RNAi抑制Smad6并在TGF-β1作用下對小鼠骨髓源樹突狀細胞生物學(xué)特性變化的研究
發(fā)布時間：2018-08-14 17:10
【摘要】： 第一部分培養(yǎng)擴增小鼠骨髓源樹突狀細胞及其生物學(xué)鑒定 目的:探討體外擴增小鼠骨髓來源樹突狀細胞(BMDC)的方法并進行形態(tài)學(xué)觀察和生物學(xué)特性鑒定。 方法:用重組小鼠粒細胞-巨噬細胞集落刺激因子(rmGM-CSF)和重組小鼠白細胞介素4(rmIL-4)體外誘導(dǎo)小鼠骨髓細胞分化為樹突狀細胞,倒置顯微鏡動態(tài)觀察形態(tài)學(xué)變化,流式細胞術(shù)分析細胞表面分子,同時進行刺激初始型T淋巴細胞增殖能力的檢測。 結(jié)果:體外培養(yǎng)9天后小鼠樹突狀細胞可達80%以上,光鏡下可見典型的樹突狀細胞形態(tài)。未成熟DC的細胞表型為CD11clow、MHCIIlow、CD86low,未成熟DC經(jīng)LPS刺激培養(yǎng)后可轉(zhuǎn)變?yōu)槌墒霥C,細胞表型為CD11chigh、MHCIIhigh、CD86high ,可顯著刺激同種異體混合淋巴細胞增殖。 結(jié)論:本方法可獲得較高純度的骨髓樹突狀細胞,避免了使用傳統(tǒng)磁珠分離方法所帶來的高成本,復(fù)雜操作,低產(chǎn)出率的弊端,為研究樹突狀細胞的功能以及運用其開展下游實驗提供材料 第二部分細胞慢病毒感染及感染后細胞中基因表達狀況檢測 目的:使用已構(gòu)建成功的小鼠Smad6基因RNA干擾(RNAi)慢病毒載體,有效沉默骨髓樹突狀細胞(BMDC)的Smad6基因表達。 方法:運用構(gòu)建好的慢病毒載體,根據(jù)前期試驗所明確的感染復(fù)數(shù)對第一部分所培養(yǎng)的小鼠BMDC進行感染,120h后收集細胞,并經(jīng)倒置熒光顯微鏡、RT-PCR,WESTERN BLOT檢測Smad6基因的表達狀況。 結(jié)果:熒光顯微鏡觀察初步推斷病毒感染效率在75%左右, PCR和WESTERN BLOT證實,通過病毒途徑,在BMDC細胞中smad6基因的沉默效約為85%。 結(jié)論:通過使用慢病毒載體,較成功抑制小鼠BMDC中Smad6基因表達。說明運用慢病毒做RNAi載體可以實現(xiàn)對DC細胞某一基因的有效沉默。 第三部分RNAi抑制Smad6并在TGF-β1作用下對小鼠骨髓源樹突狀細胞生物學(xué)特性變化的研究 目的:探討在Smad6基因被抑制并有TGF-β1刺激的情況下,小鼠骨SH髓源樹突細胞生物學(xué)特性是否存在著變化。 方法:分別以GM-CSF和IL-4誘導(dǎo)培養(yǎng)兩批小鼠BMDC ,其中一批經(jīng)用慢病毒感染,對細胞進行Smad6基因抑制,6d后同時用TGF-β1刺激,再經(jīng)過48h后分別對兩批細胞進行電鏡觀察、流式細胞術(shù)檢測細胞表型CD11C、CD80、CD86、CD40、MHCⅡ,混合淋巴細胞反應(yīng)檢測其抗原提呈功能,檢測細胞凋亡情況,收集上清液用ELISA檢測IL-6,IL-12 p70。 結(jié)果: Smad6未干擾組電鏡下DC成熟樣特征較明顯,CD11c、CD80、CD86及MHCⅡ的表達水平也較高,細胞凋亡指數(shù)較低,混合淋巴細胞反應(yīng)和分泌IL-4、IL-12 p70能力較強,Smad6干擾組DC形態(tài)成熟特征不明顯,CD11c、CD80、CD86和MHCⅡ的表達水平較低,細胞凋亡指數(shù)較高,混合淋巴細胞反應(yīng)和分泌IL-4、IL-12 p70能力較弱。 結(jié)論:在小鼠BMDC中Smad6基因被抑制并有TGF-β1刺激的情況下,細胞多呈未成熟狀態(tài),以未成熟的生物學(xué)特性為主,而Smad6基因未被抑制情況下,細胞更接近于成熟狀態(tài)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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相關(guān)期刊論文 前2條
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