本文選題：貧血 + 再生障礙性　； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：目的探討重型/極重型再生障礙性貧血(SAA/VSAA)患者免疫抑制治療(IST)前應(yīng)用粒細胞集落刺激因子(G-CSF)治療的中性粒細胞反應(yīng)(ANC反應(yīng)),對于IST早期療效的預(yù)測價值。方法回顧性分析我中心呢2005年9月至2011年11月間接受一線IST治療,并于IST前接受G-CSF治療的共125例SAA/VSAA患者臨床資料。G-CSF治療第n天內(nèi)最大△ANC≥0.5×109/L定義為G-CSF治療有ANC反應(yīng),0.5×109/L定義為無反應(yīng)。首先分別總結(jié)IST前應(yīng)用G-CSF治療有ANC反應(yīng)組和無ANC反應(yīng)組的IST近期療效,并對二者進行相關(guān)性分析。進而為細化、優(yōu)化這一預(yù)測指標,分別分析G-CSF治療第5天、7天、10天、13天、14天、16天和21天有無ANC反應(yīng)與IST早期療效的關(guān)系,尋找預(yù)測IST早期療效的最佳ANC反應(yīng)時間。進而,繪制G-CSF治療后10天內(nèi)ANC增高程度(△ANC)預(yù)測IST療效的ROC曲線,探索G-CSF治療的ANC反應(yīng)預(yù)測IST早期療效的最佳△ANC界限值。結(jié)果IST前接受G-CSF治療,獲得ANC反應(yīng)組IST后3個月血液學(xué)反應(yīng)(HR)為49%,6個月HR為61.2%,均顯著高于無ANC反應(yīng)組(3個月HR 28.9%,P=0.023;6個月HR40.8%,P=0.026)。G-CSF治療5天,7天內(nèi)ANC反應(yīng)尚不能提示IST后3個月及6個月HR,而以G-CSF治療10 d內(nèi)△ANC≥0.5×109/L作為ANC反應(yīng)界值預(yù)測IST后3個月及6個月療效最佳,ROC曲線下面積最大,且與更長時間應(yīng)用G-CSF治療ANC反應(yīng)來預(yù)測IST后HR的效能接近。G-CSF治療10 d內(nèi)獲得ANC反應(yīng)為IST后3個月療效的獨立預(yù)后因素(P=0.004),但并非IST后6個月療效的獨立預(yù)后因素。G-CSF治療ANC反應(yīng)組患者5年總生存率顯著高于無反應(yīng)組[(92.8±4.0)%對(69.5±6.5)%,P=0.025]。結(jié)論IST前G-CSF治療的ANC反應(yīng)可提示骨髓殘存造血,是方便、實用的預(yù)測IST早期療效及長期生存的指標。目的分析不同中性粒細胞計數(shù)(ANC)閾值的極重型再生障礙性貧血(VSAA)患者接受一線免疫抑制治療(IST)的早期血液學(xué)反應(yīng)(HR)和長期生存情況。方法回顧性分析我中心2006年1月至2011年10月期間連續(xù)收治的145例接受一線r-ATG聯(lián)合環(huán)孢素IST的VSAA患者臨床資料。評估不同ANC閾值下VSAA患者的早期死亡、早期HR、長期生存情況,并以受試者操作特征曲線(ROC曲線)法優(yōu)化ANC預(yù)測VSAA患者HR的臨界值。并進行VSAA患者HR及生存相關(guān)因素分析。結(jié)果VSAA患者IST后3個月HR率為35.9%,6個月HR率為46.9%,5年OS率為75.0±3.7%,均明顯劣于SAA(IST后3個月HR率為65.5%,P=0.000;6個月HR 率為 74.5%,P=0.000;5 年 OS 率為 92.5±2.6%,P=0.000)。且 VSAA 早期死亡率高達8.3%(SAA患者僅為2.7%,P=0.062)。設(shè)定VSAAIST前ANC閾值分別≤0.15×109/L、≤0.10×109/L和≤0.05×109/L,相應(yīng)群組患者早期死亡率分別為9.2%(12/130)、9.9%(11/111)和 13.4%(11/82),即 12 例早期死亡的 VSAA 中有 11 例(91.7%)IST 前 ANC≤0.05 X 109/L;IST 后 3 個月 HR 率分別為 33.8%(44/130)、27.9%(31/111)和 22.0%(18/82);6 個月 HR 率分別為 43.8%(57/130)、39.6%(44/111)和 34.1%(28/82);5 年 OS 率分別為 73.1 ±4.0%、70.3±4.4%和62.5±5.4%;5 年 EFS 率分別為 48.3±4.5%、45.1 ±4.8%和 42.3±5.5%。以 0.05×109/L為ANC界值預(yù)測VSAA患者IST后6個月HR和12個月CR+GPR最佳,預(yù)測IST后6個月HR靈敏度為58.8%、特異度為70.1%;預(yù)測12個月良好HR靈敏度60.9%、特異度72.4%。ANC≤0.05×109/L的VSAA患者早期死亡率為11/82(13.4%),占VSAA 早期死亡患者的 91.7%(11/12);IST 后 3 個月 HR 率為 18/82(22.0%);6個月 HR 率為 28/82(34.1%);5 年 OS 率為(62.5±5.4)%;5 年 EFS 率為(42.3±5.5)%。結(jié)論ANC≤0.05×109/L的VSAA患者接受一線IST早期死亡率高、療效差。目的ASXL1基因突變常見于各髓系腫瘤和骨髓衰竭性疾病的惡性轉(zhuǎn)化,且為發(fā)生白血病轉(zhuǎn)化和不良預(yù)后的相關(guān)因素。本研究旨在探討ASXL1基因突變對人白血病細胞功能的影響及其作用的分子生物學(xué)機制,為ASXL1基因突變在髓系腫瘤性疾病中的作用提供更多依據(jù),為個體化和靶向治療提供更多的線索。方法應(yīng)用CRISPR/Cas9基因編輯系統(tǒng)構(gòu)建ASXL1敲除的U937人白血病細胞系。應(yīng)用流式細胞術(shù)進行GFP陽性單個細胞分選并擴增為單個細胞來源的克隆。Sanger測序檢測各細胞系A(chǔ)SXL1基因突變情況,篩選出含ASXL1雜合及純和突變的克隆細胞系。應(yīng)用瑞士吉姆薩染色分析細胞形態(tài),G帶進行細胞染色體核型分析,應(yīng)用5-氟尿嘧啶誘導(dǎo)細胞凋亡,研究其對各細胞系增殖抑制和凋亡誘導(dǎo)情況,應(yīng)用PMA誘導(dǎo)細胞分化并比較ASXL1突變細胞系與野生型分化情況異同,應(yīng)用流式細胞術(shù)測定細胞周期、細胞凋亡和細胞分化等。進行RNA測序篩選在ASXL1突變克隆中差異性表達的基因,并進而進行信號通路分析、基因集分析等,研究ASXL1突變對下游基因表達的影響,應(yīng)用逆轉(zhuǎn)錄定量PCR和蛋白印跡法驗證差異性表達基因。結(jié)果應(yīng)用CRISPR/Cas9基因編輯技術(shù)結(jié)合單個細胞分選技術(shù)構(gòu)建單個細胞來源的人U937細胞系,共獲得56個野生型細胞系(ASXL1基因野生型的U937單個細胞來源細胞系),另外17個突變型細胞系(6個含ASXL1雜合突變和11個含ASXL1純和突變),突變細胞系均含有共同的c.594insA(Ser199Glufsx55)位點突變,此突變基因能夠編輯提前終止編碼的截短蛋白。其中3個ASXL1雜合突變克隆(MT1,MT2和MT3),3個ASXL1純和突變克隆(MT11,MT12和MT13),2個轉(zhuǎn)染后的ASXL1野生型克隆(WT1和WT2),以及未經(jīng)轉(zhuǎn)染的親代細胞系WTblk用于進一步細胞功能及RNA測序等實驗。整體來看,各個單細胞克隆之間異質(zhì)性較大,但野生組與ASXL1雜合突變組、ASXL1純和突變組,在細胞增殖,凋亡,細胞周期分布,5-氟尿嘧啶誘導(dǎo)的細胞增殖抑制和誘導(dǎo)細胞凋亡方面并無顯著差異。而ASXL1雜合突變組和ASXL1純和突變組相對于野生組均表現(xiàn)出對于PMA誘導(dǎo)的單核細胞向吞噬細胞分化能力顯著降低,表現(xiàn)為PMA誘導(dǎo)分化后96小時,CD11陽性細胞比例及CD11中位熒光指數(shù)均顯著低于野生組。RNA測序及信號通路和基因集分析顯示,ASAL1雜合及純和突變克隆,髓系分化相關(guān)基因通路整體表達下降,其中與髓系分化相關(guān)的基因CYBB和CLEC5A表達水平顯著降低。同時,與細胞存活相關(guān)的基因集包括干擾素a反應(yīng)通路、MYC靶基因通路和STAT3靶基因通路等廣泛下調(diào)。此外,染色體核型分析顯示相對于未經(jīng)轉(zhuǎn)染的親代細胞系WTblk,轉(zhuǎn)染后的ASXL1野生型克隆并未出現(xiàn)額外的染色體核型異常,而ASXL1突變克隆不同程度得獲得額外染色體異常,在4個檢測的ASXL1突變克隆中有3個獲得11號染色體長臂缺失。結(jié)論ASXL1基因突變通過下調(diào)與髓系分化密切相關(guān)的CYBB和CLEC5A等基因干擾髓系分化。此外,ASXL1突變廣泛干擾與細胞生存相關(guān)的基因通路,并增加人白血病細胞的遺傳不穩(wěn)定性。我們應(yīng)用CRISPR/Cas9基因編輯技術(shù)建立的ASXL1突變型人U937白血病細胞系可作為研究ASXL1作用分子機制的模型,并未未來研究ASXL1相關(guān)的髓系惡性腫瘤相關(guān)分子機制的研究提供可能。
[Abstract]:Objective to explore the predictive value of neutrophils response (ANC reaction) for the early application of granulocyte colony-stimulating factor (G-CSF) therapy for severe / severe aplastic anemia (SAA/VSAA) patients with granulocyte colony-stimulating factor (G-CSF) treatment. Methods a retrospective analysis was made to receive first-line IST treatment between September 2005 and November 2011. The clinical data of 125 cases of SAA/VSAA patients treated with G-CSF before IST,.G-CSF was defined as G-CSF therapy with ANC reaction in n days, and 0.5 x 109/L was defined as no response. First, the short-term efficacy of G-CSF treatment group and non reactive group before IST was summarized, and the correlation analysis of two cases was analyzed. And then to optimize this prediction index, we analyzed the relationship between G-CSF treatment for fifth days, 7 days, 10 days, 13 days, 14 days, 16 days and 21 days, in order to find the best time to predict the early effect of IST, to find the best time to predict the early effect of IST, and then to draw the ROC curve of ANC increase (delta ANC) in the 10 days after G-CSF treatment (delta ANC) to predict the IST curative effect, and explore G-C. The ANC reaction of the SF treatment predicted the best Delta ANC limit for the early effect of IST. Results before IST, G-CSF treatment was accepted, and the hematological reaction (HR) was 49% in ANC reaction group and 61.2% for HR in 6 months after IST, which was significantly higher than that without ANC reaction group (3 months HR 28.9%, 6 months%, 6 months). T 3 months and 6 months HR, and G-CSF treatment 10 d Delta ANC > 0.5 x 109/L as ANC reaction to predict IST 3 months and 6 months of the best effect, the maximum area under the ROC curve, and longer time application of G-CSF ANC reaction to predict the effectiveness of IST after IST treatment 10 The post factor (P=0.004), but not the independent prognostic factor of the 6 months after IST, the total 5 year survival rate in the ANC reaction group was significantly higher than that in the non reactive group [(92.8 + 4)% to (69.5 + 6.5)%. P=0.025]. conclusion ANC reaction before IST G-CSF therapy could suggest bone marrow remnant hematopoiesis, which is convenient and practical to predict the early curative effect and long-term survival of IST. Objective. Objective to analyze the early hematological response (HR) and long-term survival of extremely severe aplastic anemia (VSAA) patients with different neutrophils counts (ANC) threshold. Methods a retrospective analysis of 145 consecutive patients receiving first-line r-ATG associated cyclosporine from January 2006 to October 2011 The clinical data of VSAA patients with vegetarian IST. Evaluate the early death of VSAA patients under different ANC thresholds, early HR, long-term survival, and optimize the critical value of ANC prediction of HR in VSAA patients by the operator's operation characteristic curve (ROC curve) method. The HR and survival correlation factors of VSAA patients were analyzed. Results the rate of VSAA patients was 35.9%, 6 months after 3 months of VSAA. The rate of OS was 46.9%, and the rate of 5 years was 75 + 3.7%. The rate of HR was significantly inferior to that of SAA (3 months after IST, HR rate was 65.5%, P=0.000; HR rate in 6 months was 74.5%, P=0.000; OS rate of 5 years was 92.5 + 2.6%, P=0.000). And the early VSAA mortality was up to 8.3% (SAA patients only 2.7%, P=0.062). The early mortality of the corresponding group was 9.2% (12/130), 9.9% (11/111) and 13.4% (11/82), that is, 11 cases (91.7%) of 12 early deaths (91.7%) IST ANC < 0.05 X 109/L, and HR rates of 33.8% (44/130), 27.9% (31/111) and 22%, respectively, 3 months after IST, 6, 27.9% and 22% respectively. % (28/82); the rate of OS in 5 years is 73.1 + 4%, 70.3 + 4.4% and 62.5 + 5.4%, and 5 years EFS rate is 48.3 + 4.5%, 45.1 + 4.8% and 42.3 + 109/L as ANC boundary value prediction for VSAA patients IST. .9%, the early mortality of VSAA patients with specific degree of 72.4%.ANC < 0.05 x 109/L was 11/82 (13.4%), accounting for 91.7% (11/12) of early death in VSAA; HR rate in 3 months after IST was 18/82 (22%); 6 months HR rate was 28/82 (34.1%); 5 years was (62.5 + 5.4)%, and the rate was (42.3 + 5.5)% in 5 years. The early mortality of IST is high and the curative effect is poor. Objective ASXL1 gene mutation is common in the malignant transformation of various myeloid tumors and bone marrow failure diseases, and it is the related factors of leukemia transformation and poor prognosis. The aim of this study is to explore the effect of ASXL1 gene mutation on the function of human leukemia cells and the molecular biological mechanism of ASXL1 basis. It provides more evidence for the role of mutation in myeloid tumor diseases and provides more clues for individualized and targeted therapy. Methods the CRISPR/Cas9 gene editing system was used to construct a ASXL1 knockout U937 human leukemia cell line. Flow cytometry was used to select and amplify the single cell of GFP positive single cells to be cloned.Sange R sequencing was used to detect the mutation of ASXL1 gene in each cell line, screening the clones containing ASXL1 heterozygous and pure and mutated cells. Using Swiss Giemsa staining to analyze cell morphology, G band for cell chromosome karyotype analysis, 5- fluorouracil induced apoptosis, to study the proliferation inhibition and apoptosis induction of each cell line, and to apply PMA inducement. Differentiation and comparison of ASXL1 mutant cell lines and wild type differentiation, cell cycle, cell apoptosis and cell differentiation were measured by flow cytometry. RNA sequencing was used to screen differentially expressed genes in ASXL1 mutant clones, and then signal pathway analysis, gene set analysis, and so on, to study the ASXL1 mutation on the lower swimming base. The differential expression gene was verified by reverse transcriptase PCR and Western blot. Results the human U937 cell line of single cell source was constructed by CRISPR/Cas9 gene editing technique and single cell sorting technique, and 56 wild type cell lines (U937 single cell source cell lines of ASXL1 gene wild type) were obtained. 17 mutant cell lines (6 ASXL1 heterozygous mutations and 11 ASXL1 pure and mutants) with a common c.594insA (Ser199Glufsx55) mutation, which can edit the truncated protein of the early termination code. 3 ASXL1 heterozygous clones (MT1, MT2 and MT3), 3 ASXL1 pure and mutant clones (MT11, MT12, and clones) MT13), 2 transfected ASXL1 wild type clones (WT1 and WT2) and untransfected parent cell line WTblk were used for further cell function and RNA sequencing experiments. Overall, the heterogeneity of each single cell clone was larger, but the wild group and the ASXL1 heterozygous mutation group, ASXL1 pure and mutant group, in cell proliferation, apoptosis, cell cycle distribution, There was no significant difference in the inhibition of cell proliferation induced by 5- fluorouracil and the induction of apoptosis. The ASXL1 heterozygous mutation group and the ASXL1 pure and mutation group showed a significant reduction in the differentiation ability of the monocyte to the phagocytic cells induced by PMA, 96 hours after PMA induced differentiation, the proportion of CD11 positive cells and CD11. The median fluorescence index was significantly lower than that in the wild group.RNA sequencing and signal transduction and gene set analysis. ASAL1 heterozygosity and pure and mutant clones, the overall expression of the myeloid differentiation related gene pathway decreased, and the expression level of CYBB and CLEC5A related to myeloid differentiation was significantly reduced. The perturbed a pathway, the MYC target gene pathway and the STAT3 target gene pathway were widely downregulated. In addition, the chromosome karyotype analysis showed that the ASXL1 wild type clones had no extra chromosome karyotype abnormalities compared to the untransfected parent cell line WTblk, and the ASXL1 mutant clones had to obtain extra chromosomal abnormalities in different degrees, at 4 3 of the ASXL1 mutant clones detected to be detected in the long arm deletion of chromosome 11. Conclusion the ASXL1 gene mutation interferes with the myeloid differentiation by reducing the CYBB and CLEC5A genes closely related to the myeloid differentiation. Furthermore, the ASXL1 mutation widely interferes with the gene pathway associated with cell survival and increases the genetic instability of the human leukemia cells. The ASXL1 mutant human U937 leukemia cell line established by CRISPR/Cas9 gene editing technique can be used as a model to study the molecular mechanism of ASXL1, and it is not possible to study the molecular mechanism of ASXL1 related myeloid malignancies in the future.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R556.5

【相似文獻】

相關(guān)期刊論文 前10條

1 邵彬,高彥榮;骨髓增生異常綜合征的免疫抑制治療[J];醫(yī)學(xué)綜述;2005年05期

2 錢新華;;兒童獲得性再生障礙性貧血的免疫抑制治療[J];實用兒科臨床雜志;2006年03期

3 梁冬梅;聶大年;尹松梅;李益清;謝雙鋒;王秀菊;;強化免疫抑制治療重型再生障礙性貧血18例[J];實用醫(yī)學(xué)雜志;2006年23期

4 鄒緯;馮曉勤;李春富;何岳林;吳學(xué)東;石磊;李娜;;外周血T淋巴細胞亞群比例對免疫抑制治療急性再生障礙性貧血患兒預(yù)后預(yù)測的意義[J];實用兒科臨床雜志;2010年03期

5 王婉玲;黃琰;韓效林;吳隼;李敬東;;聯(lián)合免疫抑制治療重型再生障礙性貧血[J];實用兒科臨床雜志;2011年09期

6 蘇爾云;方玉華;陳輝樹;;中西醫(yī)結(jié)合治療62例免疫抑制治療無效的重型再生障礙性貧血臨床觀察[J];中國中西醫(yī)結(jié)合雜志;2012年12期

7 曹九芳;李曉明;;重型再生障礙性貧血的免疫抑制治療現(xiàn)狀[J];華西醫(yī)學(xué);2014年04期

8 李志芹;賈國榮;賀其圖;馬宏杰;云雁;;聯(lián)合免疫抑制治療重型再生障礙性貧血[J];內(nèi)蒙古醫(yī)學(xué)雜志;2006年11期

9 鄭良宏;;聯(lián)合免疫抑制治療小兒重型再生障礙性貧血研究[J];當(dāng)代醫(yī)學(xué);2013年09期

10 葉德富;;慢性原發(fā)性血小板減少性紫癜的免疫抑制治療[J];國外醫(yī)學(xué)參考資料(內(nèi)科學(xué)分冊);1975年05期

相關(guān)會議論文 前6條

1 楊世隆;宋華;湯永民;趙芬英;潘斌華;石淑文;徐衛(wèi)群;沈紅強;錢柏芹;羅春芳;;強烈免疫抑制治療兒童再生障礙性貧血的臨床研究[A];2006年浙江省血液病學(xué)學(xué)術(shù)年會論文匯編[C];2006年

2 楊世隆;宋華;湯永民;趙芬英;潘斌華;石淑文;徐衛(wèi)群;沈紅強;錢柏芹;羅春芳;;強烈免疫抑制治療兒童再生障礙性貧血的臨床研究[A];2007年浙江省兒科學(xué)、小兒外科學(xué)學(xué)術(shù)年會論文匯編[C];2007年

3 周郁鴻;郭宇;沈一平;葉寶東;胡致平;沈建平;;強化免疫抑制治療不同中醫(yī)病性重型再生障礙性貧血的療效觀察[A];2009年浙江省中醫(yī)藥學(xué)會血液病學(xué)術(shù)年會、浙江省中西醫(yī)結(jié)合學(xué)會血液病學(xué)術(shù)年會暨國家級中西醫(yī)結(jié)合血液病新進展繼續(xù)教育學(xué)習(xí)班論文匯編[C];2009年

4 楊世隆;宋華;湯永民;趙芬英;潘斌華;石淑文;徐衛(wèi)群;沈紅強;錢柏芹;羅春芳;;強烈免疫抑制治療兒童再生障礙性貧血的臨床研究[A];2006（第三屆）江浙滬兒科學(xué)術(shù)會議暨浙江省兒科學(xué)術(shù)年會論文匯編[C];2006年

5 楊世隆;宋華;湯永民;趙芬英;潘斌華;石淑文;徐衛(wèi)群;沈紅強;錢柏芹;羅春芳;;強烈免疫抑制治療兒童再生障礙性貧血的臨床研究[A];中華醫(yī)學(xué)會第十四次全國兒科學(xué)術(shù)會議論文匯編[C];2006年

6 趙洪雯;余榮杰;吳雄飛;劉宏;彭侃夫;;IgA腎病的免疫抑制治療評價[A];西南地區(qū)第12屆腎臟病學(xué)術(shù)會議暨貴州省醫(yī)學(xué)會腎臟病學(xué)分會2012年學(xué)術(shù)會議論文集[C];2012年

相關(guān)博士學(xué)位論文 前2條

1 武志潔;中性粒細胞與再生障礙性貧血免疫抑制治療療效關(guān)系的研究[D];北京協(xié)和醫(yī)學(xué)院;2017年

2 江怡;重度病理分級IgA腎病的免疫抑制治療研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

相關(guān)碩士學(xué)位論文 前5條

1 王書春;免疫抑制治療兒童獲得性重型再生障礙性貧血[D];北京協(xié)和醫(yī)學(xué)院;2007年

2 唐莉;免疫抑制治療重型再生障礙性貧血的臨床研究[D];吉林大學(xué);2014年

3 蘇淑芳;72例兒童急性再生障礙性貧血臨床分析[D];鄭州大學(xué);2012年

4 楊文睿;應(yīng)用可溶性轉(zhuǎn)鐵蛋白受體預(yù)測重型再生障礙性貧血免疫抑制治療早期療效[D];北京協(xié)和醫(yī)學(xué)院;2013年

5 周少宏;根除幽門螺桿菌在兒童慢性特發(fā)性血小板減少性紫癜中的療效觀察[D];第四軍醫(yī)大學(xué);2010年

，

本文編號：2060956