內生性一氧化氮藉由mitogen-activated protein kinase途徑調控氣喘病人先趨細胞增生能力之探討

Endogenous nitric oxide (NO) modulates the proliferation capacity via mitogen-activated protein kinase pathway in progenitor cells of asthmatics

黃國雄(Kuo-Hsiung Huang)

指導教授 ： 林建煌 ； 郭漢彬

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2002年)

摘要

我們過去的研究發現，來自氣喘患者週邊血液的CD34+先驅細胞(progenitor cells)，其誘導性一氧化氮合成酶(inducible nitric oxide synthase, iNOS)明顯上升，給予L-NAME (NG-nitro-L-arginine methyl ester)抑制NO (一氧化氮)合成，會促進先驅細胞增生，株落形成大量增加。NO也會引起細胞凋亡(apoptosis)，影響Bcl-2的表現。MAPK (Mitogen-activated protein kinases)是細胞膜與細胞核間訊號傳遞的一個重要介質，與細胞的死亡和分化息息相關，而一氧化氮會調節MAPK的活化。本論文主要在探討NO影響氣喘患者週邊血液CD34+先驅細胞增生，是否經由MAPK之訊號傳遞路徑，改變Bcl-2的表現，進一步影響先驅細胞的增生。從26個氣喘患者與32個正常人的週邊血液先驅細胞給予不同的濃度L-NAME、SNP (sodium nitorprusside)、PD98059 (MEK抑制劑)與SB203580 (p38 MAPK抑制劑)經24小時處理後，以流式細胞計數儀(flow cytometery)分析Bcl-2的蛋白質表現與Annexin-V的表現，分析先驅細胞細胞凋亡的變化。經14天細胞培養，計算細胞株落形成的數目，進一步了解各種處理對先驅細胞生長的影響；並分析細胞內DNA含量的變化，以探討細胞生長週期(cell cycle)的變化。我們的結果發現，L-NAME明顯地增加氣喘患者先驅細胞Bcl-2的表現，而正常人則無影響。相反的，當以NO提供者SNP處理氣喘病人及正常人先驅細胞 24小時後，發現其Bcl-2表現並沒有受到影響。以Annexin-V的表現分析細胞凋亡的變化可發現，L-NAME明顯地減少氣喘病人先驅細胞Annexin-V的表現，於正常人則否。而SNP對於氣喘病人及正常人先驅細胞之細胞凋亡完全沒有影響。為了觀察MAPK是否會影響Bcl-2的表現，於氣喘病人之先驅細胞加入SB203580 (10 μM)及PD98059 (18 μM)培養24小時，發現SB203580及PD98059都會明顯的減少其Bcl-2表現。相反地，不論SB203580或PD98059都不會影響正常人先驅細胞Bcl-2之表現。當以L-NAME處理氣喘病人先驅細胞，測量其ERK的磷酸化變化，發現在2分鐘時，L-NAME會促進ERK的磷酸化表現達到最大值，但對正常人先驅細胞則沒有影響。SNP對氣喘病人及正常人先驅細胞的ERK磷酸化，則完全沒有影響。以L-NAME (1mM)處理先驅細胞經14天培養後，氣喘病人株落(colony)形成相對於對照組明顯地增加，給予SNP株落形成減少，而L-NAME及SNP對正常人則無影響。氣喘病人先驅細胞經培養14天後發現，PD98059明顯抑制株落形成，由對照組的55.8 ± 4.9個(n=12)減少至46.9 ± 4.7個(n=12, p<0.05)，而SB203580則減少至34.3 ± 4.9個(n=12, p<0.01)，同時加入L-NAME 與PD98059抑制株落形成更明顯，對正常人則無影響。然而，L-NAME抑制內生性NO並不影響先驅細胞之細胞週期，但PD98059則會明顯減少S phase，而抑制細胞生長。由以上的結果發現，內生性一氧化氮會抑制氣喘病人先驅細胞的增生，其可能是經由抑制ERK及p38 MAPK的路徑，使Bcl-2的表現減少，因而導致先驅細胞的細胞凋亡之增加所產生。

關鍵字

一氧化氮 ； 氣喘 ； 先趨細胞 ； mitogen-activated protein kinase

並列摘要

Our previous report has demonstrated that progenitor cells from asthmatics possess higher expression of induced nitric oxide synthase (iNOS). Inhibition of endogenous of nitric oxide (NO) promotes the progenitor cells proliferation and increases colony formation by NG-nitro-L-arginine methyl ester (L-NAME). NO can induce cell apoptosis and affect Bcl-2 expression. Mitogen-activated protein kinases (MAPK) play an important mediator of signal transduction in regulation of cell death and proliferation. NO may modulate the activation of MAPK. It is unknown whether NO would modulate the proliferation and apoptosis of peripheral blood progenitor cells from asthmatic patient via MAPK signaling transduction pathway. Peripheral blood progenitor cells isolated from 26 asthmatics and 32 normal subjects were treated by L-NAME or SNP (sodium nitroprusside, NO donor) or PD98059 (MEK inhibitor) or SB203580 (p38 MAPK inhibitor) for 24 hours. The expression of Bcl-2 protein and Annexin-V was analysed to measure cell apoptosis by flow cytometry. Colonies of progenitor cells were scored to determine the capacity of cell proliferation after 14-day culture. Using flow cytometric analyses, cell cycle of progenitor cells was detected by DNA contents of propidium iodide staining. In asthmatics, inhibition of endogenous NO by L-NAME dose-dependently increased the expression of Bcl-2 protein in progenitor cells (1mM 345.2 ± 37.0, 10mM 415.2 ± 44.5 mean fluorescence intensity, MFI, respectively, n=10, p<0.01) compared to vehicle control (323.0 ± 37.3, MFI, n=10), while there was no effect in normal subjects. In contrast, SNP did not affect the Bcl-2 expression of progenitor cells either from asthmatics or normal subjects. In addition, L-NAME significantly decreased the Annexin-V expression of progenitor cells from asthmatics, but not normal subjects. Treatment with SNP showed no difference in the Annexin-V expression of progenitor cells from asthmatics or normal subjects. To study effect of MAPK on cell apoptosis, both PD98059 (18μM) and SB203580 (10μM) significantly decreased the Bcl-2 expression (PD98059, 45.6 ± 12.1 MFI; SB203580, 50.6 ± 12.7 MFI, n=8, p<0.05, respectively) of progenitor cells from asthmatics compared to vehicle control (86.2 ± 19.3 MFI, n=8). There were no significant differences in normal subjects. In asthmatic group, L-NAME significantly enhanced ERK phosphorylation at 2 minutes (180.9 ± 18.5 MFI, p<0.05, n=6). L-NAME potentiated and SNP inhibited the colonies growth. In normal subjects there were no effect. Both PD98059 and SB203580 significantly inhibited the colony formation from 55.2 ± 6.8 to 46.9 ± 6.7 (PD98059, n=12, p<0.05) and 34.3 ± 6.0 (SB203580 n=12, p<0.05). Combined L-NAME with PD98059 had a significant synergic effect on the colony formation. There was no significant difference in normal subjects. L-NAME did not affect cell cycle of progenitor cells, while the PD98059 decreased the S phase cell cycle to inhibit cell growth. We concluded that NO decreased the Bcl-2 expression and accelerated cell apoptosis of progenitor cells from asthmatics, which may be through signaling pathway of MAPK, in turn contributing to the inhibition of cell proliferation.

並列關鍵字

NO ； asthma ； progenitor cells ； mitogen-activated protein kinase

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