兔骨髓间充质干细胞体外扩增培养影响因素的实验研究

发布时间：2018-02-04 22:39

本文关键词： 骨髓间充质干细胞细胞扩增组织工程　出处：《广西医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:通过研究兔骨髓间充质干细胞体外扩增培养的各种条件及比较各种影响因素的差异性,探索一种简单易行、细胞扩增效率高的方法,为干细胞应用于创伤修复及组织器官再造提供必要的前提帮助。方法:1.从新西兰白兔(月龄＜3月)股骨转子间区严格无菌条件下负压抽取骨髓穿刺液5-10ml,经密度梯度离心并贴壁法或全骨髓分离法分离提纯骨髓间充质干细胞,37℃、5%CO_2培养箱中卵育。测定两种方法培养的原代细胞的细胞克隆集落数、细胞生长时间和16d细胞消化数量。2.选取月龄不同的新西兰白兔分组并在无菌条件下股骨转子间区负压抽取骨髓穿刺液5-10ml,经密度梯度离心并贴壁法分离提纯骨髓间充质干细胞,LG-DMEM(10%FBS)培养基中进行原代、传代培养。分别检测不同月龄组的骨髓间充质干细胞的原代及传代细胞的生长活性。3.选取月龄＜3月的新西兰白兔,密度梯度离心并贴壁法体外分离骨髓间充质干细胞,LG-DMEM(10%FBS)培养基培养。待细胞传代后,用LG-DMEM(10%FBS)、LG-DMEM(15%FBS)、LG-DMEM(20%FBS)、HG-DMEM(10%FBS)四种培养基重悬细胞,测定不同培养基培养细胞的扩增倍数、集落形成率、生长速度和观察细胞大体形态。4.选取生长良好的P4代细胞(动物月龄＜3月,密度梯度离心并贴壁法分离,LG-DMEM(15%FBS)培养基体外培养)条件培养基成骨诱导,显微镜下观察细胞形态变化和进行茜素红染色。结果:1.通过密度梯度离心并贴壁法分离纯化的骨髓间充质干细胞在LG-DMEM(10%FBS)培养基中生长良好。对照组中相同培养条件下由全骨髓分离法培养的骨髓间充质干细胞的培养成功率明显比密度梯度离心纯化组低。2.不同月龄组中兔骨髓间充质干细胞增殖速度不同,月龄＜3月龄的兔骨髓间充质干细胞增殖最好,细胞生长活性明显优于其它两组。3.LG-DMEM(10%FBS)、LG-DMEM(15%FBS)、LG-DMEM(20%FBS)、HG-DMEM(10%FBS)四种培养基中,LG-DMEM(15%FBS)组中细胞扩增倍数为16.20±1.60倍,第2—3天进入对数生长期,平均细胞集落形成率为6.11±1.17%。比较LG—DMEM(10%FBS)组和LG—DMEM(20%FBS)组中的细胞扩增倍数、平均细胞集落形成率和细胞生长速度,差异性不显著。细胞增殖随着FBS浓度的增加,形态发生了变化。在LG—DMEM(20%FBS)组中的细胞后期增殖过程中出现明显的老化现象。HG—DMEM(10%FBS)中细胞生长扩增倍数较其他3组小,平均11.40±2.07倍,平均细胞集落形成率2.67±1.00%,细胞增殖速度低于其他3组。4.P4兔骨髓间充质干细胞经过成骨诱导18d,硒红染色阳性,钙结节形成。结论:1.相同培养条件下密度梯度离心并贴壁法培养的兔骨髓间充质干细胞增殖活性明显优于全骨髓培养法,成功率高。2.月龄＜3月的兔骨髓中含有更多的间充质干细胞,且生物活性和增殖特性明显优于老龄组。3.LG—DMEM(15%FBS)培养基较其他三种培养基更适合骨髓间充质干细胞体外扩增培养,且更好的保持了细胞的正常形态和生物活性。
[Abstract]:Objective: to study the conditions of rabbit bone marrow mesenchymal stem cells (BMSCs) cultured in vitro and compare the differences of various influencing factors to explore a simple and efficient method for the expansion of rabbit bone marrow mesenchymal stem cells (BMSCs). To provide the necessary prerequisite for the application of stem cells in wound repair and tissue and organ reconstruction. Methods 1. Bone marrow puncture fluid (5-10ml) was extracted from New Zealand white rabbit (age < March) under strict aseptic condition of femoral intertrochanteric region. Bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent method or whole bone marrow separation method. Cell growth time and 16d cell digestibility. 2. New Zealand white rabbits of different ages were divided into groups and bone marrow puncture fluid 5-10ml was extracted under negative pressure of intertrochanteric region of femur under aseptic condition. Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified from LG-DMEM (10S) medium by density gradient centrifugation and adherent method. The growth activity of bone marrow mesenchymal stem cells (BMSCs) in different age groups was detected. 3. New Zealand white rabbits with the age of less than March were selected. Bone marrow mesenchymal stem cells were isolated from LG-DMEM (10S) medium by density gradient centrifugation and adherent method in vitro. LG-DMEM (15s / LG-DMEM (20S) HG-DMEM (10S) medium) was cultured in four kinds of medium, and the expansion times of the cultured cells in different culture medium were measured. Colony formation rate, growth rate and observed cell morphology. 4. P4 passage cells with good growth (animal age < March, density gradient centrifugation and adherent method) were selected. LG-DMEM (15s) medium was used to induce osteogenesis. Morphological changes of cells and alizarin red staining were observed under microscope. Results 1. Bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent method in LG-DMEM (10S). The success rate of bone marrow mesenchymal stem cells cultured by the whole bone marrow separation method in the control group was significantly lower than that in the density gradient centrifugation group. The proliferation rate of MSCs was different. The proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs) under 3 months old was the best, and the cell growth activity was obviously superior to that of the other two groups. 3. LG-DMEM (10S / LG-DMEM (15S)). The expansion times of LG-DMEM (15S) in LG-DMEM (10S) medium were 16.20 卤1.60 times. The logarithmic growth period entered into 2-3 days. The average colony-forming rate was 6.11 卤1.17.The cell expansion times in LG-DMEM10S group and LG-DMEM20S group were compared between LG-DMEM (10S) group and LG-DMEM (20S) group. The average cell colony formation rate and cell growth rate were not significantly different. Cell proliferation increased with the concentration of FBS. In LG-DMEM (20S) group, there were obvious aging phenomena during anaphase proliferation of LG-DMEM (20S). HG-DMEM (10S). Compared with the other three groups, the growth and amplification times of the medium cell were smaller. The average cell colony formation rate was 2.67 卤1.00 and the average cell colony formation rate was 11.40 卤2.07 times. The cell proliferation rate was lower than that of the other three groups. 4. P4 rabbit bone marrow mesenchymal stem cells were induced by osteogenesis for 18 days. Selenium red staining was positive and calcium nodules were formed. Conclusion the proliferative activity of rabbit bone marrow mesenchymal stem cells cultured by density gradient centrifugation and adherent method under the same culture conditions is obviously superior to that of whole bone marrow culture. More mesenchymal stem cells were found in bone marrow of rabbits under March. And the biological activity and proliferation characteristics were significantly better than the aging group. 3. LG-DMEM (15S) medium was more suitable for bone marrow mesenchymal stem cell culture in vitro than the other three kinds of medium. And better maintain the normal morphology and biological activity of cells.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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