β-1，4-半乳糖基转移酶Ⅰ和Ⅴ在两型星形胶质和施万细胞中的生物学作用

发布时间：2018-06-13 16:43

本文选题：β-1 + 4-半乳糖基转移酶I、V　；参考：《苏州大学》2009年博士论文

【摘要】： 目的:观察β-1,4-半乳糖基转移酶I、V(β1,4 galactosyltransferase I、V,β-1,4-GalT I、V)在生理病理状态下两型星型胶质细胞和体内外施万细胞中的表达情况,探讨β-1,4- GalT I、V在两型星形胶质细胞和体内外施万细胞中可能存在的生物学作用。方法:体外培养两型星形胶质细胞和施万细胞,从炎性细胞因子TNFα着手,不同浓度、不同时间用LPS刺激两型星形胶质细胞或施万细胞,用酶联免疫吸附实验(ELISA)检测上清中分泌的TNFα的表达量;RT- PCR检测细胞中TNFα、TNFR1及TNFR2 mRNA的表达;同时用免疫荧光细胞化学染色检测TNFR1和TNFR2的细胞定位,real-time PCR检测β-1,4-GalT I和β-1,4-GalT V mRNA的表达变化。制备大鼠坐骨神经夹伤和切断模型,利用足迹试验观察坐骨神经功能指数(sciatic functional index, SFI);应用腹腔注射LPS建立炎症模型。通过real-time PCR方法,分析β-1,4-GalT-I、V mRNA在损伤及炎症性大鼠坐骨神经中的表达。利用RT-PCR扩增β-1,4-GalT I、V基因,克隆至pGEM-T载体,经体外转录法合成地高辛标记的正、反义β-1,4-GalT I、V RNA探针。通过原位杂交及图像分析,观察β-1,4-GalT I、V mRNA在大鼠正常、损伤及炎症坐骨神经中的表达变化。采用原位杂交与免疫组化相结合的方法检测β-1,4-GalT I、V的具体细胞定位。利用RNAi和细胞转染等技术干扰β-1,4-GalT V在施万细胞中的表达,运用Lectin Blot检测施万细胞在生理和病理状态N寡糖链合成情况。分别采用丝裂原活化的蛋白激酶(mitogen-acitivated protein kinase, MAPK)的特异性抑制剂(U0126、SB202190和SP600125)预处理细胞后观察MAPK信号通路在炎症过程中对β-1,4-GalT I、V的影响。结果:(1)TNFα和LPS作用于2型星形胶质细胞后β-1,4-GalT I mRNA的表达发生变化,且这种变化与TNFα和LPS作用的时间和浓度有关,呈时间和剂量依赖性。RT-PCR检测发现TNFα和LPS作用后2型星形胶质细胞中TNFα及TNFR2表达水平均显著升高,TNFR1的水平也有所提高。ELISA检测发现LPS作用后TNFα的分泌水平增加,免疫荧光双标检测结果发现,在正常未处理的2型星形胶质细胞中TNFR1广泛分布于细胞核、细胞浆以及细胞突起中,信号较弱,而在10 ng/ml LPS处理后TNFR1的表达信号明显增强,且其在核中的表达减少,主要定位于细胞浆与细胞突起中,在正常未处理的2型星形胶质细胞中TNFR2主要分布于细胞核中,10 ng/ml LPS处理后TNFR1在核中的表达显著降低,几乎检测不到,主要定位于细胞膜与细胞突起中。TNFR1抗体或TNFR2抗体可抑制LPS诱导的β-1,4-GalT I mRNA表达水平的升高。 (2) TNFα和LPS作用于1型星形胶质细胞后β-1,4-GalT I和β-1,4-GalT V mRNA的表达发生变化,且这种变化与TNFα和LPS作用的时间和浓度有关,呈时间和剂量依赖性。免疫细胞化学染色检测发现在正常的1型星形胶质细胞中,TNFR1有表达,主要定位于核周的细胞浆中,在LPS处理后TNFR1的表达强度增强,且广泛分布于胞浆中;在正常的1型星形胶质细胞中检测不到TNFR2的表达,而在LPS处理后可检测到TNFR2在1型星形胶质细胞中有表达,且信号强度较高,广泛分布于细胞浆中。TNFR抗体可逆转β-1,4-GalT I和V mRNAs因TNF-α作用后的表达下调及抑制因LPS作用后的表达上调。 (3)β-1,4-GalT I和β-1,4-GalT V mRNA在坐骨神经夹伤后2 w与切断1 w时表达水平明显增高,与正常对照组及其他各组相比,差异有统计学意义(P0.05),原位杂交结果显示β-1,4-GalT I和β-1,4-GalT V主要表达于S100阳性的施万细胞中。β-1,4-GalT I和β-1,4-GalT V mRNA的表达水平与体内炎症模型中LPS作用的浓度和作用时间有关,具有剂量和时间依赖性,同样体外细胞培养时β-1,4-GalT I、β-1,4-GalT V mRNA的表达水平及N糖链的合成也与LPS的作用时间和作用浓度有关。干扰β-1,4-GalT V的表达后可明显抑制N糖链的合成。使用MAPK信号通路的抑制剂,U0126,SB202190以及SP600125处理施万细胞后,通过real-time PCR检测发现3种抑制剂均可不同程度地抑制LPS诱导的β1, 4-GalT I和β1, 4-GalT V的表达水平。结论:(1)在2型星形胶质细胞中,TNFα是通过TNFR1和TNFR2由细胞核向细胞浆和细胞突起的转位来诱导β-1,4-GalT I表达的,除了外源性TNFα之外,2型星形胶质细胞还可通过自分泌方式产生TNFα对β-1,4-GalT I的表达产生影响,说明TNFα可以通过自分泌循环通路在炎症反应过程中发挥作用。 (2)在炎性因子作用下1型星形胶质细胞可通过自分泌方式产生的TNFα影响β-1,4-GalT I和β-1,4-GalT V的表达。 (3)对生理病理状态下的施万细胞,无论是在体内还是在体外β-1,4-GalT I和β-1,4-GalT V的表达均受到影响,提示β-1,4-GalT I和β-1,4-GalT V在施万细胞的生理病理过程均发挥重要的作用。干扰了施万细胞中β-1,4-GalT V表达后其N糖链的合成也显著减少,证实了β-1,4-GalT V与N糖链的合成密切相关。此外,β-1,4-GalT I和β-1,4-GalT V的表达是受ERK、P38以及SAPK/JNK这些MAPK信号通路来调节的。
[Abstract]:Objective: To observe the expression of beta -1,4- galactotransferase I, V (beta 1,4 galactosyltransferase I, V, I, V) in physiological and pathological state of astrocytes and Schwann cells in vivo and in vitro, and explore the possible biological effects of beta -1,4- GalT in astrocytes and Schwann cells in type two and in vivo and in vitro.
Methods: in vitro culture of type two astrocytes and Schwann cells, from inflammatory cytokine TNF alpha, different concentrations, different time to stimulate type two astrocytes or Schwann cells with LPS, and enzyme linked immunosorbent assay (ELISA) to detect the expression of TNF alpha in the supernatant; RT- PCR was used to detect TNF alpha, TNFR1 and TNFR2 mRNA. At the same time, the cell location of TNFR1 and TNFR2 was detected by immunofluorescent cytochemical staining, and the expression of V mRNA in beta -1,4-GalT I and beta -1,4-GalT was detected by real-time PCR. The model of sciatic nerve clamp injury and cut off in rats was prepared, and the sciatic nerve function index (sciatic functional index) was observed by footprints test. Real-time PCR method was used to analyze the expression of beta -1,4-GalT-I and V mRNA in the sciatic nerve of the injured and inflammatory rats. Using RT-PCR to amplify beta -1,4-GalT I, V gene, clone to pGEM-T vector, and synthesize digoxin labeled positive, antisense beta -1,4-GalT I, through in vitro transcription, through in situ hybridization and image analysis. The expression of beta -1,4-GalT I and V mRNA in normal, injured and inflammatory sciatic nerves was detected. The specific cell location of beta -1,4-GalT I, V was detected by the combination of in situ hybridization and immunohistochemistry. The expression of beta -1,4-GalT V in Schwann cells was interfered with RNAi and cell transfection, and the Schwann cells were detected by Lectin Blot. N oligosaccharide chain synthesis in physiological and pathological state. The specific inhibitors (U0126, SB202190 and SP600125) of mitogen activated protein kinase (U0126, SB202190 and SP600125) were pretreated by mitogen activated protein kinase (U0126, SB202190 and SP600125) respectively. The effect of MAPK signaling pathway on beta -1,4-GalT I and V was observed in the process of inflammation.
Results: (1) the expression of beta -1,4-GalT I mRNA after the action of TNF alpha and LPS on type 2 astrocytes was related to the time and concentration of TNF alpha and LPS action. The time and dose dependent.RT-PCR detected the significant increase in TNF alpha and TNFR2 expression level in the 2 astrocytes after the action of TNF alpha and LPS. The secretion level of TNF alpha was increased after.ELISA detection, and the results of double immunofluorescence double labeling showed that TNFR1 was widely distributed in nucleus, cytoplasm and cell protuberance in normal untreated type 2 astrocytes, and the signal was weaker in the cytoplasm and cell protuberance, and the expression of TNFR1 was obviously enhanced after 10 ng/ ml LPS treatment. The expression in the nucleus is reduced, mainly located in the cytoplasm and cell protuberance. In the normal untreated type 2 astrocytes, TNFR2 is mainly distributed in the nucleus. After 10 ng/ml LPS treatment, the expression of TNFR1 in the nucleus is significantly reduced, almost undetected, mainly located in the cell membrane and the cell protuberance of the.TNFR1 or TNFR2 antibodies. The expression level of beta -1,4-GalT I mRNA induced by LPS was increased.
(2) the expression of beta -1,4-GalT I and beta -1,4-GalT V mRNA after the action of TNF alpha and LPS on type 1 astrocytes, and this change is dependent on the time and concentration of TNF alpha and LPS, and is time and dose dependent. Immunocytochemical staining shows that TNFR1 is expressed in normal type 1 astrocytes, mainly located in the expression of TNFR1. In the cytoplasm of the perinuclear cell, the expression of TNFR1 was enhanced after LPS treatment and was widely distributed in the cytoplasm. The expression of TNFR2 was not detected in the normal type 1 astrocytes, and the expression of TNFR2 in type 1 astrocytes was detected after LPS treatment, and the signal intensity was higher, and the.TNFR antibody in the cytoplasm was widely distributed in the cytoplasm. The expression of beta -1,4-GalT I and V mRNAs was down regulated after the action of TNF- alpha, and the expression of LPS was inhibited by LPS.
(3) the expression level of beta -1,4-GalT I and beta -1,4-GalT V mRNA increased significantly at 2 W and 1 W after sciatic nerve clamp injury. Compared with the normal control group and other groups, the difference was statistically significant (P0.05). The results of in situ hybridization showed that beta -1,4-GalT I and beta -1,4-GalT V were mainly expressed in the positive Schwann cells. The expression level of T V mRNA is related to the concentration and time of the action of LPS in the inflammatory model in the body. It is dependent on the dose and time. The expression of beta -1,4-GalT I, the expression level of beta -1,4-GalT V mRNA and the synthesis of N sugar chain are also related to the LPS action time and concentration degree. Inhibition of the synthesis of N sugar chains. After treating Schwann cells with MAPK signaling pathway inhibitors, U0126, SB202190 and SP600125, the 3 inhibitors can be detected by real-time PCR to varying degrees of inhibition of LPS induced beta 1, 4-GalT I and beta 1, 4-GalT V.
Conclusion: (1) in type 2 astrocytes, TNF alpha induces the expression of beta -1,4-GalT I through the transposition of the nucleus to the cytoplasm and cell protrusion by TNFR1 and TNFR2. In addition to the exogenous TNF alpha, the type 2 astrocytes can also produce the effect of TNF a on the expression of the I of beta -1,4-GalT by autocrine mode, indicating that TNF a can pass by itself. Secretory circulation pathway plays an important role in inflammatory reaction.
(2) under the action of inflammatory factors, type TNF astrocytes can affect the expression of beta -1,4-GalT I and beta -1,4-GalT V by the production of TNF alpha by autocrine.
(3) the expression of Schwann cells in physiological and pathological conditions, both in vivo and in vitro, are affected by the expression of beta -1,4-GalT I and beta -1,4-GalT V, suggesting that beta -1,4-GalT I and beta -1,4-GalT V play an important role in the physiological and pathological processes of Schwann cells. The synthesis of N sugar chains after the expression of beta -1,4-GalT V in Schwann cells also shows a significant effect on the synthesis of N sugar chains. The reduction has proved that beta -1,4-GalT V is closely related to the synthesis of N sugar chains. In addition, the expression of beta -1,4-GalT I and beta -1,4-GalT V is regulated by ERK, P38, and SAPK/JNK MAPK signaling pathways.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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