五羟色胺对骨髓间充质干细胞增殖及其分泌IL-11及IL-6的影响

发布时间：2018-06-28 02:11

本文选题：五羟色胺 + 骨髓间充质干细胞　；参考：《汕头大学》2007年硕士论文

【摘要】： 背景与目的：血小板减少症是大剂量化疗、放疗以及造血干细胞移植的主要副作用和并发症，是导致病人死亡的重要原因之一。目前主要通过输注血小板进行治疗，但是反复输注血小板可诱生血小板抗体，导致血小板输注无效。近年来小板生成素(thrombopoietin TPO)、重组人白介素-11(recombinant human interleukin-11 rhIL-11)相继开发应用于临床，使血小板减少症的治疗取得一定的效果。但是，进一步的临床研究发现TPO能够诱导机体产生抗TPO抗体，激活血小板形成血栓等不良反应，并且其临床效果也不及先前的预料，这也就限制了TPO在临床上的应用。此外，rhIL-11也存在起效慢、疗效不佳、费用昂贵等缺点。血小板生成是在巨核细胞增殖分化的基础上进行的，因此，寻找一种物质代替或是辅助促血小板生成因子，促进巨核系造血，以快速增加循环中的血小板数量，就显得非常重要。我们可以借助某种物质，从其促进巨核细胞增殖分化入手，明确该物质促进巨核细胞增殖分化的特点及机制，了解巨核系造血的调控网络特点，为研究新药物提供新的思路。这种作为研究媒介的物质必须是可以直接促进巨核细胞分化增殖，或是通过影响骨髓造血微环境中的间充质干细胞而间接促进巨核系造血，或是既可以直接促进巨核系造血，又可同时影响骨髓微环境中的间充质干细胞而间接促进巨核细胞分化。本研究组在前期工作中，已经证实了五羟色胺(5-Hydroxtryptamine／seroto 5-HT)有促进巨核细胞增殖和分化功能，同时，5-HT也有显著的抗巨核细胞凋亡作用。这说明5-HT具有直接促进巨核系造血的作用，那么，5-HT是否也存在影响骨髓间充质干细胞从而发挥间接促进巨核细胞增殖分化的作用? 本实验拟通过观察5-HT对体外培养骨髓间充质干细胞的促增殖作用，及对其分泌分泌细胞因子IL—11、IL—6的影响，探讨5-HT能否促进骨髓间充质干细胞增殖，并同时增进IL-11、IL-6的分泌，从而间接促进巨核细胞增殖。材料和方法：取3—5周龄BABL／c小鼠的股骨和胫骨骨髓悬液，应用Percoll分离液分离出骨髓间充质干细胞，体外有血清培养P0及P1，P2代转入无血清培养，培养3天后进行实验。将细胞分为5—HT100nmol／L组、200nmol／L组、400nmol／L组和5—HT 0nmol／L的对照组共4组。采用倒置显微镜观察不同浓度5-HT作用下0小时、6小时、12小时、24小时及48小时等不同时间段巨核细胞的增殖情况，采用激光共聚焦显微镜研究不同浓度5-HT作用下和对照组在5个不同时间段时细胞胞浆IL-11、IL-6量的变化，以及通过应用ELISA方法检测相对应时间段细胞培养上清液中上述两种细胞因子量的变化情况。结果：各5-HT浓度组与对照组比较显示其对骨髓间充质干细胞均具有促进增殖作用，其中200nmoml／L时的促增殖作用最强(P＜0.01)，并以24小时时间段时作用效果最明显。激光共聚焦显微镜检测不同浓度组不同时间段细胞浆内IL-11、IL-6的量发现，200nmol／L浓度组在6小时、12小时、24小时、48小时时与对照组同时间段比较具有显著性统计学意义(P＜0.01)，400nmol／L组各时间段IL-11、IL-6的浓度均比200nmol／L组下降，，但无统计学意义(P＞0.05)，100nmol／l组和对照组各时间段比较有统计学意义(P＜0.05)。对照组各时间段比较差异无统计学意义(P＞0.05)。各5-HT浓度组在6小时及12小时时间段时与0小时比较有统计学意义，24小时与0小时比较有显著性差异，48小时与24小时比较差异无统计学意义。ELISA的结果与激光共聚焦结果相类似，验证了激光共聚焦显微镜的检测结果。结论：本研究结果提示5-HT有促进骨髓间充质干细胞增殖作用，对其分泌IL-11、IL-6也有促进作用。
[Abstract]:Background and purpose: thrombocytopenia is one of the major side effects and complications of large dose chemotherapy, radiotherapy and hematopoietic stem cell transplantation. It is one of the important causes of death in patients. At present, platelet transfusion is mainly used in the treatment of platelet transfusion, but repeated infusion of platelets can induce blood platelets ineffective. In recent years, platelet transfusion is not effective. Thrombopoietin TPO and recombinant human interleukins -11 (recombinant human interleukin-11 rhIL-11) have been developed and applied to the clinic to make a certain effect on the treatment of thrombocytopenia. However, further clinical studies have found that TPO can induce the body to produce anti TPO antibodies and activate platelets to form thrombus and other adverse reactions. And its clinical effect is less than previously expected, which also restricts the clinical application of TPO. In addition, rhIL-11 also has the disadvantages of slow effect, poor curative effect, expensive cost and so on.
The formation of platelets is based on the proliferation and differentiation of megakaryocytes. Therefore, it is very important to find a substance instead of or assist in promoting the hematopoiesis of the megakaryocyte to rapidly increase the number of platelets in the megakaryocyte, and it is very important to use certain substances to promote the proliferation and differentiation of megakaryocytes. It is true that the substance promotes the proliferation and differentiation of megakaryocyte, and understands the characteristics of the regulation network of megakaryocyte hematopoiesis to provide new ideas for the study of new drugs. This substance, as a medium, must directly promote the differentiation and proliferation of megakaryocytes, or indirectly through the influence of mesenchymal stem cells in the hematopoietic microenvironment of the bone marrow. Promoting megakaryocyte hematopoiesis, either directly promotes megakaryocyte hematopoiesis, but simultaneously affects mesenchymal stem cells in bone marrow microenvironment and indirectly promotes megakaryocyte differentiation.
In our previous work, five serotonin (5-Hydroxtryptamine / seroto 5-HT) has been proved to promote the proliferation and differentiation of megakaryocyte, and 5-HT also has a significant anti megakaryocyte apoptosis effect. This indicates that 5-HT has the effect of directly promoting megakaryocyte hematopoiesis, and whether 5-HT also affects bone marrow mesenchymal stem cells. Cells play an indirect role in promoting proliferation and differentiation of megakaryocytes.
This experiment is to observe the effect of 5-HT on the proliferation of bone marrow mesenchymal stem cells in vitro, and the effect on the secretion of cytokines IL - 11, IL - 6, and to explore whether 5-HT can promote the proliferation of mesenchymal stem cells and enhance the secretion of IL-11 and IL-6, thus indirectly promoting the proliferation of megakaryocytes.
Materials and methods: bone marrow suspension of femur and tibia in BABL / c mice of 3 to 5 weeks of age was taken and bone marrow mesenchymal stem cells were separated by Percoll separation solution. In vitro, P0 and P1 were cultured with serum, and P2 generation was transferred into serum-free culture for 3 days. The cells were divided into 5 HT100nmol / L group, 200nmol / L group, 400nmol / L group and 5 In the control group, 4 groups were used to observe the proliferation of megakaryocytes at different time periods, such as 0 hours, 6 hours, 12 hours, 24 hours and 48 hours at different concentrations of 5-HT, using laser confocal microscopy to study the changes of IL-11 and IL-6 in the cytoplasm of the cells under the action of different concentrations of 5-HT and the control group at 5 different time periods. And the changes of the above two cytokines in the cell culture supernatant were detected by ELISA.
Results: the 5-HT concentration group compared with the control group showed that it could promote the proliferation of bone marrow mesenchymal stem cells, and the proliferation promoting effect of 200nmoml / L was the strongest (P < 0.01), and the effect was most obvious at 24 hours period. The amount of IL-11 and IL-6 in different concentration groups of different time segments was detected by confocal laser confocal microscope. It was found that the 200nmol / L concentration group had significant statistical significance compared with the control group at 6 hours, 12 hours, 24 hours and 48 hours (P < 0.01). The concentration of IL-6 in 400nmol / L group was lower than that of 200nmol / L group, but there was no statistical significance (P > 0.05). 100nmol / L and control groups were statistically significant. Significance (P < 0.05). There was no significant difference in the time interval of the control group (P > 0.05). The 5-HT concentration group was statistically significant compared with 0 hours at 6 hours and 12 hours, 24 hours and 0 hours compared with 0 hours, and 48 hours and 24 hours compared with the results of.ELISA and laser confocal results. Similarly, the results of confocal laser scanning microscopy were verified.
Conclusion: the results suggest that 5-HT can promote the proliferation of bone marrow mesenchymal stem cells and promote the secretion of IL-11 and IL-6.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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