本文選題：五羥色胺 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《汕頭大學(xué)》2007年碩士論文

【摘要】： 背景與目的：血小板減少癥是大劑量化療、放療以及造血干細(xì)胞移植的主要副作用和并發(fā)癥，是導(dǎo)致病人死亡的重要原因之一。目前主要通過輸注血小板進(jìn)行治療，但是反復(fù)輸注血小板可誘生血小板抗體，導(dǎo)致血小板輸注無效。近年來小板生成素(thrombopoietin TPO)、重組人白介素-11(recombinant human interleukin-11 rhIL-11)相繼開發(fā)應(yīng)用于臨床，使血小板減少癥的治療取得一定的效果。但是，進(jìn)一步的臨床研究發(fā)現(xiàn)TPO能夠誘導(dǎo)機(jī)體產(chǎn)生抗TPO抗體，激活血小板形成血栓等不良反應(yīng)，并且其臨床效果也不及先前的預(yù)料，這也就限制了TPO在臨床上的應(yīng)用。此外，rhIL-11也存在起效慢、療效不佳、費(fèi)用昂貴等缺點。 血小板生成是在巨核細(xì)胞增殖分化的基礎(chǔ)上進(jìn)行的，因此，尋找一種物質(zhì)代替或是輔助促血小板生成因子，促進(jìn)巨核系造血，以快速增加循環(huán)中的血小板數(shù)量，就顯得非常重要。我們可以借助某種物質(zhì)，從其促進(jìn)巨核細(xì)胞增殖分化入手，明確該物質(zhì)促進(jìn)巨核細(xì)胞增殖分化的特點及機(jī)制，了解巨核系造血的調(diào)控網(wǎng)絡(luò)特點，為研究新藥物提供新的思路。這種作為研究媒介的物質(zhì)必須是可以直接促進(jìn)巨核細(xì)胞分化增殖，或是通過影響骨髓造血微環(huán)境中的間充質(zhì)干細(xì)胞而間接促進(jìn)巨核系造血，或是既可以直接促進(jìn)巨核系造血，又可同時影響骨髓微環(huán)境中的間充質(zhì)干細(xì)胞而間接促進(jìn)巨核細(xì)胞分化。 本研究組在前期工作中，已經(jīng)證實了五羥色胺(5-Hydroxtryptamine／seroto 5-HT)有促進(jìn)巨核細(xì)胞增殖和分化功能，同時，5-HT也有顯著的抗巨核細(xì)胞凋亡作用。這說明5-HT具有直接促進(jìn)巨核系造血的作用，那么，5-HT是否也存在影響骨髓間充質(zhì)干細(xì)胞從而發(fā)揮間接促進(jìn)巨核細(xì)胞增殖分化的作用? 本實驗擬通過觀察5-HT對體外培養(yǎng)骨髓間充質(zhì)干細(xì)胞的促增殖作用，及對其分泌分泌細(xì)胞因子IL—11、IL—6的影響，探討5-HT能否促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖，并同時增進(jìn)IL-11、IL-6的分泌，從而間接促進(jìn)巨核細(xì)胞增殖。 材料和方法：取3—5周齡BABL／c小鼠的股骨和脛骨骨髓懸液，應(yīng)用Percoll分離液分離出骨髓間充質(zhì)干細(xì)胞，體外有血清培養(yǎng)P0及P1，P2代轉(zhuǎn)入無血清培養(yǎng)，培養(yǎng)3天后進(jìn)行實驗。將細(xì)胞分為5—HT100nmol／L組、200nmol／L組、400nmol／L組和5—HT 0nmol／L的對照組共4組。采用倒置顯微鏡觀察不同濃度5-HT作用下0小時、6小時、12小時、24小時及48小時等不同時間段巨核細(xì)胞的增殖情況，采用激光共聚焦顯微鏡研究不同濃度5-HT作用下和對照組在5個不同時間段時細(xì)胞胞漿IL-11、IL-6量的變化，以及通過應(yīng)用ELISA方法檢測相對應(yīng)時間段細(xì)胞培養(yǎng)上清液中上述兩種細(xì)胞因子量的變化情況。 結(jié)果：各5-HT濃度組與對照組比較顯示其對骨髓間充質(zhì)干細(xì)胞均具有促進(jìn)增殖作用，其中200nmoml／L時的促增殖作用最強(qiáng)(P＜0.01)，并以24小時時間段時作用效果最明顯。激光共聚焦顯微鏡檢測不同濃度組不同時間段細(xì)胞漿內(nèi)IL-11、IL-6的量發(fā)現(xiàn)，200nmol／L濃度組在6小時、12小時、24小時、48小時時與對照組同時間段比較具有顯著性統(tǒng)計學(xué)意義(P＜0.01)，400nmol／L組各時間段IL-11、IL-6的濃度均比200nmol／L組下降，，但無統(tǒng)計學(xué)意義(P＞0.05)，100nmol／l組和對照組各時間段比較有統(tǒng)計學(xué)意義(P＜0.05)。對照組各時間段比較差異無統(tǒng)計學(xué)意義(P＞0.05)。各5-HT濃度組在6小時及12小時時間段時與0小時比較有統(tǒng)計學(xué)意義，24小時與0小時比較有顯著性差異，48小時與24小時比較差異無統(tǒng)計學(xué)意義。ELISA的結(jié)果與激光共聚焦結(jié)果相類似，驗證了激光共聚焦顯微鏡的檢測結(jié)果。 結(jié)論：本研究結(jié)果提示5-HT有促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖作用，對其分泌IL-11、IL-6也有促進(jìn)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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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相關(guān)期刊論文 前3條

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