【摘要】：目的： 探討五羥色胺(serotonin,5-HT)對(duì)人骨髓間充質(zhì)干細(xì)胞(Bone Mesenchymal Stem Cells,BMSCs)體外增殖的影響。 方法： 從捐贈(zèng)者髂骨穿刺抽取督髓細(xì)胞,采用密度梯度離心和貼壁培養(yǎng)法進(jìn)行培養(yǎng)和純化BMSCs,取生長(zhǎng)良好的P3或P4代BMSCs,通過(guò)用含不同濃度5-HT的條件培養(yǎng)基分組對(duì)BMSCs進(jìn)行干預(yù)。光學(xué)顯微鏡下觀察細(xì)胞24、48及72小時(shí)的形態(tài)變化；MTS檢測(cè)評(píng)估細(xì)胞增殖情況；流式細(xì)胞儀(FCM)檢測(cè)BMSCs凋亡率。 結(jié)果： 1、細(xì)胞形態(tài)學(xué)觀察：剛接種細(xì)胞時(shí),BMSCs呈圓形,胞體透亮,折光性好。常規(guī)培養(yǎng)24、48、72小時(shí)后,BMSCs開(kāi)始貼壁,細(xì)胞分布均勻,輪廓清楚,形態(tài)均-,呈多突狀或長(zhǎng)梭狀,細(xì)胞增殖速度加快并逐漸相互融合。加入5-HT培養(yǎng)24、48、72小時(shí)后,觀察細(xì)胞形態(tài)逐漸發(fā)生改變,細(xì)胞變圓、皺縮,與鄰近細(xì)胞分離；核染色質(zhì)固縮集聚、呈塊或碎裂狀改變；胞質(zhì)濃縮,中心透光度降低。2、MTS法檢測(cè)：在不同5-HT(0.01,0.1,1,10,100,500,1000μM)濃度作用下,在血清濃度分別為0%和10%時(shí),與對(duì)照組相比,酶標(biāo)儀檢測(cè)OD值均有所降低。檢測(cè)結(jié)果顯示：在24、48小時(shí)時(shí),5-HT(0.01,0.1,1,10,100μM)濃度對(duì)BMSCs的作用與對(duì)照組比較P0.05,5-HT濃度在500μM和1000μM時(shí),對(duì)BMSCs的作用與對(duì)照組比較P0.05；在72小時(shí)時(shí),5-HT(0.01,0.1,1,10μM)濃度對(duì)BMSCs的作用與對(duì)照組比較P0.05,5-HT濃度在100μM、500μM和1000μM時(shí),對(duì)BMSCs的作用與對(duì)照組比較P0.05。檢測(cè)結(jié)果顯示隨著觀察時(shí)間延長(zhǎng),5-HT濃度增加,細(xì)胞存活率逐漸降低。在無(wú)血清情況下,存活率最低可達(dá)59.5%(P0.05)。3、流式細(xì)胞儀檢測(cè)：BMSCs在各組5-HT(1,10,100,1000μM)藥物濃度作用48小時(shí)后,與對(duì)照組相比,檢測(cè)結(jié)果顯示5-HT濃度在10μM、100μM和1000μM時(shí),隨著濃度增加,細(xì)胞凋亡率升高(P0.05),在5-HT為1000μM時(shí),凋亡率為21.42%。 結(jié)論： 5-HT對(duì)體外培養(yǎng)的人BMSCs的增殖具有凋亡作用,無(wú)論在有血清或無(wú)血清情況下,5-HT作用時(shí)間越長(zhǎng),細(xì)胞存活率越低；5-HT藥物濃度越高,細(xì)胞凋亡率越大；且在無(wú)血清這種低營(yíng)養(yǎng)環(huán)境下,5-HT后期的凋亡作用愈明顯。
[Abstract]:Aim: to investigate the effect of serotonin (serotonin,5-HT) on the proliferation of human bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells,BMSCs) in vitro. Methods: medullary cells were extracted from donor iliac bone puncture and cultured with density gradient centrifugation and adherent culture. The BMSCs, of P3 or P4 passages were obtained from BMSCs, by using the method of density gradient centrifugation and adherent culture. BMSCs was treated with conditioned medium containing different concentrations of 5-HT. The morphological changes of the cells at 48 and 72 hours were observed under optical microscope, the proliferation of cells was evaluated by MTS assay, and the apoptosis rate of BMSCs was detected by flow cytometry (FCM). Results: 1. Observation of cell morphology: when the cells were first inoculated, the BMSCs was round, the cell body was bright, and the refraction was good. After 72 hours of conventional culture, BMSCs began to adhere to the wall. The cells were evenly distributed, with clear outline, uniform shape, multi-protruding or long fusiform, and the proliferation rate of the cells was accelerated and gradually fused with each other. After being cultured with 5-HT for 72 hours, the morphology of the cells was gradually changed, the cells became round, shrinked and separated from the adjacent cells, the chromatin was pyknosis and accumulated, and the nuclear chromatin was changed in the shape of block or fragmentation. The concentration of cytoplasm and the decrease of central transmittance. 2Detection by 5-HT: under the action of different concentration of 5-HT (0.01 ~ (0.1) ~ (0.1) ~ (10 ~ (10) ~ (100) ~ (500) 渭 M, when the serum concentration was 0% and 10 ~ (5) 渭 M, compared with the control group, the value of OD detected by enzymatic marker was lower than that of the control group. The results showed that the effect of 5-HT on BMSCs was significantly higher than that of the control group (P 0.05 渭 M and 1000 渭 M), and the effect of BMSCs was higher than that of the control group (P 0.05 渭 M and P 0.05 渭 M) at 24 h for 48 h. The effect of 5-HT (0.01g / ml) on BMSCs was higher than that of the control group (P 0.05 渭 M and 1000 渭 M). At 72 hours, the effect of 5-HT (0.01 渭 m) on BMSCs was higher than that of control group (100 渭 M, 1000 渭 M), and that of BMSCs was higher than that of control group (P 0.05). The results showed that the concentration of 5-HT increased and the cell survival rate decreased with the increase of observation time. In serum-free condition, the lowest survival rate was 59.5% (P0.05). Flow cytometry showed that the concentration of BMSCs was 10 渭 m after 48 hours of exposure to the drug concentration of 5-HT (100 渭 M). Compared with the control group, the results showed that the concentration of 5-HT was 10 渭 m. When the concentration of 5-HT was 1000 渭 M, the apoptotic rate was 21.42 渭 M, and the apoptosis rate increased with the increase of the concentration of 100 渭 M and 1000 渭 M (P0.05). Conclusion: 5-HT has apoptotic effect on the proliferation of human BMSCs in vitro. The longer the 5-HT is, the lower the cell survival rate is, and the higher the concentration of 5-HT is, the higher the apoptosis rate is. Moreover, in the low nutrition environment of serum-free, the effect of apoptosis in the late stage of 5-HT was more obvious.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 沈亞莉;徐酉華;羅慶;郭振華;鄭改煥;郭玉霞;;wnt5a基因修飾bMSCs對(duì)HL60細(xì)胞生長(zhǎng)分化影響的研究[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年06期

2 李小川,李雷,王歡,崔少千,白倫浩;Fas/FasL基因在退變腰椎間盤(pán)組織中的表達(dá)及誘導(dǎo)凋亡作用[J];中華醫(yī)學(xué)雜志;2005年24期

3 趙梓汝;吳小濤;祁亞斌;付文芹;王剛;;TGF-β1干預(yù)下體內(nèi)兔骨髓間充質(zhì)干細(xì)胞對(duì)退變椎間盤(pán)治療的實(shí)驗(yàn)研究[J];中國(guó)矯形外科雜志;2006年13期

4 易豈建,楊默,龐雅軒,李桂霞,夏文杰,霍泰輝,宋銀子;五羥色胺促進(jìn)骨髓基質(zhì)細(xì)胞生長(zhǎng)初探[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2005年02期

5 張琦;錢(qián)濟(jì)先;文艷華;裘秀春;馬保安;范清宇;;SOX9基因真核表達(dá)載體轉(zhuǎn)染誘導(dǎo)BMSCs分化為類(lèi)髓核細(xì)胞[J];中國(guó)修復(fù)重建外科雜志;2010年07期

，

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