本文選題：β-1 + 4-半乳糖基轉(zhuǎn)移酶I、V��； 參考：《蘇州大學(xué)》2009年博士論文

【摘要】： 目的:觀察β-1,4-半乳糖基轉(zhuǎn)移酶I、V(β1,4 galactosyltransferase I、V,β-1,4-GalT I、V)在生理病理狀態(tài)下兩型星型膠質(zhì)細(xì)胞和體內(nèi)外施萬(wàn)細(xì)胞中的表達(dá)情況,探討β-1,4- GalT I、V在兩型星形膠質(zhì)細(xì)胞和體內(nèi)外施萬(wàn)細(xì)胞中可能存在的生物學(xué)作用。 方法:體外培養(yǎng)兩型星形膠質(zhì)細(xì)胞和施萬(wàn)細(xì)胞,從炎性細(xì)胞因子TNFα著手,不同濃度、不同時(shí)間用LPS刺激兩型星形膠質(zhì)細(xì)胞或施萬(wàn)細(xì)胞,用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測(cè)上清中分泌的TNFα的表達(dá)量;RT- PCR檢測(cè)細(xì)胞中TNFα、TNFR1及TNFR2 mRNA的表達(dá);同時(shí)用免疫熒光細(xì)胞化學(xué)染色檢測(cè)TNFR1和TNFR2的細(xì)胞定位,real-time PCR檢測(cè)β-1,4-GalT I和β-1,4-GalT V mRNA的表達(dá)變化。制備大鼠坐骨神經(jīng)夾傷和切斷模型,利用足跡試驗(yàn)觀察坐骨神經(jīng)功能指數(shù)(sciatic functional index, SFI);應(yīng)用腹腔注射LPS建立炎癥模型。通過(guò)real-time PCR方法,分析β-1,4-GalT-I、V mRNA在損傷及炎癥性大鼠坐骨神經(jīng)中的表達(dá)。利用RT-PCR擴(kuò)增β-1,4-GalT I、V基因,克隆至pGEM-T載體,經(jīng)體外轉(zhuǎn)錄法合成地高辛標(biāo)記的正、反義β-1,4-GalT I、V RNA探針。通過(guò)原位雜交及圖像分析,觀察β-1,4-GalT I、V mRNA在大鼠正常、損傷及炎癥坐骨神經(jīng)中的表達(dá)變化。采用原位雜交與免疫組化相結(jié)合的方法檢測(cè)β-1,4-GalT I、V的具體細(xì)胞定位。利用RNAi和細(xì)胞轉(zhuǎn)染等技術(shù)干擾β-1,4-GalT V在施萬(wàn)細(xì)胞中的表達(dá),運(yùn)用Lectin Blot檢測(cè)施萬(wàn)細(xì)胞在生理和病理狀態(tài)N寡糖鏈合成情況。分別采用絲裂原活化的蛋白激酶(mitogen-acitivated protein kinase, MAPK)的特異性抑制劑(U0126、SB202190和SP600125)預(yù)處理細(xì)胞后觀察MAPK信號(hào)通路在炎癥過(guò)程中對(duì)β-1,4-GalT I、V的影響。 結(jié)果:(1)TNFα和LPS作用于2型星形膠質(zhì)細(xì)胞后β-1,4-GalT I mRNA的表達(dá)發(fā)生變化,且這種變化與TNFα和LPS作用的時(shí)間和濃度有關(guān),呈時(shí)間和劑量依賴性。RT-PCR檢測(cè)發(fā)現(xiàn)TNFα和LPS作用后2型星形膠質(zhì)細(xì)胞中TNFα及TNFR2表達(dá)水平均顯著升高,TNFR1的水平也有所提高。ELISA檢測(cè)發(fā)現(xiàn)LPS作用后TNFα的分泌水平增加,免疫熒光雙標(biāo)檢測(cè)結(jié)果發(fā)現(xiàn),在正常未處理的2型星形膠質(zhì)細(xì)胞中TNFR1廣泛分布于細(xì)胞核、細(xì)胞漿以及細(xì)胞突起中,信號(hào)較弱,而在10 ng/ml LPS處理后TNFR1的表達(dá)信號(hào)明顯增強(qiáng),且其在核中的表達(dá)減少,主要定位于細(xì)胞漿與細(xì)胞突起中,在正常未處理的2型星形膠質(zhì)細(xì)胞中TNFR2主要分布于細(xì)胞核中,10 ng/ml LPS處理后TNFR1在核中的表達(dá)顯著降低,幾乎檢測(cè)不到,主要定位于細(xì)胞膜與細(xì)胞突起中。TNFR1抗體或TNFR2抗體可抑制LPS誘導(dǎo)的β-1,4-GalT I mRNA表達(dá)水平的升高。 (2) TNFα和LPS作用于1型星形膠質(zhì)細(xì)胞后β-1,4-GalT I和β-1,4-GalT V mRNA的表達(dá)發(fā)生變化,且這種變化與TNFα和LPS作用的時(shí)間和濃度有關(guān),呈時(shí)間和劑量依賴性。免疫細(xì)胞化學(xué)染色檢測(cè)發(fā)現(xiàn)在正常的1型星形膠質(zhì)細(xì)胞中,TNFR1有表達(dá),主要定位于核周的細(xì)胞漿中,在LPS處理后TNFR1的表達(dá)強(qiáng)度增強(qiáng),且廣泛分布于胞漿中;在正常的1型星形膠質(zhì)細(xì)胞中檢測(cè)不到TNFR2的表達(dá),而在LPS處理后可檢測(cè)到TNFR2在1型星形膠質(zhì)細(xì)胞中有表達(dá),且信號(hào)強(qiáng)度較高,廣泛分布于細(xì)胞漿中。TNFR抗體可逆轉(zhuǎn)β-1,4-GalT I和V mRNAs因TNF-α作用后的表達(dá)下調(diào)及抑制因LPS作用后的表達(dá)上調(diào)。 (3)β-1,4-GalT I和β-1,4-GalT V mRNA在坐骨神經(jīng)夾傷后2 w與切斷1 w時(shí)表達(dá)水平明顯增高,與正常對(duì)照組及其他各組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05),原位雜交結(jié)果顯示β-1,4-GalT I和β-1,4-GalT V主要表達(dá)于S100陽(yáng)性的施萬(wàn)細(xì)胞中。β-1,4-GalT I和β-1,4-GalT V mRNA的表達(dá)水平與體內(nèi)炎癥模型中LPS作用的濃度和作用時(shí)間有關(guān),具有劑量和時(shí)間依賴性,同樣體外細(xì)胞培養(yǎng)時(shí)β-1,4-GalT I、β-1,4-GalT V mRNA的表達(dá)水平及N糖鏈的合成也與LPS的作用時(shí)間和作用濃度有關(guān)。干擾β-1,4-GalT V的表達(dá)后可明顯抑制N糖鏈的合成。使用MAPK信號(hào)通路的抑制劑,U0126,SB202190以及SP600125處理施萬(wàn)細(xì)胞后,通過(guò)real-time PCR檢測(cè)發(fā)現(xiàn)3種抑制劑均可不同程度地抑制LPS誘導(dǎo)的β1, 4-GalT I和β1, 4-GalT V的表達(dá)水平。 結(jié)論:(1)在2型星形膠質(zhì)細(xì)胞中,TNFα是通過(guò)TNFR1和TNFR2由細(xì)胞核向細(xì)胞漿和細(xì)胞突起的轉(zhuǎn)位來(lái)誘導(dǎo)β-1,4-GalT I表達(dá)的,除了外源性TNFα之外,2型星形膠質(zhì)細(xì)胞還可通過(guò)自分泌方式產(chǎn)生TNFα對(duì)β-1,4-GalT I的表達(dá)產(chǎn)生影響,說(shuō)明TNFα可以通過(guò)自分泌循環(huán)通路在炎癥反應(yīng)過(guò)程中發(fā)揮作用。 (2)在炎性因子作用下1型星形膠質(zhì)細(xì)胞可通過(guò)自分泌方式產(chǎn)生的TNFα影響β-1,4-GalT I和β-1,4-GalT V的表達(dá)。 (3)對(duì)生理病理狀態(tài)下的施萬(wàn)細(xì)胞,無(wú)論是在體內(nèi)還是在體外β-1,4-GalT I和β-1,4-GalT V的表達(dá)均受到影響,提示β-1,4-GalT I和β-1,4-GalT V在施萬(wàn)細(xì)胞的生理病理過(guò)程均發(fā)揮重要的作用。干擾了施萬(wàn)細(xì)胞中β-1,4-GalT V表達(dá)后其N糖鏈的合成也顯著減少,證實(shí)了β-1,4-GalT V與N糖鏈的合成密切相關(guān)。此外,β-1,4-GalT I和β-1,4-GalT V的表達(dá)是受ERK、P38以及SAPK/JNK這些MAPK信號(hào)通路來(lái)調(diào)節(jié)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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本文編號(hào)：2014681