低强度脉冲超声促进骨髓来源细胞增殖的研究

发布时间：2018-01-29 11:08

本文关键词： 低强度脉冲超声骨髓间充质干细胞骨髓有核细胞细胞增殖　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：骨髓间充质干细胞（Bone Mesenchymal Stem Cells, BMSCs）和骨髓有核细胞均来源于骨髓，对医学的发展起着非常重要的作用，现已成为医学领域研究热点。骨髓间充质干细胞作为骨髓来源细胞的一种，具有取材方便，体外易培养，增殖快等优点，被认为是组织工程学中修复和替换病损组织和器官、以及功能重建理想的种子细胞。但是目前种子细胞数量不足，体外常规扩增无法满足临床研究及组织工程学产业化需求，这极大地制约其发展。骨髓有核细胞作为骨髓中的主要成份，对人体造血、免疫等有重要作用，当骨髓有核细胞减少或者增生低下时，会引起严重的血液、免疫系统疾病。由此可见骨髓来源细胞不足或者增生低下时，对人体多个组织器官系统会产生极大的影响。目前很多学者已经发现了一些促进骨髓来源细胞增殖的方法和药物，但都存在不完善之处。因此，寻求一种更安全、更有效的技术手段以获得足够数量、具有再生活力的骨髓来源细胞已成为当前医学领域研究中急需解决的关键问题。近年来，低强度脉冲超声（Low-Intensity Pulsed Ultrasound, LIPUS）作为一种安全的纯“绿色”物理技术手段，具有操作安全简便、费用低廉等特点。目前，已应用于临床骨与软组织创伤的愈合，使用安全，已经取得显著效果。前期实验初步探究了LIPUS促进BMSCs增殖的可行性。由此，本课题在前期实验的基础上继续深入研究，并着眼于骨髓，将LIPUS分别作用于离体BMSCs和活体兔骨髓有核细胞，进一步探索能否找到扩增骨髓来源细胞的新途径。目的 1.观察不同参数的低强度脉冲超声对体外分离培养的BMSCs增殖的影响，筛选能提高BMSCs增殖效率的适宜的低强度脉冲超声参数。 2.进一步验证适宜参数LIPUS对体外分离培养BMSCs增殖效应的影响，探讨LIPUS促进BMSCs增殖的有效性和安全性。 3.观察LIPUS对活体兔骨髓来源有核细胞增殖效应的影响，初步探讨LIPUS在促进骨髓有核细胞增殖方面的作用。方法 1.为筛选后续研究适宜的LIPUS参数，实验选用频率为0.6MHz，强度为0.03、0.1、0.2、0.3W/cm2的LIPUS分别辐照体外培养的第三代兔骨髓间充质干细胞5、10、15、20、30min，各参数组辐照一次，24h后倒差显微镜观察细胞生长情况，四甲基偶氮唑蓝（MTT）法和流式细胞仪检测LIPUS辐照后细胞增殖活性和细胞周期。 2.将体外分离培养的第三代BMSCs随机分为2组：LIPUS辐照组和对照组。采用前期实验筛选出的适宜参数0.2W/cm2，10min进行辐照，连续辐照8d。辐照后每天镜下观察细胞生长情况、四甲基偶氮唑蓝法检测细胞增殖活性；流式细胞仪检测各组细胞周期及增殖指数（PI）。 3.将120只新西兰大白兔随机分为LIPUS组、自身对照组和正常组。LIPUS连续辐照5d后，分别于辐照前，辐照5d后⑴每天观察兔的生长情况；⑵第6d、8d、10d、12d进行骨髓有核细胞计数、观察骨髓增生程度变化；⑶辐照处进行皮肤、肌肉组织病理切片。结果 1．各参数组经过LIPUS辐照后24h观察细胞分裂增殖均较空白对照组明显活跃，细胞集落更为密集。细胞增殖活性和细胞增殖指数均明显高于空白对照组（P0.05），其中以0.2W/cm2,10min辐照后促进增殖效果最为显著。 2. LIPUS能够明显促进BMSCs增殖，LIPUS组于辐照后各天细胞分裂增殖活性较对照组明显活跃，细胞集落更为密集；MTT法检测LIPUS组于辐照前、辐照后1d、3d、5d、7d的细胞增殖活性均明显高于对照组（P0.05)；流式细胞仪检测细胞增殖指数提示LIPUS组于辐照后各天均明显高于对照组（P0.05）。 3.辐照5d后每天观察兔的生活习惯与正常组比较，未发现明显异常。LIPUS组辐照5d后，6d、8d的骨髓增生程度、骨髓有核细胞数明显高于自身对照组和正常组（P0.05）。自身对照组骨髓有核细胞数与正常组比较，没有统计学差异（P0.05）。辐照处的皮肤和肌肉组织病理切片：LIPUS辐照后皮肤表皮及真皮层均未见异常，肌肉纹理清晰，染色较浅，其内均未见炎性细胞。结论 1. LIPUS对体外分离培养的BMSCs增殖具有明显的促进作用。 2. LIPUS辐照一定时间后能够促进BMSCs增殖，其中强度为0.2W/cm2，辐照10min剂量促进增殖效率最佳。 3. LIPUS对活体兔骨髓来源有核细胞的增殖具有促进作用。并且可能是一种较为安全有效的技术方法。
[Abstract]:Bone Mesenchymal Stem Cells (BMSCs) and bone marrow nucleated cells are all derived from bone marrow. They play a very important role in the development of medicine, and have become a research focus in the field of medicine.
Bone marrow mesenchymal stem cells as a source of bone marrow cells, it is a convenient, easily cultured in vitro, the advantages of rapid proliferation, is considered to be the replacement and repair of damaged tissues and organs in tissue engineering, seed cells and functional reconstruction of the ideal. But at present insufficient number of seed cells, in vitro amplification can not meet the clinical research and tissue engineering industrialization needs, which greatly restricts its development. As the main component of bone marrow mononuclear cells in the bone marrow, on human hematopoiesis, immunity plays an important role, when the bone marrow nucleated cells to reduce or increase students' low, will cause serious blood diseases of the immune system.
This lack of bone marrow cells or low proliferation, multiple organ system will greatly influence. Many scholars have found some methods to promote the proliferation of bone marrow cells and drugs, but are not perfect. Therefore, to seek a safer and more effective techniques to obtain a sufficient number of bone marrow derived cells, with regeneration has become a key problem needs to be solved urgently in medical research.
In recent years, the low intensity pulsed ultrasound (Low-Intensity Pulsed, Ultrasound, LIPUS) as a kind of pure "green" physical security technology, has the characteristics of simple and safe operation, low cost and so on. At present, has been applied to clinical healing of bone and soft tissue trauma, safe use, have achieved remarkable results. The preliminary inquiry the feasibility of LIPUS to promote the proliferation of BMSCs. Therefore, this topic further research based on the previous experiment, and focus on the bone marrow, LIPUS were exposed to BMSCs in vitro and in vivo rabbit bone marrow nucleated cells, further exploration can find a new way of expansion of bone marrow cells.
objective
1., we observed the effect of low intensity pulsed ultrasound with different parameters on the proliferation of BMSCs in vitro, and screened suitable low intensity pulsed ultrasound parameters that could increase BMSCs proliferation efficiency.
2. to further verify the effect of the suitable parameter LIPUS on the proliferation of BMSCs in vitro, and to explore the effectiveness and safety of LIPUS to promote the proliferation of BMSCs.
3. the effect of LIPUS on the proliferation of nucleated cells from bone marrow of living rabbits was observed, and the effect of LIPUS on the proliferation of bone marrow nucleated cells was preliminarily investigated.
Method
The 1. parameter selection for LIPUS follow-up study for the frequency selection experiment for 0.6MHz, intensity of 0.03,0.1,0.2,0.3W/cm2 LIPUS respectively in vitro irradiation of the third generation of rabbit bone marrow mesenchymal stem cells 5,10,15,20,30min, each array irradiation time, 24h inverted contrast microscope to observe cell growth, four methyl thiazolyl tetrazolium (MTT) method flow cytometry and LIPUS after irradiation, cell proliferation and cell cycle.
2. the third generation of BMSCs cultured in vitro were randomly divided into 2 groups: LIPUS groups and control groups. The optimal parameters of 0.2W/cm2,10min irradiation experiment screened by continuous irradiation after irradiation with 8D. every day under the microscope to observe the cell growth, cell proliferation activity of four methyl thiazolyl tetrazolium assay; cell cycle and proliferation index of flow cytometry (PI).
3. 120 New Zealand white rabbits were randomly divided into LIPUS group, control group and normal group.LIPUS 5D after continuous irradiation, respectively before irradiation after irradiation was observed every day after 5D growth of rabbits; the 6D, 8D, 10d, 12D, bone marrow nucleated cell count, observe the change of the degree of bone marrow hyperplasia; irradiation skin, muscle tissue pathological sections.
Result
1. the parameter array after LIPUS irradiation on 24h cell proliferation were significantly more active than the control group, cell colony was more intensive. The index of cell proliferation activity and cell proliferation were significantly higher than control group (P0.05), of which 0.2W/cm2,10min after irradiation can promote the proliferation of the most significant effect.
2. LIPUS can significantly promote the proliferation of BMSCs in group LIPUS after irradiation the cell proliferation activity compared to the control group was active, the colony was more intensive; MTT assay in LIPUS group before irradiation, irradiation after 1D, 3D, 5D, cell proliferation activity of 7D were significantly higher than the control group (P0.05); cell the proliferation index of flow cytometry showed that LIPUS group in the days after irradiation were significantly higher than the control group (P0.05).
The 3. day after irradiation 5D habits compared with the normal group of rabbits, no obvious abnormality in group.LIPUS irradiation after the discovery of 5D, 6D, 8D and the degree of bone marrow hyperplasia, bone marrow nucleated cell number was significantly higher than that of the control group and normal group (P0.05). The control group the number of bone marrow cells and normal groups. No statistically significant difference (P0.05). The skin and muscle tissue pathological sections at LIPUS after irradiation irradiation: skin epidermis and dermis showed no abnormality of muscle, texture clear, lighter staining, in which there was no inflammatory cell.
conclusion
1. LIPUS has an obvious promoting effect on the proliferation of BMSCs in vitro.
2. LIPUS can promote the proliferation of BMSCs after a certain time, of which the intensity is 0.2W/cm2, and the dose of irradiated 10min is the best to promote the proliferation efficiency.
3. LIPUS can promote the proliferation of nucleated cells from the bone marrow of living rabbits, and may be a safer and more effective technique.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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