陳 謹, 王曉明, 周敏然, 孫 婷, 陳忠敏, 陳春燕△
(1安徽醫(yī)學(xué)高等??茖W(xué)校,安徽 合肥 230601; 2山東大學(xué)齊魯醫(yī)院血液科,山東 濟南 250012; 3重慶理工大學(xué)藥學(xué)與生物工程學(xué)院,重慶 400054)
FoxM1靶向調(diào)控bcl-2促進急性髓系白血病發(fā)生*
陳 謹1, 王曉明2, 周敏然2, 孫 婷2, 陳忠敏3, 陳春燕2△
(1安徽醫(yī)學(xué)高等??茖W(xué)校,安徽 合肥 230601;2山東大學(xué)齊魯醫(yī)院血液科,山東 濟南 250012;3重慶理工大學(xué)藥學(xué)與生物工程學(xué)院,重慶 400054)
目的:探討叉頭框蛋白M1(FoxM1)及其調(diào)控的原癌基因B細胞白血病/淋巴瘤-2(bcl-2)在急性髓系白血病(AML)發(fā)生中的作用。方法: RT-qPCR和免疫熒光方法檢測17例AML初診患者、17例治療后達完全緩解(CR)患者、17例難治復(fù)發(fā)(RR)患者和15例正常骨髓標本中FoxM1的mRNA和蛋白表達;轉(zhuǎn)染FoxM1 siRNA和對照siRNA至白血病HL60細胞和K562細胞,觀察FoxM1對細胞生長和克隆形成的影響,流式細胞術(shù)檢測FoxM1對凋亡的影響,RT-qPCR和Western blot法檢測FoxM1對Bcl-2表達的影響,雙螢光素酶活性實驗檢測FoxM1是否可以靶向作用于bcl-2啟動子區(qū)域進而影響B(tài)cl-2的表達。結(jié)果: AML初診患者骨髓標本的FoxM1表達較正常對照顯著升高,CR患者的FoxM1表達較初診組降低,RR組的FoxM1表達較初診組進一步升高。轉(zhuǎn)染FoxM1 siRNA沉默HL60細胞和K562細胞的FoxM1表達后,細胞生長速率顯著下降,細胞克隆形成能力顯著降低,細胞凋亡率顯著升高,bcl-2表達降低,F(xiàn)oxM1可靶向調(diào)控bcl-2。結(jié)論: 初步證實FoxM1可通過靶向調(diào)控bcl-2促進AML發(fā)生發(fā)展,干擾FoxM1表達可抑制細胞增殖并促進細胞凋亡,提示FoxM1是治療AML的潛在靶標。
急性髓系白血??; 叉頭框蛋白M1; Bcl-2
急性髓系白血病(acute myeloid leukemia,AML)是成年人最常見的急性白血病,現(xiàn)有的治療方案效果不佳,如以蒽環(huán)類和阿糖胞苷為主的聯(lián)合化療是非急性早幼粒細胞白血病的AML的主要治療方法,60歲以下患者的5年生存率僅為30%~40%,60歲以上的5年生存率<10%[1]。因此,深入研究AML發(fā)生發(fā)展的機制,發(fā)現(xiàn)新的潛在靶標是重要基礎(chǔ)性科學(xué)問題。
叉頭框蛋白M1(Forkhead box M1,F(xiàn)oxM1)是在有絲分裂G1/S和G2/M期中起正向調(diào)控的轉(zhuǎn)錄因子,促進細胞有絲分裂和增殖,已發(fā)現(xiàn)在一些實體瘤中FoxM1過度表達,通過誘發(fā)細胞異常增殖參與惡性轉(zhuǎn)化,由于FoxM1在正常成熟組織細胞不表達,因此FoxM1被認為是抗腫瘤的潛在靶標[2],然而,F(xiàn)oxM1在急性白血病中作用的研究報道較為少見[1, 3-4]。Bcl-2通過抑制細胞凋亡促進惡性進展,是細胞凋亡研究中最受重視的癌基因之一,F(xiàn)oxM1能否調(diào)控Bcl-2發(fā)揮生物學(xué)效應(yīng)值得關(guān)注。有鑒于此,本研究擬探討FoxM1及其調(diào)控Bcl-2的機制及其在AML發(fā)生發(fā)展中的作用,為發(fā)現(xiàn)AML的新靶標提供初步依據(jù)。
1 主要試劑
人淋巴細胞分離液購自中國上海泛柯實業(yè)有限公司;胎牛血清(fetal bovine serum,F(xiàn)BS)和RPMI-1640培養(yǎng)基購自Gibco;HL60細胞株由本實驗室保存;K562細胞株購自北京北納創(chuàng)聯(lián)生物技術(shù)研究院;FoxM1 siRNA和對照siRNA購自Sigma;轉(zhuǎn)染試劑LipofectamineTM2000和RNA提取試劑TRIzol購自Invitrogen;逆轉(zhuǎn)錄試劑盒購自Fermentas;PCR引物由深圳華大基因科技有限公司合成;RT-qPCR試劑盒購自TaKaRa;Western blot相關(guān)試劑購自北京索萊寶科技有限公司;FoxM1和Bcl-2抗體購自Santa Cruz;ECL化學(xué)發(fā)光檢測試劑盒購自Millipore;細胞免疫熒光試劑丙酮購自中國天津市灝洋生物制品科技有限責(zé)任公司;Triton X-100購自中國北京索萊寶科技有限公司;抗兔辣根過氧化物酶標記的 II 抗購自Abcam;凋亡檢測試劑盒購自BD。
2 急性髓系白血病患者骨髓標本的獲取
隨機采集17例AML初診(denovo)患者、17例治療后達完全緩解(complete remission,CR)患者、17例難治復(fù)發(fā)(refractoriness and relapse,RR)患者和15例正常骨髓標本4~5 mL,置于EDTA抗凝管,使用人淋巴細胞分離液處理標本,分離單個核細胞后存放于-80 ℃冰箱中待用。
3 方法
3.1 RT-qPCR檢測FoxM1和Bcl-2的mRNA表達水平 0.5~1 mL TRIzol處理骨髓單個核細胞或白血病細胞系,提取總RNA。使用隨機引物和MMLV逆轉(zhuǎn)錄酶進行反轉(zhuǎn)錄,按照程序65 ℃ 5 min、25 ℃ 5 min、42 ℃ 60 min、70 ℃ 5 min合成cDNA。獲得cDNA后,使用SYBR Green RT-qPCR試劑盒建立PCR體系,按照程序95 ℃ 10 s;95℃ 5 s、60 ℃ 31 s,40個循環(huán)進行PCR。qPCR的引物序列見表1。
表1 引物序列和siRNA序列
Table 1.The sequences of the primers for RT-qPCR and the sequences of the siRNA
NC: negative control.
3.2 細胞免疫熒光實驗檢測骨髓標本FoxM1和Bcl-2蛋白表達 將提取的AML患者和正常對照骨髓中單個核細胞,涂片,丙酮固定,置于4 ℃冰箱,PBS潤洗、細胞打孔、山羊血清封閉30 min,加 I 抗FoxM1(1∶150)和Bcl-2(1∶100)4 ℃孵育過夜,PBS潤洗, II 抗孵育(1∶1 000),PBS潤洗,DAPI孵育,然后抗淬滅劑孵育,最后拍照保存。
3.3 細胞培養(yǎng)及轉(zhuǎn)染實驗 白血病細胞系HL60和K562用10% FBS和RPMI-1640培養(yǎng)基,置于37 ℃、5% CO2細胞培養(yǎng)箱中培養(yǎng)。將HL60細胞和K562細胞以每孔1×105接種到6孔板中后,配置混合液(250 μL Opti-MEM+5 μL LipofectamineTM2000;250 μL Opti-MEM+5 μLFoxM1/NC siRNA),5 min后將兩者混合形成轉(zhuǎn)染復(fù)合物(500 μL Opti-MEM+5 μL LipofectamineTM2000+5 μLFoxM1/NC siRNA),20 min后將轉(zhuǎn)染復(fù)合物加入每孔細胞中,孵育72 h后,收集細胞進行相關(guān)檢測。siRNA序列見表1。
3.4 細胞生長曲線測定 分別轉(zhuǎn)染FoxM1 siRNA或?qū)φ誷iRNA到白血病細胞HL60和K562,在轉(zhuǎn)染0 h、24 h、48 h、72 h時分別進行細胞計數(shù),繪制細胞生長曲線。
3.5 軟瓊脂克隆形成實驗 將1%下層瓊脂與20% FBS和2×RPMI-1640培基按照1∶1比例配置13 mL,均勻加入6孔板(每孔2 mL)。待下層瓊脂培養(yǎng)基凝固后,將0.3%或0.4%上層瓊脂與HL60/K562細胞懸液按照1∶1的比例配制7 mL,將含有細胞的上層培養(yǎng)基均勻加入6孔板。待上層瓊脂凝固后,將6孔板置于37 ℃、5% CO2細胞培養(yǎng)箱中孵育,約15 d后觀察白色致密不透光細胞團的數(shù)目及大小。
3.6 流式細胞術(shù)測定細胞凋亡率 將1×106已轉(zhuǎn)染FoxM1/NC siRNA的HL60細胞和K562細胞用PBS清洗后,用100 μL 結(jié)合緩沖液重懸細胞后放入5 mL流式管中。加入PE標記的5 μL Annexin V,再加入5 μL 7-AAD,15 min后再加入400 μL 結(jié)合緩沖液均勻混合后用流式細胞儀測定細胞凋亡率。
3.7 雙螢光素酶活性檢測 使用Lipofectamine 2000分別將bcl-2啟動子質(zhì)粒和FoxM1/NC siRNA及內(nèi)參照pRL-TK質(zhì)粒同時轉(zhuǎn)染細胞,作用48 h后,進行雙螢光素酶活性檢測。雙螢光素酶活性檢測按雙螢光素酶活性檢測試劑盒說明進行。
4 統(tǒng)計學(xué)處理
采用SPSS 13.0統(tǒng)計軟件對實驗數(shù)據(jù)進行分析。數(shù)據(jù)用均數(shù)±標準差(mean±SD)表示。多組間的差異采用單因素方差分析進行比較,兩組均數(shù)間比較采用t檢驗,以P<0.05為差異有統(tǒng)計學(xué)意義。
1 急性髓系白血病初診和難治復(fù)發(fā)患者FoxM1表達升高
用RT-qPCR方法和免疫熒光技術(shù)檢測17例AML-denovo患者、17例AML-CR患者、17例AML-RR患者和15例正常骨髓標本中FoxM1的mRNA和蛋白表達。結(jié)果顯示,與正常健康對照骨髓標本相比,AML-denovo組患者骨髓標本中FoxM1表達顯著升高,而AML-CR組 FoxM1表達較初診組降低,AML-RR組 FoxM1表達較初診組進一步升高,表明FoxM1與AML的發(fā)生發(fā)展和治療轉(zhuǎn)歸密切相關(guān),見圖1。
Figure 1. FoxM1 was over-expressed in AML. A: the mRNA expression of FoxM1 in BM samples from the patients in different groups detected by RT-qPCR analysis; B: the protein expression of FoxM1 in BM samples from the patients in different groups determined by cellular immunofluorescence analysis. Mean±SD.n=17.*P<0.05vscontrol;#P<0.05vsAML-denovo.
圖1 急性髓系白血病患者骨髓標本中FoxM1表達升高
2 干擾FoxM1表達抑制白血病HL60和K562細胞增殖
轉(zhuǎn)染FoxM1 siRNA到HL60和K562細胞,轉(zhuǎn)染0 h、24 h、48 h、72 h后分別計數(shù)活細胞,繪制細胞生長曲線,結(jié)果表明,轉(zhuǎn)染FoxM1 siRNA組較轉(zhuǎn)染對照siRNA組的HL60和K562細胞增殖速率顯著降低。轉(zhuǎn)染FoxM1 siRNA 72 h后進行軟瓊脂克隆形成實驗,14 d后觀察結(jié)果顯示,轉(zhuǎn)染FoxM1 siRNA組較轉(zhuǎn)染對照siRNA組的HL60細胞和K562細胞克隆形成能力顯著降低,提示干擾FoxM1表達可抑制白血病細胞增殖,見圖2。
3 干擾FoxM1表達促進白血病HL60和K562細胞凋亡
轉(zhuǎn)染FoxM1 siRNA到HL60細胞和K562細胞,72 h后收集細胞,流式細胞術(shù)檢測發(fā)現(xiàn)與對照siRNA組相比較,F(xiàn)oxM1 siRNA組的凋亡率明顯升高,表明干擾FoxM1表達可促進白血病細胞凋亡,見圖3。
Figure 2.FoxM1 specific siRNA inhibited the proliferation of HL60 cells (A) and K562 cells (B). Foci formation (C) of HL60 cells and K562 cells after transfection. Mean±SD.n=3.*P<0.05,**P<0.01vsNC siRNA.
圖2 干擾FoxM1表達可抑制HL60和K562細胞增殖
Figure 3.Silencing ofFoxM1 promoted the apoptosis of HL60 cells and K562 cells.FoxM1 specific siRNA was transfected into the HL60 cells and K562 cells. The cells were harvested 72 h after transfection. Flow cytometry was performed with Annexin V-phycoerythrin (PE) and 7-aminoactinomycin (7-AAD) double staining.Mean±SD.n=3.**P<0.01vsNC siRNA.
圖3 干擾FoxM1表達促進HL60細胞和K562細胞凋亡
4 FoxM1靶向調(diào)控原癌基因bcl-2
轉(zhuǎn)染FoxM1 siRNA至HL60細胞和K562細胞后,RT-qPCR和Western blot檢測證實FoxM1 siRNA轉(zhuǎn)染是有效的,F(xiàn)oxM1的mRNA和蛋白水平均明顯降低。同時發(fā)現(xiàn)Bcl-2的mRNA和蛋白水平隨之降低。這提示bcl-2可能是FoxM1的靶基因。進一步共轉(zhuǎn)染bcl-2啟動子質(zhì)粒和FoxM1/NC siRNA及內(nèi)參照pRL-TK質(zhì)粒至HL60細胞和K562細胞,雙螢光素酶活性檢測發(fā)現(xiàn)干擾FoxM1表達后,bcl-2啟動子活性降低。這說明FoxM1通過結(jié)合于bcl-2啟動子直接靶向調(diào)控bcl-2表達,見圖4。
5 急性髓系白血病初診患者FoxM1表達與Bcl-2表達呈正相關(guān)
用RT-qPCR方法和免疫熒光技術(shù)檢測14例AML-denovo患者、14例正常骨髓標本中FoxM1和Bcl-2的mRNA水平和蛋白表達。結(jié)果顯示,與正常健康對照相比,AML-denovo組患者骨髓標本中FoxM1 mRNA水平顯著升高,同時伴隨著Bcl-2 mRNA水平顯著升高,二者表達呈正相關(guān)。此外,免疫熒光結(jié)果顯示,AML-denovo組患者骨髓標本中Bcl-2蛋白表達明顯升高,表明Bcl-2在急性髓性白血病中表達升高,且與FoxM1表達呈正相關(guān),見圖5。
化學(xué)治療是急性髓系白血病的主要干預(yù)策略,但由于其在白血病細胞和正常細胞之間無選擇性,因此不可避免會傷害正常細胞,引起毒副作用,因此,針對AML發(fā)生發(fā)展中關(guān)鍵分子為靶點的精準靶向治療將成為AML治療的新趨勢。目前腫瘤的分子靶向治療已取得巨大進步,如酪氨酸激酶抑制劑、CD20單抗等分子靶向藥物已顯示良好臨床療效。然而,AML的發(fā)生發(fā)展是一個多因素、多階段、多步驟的過程,涉及到大量分子參與的復(fù)雜網(wǎng)絡(luò)調(diào)控,現(xiàn)有的分子靶向藥物難以滿足臨床治療的需要[5-6]。因此,挖掘AML發(fā)生發(fā)展新的節(jié)點分子,確定其做為AML干預(yù)的潛在靶點具有十分重要的價值。
Figure 4. FoxM1 directly regulatedbcl-2 expression. A: RT-qPCR analysis of the mRNA expression of FoxM1 and Bcl-2 in the HL60 cells and K562 cells transfected withFoxM1 specific siRNA; B: the protein expression of FoxM1 and Bcl-2 in the HL60 cells and K562 cells transfected withFoxM1 specific siRNA determined by Western blot analysis; C:bcl-2 promoter activity withFoxM1 siRNA transfection in the HL60 cells and K562 cells. Luciferase activities were measured at 72 h and normalized byRenillaluciferase activity. Mean±SD.n=3.*P<0.05,**P<0.01vsNC siRNA.
圖4 FoxM1靶向調(diào)控bcl-2表達
本研究通過對AML初診患者、完全緩解患者、難治復(fù)發(fā)患者骨髓標本中FoxM1表達的測定,發(fā)現(xiàn)初診患者FoxM1水平較正常骨髓標本明顯增高,完全緩解患者下降,難治復(fù)發(fā)組FoxM1表達水平較初診患者升高更為明顯,提示FoxM1與AML發(fā)生發(fā)展及治療效果相關(guān)。由于FoxM1在健康人骨髓標本不表達,因此,F(xiàn)oxM1是干預(yù)AML的潛在靶標。
FoxM1主要通過調(diào)控細胞周期相關(guān)分子轉(zhuǎn)錄介導(dǎo)細胞異常增殖,從而參與腫瘤發(fā)生[7]。新近發(fā)現(xiàn)FoxM1與細胞凋亡有關(guān),如FoxM1可通過抑制蛋白激酶JNK活性發(fā)揮抗凋亡作用[8],蛋白酶體抑制劑Bortezomib處理人腫瘤細胞系FoxM1水平下調(diào)后,促進細胞凋亡[9]。我們以白血病細胞株HL60和K562為研究對象,用FoxM1特異性siRNA轉(zhuǎn)染HL60和K562細胞后,觀察到白血病細胞生長受抑、凋亡增加,表明抑制AML細胞FoxM1表達,不僅可抑制白血病細胞生長,還可以促進白血病細胞凋亡。凋亡抑制分子Bcl-2是細胞凋亡研究中最受關(guān)注的癌基因之一。研究發(fā)現(xiàn)AML中存在Bcl-2高表達,與AML凋亡受阻、對化療耐藥相關(guān),有IDH1和IDH1異常的AML細胞依賴Bcl-2的高表達,用Bcl-2抑制劑治療效果好[10]。鑒于FoxM1和Bcl-2在AML中都有重要作用,為此,我們研究AML細胞中Bcl-2的表達,發(fā)現(xiàn)用特異性siRNA抑制FoxM1表達時Bcl-2的表達同步降低,進一步通過雙螢光素酶活性檢測觀察到FoxM1影響bcl-2啟動子活性。而在急性髓性白血病患者標本中也檢測到Bcl-2的表達升高,與FoxM1表達呈正相關(guān),表明FoxM1和Bcl-2共同參與AML的發(fā)生。因此,我們推測FoxM1通過靶向調(diào)控bcl-2的表達,抑制細胞凋亡和促進細胞增殖,從而促進AML發(fā)生發(fā)展;沉默F(xiàn)oxM1表達則可能靶向抑制bcl-2的表達進而干預(yù)AML發(fā)生。
Figure 5. Bcl-2 was over-expressed in AML and its expression was positively correlated with FoxM1 expression. A: the mRNA expression of FoxM1 and Bcl-2 in BM samples from the patients in different groups detected by RT-qPCR analysis; B: FoxM1 and Bcl-2 expression was positively correlated; C: the protein expression of Bcl-2 in BM samples from the patients in different groups determined by cellular immunofluorescence analysis. Mean±SD.n=14.*P<0.05vscontrol.
圖5 急性髓性白血病初診患者FoxM1表達與Bcl-2表達呈正相關(guān)
綜上所述,我們的研究表明FoxM1與AML發(fā)生發(fā)展相關(guān),沉默F(xiàn)oxM1可抑制bcl-2表達進而影響到白血病細胞的增殖和凋亡,此為探索基于FoxM1為靶點干預(yù)AML發(fā)生發(fā)展提供了初步依據(jù)。
[1] Zhang X, Zeng J, Zhou M, et al. The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia[J]. Mol Cancer, 2012, 11(1):56-67.
[2] Wierstra I. The transcription factor FoxM1 (Forkhead box M1): proliferation-specific expression, transcription factor function, target genes, mouse models, and normal biological roles[J]. Adv Cancer Res, 2013, 118:97-398.
[3] Buchner M, Park E, Geng H, et al. Identification of FoxM1 as a therapeutic target in B-cell lineage acute lymphoblastic leukaemia[J]. Nat Commun, 2015, 6: 6471.
[4] Nakamura S, Hirano I, Okinaka K, et al. The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia[J]. Carcinogenesis, 2010, 31(11):2012-2021.
[5] D?hner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet[J]. Blood, 2010, 115(3):453-474.
[6] L?wenberg B, Beck J, Graux C, et al. Gemtuzumab ozogamicin as postremission treatment in AML at 60 years of age or more: results of a multicenter phase 3 study[J]. Blood, 2010, 115(13):2586-2591.
[7] 陳 謹,周敏然,孫 婷,等. 抑制白血病K562細胞FoxM1表達可增強高三尖杉酯堿的藥物敏感性[J]. 中國病理生理雜志,2015,31(11):1928-1932.
[8] Liu Y, Chen X, Gu Y, et al. FoxM1 overexpression is associated with cisplatin resistance in non-small cell lung cancer and mediates sensitivity to cisplatin in A549 cells via the JNK/mitochondrial pathway[J]. Neoplasma, 2015, 62(1):61-71.
[9] Gartel AL. Suppression of the oncogenic transcription factor FoxM1 by proteasome inhibitors[J]. Scientifica (Cairo), 2014, 2014:596528.
[10] Chan SM, Thomas D, Corces-Zimmerman MR, et al. Isocitrate dehydrogenase 1 and 2 mutations induce Bcl-2 dependence in acute myeloid leukemia[J].Nat Med, 2015, 21(2):178-184.
(責(zé)任編輯: 盧 萍, 羅 森)
FoxM1 promotes development of AML by regulating Bcl-2 expression
CHEN Jin1, WANG Xiao-ming2, ZHOU Min-ran2, SUN Ting2, CHEN Zhong-min3, CHEN Chun-yan2
(1AnhuiMedicalCollege,Hefei230601,China;2DepartmentofHematology,QiluHospital,ShandongUniversity,Jinan250012,China;3SchoolofPharmacyandBioengineering,ChongqingUniversityofTechnology,Chongqing400054,China.E-mail:chency@sdu.edu.cn)
AIM: To investigate the role of Forkhead box M1 (FoxM1) and B-cell leukemia/lymphoma-2 (Bcl-2) in the pathogenesis of acute myeloid leukemia (AML). METHODS: RT-qPCR and immunofluorescence analysis were used to determine the expression of FoxM1 at mRNA and protein levels in AML-denovopatients, AML-complete remission (CR) patients, AML-refractoriness and relapse (RR) patients and healthy controls. HL60 cells and K562 cells were transfected withFoxM1 siRNA. The cell proliferation was detected by cell proliferation assay and colony formation assay on soft agar, and the cell apoptosis was determined by flow cytometry. The expression of FoxM1 and Bcl-2 at mRNA and protein levels was detected by RT-qPCR and Western blotting. The activity ofbcl-2 promoter was examined by luciferase reporter assay with FoxM1 targetting. RESULTS: FoxM1 expression level in the AML-denovopatients was significantly higher than that in the healthy controls. As compared with the AML-denovopatients, FoxM1 expression in the AML-CR patients was reduced, and the FoxM1 expression level was the highest in the AML-RR patients. FoxM1 expression was inhibited in the HL60 cells and K562 cells transfected withFoxM1 siRNA. Transfection withFoxM1 siRNA in the HL60 cells and K562 cells inhibited the proliferation as compared with NC siRNA transfection, and impaired the colony formation ability. On the contrary, transfection withFoxM1 siRNA promoted the cell apoptosis. FoxM1 regulatedbcl-2 expression positively. CONCLUSION: FoxM1 promotes the development of AML by regulatingbcl-2 expression. Silencing ofFoxM1 expression suppresses cell proliferation and promotes cell apoptosis. FoxM1 is a potential target for AML treatment.
Acute myeloid leukemia; Forkhead box M1; Bcl-2
1000- 4718(2016)08- 1383- 06
2016- 02- 17
2016- 07- 05
國家自然科學(xué)基金資助項目(No. 81170514)
R730.23
A
10.3969/j.issn.1000- 4718.2016.08.007
雜志網(wǎng)址: http://www.cjpp.net
△通訊作者 Tel: 0531-82169867; E-mail: chency@sdu.edu.cn