【摘要】：目的:探討Ruxolitinib對JAK2V617F突變陽性骨髓增殖性腫瘤細(xì)胞內(nèi)基質(zhì)金屬蛋白酶調(diào)控的影響。觀察Ruxolitinib對人紅白血病HEL細(xì)胞(JAK2V617F突變陽性)增殖、凋亡、遷移及JAK2、MMP-2、MMP-9基因、蛋白表達(dá)水平的影響。探討Ruxolitinib對HEL細(xì)胞是否存在抑制增殖、遷移,誘導(dǎo)凋亡作用,以及對骨髓增殖性腫瘤原代細(xì)胞及HEL細(xì)胞抑制JAK2、MMP-2、MMP-9蛋白、基因表達(dá)的作用,為Ruxolitinib應(yīng)用于臨床治療骨髓增殖性腫瘤提供理論依據(jù)。方法:1臨床資料:保定市第一醫(yī)院在2012年1月至2015年12月收治的40例初診JAK2V617F突變陽性(排除MPLW515K/L及CALR突變)的MPN患者納入研究(MPN組),男18例、女22例,中位年齡59(34~72)歲,真性紅細(xì)胞增多癥(PV)13例,原發(fā)性血小板增多癥(ET)10例,原發(fā)性骨髓纖維化PMF 17例,以15名健康志愿者作為對照組,其中男8名、女7名,中位年齡55(36~78)歲。所選取的患者診斷均符合《血液病診斷及療效標(biāo)準(zhǔn)》[1],患者均簽署了知情同意。本研究得到保定市第一醫(yī)院倫理委員會的批準(zhǔn)。2基礎(chǔ)細(xì)胞實驗分組選用人紅白血病HEL細(xì)胞株(JAK2V617F突變陽性)。實驗分為:Ruxolitinib處理組、對照組。對照組以含10%新生牛血清的RPMI1640培養(yǎng)基培養(yǎng)。Ruxolitinib處理組以含有不同濃度的Ruxolitinib 10%新生牛血清RPMI1640培養(yǎng)基培養(yǎng)。Ruxolitinib的終濃度分別為(0 nmol/L,50nmol/L,100 nmol/L,250 nmol/L,500 nmol/L,1000 nmol/L)。3應(yīng)用實時熒光定量PCR檢測MPN患者JAK2V617F突變量、MMP-2、MMP-9的m RNA表達(dá)。4采用免疫組化檢測患者骨髓病理組織p-JAK2、MMP-2、MMP-9蛋白的表達(dá)。5 Cell Counting Kit(CCK-8)方法,檢測Ruxolitinib作用后HEL細(xì)胞增殖抑制情況。6流式細(xì)胞術(shù),檢測18例MPN Ruxolitinib作用24h后細(xì)胞CD34+表達(dá)的影響。7 Transwell小室實驗,檢測Ruxolitinib作用24h后MPN細(xì)胞、HEL細(xì)胞遷移的影響8采用蛋白印跡(Western blot)技術(shù)檢測Ruxolitinib作用于MPN細(xì)胞、HEL細(xì)胞后,各組細(xì)胞p-JAK2、MMP-2、MMP-9蛋白的表達(dá)情況。結(jié)果:1應(yīng)用熒光定量聚合酶鏈反應(yīng)(Quantitative Real-time PCR,q PCR)檢測MPN患者JAK2V617F突變量在26.8%-75.4%之間。Ruxolitinib對HEL細(xì)胞JAK2、MMP-2、MMP-9基因m RNA表達(dá)的影響:不同濃度(0nmol/l,50 nmol/l,100 nmol/l,250 nmol/l,500 nmol/l,1000 nmol/L)Ruxolitinib處理HEL細(xì)胞48 h后,JAK2基因表達(dá)分別為(0.431±0.043)、(0.362±0.032)、(0.254±0.031)、(0.181±0.022)、(0.143±0.022)、(0.143±0.022);MMP-2基因表達(dá)分別為(0.397±0.031)、(0.362±0.030)、(0.235±0.023)、(0.188±0.018)、(0.155±0.010)、(0.098±0.009);MMP-9基因表達(dá)分別為(0.321±0.032)、(0.252±0.025)、(0.245±0.024)、(0.215±0.025)、(0.155±0.018)、(0.098±0.009)。JAK2、MMP-2、MMP-9 m RNA表達(dá)水平均隨Ruxolitinib濃度的增加而逐漸減低(P0.001)。2免疫組織化學(xué)檢測結(jié)果顯示:MPN組p-JAK2、MMP-2、MMP-9蛋白表達(dá)均高于對照組[(78.56±24.55)%對(41.59±17.29)%、(48.25±18.74)%對(22.79±13.89)%、(53.29±19.28)%對(15.56±14.96)%,P值均0.05]。Spearman相關(guān)分析顯示MMP-2、MMP-9蛋白水平與JAK2V617F突變量正相關(guān)(r=0.526,P0.05;r=0.543,P0.05)]。3 CCK-8結(jié)果顯示:不同濃度(0nmol/l,50 nmol/l,100 nmol/l,250nmol/l,500 nmol/l,1000 nmol/L)Ruxolitinib處理組HEL細(xì)胞的存活率,24h時分別為(76.10±3.09)%、(66.04±3.14)%、(60.06±2.71)%、(59.27±2.86)%、(57.86±2.32)%;48h時分別為(70.14±3.39)%、(59.71±3.34)%、(52.80±2.61)%、(43.30±2.96)%(38.56±2.15)%;72h時分別為(55.83±3.43)%、(47.63±3.12)%、(36.43±2.98)%、(31.05±2.78)%、(14.48±2.86)%。與空白對照組相比,不同濃度Ruxolitinib在作用HEL細(xì)胞不同時間(24h、48h、72h)后,HEL細(xì)胞的活力明顯受到抑制,差別有統(tǒng)計學(xué)意義(P0.05)。延長Ruxolitinib作用的時間和增加藥物的劑量,細(xì)胞活力亦顯著減低(P0.05)。4 Ruxolitinib對JAK2V617F陽性MPN患者骨髓CD34+細(xì)胞影響:18例初診MPN患者中CD34+細(xì)胞比例高于對照組[(0.29±0.13)%對(0.23±0.05)%,t=0.286,P0.001],250 nmol/L Ruxolitinib干預(yù)組MPN患者CD34+細(xì)胞水平較低于未干預(yù)組[(0.29±0.13)%對(0.14±0.07)%,P0.001]。PV(5例)、ET(6例)、PMF(7例)患者250 nmol/L Ruxolitinib干預(yù)組CD34+細(xì)胞表達(dá)均低于未干預(yù)組[(0.18±0.05)對(0.26±0.05)、(0.19±0.04)對(0.25±0.05)、(0.14±0.08)對(0.35±0.08),P值均0.05]。5 Transwell遷移實驗觀察Ruxolitinib對MPN原代細(xì)胞及HEL細(xì)胞遷移的影響:5 nmol/L Ruxolitinib作用MPN原代細(xì)胞24 h后遷移至下室細(xì)胞數(shù)少于無Ruxolitinib的空白組(154.7±27.5對320.3±67.3,t=13.47,P0.01)。5 nmol/L Ruxolitinib處理HEL細(xì)胞24 h后遷移至下室細(xì)胞數(shù)少于無Ruxolitinib對照組(70.7±10.5對135.3±16.7,t=13.89,P0.01)。6 Western blot檢測顯示:Ruxolitnib作用于HEL細(xì)胞48h后,空白對照組不同濃度(0nmol/l,50 nmol/l,100 nmol/l,250 nmol/l,500 nmol/l,1000nmol/L)Ruxolitnib作用后HEL細(xì)胞檢測p-JAK2蛋白相對表達(dá)(條帶的相對灰度比值)分別為(1.52±0.12)、(1.21.±0.10)、(1.12±0.09)、(0.98±0.07)、(0.45±0.04)、(0.15±0.01)。MMP-2蛋白相對表達(dá)分別為(0.53±0.04)、(0.42±0.03)、(0.31±0.03)、(0.27±0.02)、(0.23±0.01)、(0.19±0.01)。MMP-9蛋白相對表達(dá)分別為(0.78±0.06)、(0.65±0.04)、(0.54±0.04)、(0.30±0.02)、(0.27±0.02)、(0.23±0.01)。β-actin蛋白的表達(dá)在三組中無顯著差異。Ruxolitinib處理組p-JAK2、MMP-2、MMP-9蛋白的表達(dá)與空白對照組相比,均受到抑制(P0.05)。藥物濃度增加,蛋白表達(dá)量明顯減少,差異存在統(tǒng)計學(xué)意義(P0.05)。Western blot檢測Ruxolitinib對MPN原代細(xì)胞p-JAK2、MMP-2、MMP-9蛋白表達(dá)的影響:250 nmol/L Ruxolitinib作用48 h能明顯抑制MPN原代細(xì)胞p-JAK2、MMP-2、MMP-9表達(dá)。結(jié)果顯示,p-JAK2、MMP-2和MMP-9蛋白的表達(dá)明顯降低,在不同濃度的Ruxolitnib作用后。結(jié)論:1在JAK2V617F突變陽性初治組MPN患者中p-JAK2、MMP-2、MMP-9蛋白表達(dá)水平明顯高于對照組,應(yīng)用Ruxolitnib后MPN患者p-JAK2、MMP-2、MMP-9表達(dá)水平明顯降低。2應(yīng)用Ruxolitnib可明顯抑制HEL細(xì)胞增殖,并隨著Ruxolitnib濃度的增加時間的延長HEL細(xì)胞活力明顯下降。3體外應(yīng)用Ruxolitnib對MPN細(xì)胞CD34+表達(dá)水平明顯抑制作用。4應(yīng)用Ruxolitnib能下調(diào)HEL細(xì)胞JAK2、MMP-2、MMP-9 m RNA及其p-JAK2、MMP-2、MMP-9蛋白的表達(dá)水平,并隨Ruxolitnib的濃度增加而進(jìn)行性減低。5 Ruxolitnib對MPN原代細(xì)胞及HEL細(xì)胞遷移有明顯抑制作用。
[Abstract]:AIM: To investigate the effect of Ruxolitinib on the regulation of matrix metalloproteinases (MMPs) in JAK2V617F mutant positive myeloproliferative tumor cells. To observe the effect of Ruxolitinib on proliferation, apoptosis, migration, JAK2, MMP-2, MMP-9 gene and protein expression of human erythroleukemia HEL cells (JAK2V617F mutant positive). Inhibition of proliferation, migration, induction of apoptosis, and inhibition of JAK2, MMP-2, MMP-9 protein and gene expression in primary cells and HEL cells of myeloproliferative tumors provide theoretical basis for the clinical application of Ruxolitinib in the treatment of myeloproliferative tumors. Methods: 1 Clinical data: Baoding First Hospital from January 2012 to December 2015. Forty MPN patients with positive JAK2V617F mutation (excluding MPLW515K/L and CALR mutations) were included in the study (MPN group), including 18 males and 22 females, with a median age of 59 (34-72), 13 with polycythemia vera (PV), 10 with primary thrombocytosis (ET), 17 with primary myelofibrosis (PMF), and 15 healthy volunteers as control group. The study was approved by the ethical committee of Baoding First Hospital. 2 Basic cell experiment group was selected HEL cell line (JAK2V617F mutation positive). The experiment was divided into: Ruxolitini. The control group was cultured on RPMI1640 medium containing 10% bovine serum. The Ruxolitinib group was cultured on RPMI1640 medium containing 10% bovine serum of different concentrations. The final concentrations of Ruxolitinib were (0 nmol/L, 50 nmol/L, 100 nmol/L, 250 nmol/L, 500 nmol/L, 1000 nmol/L) respectively. 3 were determined by real-time fluorescence. The expression of JAK2, MMP-2 and MMP-9 was detected by immunohistochemistry. 5 Cell Counting Kit (CCK-8) assay was used to detect the proliferation inhibition of HEL cells after Ruxolitinib treatment. 6 Flow cytometry was used to detect the expression of MMP-2 and MMP-9 m RNA in 18 MPN Ruxolitinib treated HEL cells 24 hours later. The effect of Ruxolitinib on MPN cell migration was detected by Western blot. The expression of p-JAK2, MMP-2 and MMP-9 in MPN cells and HEL cells was detected by Western blot. Results: 1. Quantitative polymerase chain reaction (Quantita) was used. The mutations of JAK2V617F in MPN patients were 26.8% - 75.4%. The effects of Ruxolitinib on the expression of JAK2, MMP-2, MMP-9 gene m RNA in HEL cells were (0.431 [0.043], (0.043]) at different concentrations (0 nmol/l, 50 nmol/l, 100 nmol/l, 250 nmol/l, 500 nmol/l, 1000 nmol/L) Ruxolitinib for 48 hours. The expression of MMP-2 gene was (0.397 [(0.397 [(0.031), (0.362 [(0.362 [0.032), (0.235 [(0.235 [0.023), (0.188 [0.188 [0.018], (0.1885 [0.010.018, (0.155 [0.010 0 0.010 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.010), (0.143 [0.098 [0.098 [0.090.090.143 [0.143 [0.022]], (0.143 [0.14(0.025), (0.245 + 0.024), (0.215 + 0.025), (0.155 + 0.018), (0.098 + 0.0) 09). JAK2, MMP-2, and MMP-9 m RNA expression levels gradually decreased with the increase of Ruxolitinib concentration (P 0.001). 2 Immunohistochemical results showed that the expression of p-JAK2, MMP-2, and MMP-9 protein in MPN group was higher than that in control group [(78.56 24.55)% vs. (41.59 17.29)%, (48.25 18.74)% vs. (22.79 13.89)%, (53.29 (19.28)% vs. (15.56 14.96)%, P Spearman correlation analysis showed that MMP-2 and MMP-9 protein levels were positively correlated with JAK2V617F mutation (r = 0.526, P 0.05; r = 0.543, P 0.05). 3 CCK-8 results showed that the survival rates of HEL cells treated with different concentrations (0 nmol/l, 50 nmol/l, 100 nmol/l, 250 nmol/l, 500 nmol/l, 1000 nmol/L) Ruxolitinib were (76.10 (+3.09)%, (66.04 (+3.14)%, (60.14)% respectively at 24 h. 06 [(56.27 [2.71)%, (59.27 [2.86)%, (57.86 [2.32)%, (57.86 [2.32)%, (57.86 [2.32)%, (70.14 [3.39)%, (59.71 [3.34%, (59.71 [59.71 [3.34)%, (52.80 [2.61%, (52.80 [(52.80 [2.61)%, (43.30 [2.96%, (43.30 [38.30 [38.56 [2.56 [38.56 [2.56 [2.15)%, (38.56 [[38.56 [38.56 [ib acted on HEL cells at different time. After 24h, 48h, 72h, the activity of HEL cells was significantly inhibited, the difference was statistically significant (P 0.05). prolonging the time of action of Ruxolitinib and increasing the dosage of the drug, the cell viability was also significantly decreased (P 0.05). 4 Ruxolitinib on JAK2V617F positive MPN patients with bone marrow CD34 + cells: 18 patients with newly diagnosed MPN CD34 + cell ratio was higher than the control group [(0.05). The levels of CD34+ cells in MPN patients with 250 nmol/L Ruxolitinib intervention group were lower than those in non-intervention group [(0.29+0.13)% vs. (0.14+0.07)%, P 0.001]. The expression of CD34+ cells in PV (5 cases), ET (6 cases) and PMF (7 cases) patients with 250 nmol/L Ruxolitinib intervention group was lower than that in non-intervention group [(0.18+0.05)% vs. (0.24+0.07)%, P 0.001]. 5 Transwell migration assay was used to observe the effect of Ruxolitinib on the migration of MPN primary cells and HEL cells. The number of MPN primary cells migrated to the lower chamber after 24 hours of treatment with 5 nmol/L Ruxolitinib was less than that without Ruxolitinib (154.7+27.5 vs 320.3+67.3, t=13.47, P 0.01). The number of HEL cells migrated to the lower chamber after 24 hours of treatment with l/L Ruxolitinib was less than that of the control group without Ruxolitinib (70.7+10.5 vs 135.3+16.7, t=13.89, P 0.01). 6 Western blot assay showed that the effect of Ruxolitnib on HEL cells at different concentrations (0 nmol/l, 50 nmol/l, 100 nmol/l, 250 nmol/l, 500 nmol/l, 1000 nmol/L) was observed in the blank control group. The relative expression of p-JAK2 protein in HEL cells was (1.52 (+ 0.12), (1.21 (+ 0.10), (1.21 (+ (+0.10), (1.12 + (+0.10), (1.12 (+0.09), (0.12 (+0.09,,,, (0.98 +0.07,,,,, (0.98 +,,,,, (0.45 +,,,, (0.04,, (0.45 +,,,, (0.15 +0.01), (0.MMP-2 protein relative expression was (0.53 ((0.53 + (0.42 (0.42 +0.03,,, (0.Express differently The expression of P-JAK2, MMP-2 and MMP-9 protein in Ruxolitinib treatment group was inhibited compared with the blank control group (P 0.05). Drug concentration increased, protein expression decreased significantly, and the difference was statistically significant. Western blot analysis of Ruxolitinib on MPN primary cells p-JAK2, MMP-2, MMP-9 protein expression: 250 nmol/L Ruxolitinib 48 hours can significantly inhibit the expression of p-JAK2, MMP-2, MMP-9 in MPN primary cells. The results showed that the expression of p-JAK2, MMP-2 and MMP-9 protein was significantly reduced, after the effect of different concentrations of Ruxolitinib. The expression levels of p-JAK2, MMP-2 and MMP-9 in MPN patients with positive JAK2V617F mutation were significantly higher than those in the control group. The expression levels of p-JAK2, MMP-2 and MMP-9 in MPN patients with positive JAK2V617F mutation were significantly decreased after the application of Ruxolitnib. 3. Ruxolitnib significantly inhibited the expression of CD34+ in MPN cells in vitro. 4 Ruxolitnib could down-regulate the expression of JAK2, MMP-2, MMP-9m RNA and its p-JAK2, MMP-2 and MMP-9 proteins in HEL cells. Ruxolitnib significantly inhibited the migration of primary MPN cells and HEL cells.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.3

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