不同浓度罗哌卡因对脂肪干细胞生长增殖与成脂分化的影响

发布时间：2018-01-27 03:02

本文关键词： 脂肪干细胞增殖成脂化倍增　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：1目的研究表明在实验室进行体外培养时,人脂肪干细胞(adipose-derived stem cells,ADSCs)与骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)有类似的生物学特性,即同样具有强大的自体复制、跨胚层或跨系分化的能力,在长期体外培养过程中仍然保持多向分化的能力,同时遗传背景稳定,移植后排异反应较少等优点,并且脂肪干细胞具有取材方便、自体来源即可满足需要等优点,已成为近年来研究的热点。目前临床应用中获取人脂肪干细胞的方法,主要是将脂肪抽吸术后得到的脂肪混合液用Ⅰ型胶原酶消化法进行分离、培养,但在吸脂术肿胀麻醉液使用过程中,麻醉药及药物浓度对脂肪细胞及分离培养得到的脂肪干细胞的生物学特性是否存在负面影响,知之甚少,国内外也少见相关研究报道。大鼠的脂肪组织来源相对丰富,同时涉及的伦理问题及试验项目限制均较少,是良好的试验材料。本试验在现有基本成熟的人和动物体内提取脂肪组织并由此培养脂肪干细胞的操作基础上,在大鼠身上提取脂肪组织进而培养脂肪干细胞,并探寻不同浓度罗哌卡因对大鼠脂肪干细胞的增殖能力及成脂化的影响,对脂肪干细胞获取过程中麻醉方案的最佳方式提供基础研究依据,为临床上高效能获取脂肪干细胞,减少细胞损耗,提高脂肪干细胞的利用率,并利用其移植修复皮肤软组织缺损和在整形外科其他方面的应用提供理论依据。2材料与方法2.1设计:细胞学体外观察2.2材料:来源于4周龄大鼠双侧腹股沟脂肪组织2.3试验方法:脂肪干细胞的分离培养→麻醉肿胀液干预→CCK-8试剂盒检测脂肪干细胞增殖能力→成脂能力检测2.4观察指标(1)大鼠脂肪干细胞形态学特征(2)脂肪干细胞的增殖能力(3)细胞成脂分化率结果原代培养的细胞呈较大的类球形单个核细胞,消化传代后,细胞生长速度明显较原代细胞增快,2~3天即可覆盖约85%以上。CCK-8实验发现,25μmol/L、50μmol/L、100μmol/L的罗哌卡因处理脂肪干细胞24h,48h,72h后吸光度值明显下降,与对照组相比,差异有统计学意义。实验说明罗哌卡因处理可显著降低脂肪干细胞的增殖能力,在24h时,浓度越高,抑制干细胞增值能力越强,在48h和72h,浓度高低与抑制作用无明显差别。对照组脂肪干细胞成脂分化比例为(44.61±4.43)%,25μmol/L罗哌卡因处理组为(46.30±3.25)%,50μmol/L罗哌卡因处理组为(49.46±2.34)%,100μmol/L罗哌卡因处理组为(50.07±5.29)%,实验说明低浓度罗哌卡因处理后对脂肪干细胞的成脂化能力无明显影响,高浓度罗哌卡因处理后对脂肪干细胞的成脂化能力存在影响。当浓度上升至一定数值时,脂肪干细胞成脂化率不随浓度增加而改变,但仍需进一步试验证实。结论罗哌卡因处理可显著降低脂肪干细胞的增殖能力,在24h时,浓度越高,抑制干细胞增值能力越强,在48h和72h,浓度高低与抑制作用无明显差别。低浓度罗哌卡因处理后对脂肪干细胞的成脂化能力无明显影响,高浓度罗哌卡因处理后对脂肪干细胞的成脂化能力存在影响。当浓度上升至一定数值时,脂肪干细胞成脂化率不随浓度增加而改变,但仍需进一步试验证实。
[Abstract]:Objective to study the 1 that were cultured in the laboratory, human adipose derived stem cells (adipose-derived stem cells, ADSCs) and bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) have similar biological properties, which also has the powerful ability of self replication, cross germ layer or transdifferentiation, in the long term in vitro culture still retain the capacity for multilineage differentiation process, and stable genetic background, rejection of the advantages of less, and adipose stem cells have convenient, autologous can satisfy the needs and other advantages, has become a research hotspot in recent years. The current clinical application of human adipose derived stem cells access method, which is used primarily by type collagenase digestion fat mixture obtained after liposuction culture were isolated, but in the process of using liposuction tumescent fluid, drug and drug concentration on fat Whether or not the culture has negative impact on the biological characteristics of adipose derived stem cells and isolated, poorly understood, at home and abroad are seldom reported. Relatively abundant adipose tissue derived from rat, and relates to the project problems and test ethical limits are less, is a good test material in this experiment. The existing basic mature man and in vivo animal adipose tissue and adipose derived stem cells on the basis of operational training, in rats from the adipose tissue and cultured stem cells, and explore the proliferation capacity of different concentrations of ropivacaine on rat adipose derived stem cells and the effects of fat, and provide basic evidence for the best way of anesthesia scheme fat stem cells in the process of acquiring the clinical effective acquisition of adipose derived stem cells, reduce the cell loss, improve the utilization rate of adipose derived stem cells, and the transplantation for repairing skin and soft Provide a theoretical basis for the design of.2 materials and methods 2.1 tissue defect and other aspects of the application in plastic surgery: the cytology in vitro observation of 2.2 materials: from bilateral inguinal adipose tissue of 4 week old rats 2.3 test methods: culture, anesthetic liquid intervention CCK-8 kit, adipose stem cell proliferation and adipogenic ability to detect 2.4 observation the separation index of adipose derived stem cells (1) cell morphological characteristics of rat adipose derived stem (2) adipose derived stem cells (3) cell proliferation and adipogenic differentiation rate of the cultured cells were spherical single nuclear cells large, after digestion and passage, the cell growth rate was significantly faster than primary cells, 2~3 days covered above about 85%.CCK-8 experiment, 25 mol/L, 50 mol/L, 100 mol/L ropivacaine treatment of adipose derived stem cells 24h, 48h, 72h after the absorbance value decreased significantly, compared with the control group, there was statistically significant difference Yi. Experiment shows that ropivacaine treatment can significantly reduce the fat stem cell proliferation in the 24h, the higher the concentration, inhibition of stem cells proliferation is stronger in 48h and 72h, concentration and inhibition of no significant difference. Group of adipose derived stem cells into fat differentiation ratio as control (44.61 + 4.43)%, 25 mol/L ropivacaine treatment group (46.30 + 3.25)%, 50 mol/L ropivacaine group was (49.46 + 2.34)%, 100 mol/L ropivacaine group was (50.07 + 5.29)%. The experiment shows that low concentration of ropivacaine after treatment of adipogenic ability has no obvious effect on adipose derived stem cells, influence on fat the adipogenic ability of stem cells in the presence of high concentrations of ropivacaine after treatment. When the concentration increased to a certain value, and the change of adipose derived stem cells into fat rate with the increase of concentration, but still need to be further confirmed. Conclusion ropivacaine treatment can significantly reduce the fat stem cells The ability of proliferation in the 24h, the higher the concentration, inhibition of stem cells proliferation is stronger in 48h and 72h, concentration and inhibition of no significant difference. After treatment with low concentration of ropivacaine on adipogenic ability has no obvious effect on adipose derived stem cells, adipose stem cells effects on adipogenic ability of high concentration ropivacaine after treatment. When the concentration increased to a certain value, and the change of adipose derived stem cells into fat rate with the increase of concentration, but still need to be further confirmed.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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