RNAi抑制Smad6并在TGF-β1作用下对小鼠骨髓源树突状细胞生物学特性变化的研究
发布时间：2018-08-14 17:10
【摘要】： 第一部分培养扩增小鼠骨髓源树突状细胞及其生物学鉴定 目的:探讨体外扩增小鼠骨髓来源树突状细胞(BMDC)的方法并进行形态学观察和生物学特性鉴定。 方法:用重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组小鼠白细胞介素4(rmIL-4)体外诱导小鼠骨髓细胞分化为树突状细胞,倒置显微镜动态观察形态学变化,流式细胞术分析细胞表面分子,同时进行刺激初始型T淋巴细胞增殖能力的检测。 结果:体外培养9天后小鼠树突状细胞可达80%以上,光镜下可见典型的树突状细胞形态。未成熟DC的细胞表型为CD11clow、MHCIIlow、CD86low,未成熟DC经LPS刺激培养后可转变为成熟DC,细胞表型为CD11chigh、MHCIIhigh、CD86high ,可显著刺激同种异体混合淋巴细胞增殖。 结论:本方法可获得较高纯度的骨髓树突状细胞,避免了使用传统磁珠分离方法所带来的高成本,复杂操作,低产出率的弊端,为研究树突状细胞的功能以及运用其开展下游实验提供材料 第二部分细胞慢病毒感染及感染后细胞中基因表达状况检测 目的:使用已构建成功的小鼠Smad6基因RNA干扰(RNAi)慢病毒载体,有效沉默骨髓树突状细胞(BMDC)的Smad6基因表达。 方法:运用构建好的慢病毒载体,根据前期试验所明确的感染复数对第一部分所培养的小鼠BMDC进行感染,120h后收集细胞,并经倒置荧光显微镜、RT-PCR,WESTERN BLOT检测Smad6基因的表达状况。 结果:荧光显微镜观察初步推断病毒感染效率在75%左右, PCR和WESTERN BLOT证实,通过病毒途径,在BMDC细胞中smad6基因的沉默效约为85%。 结论:通过使用慢病毒载体,较成功抑制小鼠BMDC中Smad6基因表达。说明运用慢病毒做RNAi载体可以实现对DC细胞某一基因的有效沉默。 第三部分RNAi抑制Smad6并在TGF-β1作用下对小鼠骨髓源树突状细胞生物学特性变化的研究 目的:探讨在Smad6基因被抑制并有TGF-β1刺激的情况下,小鼠骨SH髓源树突细胞生物学特性是否存在着变化。 方法:分别以GM-CSF和IL-4诱导培养两批小鼠BMDC ,其中一批经用慢病毒感染,对细胞进行Smad6基因抑制,6d后同时用TGF-β1刺激,再经过48h后分别对两批细胞进行电镜观察、流式细胞术检测细胞表型CD11C、CD80、CD86、CD40、MHCⅡ,混合淋巴细胞反应检测其抗原提呈功能,检测细胞凋亡情况,收集上清液用ELISA检测IL-6,IL-12 p70。 结果: Smad6未干扰组电镜下DC成熟样特征较明显,CD11c、CD80、CD86及MHCⅡ的表达水平也较高,细胞凋亡指数较低,混合淋巴细胞反应和分泌IL-4、IL-12 p70能力较强,Smad6干扰组DC形态成熟特征不明显,CD11c、CD80、CD86和MHCⅡ的表达水平较低,细胞凋亡指数较高,混合淋巴细胞反应和分泌IL-4、IL-12 p70能力较弱。 结论:在小鼠BMDC中Smad6基因被抑制并有TGF-β1刺激的情况下,细胞多呈未成熟状态,以未成熟的生物学特性为主,而Smad6基因未被抑制情况下,细胞更接近于成熟状态。
[Abstract]:Part 1 culture and amplification of dendritic cells from mouse bone marrow and their biological identification
OBJECTIVE: To investigate the method of amplifying murine bone marrow-derived dendritic cells (BMDC) in vitro and identify its morphological and biological characteristics.
METHODS: Mouse bone marrow cells were induced to differentiate into dendritic cells by recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4) in vitro. Morphological changes were observed dynamically under inverted microscope, cell surface molecules were analyzed by flow cytometry, and initial T-lymphocyte proliferation was stimulated. Force detection.
Results: After 9 days of culture in vitro, more than 80% of the dendritic cells in mice could be seen. Typical morphology of dendritic cells was observed under light microscope. Immature DCs were CD11clow, MHCIIlow, CD86low. Immature DCs could be transformed into mature DCs after LPS stimulation culture. The phenotype of immature DCs was CD11chigh, MHCIIhigh, CD86high, which could significantly stimulate allogeneic mixed lymphoma. Proliferation of Ba cells.
CONCLUSION: This method can obtain high purity bone marrow dendritic cells and avoid the disadvantages of high cost, complicated operation and low yield caused by traditional magnetic bead separation method. It provides materials for studying the function of dendritic cells and developing downstream experiments.
The second part is the detection of lentivirus infection and the expression of genes in infected cells.
AIM: To effectively silence the expression of Smad6 gene in bone marrow dendritic cells (BMDC) by using the constructed mouse Smad6 gene RNA interference (RNAi) lentiviral vector.
METHODS: The constructed lentiviral vector was used to infect the BMDC of the first part of the experiment according to the specific infection complex. The cells were collected 120 hours later and the expression of Smad6 gene was detected by inverted fluorescence microscope, RT-PCR and WESTERN BLOT.
Results: Fluorescence microscopy showed that the viral infection rate was about 75%. PCR and WESTERN BLOT confirmed that the silencing effect of Smad6 gene in BMDC cells was about 85%.
Conclusion: The expression of Smad6 gene in BMDC of mice was inhibited successfully by using lentiviral vector, indicating that lentiviral vector as RNAi vector can effectively silence a certain gene in DC cells.
The third part RNAi inhibits Smad6 and changes the biological characteristics of murine bone marrow-derived dendritic cells in the presence of TGF-beta 1
AIM: To investigate whether the biological characteristics of bone SH myeloid derived dendritic cells (BMDCs) in mice have been altered when the Smad 6 gene is inhibited and stimulated by TGF-beta 1.
Methods: Two batches of BMDC were induced and cultured by GM-CSF and IL-4 respectively. One batch of BMDC was inhibited by lentiviral infection and stimulated by TGF-beta 1 6 days later. After 48 hours, the two batches of cells were observed by electron microscope. The phenotypes of CD11C, CD80, CD86, CD40, MHC II and mixed lymphocyte reaction were detected by flow cytometry. The antigen presenting function was detected, the apoptosis was detected, and the supernatant was collected. IL-6, IL-12 p70. were detected by ELISA.
Results: Smad6 did not interfere with the expression of CD11c, CD80, CD86 and MHC II, the expression of CD11c, CD80, CD86 and MHC II were higher, the apoptosis index was lower, the ability of mixed lymphocyte to react and secrete IL-4, IL-12 p70 was stronger, the morphological maturation of DC in Smad6 interfered group was not obvious, the expression levels of CD11c, CD80, CD86 and MHC II were lower, and the apoptosis was stronger. The index is higher, mixed lymphocyte reaction and secretion of IL-4, IL-12 p70 ability is weak.
CONCLUSION: When the Smad6 gene is inhibited and stimulated by TGF-beta 1 in BMDC, the cells are mostly immature, mainly with immature biological characteristics, while the Smad6 gene is not inhibited, the cells are closer to the mature state.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2183543
[Abstract]:Part 1 culture and amplification of dendritic cells from mouse bone marrow and their biological identification
OBJECTIVE: To investigate the method of amplifying murine bone marrow-derived dendritic cells (BMDC) in vitro and identify its morphological and biological characteristics.
METHODS: Mouse bone marrow cells were induced to differentiate into dendritic cells by recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4) in vitro. Morphological changes were observed dynamically under inverted microscope, cell surface molecules were analyzed by flow cytometry, and initial T-lymphocyte proliferation was stimulated. Force detection.
Results: After 9 days of culture in vitro, more than 80% of the dendritic cells in mice could be seen. Typical morphology of dendritic cells was observed under light microscope. Immature DCs were CD11clow, MHCIIlow, CD86low. Immature DCs could be transformed into mature DCs after LPS stimulation culture. The phenotype of immature DCs was CD11chigh, MHCIIhigh, CD86high, which could significantly stimulate allogeneic mixed lymphoma. Proliferation of Ba cells.
CONCLUSION: This method can obtain high purity bone marrow dendritic cells and avoid the disadvantages of high cost, complicated operation and low yield caused by traditional magnetic bead separation method. It provides materials for studying the function of dendritic cells and developing downstream experiments.
The second part is the detection of lentivirus infection and the expression of genes in infected cells.
AIM: To effectively silence the expression of Smad6 gene in bone marrow dendritic cells (BMDC) by using the constructed mouse Smad6 gene RNA interference (RNAi) lentiviral vector.
METHODS: The constructed lentiviral vector was used to infect the BMDC of the first part of the experiment according to the specific infection complex. The cells were collected 120 hours later and the expression of Smad6 gene was detected by inverted fluorescence microscope, RT-PCR and WESTERN BLOT.
Results: Fluorescence microscopy showed that the viral infection rate was about 75%. PCR and WESTERN BLOT confirmed that the silencing effect of Smad6 gene in BMDC cells was about 85%.
Conclusion: The expression of Smad6 gene in BMDC of mice was inhibited successfully by using lentiviral vector, indicating that lentiviral vector as RNAi vector can effectively silence a certain gene in DC cells.
The third part RNAi inhibits Smad6 and changes the biological characteristics of murine bone marrow-derived dendritic cells in the presence of TGF-beta 1
AIM: To investigate whether the biological characteristics of bone SH myeloid derived dendritic cells (BMDCs) in mice have been altered when the Smad 6 gene is inhibited and stimulated by TGF-beta 1.
Methods: Two batches of BMDC were induced and cultured by GM-CSF and IL-4 respectively. One batch of BMDC was inhibited by lentiviral infection and stimulated by TGF-beta 1 6 days later. After 48 hours, the two batches of cells were observed by electron microscope. The phenotypes of CD11C, CD80, CD86, CD40, MHC II and mixed lymphocyte reaction were detected by flow cytometry. The antigen presenting function was detected, the apoptosis was detected, and the supernatant was collected. IL-6, IL-12 p70. were detected by ELISA.
Results: Smad6 did not interfere with the expression of CD11c, CD80, CD86 and MHC II, the expression of CD11c, CD80, CD86 and MHC II were higher, the apoptosis index was lower, the ability of mixed lymphocyte to react and secrete IL-4, IL-12 p70 was stronger, the morphological maturation of DC in Smad6 interfered group was not obvious, the expression levels of CD11c, CD80, CD86 and MHC II were lower, and the apoptosis was stronger. The index is higher, mixed lymphocyte reaction and secretion of IL-4, IL-12 p70 ability is weak.
CONCLUSION: When the Smad6 gene is inhibited and stimulated by TGF-beta 1 in BMDC, the cells are mostly immature, mainly with immature biological characteristics, while the Smad6 gene is not inhibited, the cells are closer to the mature state.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 熊志红;杨丽萍;李国利;庄玉辉;;Smad家族蛋白在真核细胞中的表达[J];实用医学杂志;2009年07期
2 薛爱民;吴慧娟;张志刚;刘学光;陈琦;郭慕依;;γ-干扰素对大鼠肾系膜细胞转化生长因子β/Smad信号通路和基质金属蛋白酶2及其组织抑制因子2表达的影响[J];中华病理学杂志;2007年06期
，本文编号：2183543
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2183543.html
最近更新
教材专著
