O染色实验、成脂率检测实验，及细胞增殖和迁移实验。 5、用超滤管将ATE按分子量富集为5个不同的组分，分别对第四代脂肪干细胞进行诱导，观察。并进行western blot实验，荧光免疫染色实验。 6、以脂肪干细胞分泌蛋白作为对照，将脂肪组织分泌物进行质谱分析，通过信息学分析探索ATE中的成脂因子。并用western blot实验验证。 结果： 1、脂肪组织块法获得的细胞生长状态良好，能向脂肪细胞、成骨细胞及神经细胞系方向分化。 2、ATE诱导二天时，可见个别第四代大鼠脂肪干细胞胞浆内有脂滴形成，第七天时，脂滴融合变大。阴性对照组未见脂滴形成。 3、获取ATE时添加或不添加FBS，在第七天时ADSCs胞浆中均有成熟脂滴形成，两组成脂率无统计学差别(P0.05)。 4、500μg/ml组分较100μg/ml、250μg/ml组分成脂率高。不同浓度的ATE对大鼠脂肪干细胞的增殖均有抑制作用，但是各组间抑制程度相仿，无统计学意义(P0.01)。ATE对大鼠脂肪干细胞的迁移没有影响。 5、分子量100KDa组分诱导大鼠脂肪干细胞，可见细胞脂向分化，而其它组未见成脂。western blot，荧光免疫实验结果表明分子量100KDa诱导组细胞,成脂相关蛋白表达阳性。 6、通过质谱分析找出差异表达蛋白中，分子量大于100KDa的蛋白有Col I、CP、IGg2a、FN，western bolt实验再次验证结果的可靠性。 结论： 1、脂肪组织块法获得的细胞有脂肪干细胞的生物学特性，具有良好的多向分化能力。 2、ATE能诱导脂肪干细胞成脂分化。 3、FBS对ATE获取的成脂能力无影响。 4、浓度较高的500μg/ml组较浓度较低组的成脂能力更强。ATE能够抑制细胞增殖，与干细胞成脂分化机制相符，但这种抑制不是无限制的。ATE对细胞迁移无影响。 5、脂肪组织块分泌物中具有成脂分化能力的因子集中在分子量100KDa组分，为进一步研究新的生理性的成脂因子奠定了基础。 6、通过我们的实验证实，，Col I、CP、IGg2a、FN的确存在于脂肪组织分泌物中，其表达量及种类与单纯的脂肪干细胞分泌蛋白不同，是可能的成脂因子。
[Abstract]:A large number of adipose tissues are needed to repair soft tissue defects in patients with trauma, tumor surgery, infection and congenital defects. However, the commonly used autologous fat transplantation has many limitations. Adipose tissue engineering is a new technique for the application of stem cells to the reconstruction of soft tissue. Stem cells and lipid factors play a key role in it. Adipose stem cells are widely used in tissue engineering research. Adipose tissue is not only an organ of energy storage, but also a large number of cytokines. We found fat stem cells in fat tissue blocks and found fat stem cells to fat. The secretions of adipose tissue may be contained in the adipose tissue, which can induce fat stem cell fat to differentiate into fat forming factors. Now the mature in vitro lipid inducing factors include insulin, indomethacin, and less research on physiological lipid factors. The relationship between the secretions of the adipose tissue and the fat formation and the possible lipid factors in the secretions of the adipose tissue. The study of the physiological lipid factors will lay the foundation for the clinical application of adipose tissue engineering.
Method:
1, the fat tissue block was used to culture the rat fat stem cells by primary culture, and the morphology and growth status of the cells were observed by inverted microscope. The original cells were cultured and purified to fourth generations. The cells were induced and cultured with fat, bone and neurochemical inducers respectively. Then the oil red O staining, alizarin red staining and fluorescence immunization were carried out. Test.
2, the fat tissue block adherence method was used to collect the culture medium for the first three days as ATE to induce the fat stem cells of the fourth generation healthy rats, and then the oil red O staining experiment was carried out.
3, the fat tissue block was cultured and ATE was collected with the medium containing FBS and no FBS, and the fat stem cells of the rat were induced and the oil red O staining experiment was carried out.
4, the ATE obtained from the FBS medium was collected, which was divided into 100 g/ml, 250 g/ml and 500 mu g/ml components. The fourth generation of rat fat stem cells were induced, respectively, to observe the induced composition of the lipids, the oil red O staining experiment, the test of the fat percentage and the proliferation and migration of the cells.
5, the molecular weight of ATE was enriched into 5 different components by the ultrafiltration tube, and the fourth generation of fat stem cells were induced and observed respectively. The Western blot experiment and the fluorescence immunostaining experiment were carried out.
6, using the secretory protein of adipose stem cells as the control, the fat tissue secretions were analyzed by mass spectrometry, and the lipid forming factors in ATE were explored by informatics analysis. The results were verified by Western blot test.
Result:
1. Adipose tissue block method can induce cell growth in good condition and differentiate into adipocytes, osteoblasts and neural cell lines.
2, when ATE was induced for two days, the lipid droplets in the fat stem cells of the fourth generation rats were found to form, and the lipid droplets became larger at the seventh day. No lipid droplets were formed in the negative control group.
3. Added or not added FBS to ATE, mature lipid droplets were formed in the cytoplasm of ADSCs on the seventh day, and there was no significant difference between the two groups (P 0.05).
The 4500 u g/ml components were 100 g/ml and 250 micron g/ml group had high fat percentage. Different concentrations of ATE had inhibitory effect on the proliferation of rat fat stem cells, but the degree of inhibition was similar, and there was no statistical significance (P0.01).ATE had no effect on the migration of fat stem cells in rats.
5, the molecular weight 100KDa component induced rat fat stem cells, which showed that the cell fat was differentiated, but the other groups did not have fat.Western blot. The results of fluorescence immunoassay showed that the molecular weight of 100KDa induced group cells, and the expression of lipid related protein was positive.
6. Mass spectrometry analysis showed that the proteins with molecular weight greater than 100KDa were ColI, CP, IGg2a, FN and Western bolt.
Conclusion:
1. The cells obtained by adipose tissue block method have the biological characteristics of adipose-derived stem cells and have good multidirectional differentiation ability.
2, ATE can induce adipose differentiation of fat stem cells.
3, FBS had no effect on the fat forming ability of ATE.
4, the higher concentration of 500 mu g/ml group was stronger than the lower concentration group and.ATE could inhibit cell proliferation, which was consistent with the mechanism of lipid differentiation of stem cells, but this inhibition was not unrestricted.ATE on cell migration.
5, the factor of lipid differentiation in the secretions of fat tissue is concentrated in the molecular weight 100KDa component, which lays the foundation for further study of the new physiological fat forming factors.
6, our experiments show that Col I, CP, IGg2a, FN do exist in the secretions of adipose tissue, and the amount and type of expression are different from those of pure adipose stem cells, which are possible lipid factors.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
本文编号：2141497
[Abstract]:A large number of adipose tissues are needed to repair soft tissue defects in patients with trauma, tumor surgery, infection and congenital defects. However, the commonly used autologous fat transplantation has many limitations. Adipose tissue engineering is a new technique for the application of stem cells to the reconstruction of soft tissue. Stem cells and lipid factors play a key role in it. Adipose stem cells are widely used in tissue engineering research. Adipose tissue is not only an organ of energy storage, but also a large number of cytokines. We found fat stem cells in fat tissue blocks and found fat stem cells to fat. The secretions of adipose tissue may be contained in the adipose tissue, which can induce fat stem cell fat to differentiate into fat forming factors. Now the mature in vitro lipid inducing factors include insulin, indomethacin, and less research on physiological lipid factors. The relationship between the secretions of the adipose tissue and the fat formation and the possible lipid factors in the secretions of the adipose tissue. The study of the physiological lipid factors will lay the foundation for the clinical application of adipose tissue engineering.
Method:
1, the fat tissue block was used to culture the rat fat stem cells by primary culture, and the morphology and growth status of the cells were observed by inverted microscope. The original cells were cultured and purified to fourth generations. The cells were induced and cultured with fat, bone and neurochemical inducers respectively. Then the oil red O staining, alizarin red staining and fluorescence immunization were carried out. Test.
2, the fat tissue block adherence method was used to collect the culture medium for the first three days as ATE to induce the fat stem cells of the fourth generation healthy rats, and then the oil red O staining experiment was carried out.
3, the fat tissue block was cultured and ATE was collected with the medium containing FBS and no FBS, and the fat stem cells of the rat were induced and the oil red O staining experiment was carried out.
4, the ATE obtained from the FBS medium was collected, which was divided into 100 g/ml, 250 g/ml and 500 mu g/ml components. The fourth generation of rat fat stem cells were induced, respectively, to observe the induced composition of the lipids, the oil red O staining experiment, the test of the fat percentage and the proliferation and migration of the cells.
5, the molecular weight of ATE was enriched into 5 different components by the ultrafiltration tube, and the fourth generation of fat stem cells were induced and observed respectively. The Western blot experiment and the fluorescence immunostaining experiment were carried out.
6, using the secretory protein of adipose stem cells as the control, the fat tissue secretions were analyzed by mass spectrometry, and the lipid forming factors in ATE were explored by informatics analysis. The results were verified by Western blot test.
Result:
1. Adipose tissue block method can induce cell growth in good condition and differentiate into adipocytes, osteoblasts and neural cell lines.
2, when ATE was induced for two days, the lipid droplets in the fat stem cells of the fourth generation rats were found to form, and the lipid droplets became larger at the seventh day. No lipid droplets were formed in the negative control group.
3. Added or not added FBS to ATE, mature lipid droplets were formed in the cytoplasm of ADSCs on the seventh day, and there was no significant difference between the two groups (P 0.05).
The 4500 u g/ml components were 100 g/ml and 250 micron g/ml group had high fat percentage. Different concentrations of ATE had inhibitory effect on the proliferation of rat fat stem cells, but the degree of inhibition was similar, and there was no statistical significance (P0.01).ATE had no effect on the migration of fat stem cells in rats.
5, the molecular weight 100KDa component induced rat fat stem cells, which showed that the cell fat was differentiated, but the other groups did not have fat.Western blot. The results of fluorescence immunoassay showed that the molecular weight of 100KDa induced group cells, and the expression of lipid related protein was positive.
6. Mass spectrometry analysis showed that the proteins with molecular weight greater than 100KDa were ColI, CP, IGg2a, FN and Western bolt.
Conclusion:
1. The cells obtained by adipose tissue block method have the biological characteristics of adipose-derived stem cells and have good multidirectional differentiation ability.
2, ATE can induce adipose differentiation of fat stem cells.
3, FBS had no effect on the fat forming ability of ATE.
4, the higher concentration of 500 mu g/ml group was stronger than the lower concentration group and.ATE could inhibit cell proliferation, which was consistent with the mechanism of lipid differentiation of stem cells, but this inhibition was not unrestricted.ATE on cell migration.
5, the factor of lipid differentiation in the secretions of fat tissue is concentrated in the molecular weight 100KDa component, which lays the foundation for further study of the new physiological fat forming factors.
6, our experiments show that Col I, CP, IGg2a, FN do exist in the secretions of adipose tissue, and the amount and type of expression are different from those of pure adipose stem cells, which are possible lipid factors.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
【共引文献】
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2 张慧;郑红光;张德伟;梅煜明;;雌激素影响冻存肾脂肪囊来源脂肪间充质干细胞的成脂分化[J];中国组织工程研究;2013年27期
3 汤佳城;蔡秀军;;脐带间充质干细胞在器官移植中的研究进展[J];中国实用外科杂志;2013年09期
4 陈津;马予洁;郭子宽;谭建明;;临床级间充质干细胞培养规范[J];中华细胞与干细胞杂志(电子版);2013年03期
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