MSCs对辐射诱导胸腺瘤的抑制作用及其机制的探讨

发布时间：2018-01-13 14:00

本文关键词：MSCs对辐射诱导胸腺瘤的抑制作用及其机制的探讨　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：随着放疗技术及设备的不断进步，肿瘤的治疗效果日益提高，患者的五年生存率得到明显提高，但其诱发第二恶性肿瘤的报道也逐年增多，包括白血病、直肠癌、膀胱癌等。胸腺作为机体主要的免疫器官，是对辐射敏感的组织之一，放射线照射诱发的胸腺淋巴瘤是一种高度浸润甚至能够转移的肿瘤，预后较差。因此，寻找一种有效的预防和治疗辐射诱导第二恶性肿瘤的方法对提高放疗的治疗效果具有实际意义。骨髓间充质干细胞（Bone Marrow Mesenchymal Stem cells，MSCs）是一类具有多向分化潜能、再生特性、免疫调节能力、靶向归巢能力等作用的多能干细胞。有研究表明，MSCs对脑损伤、脊髓损伤、肾损伤、胰腺损伤以及皮肤损伤等具有组织修复作用。本研究采用全骨髓贴壁法在体外分离培养MSCs，通过流式细胞仪检测MSCs细胞周期及细胞表面标志物来鉴定MSCs。选用健康幼年的C57BL/6J小鼠随机分成正常组、辐射组、MSCs治疗组。按照Kaplan经典方法制备胸腺淋巴瘤动物模型，在照射后当天及1周后对MSCs治疗组小鼠经尾静脉输注2×106个/ml的细胞悬液0.2ml。每日观察各只小鼠的一般状况，末次照射6个月后处死小鼠，无菌取胸腺。通过HE染色进行病理组织学观察，通过流式细胞仪检测胸腺细胞及其各亚群细胞的细胞周期和细胞增殖动力学以及各亚群中CD3的表达和TCR Mf（TCR突变频率），通过RT-PCR检测CyclinD1上游以及下游的相关基因P15(INK4B)、P16(INK4A)、P18(INK4C)、P19(INK4D)和Rb的表达。本研究结果显示：用全骨髓贴壁法分离培养的MSCs生长状态良好。流式细胞仪检测结果表明大多数MSCs处于相对不活跃的静止期G0/G1期，CD44的表达呈阳性，而不表达CD34，符合MSCs的特性。辐射组小鼠在照射完3个月后毛发无光泽，部分小鼠出现毛色灰白，蓬松逆立和精神较差等症状，MSCs治疗组小鼠状态明显有所改善。组织病理学观察结果显示，正常组小鼠胸腺皮髓质结构清晰，淋巴细胞形态规则，，大小均一，呈圆形或椭圆形，辐射组小鼠胸腺结构完全被破坏，淋巴样肿瘤细胞弥漫分布，形状不规则，大小不一，核大深染，出现镜影细胞和满天星现象，存在核分裂象，MSCs治疗组小鼠胸腺结构趋于正常。胸腺细胞中CD4-CD8-亚群细胞比例在辐射组明显高于正常组，MSCs治疗组趋于正常组；辐射组中CD4+CD8+亚群细胞比例明显低于正常组和MSCs治疗组；MSCs治疗组CD4+CD8-亚群细胞比例高于辐射组，接近于正常组；CD4-CD8+亚群细胞比例很低，接近于0，在三组中无明显差异。胸腺细胞和CD4-CD8-亚群细胞中，辐射组G1期细胞数明显高于正常组和MSCs治疗组，而辐射组S期细胞比例明显低于正常组，MSCs治疗组趋于正常水平；在CD4+CD8+和CD4+CD8-亚群细胞中，辐射组S期细胞数明显高于正常组和MSCs治疗组。胸腺细胞、CD4+CD8+和CD4+CD8-亚群细胞中，正常组和MSCs治疗组中parent、G2或G3、G4代细胞比例明显高于辐射组，而G5、G6或G8、G9、G10代细胞以及增殖指数（PI）明显低于辐射组；在胸腺CD4-CD8-亚群细胞中，辐射组parent、G2和G3代细胞比例明显高于正常组和MSCs治疗组，辐射组中G4、G5、G6代细胞比例以及PI明显低于正常组和MSCs治疗组。正常组中CD4CD8各亚群细胞中CD3+细胞的表达明显高于辐射组和MSCs治疗组，MSCs移植后CD3+细胞的表达高于辐射组，但仍显著低于正常组。其中CD3-细胞的表达与CD3+细胞的表达相反。辐射组和MSCs治疗组中TCR突变频率（TCR Mf）明显高于正常组。 RT-PCR结果显示，调控细胞周期蛋白CyclinD1上游基因P15(INK4B)、P16(INK4A)和P18(INK4C)在辐射组的mRNA表达高于正常组，MSCs移植后表达下调。与正常组相比，辐射组和MSCs治疗组P19(INK4D)基因mRNA相对表达量明显增加，和辐射组相比，MSCs治疗组胸腺组织中P19(INK4D)的相对表达量也明显增加。而Rb在辐射组的表达明显低于正常组和MSCs治疗组，差异有统计学差异。结论： MSCs移植能够使胸腺淋巴瘤细胞周期阻滞缓解，对辐射诱导的胸腺淋巴瘤具有一定的抑制作用，其机制可能与MSCs下调CyclinD1上游相关基因P15(INK4B)、P16(INK4A)、P18(INK4C)，和上调CyclinD1上游相关基因P19(INK4D)和CyclinD1下游基因Rb的表达有关。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R736.3

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