本文關(guān)鍵詞：MSCs對輻射誘導(dǎo)胸腺瘤的抑制作用及其機(jī)制的探討　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胸腺淋巴瘤 MSCs 電離輻射 細(xì)胞移植 細(xì)胞周期 細(xì)胞增殖

【摘要】：隨著放療技術(shù)及設(shè)備的不斷進(jìn)步，腫瘤的治療效果日益提高，患者的五年生存率得到明顯提高，但其誘發(fā)第二惡性腫瘤的報道也逐年增多，包括白血病、直腸癌、膀胱癌等。胸腺作為機(jī)體主要的免疫器官，是對輻射敏感的組織之一，放射線照射誘發(fā)的胸腺淋巴瘤是一種高度浸潤甚至能夠轉(zhuǎn)移的腫瘤，預(yù)后較差。因此，尋找一種有效的預(yù)防和治療輻射誘導(dǎo)第二惡性腫瘤的方法對提高放療的治療效果具有實際意義。 骨髓間充質(zhì)干細(xì)胞（Bone Marrow Mesenchymal Stem cells，MSCs）是一類具有多向分化潛能、再生特性、免疫調(diào)節(jié)能力、靶向歸巢能力等作用的多能干細(xì)胞。有研究表明，MSCs對腦損傷、脊髓損傷、腎損傷、胰腺損傷以及皮膚損傷等具有組織修復(fù)作用。 本研究采用全骨髓貼壁法在體外分離培養(yǎng)MSCs，通過流式細(xì)胞儀檢測MSCs細(xì)胞周期及細(xì)胞表面標(biāo)志物來鑒定MSCs。選用健康幼年的C57BL/6J小鼠隨機(jī)分成正常組、輻射組、MSCs治療組。按照Kaplan經(jīng)典方法制備胸腺淋巴瘤動物模型，在照射后當(dāng)天及1周后對MSCs治療組小鼠經(jīng)尾靜脈輸注2×106個/ml的細(xì)胞懸液0.2ml。每日觀察各只小鼠的一般狀況，末次照射6個月后處死小鼠，無菌取胸腺。通過HE染色進(jìn)行病理組織學(xué)觀察，通過流式細(xì)胞儀檢測胸腺細(xì)胞及其各亞群細(xì)胞的細(xì)胞周期和細(xì)胞增殖動力學(xué)以及各亞群中CD3的表達(dá)和TCR Mf（TCR突變頻率），通過RT-PCR檢測CyclinD1上游以及下游的相關(guān)基因P15(INK4B)、P16(INK4A)、P18(INK4C)、P19(INK4D)和Rb的表達(dá)。 本研究結(jié)果顯示：用全骨髓貼壁法分離培養(yǎng)的MSCs生長狀態(tài)良好。流式細(xì)胞儀檢測結(jié)果表明大多數(shù)MSCs處于相對不活躍的靜止期G0/G1期，CD44的表達(dá)呈陽性，而不表達(dá)CD34，符合MSCs的特性。輻射組小鼠在照射完3個月后毛發(fā)無光澤，部分小鼠出現(xiàn)毛色灰白，蓬松逆立和精神較差等癥狀，MSCs治療組小鼠狀態(tài)明顯有所改善。組織病理學(xué)觀察結(jié)果顯示，正常組小鼠胸腺皮髓質(zhì)結(jié)構(gòu)清晰，淋巴細(xì)胞形態(tài)規(guī)則，，大小均一，呈圓形或橢圓形，輻射組小鼠胸腺結(jié)構(gòu)完全被破壞，淋巴樣腫瘤細(xì)胞彌漫分布，形狀不規(guī)則，大小不一，核大深染，出現(xiàn)鏡影細(xì)胞和滿天星現(xiàn)象，存在核分裂象，MSCs治療組小鼠胸腺結(jié)構(gòu)趨于正常。 胸腺細(xì)胞中CD4-CD8-亞群細(xì)胞比例在輻射組明顯高于正常組，MSCs治療組趨于正常組；輻射組中CD4+CD8+亞群細(xì)胞比例明顯低于正常組和MSCs治療組；MSCs治療組CD4+CD8-亞群細(xì)胞比例高于輻射組，接近于正常組；CD4-CD8+亞群細(xì)胞比例很低，接近于0，在三組中無明顯差異。 胸腺細(xì)胞和CD4-CD8-亞群細(xì)胞中，輻射組G1期細(xì)胞數(shù)明顯高于正常組和MSCs治療組，而輻射組S期細(xì)胞比例明顯低于正常組，MSCs治療組趨于正常水平；在CD4+CD8+和CD4+CD8-亞群細(xì)胞中，輻射組S期細(xì)胞數(shù)明顯高于正常組和MSCs治療組。 胸腺細(xì)胞、CD4+CD8+和CD4+CD8-亞群細(xì)胞中，正常組和MSCs治療組中parent、G2或G3、G4代細(xì)胞比例明顯高于輻射組，而G5、G6或G8、G9、G10代細(xì)胞以及增殖指數(shù)（PI）明顯低于輻射組；在胸腺CD4-CD8-亞群細(xì)胞中，輻射組parent、G2和G3代細(xì)胞比例明顯高于正常組和MSCs治療組，輻射組中G4、G5、G6代細(xì)胞比例以及PI明顯低于正常組和MSCs治療組。 正常組中CD4CD8各亞群細(xì)胞中CD3+細(xì)胞的表達(dá)明顯高于輻射組和MSCs治療組，MSCs移植后CD3+細(xì)胞的表達(dá)高于輻射組，但仍顯著低于正常組。其中CD3-細(xì)胞的表達(dá)與CD3+細(xì)胞的表達(dá)相反。輻射組和MSCs治療組中TCR突變頻率（TCR Mf）明顯高于正常組。 RT-PCR結(jié)果顯示，調(diào)控細(xì)胞周期蛋白CyclinD1上游基因P15(INK4B)、P16(INK4A)和P18(INK4C)在輻射組的mRNA表達(dá)高于正常組，MSCs移植后表達(dá)下調(diào)。與正常組相比，輻射組和MSCs治療組P19(INK4D)基因mRNA相對表達(dá)量明顯增加，和輻射組相比，MSCs治療組胸腺組織中P19(INK4D)的相對表達(dá)量也明顯增加。而Rb在輻射組的表達(dá)明顯低于正常組和MSCs治療組，差異有統(tǒng)計學(xué)差異。結(jié)論： MSCs移植能夠使胸腺淋巴瘤細(xì)胞周期阻滯緩解，對輻射誘導(dǎo)的胸腺淋巴瘤具有一定的抑制作用，其機(jī)制可能與MSCs下調(diào)CyclinD1上游相關(guān)基因P15(INK4B)、P16(INK4A)、P18(INK4C)，和上調(diào)CyclinD1上游相關(guān)基因P19(INK4D)和CyclinD1下游基因Rb的表達(dá)有關(guān)。
[Abstract]:With the continuous progress of technology and equipment of radiotherapy, the treatment of cancer is increasing, the five year survival of patients has been significantly improved, but the induced second malignant tumor report also increased year by year, including leukemia, cancer, bladder cancer and so on. As the main body of the immune organs thymus, is one of the radiation sensitive tissues, put radiation induced thymic lymphoma is a highly invasive and metastatic tumor, the prognosis is poor. Therefore, a method for effective prevention and treatment of radiation induced second malignant tumors has practical significance for improving the therapeutic effect of radiotherapy.
Bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Stem cells, MSCs) is a kind of differentiation, regeneration characteristics, immune regulation ability, homing ability of pluripotent stem cells. Studies have shown that MSCs of brain injury, spinal cord injury, renal injury, repair of pancreatic injury and skin tissue injury.
The study of adherent method in vitro MSCs were cultured by whole bone marrow by flow cytometry in MSCs cell cycle and cell surface markers to identify MSCs. healthy young C57BL/6J mice were randomly divided into normal group, radiation group, MSCs treatment group. According to the classic Kaplan method for preparation of thymic lymphoma animal model, the general situation in the irradiation the day after and 1 weeks after treatment of MSCs mice by tail vein infusion of 2 x 106 /ml cell suspension of 0.2ml. mice were observed daily for 6 months at the time of irradiation, the mice were killed after sterile thymus. Samples of tissue were stained by HE, the cell cycle and cell proliferation kinetics detection thymus cells and its subsets by flow cytometry and subsets in the expression of CD3 and TCR Mf (TCR mutation frequency), the related gene P15 RT-PCR detection CyclinD1 upstream and downstream (INK4B), P16 (INK4A) The expression of P18 (INK4C), P19 (INK4D) and Rb.
The results of this study show that: isolated and cultured by whole bone marrow adherent method MSCs growth in good condition. The results of flow cytometry showed that most of the MSCs in a relatively inactive quiescent G0/G1 phase, the expression of CD44 was positive, and the expression of CD34 in line with the characteristics of MSCs in mice after irradiation. The radiation group after 3 months hair dull, some mice appeared color grey, fluffy and poor mental symptoms such as inverse, MSCs treated mice significantly improved. Histopathological results showed that normal mice thymus cortex and medulla structure clear, lymphocyte morphology rules, uniform size, round or oval, radiation mice thymus structure completely destruction of lymphoid tumor cells were diffusely distributed, irregular shape, size, big nucleus anachromasis, appear to mirror cells and stars exist phenomenon, mitotic MSCs treated mice thymus structure tends to be positive Often.
Thymus cells of CD4-CD8- cell subsets in proportion in the radiation group was significantly higher than the normal group, MSCs treatment group, normal group; radiation group in CD4+CD8+ cell subsets was significantly lower than that of normal group and MSCs treatment group; MSCs treatment group, CD4+CD8- cell subsets proportion is higher than the radiation group, often close to the Yu Zheng group; CD4-CD8+ cell subsets proportion is very low that is close to 0, there is no obvious difference in the three groups.
Thymus cells and CD4-CD8- cell subsets, G1 cell number of radiation group was significantly higher than that in normal group and MSCs treatment group, radiation group and the percentage of cells in S phase was significantly lower than that in normal group, MSCs treatment group to the normal level; in CD4+CD8+ and CD4+CD8- cell subsets in the radiation group, the cell number of S phase was significantly higher than the normal group and the MSCs treatment group.
Thymocytes, CD4+CD8+ and CD4+CD8- cell subsets, parent normal group and MSCs treatment group, G2 or G3, G4 cell ratio was significantly higher than that of radiation group, and G5, G6 or G8, G9, G10 and cell proliferation index (PI) was significantly lower than that of the radiation group; in the thymus of CD4-CD8- cell subsets. Parent radiation group, on behalf of the proportion of cells in G2 and G3 were significantly higher than normal group and MSCs treatment group, radiation group in G4, G5, G6 and PI cells ratio was significantly lower than that in normal group and MSCs treatment group.
In the normal group the expression of CD3+ cell subsets of CD4CD8 cells was significantly higher than that in the radiation group and MSCs treatment group, MSCs after transplantation of CD3+ cells was higher than the radiation group, but still significantly lower than the normal group. The expression of CD3- cells and CD3+ cells. On the contrary the radiation group and MSCs treatment group in the frequency of TCR mutation (TCR Mf) was significantly higher than the normal group.
RT-PCR results showed that the regulation of cyclin CyclinD1 gene upstream of P15 (INK4B), P16 (INK4A) and P18 (INK4C) is higher than the normal group the expression of mRNA in radiation group, MSCs expression after transplantation. Compared with the normal group, radiation group and MSCs treatment group P19 (INK4D) mRNA on gene expression was significantly increased compared with radiation group, MSCs treatment group, the thymus tissue P19 (INK4D) expression also increased significantly. While the expression of Rb in radiation group was significantly lower than that in normal group and MSCs treatment group, the difference was statistically significant. Conclusion:
MSCs transplantation can make thymic lymphoma cell cycle arrest remission, has a certain inhibitory effect on radiation induced thymic lymphoma and its mechanism may be related to the down-regulation of MSCs upstream of the CyclinD1 gene P15 (INK4B), P16 (INK4A), P18 (INK4C), and the upregulation of CyclinD1 upstream related gene P19 (INK4D) on the expression of CyclinD1 and downstream the gene of Rb.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R736.3

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