本文選題：急性淋巴細(xì)胞白血病 + 基因突變�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文

【摘要】：急性淋巴細(xì)胞白血病(Acute lymphoblastic leukemia,ALL)是造血干細(xì)胞或淋巴前體細(xì)胞發(fā)育停滯引起的惡性克隆性疾病,遺傳學(xué)異常在ALL的發(fā)病過程中扮演重要角色,不僅參與驅(qū)動疾病的發(fā)生,同時也與復(fù)發(fā)和疾病進(jìn)展相關(guān),可用于危險度分級和靶向治療。ALL是兒童最常見的惡性腫瘤,兒童ALL對常規(guī)化療反應(yīng)敏感,預(yù)后較好,5年無病生存率可達(dá)到90%。由于隨年齡增加預(yù)后不良的遺傳學(xué)異常更多見,且成人對化療的敏感性和治療效果都遠(yuǎn)不如兒童,成人ALL預(yù)后仍較差,總體生存率僅為30%-40%,白血病相關(guān)并發(fā)癥的發(fā)生率和復(fù)發(fā)風(fēng)險也都相對較高。而可應(yīng)用于ALL的靶向藥十分有限,目前只有針對BCR-ABL1的酪氨酸激酶抑制劑。因此進(jìn)一步了解ALL的遺傳學(xué)特征,發(fā)現(xiàn)可能導(dǎo)致疾病發(fā)生或促進(jìn)疾病進(jìn)展和復(fù)發(fā)的新的遺傳學(xué)變異,找到有效的新靶點和新的治療手段,是目前ALL治療研究的熱點。在前期工作中,本課題組利用全外顯子組測序的方法在4例成人B-ALL患者中檢測到體細(xì)胞獲得性JAK1突變,并在53例患者中進(jìn)行驗證,獲得三個新的JAK1雜合性突變,分別為S646P,A639G和P960S。明確JAK1突變在ALL中的作用,對豐富ALL的發(fā)病機制和發(fā)現(xiàn)新的治療靶點有著重要意義。JAK基因編碼一種非受體型酪氨酸激酶(JAK,Janus Kinase),包括JAK1、JAK2、JAK3和TYK2四個成員,通過JAK/STAT通路傳導(dǎo)細(xì)胞因子下游信號,調(diào)控靶基因的表達(dá),參與細(xì)胞增殖、分化和免疫調(diào)控等過程。JAKs激酶在造血生成中也有重要作用,研究發(fā)現(xiàn)一半以上的骨髓增殖性腫瘤(Myeloproliferative neoplasms,MPNs)患者存在JAK2V617F突變,經(jīng)體內(nèi)外功能實驗證實該突變是MPNs發(fā)生的一個關(guān)鍵因素,而且JAK1/2激酶抑制劑ruxolitinib已被FDA批準(zhǔn)用于中、高危骨髓纖維化的治療。在JAK2 V617F陰性的部分MPNs患者中也發(fā)現(xiàn)存在JAK2 12號外顯子上的其他突變,提示JAK2異常是MPNs發(fā)生的重要機制和有效的治療靶點�；谠贏LL中的新發(fā)現(xiàn),作為同家族成員的JAK1激酶是否也存在不可忽視的作用,是本研究關(guān)注的重點。為明確新發(fā)現(xiàn)的JAK1突變是否是ALL發(fā)生的一個重要機制,在本研究中重點對JAK1突變在ALL的發(fā)生頻率和與預(yù)后的相關(guān)性,以及在體外和體內(nèi)的惡性轉(zhuǎn)化功能進(jìn)行深入的探討研究。首先,本課題組再次收集152例ALL患者骨髓DNA樣本,由于前期發(fā)現(xiàn)的JAK1突變集中在14和21號外顯子,針對這兩個外顯子設(shè)計特異的引物,利用PCR擴增聯(lián)合sanger測序的方法,在2例患者中分別檢測到j(luò)ak1y652h和n973k突變。結(jié)合前期結(jié)果,在209例all患者中共檢測到5例jak1點突變,2例為t-all,3例為b-all。其中,y652h和p960s在cosmic中已有報道,n973k為dbsnp數(shù)據(jù)庫中已報道的snp(singlenucleotidepolymorphism),s646p和a639g為首次發(fā)現(xiàn)的突變位點,通過blast比對不同物種jak1蛋白氨基酸序列,發(fā)現(xiàn)5個突變所影響的殘基在進(jìn)化過程中具有高度保守性�？偨Y(jié)jak1突變在all中發(fā)生率約為2.4%,其中在t-all發(fā)生率為10.5%,明顯高于b-all中的發(fā)生率1.6%(p0.05)�；仡櫡治鐾蛔冴栃曰颊叩呐R床特征,觀察到5例患者中4例(80%)出現(xiàn)了早期或晚期復(fù)發(fā),且對化療耐藥,提示jak1突變可能與all的復(fù)發(fā)和耐藥存在一定相關(guān)性。隨后選擇3個突變位點(a639g、s646p和p960s)進(jìn)行體外功能實驗驗證,以已知的獲得功能性突變jak1v658f作為陽性對照,構(gòu)建插入野生型或突變型jak1的慢病毒表達(dá)載體plv-egfp(2a)puro,利用轉(zhuǎn)染試劑將質(zhì)粒轉(zhuǎn)入hek293v細(xì)胞中,通過westernblot檢測jak1突變對下游信號通路的影響,結(jié)果表明,jak1s646p、p960s和v658f突變能夠組成性激活jak1自身和下游stat3、erk1/2信號分子的磷酸化。通過慢病毒包裝、濃縮和純化,感染目的細(xì)胞,構(gòu)建穩(wěn)定表達(dá)野生型或突變型jak1的baf3和nalm-6細(xì)胞系,利用cck-8檢測細(xì)胞增殖和生長曲線,提示s646p和v658f突變能導(dǎo)致baf3細(xì)胞不依賴細(xì)胞因子生長,臺盼藍(lán)拒染結(jié)果提示s646p和v658f突變能抵抗撤去il-3引起的細(xì)胞凋亡,利用流式細(xì)胞術(shù)檢測細(xì)胞周期發(fā)現(xiàn),s646p突變組baf3細(xì)胞和nalm-6細(xì)胞s/g2期比例增加,促進(jìn)細(xì)胞的增殖。以上細(xì)胞學(xué)實驗結(jié)果證新發(fā)現(xiàn)的jak1s646p突變是一種獲得功能性突變,與jak1v658f作用類似,能夠?qū)е录?xì)胞發(fā)生惡性轉(zhuǎn)化和下游信號通路的過度活化。此外,在細(xì)胞學(xué)實驗中觀察了jak1突變對jak1/2激酶抑制劑ruxolitinib的敏感性。westernblot結(jié)果顯示ruxolitinib能抑制jak1自身和stat3、erk1/2蛋白的磷酸化。用不同濃度的ruxolitinib處理穩(wěn)定轉(zhuǎn)染的baf3細(xì)胞,cck-8檢測細(xì)胞增殖,結(jié)果提示ruxolitinib能夠抑制野生型和突變型baf3細(xì)胞的增殖,而對攜帶bcr-abl1突變的k562細(xì)胞無影響,表達(dá)s646p和v658f突變的baf3細(xì)胞對ruxolitinib的敏感性高于野生型和其他突變組,提示jak1基因可能成為有效的治療靶點。最后,為了研究表達(dá)s646p突變的baf3細(xì)胞在體內(nèi)的成瘤作用,將穩(wěn)定轉(zhuǎn)染空載體,野生型JAK1和S646P突變的BaF3細(xì)胞通過尾靜脈分別接種給三組Balb/c裸鼠。接種30天后,S646P組裸鼠外周血中白細(xì)胞計數(shù)和GFP陽性細(xì)胞比例明顯高于空載體和野生型對照組,在裸鼠處死后,觀察到S646P突變導(dǎo)致裸鼠肝、脾明顯增大,骨髓中原始細(xì)胞和GFP陽性細(xì)胞比例增高,提示S646P突變作為一種獲得功能性突變導(dǎo)致細(xì)胞發(fā)生惡性轉(zhuǎn)化,促進(jìn)其在體內(nèi)發(fā)生類惡性腫瘤樣浸潤和侵襲。以上結(jié)果證實在本實驗新發(fā)現(xiàn)的體細(xì)胞獲得性JAK1 S646P突變是一種獲得功能性突變,體外和體內(nèi)實驗中均證實存在惡性轉(zhuǎn)化能力,可能影響疾病的進(jìn)展和復(fù)發(fā),而且對JAK1/2激酶抑制劑敏感,可能會成為一個新的治療靶點�；谂R床和基礎(chǔ)研究的結(jié)果,將JAK1基因擴展至ALL患者分子學(xué)檢測中將有助于預(yù)后評估和靶向治療的發(fā)展,尤其是對于復(fù)發(fā)難治的患者,JAK抑制劑很可能會成為一種新的有效的治療手段。
[Abstract]:Acute lymphoblastic leukemia (ALL) is a malignant clonogenic disease caused by stagnation of hematopoietic stem cells or lymphoid cells. Genetic abnormalities play an important role in the pathogenesis of ALL. They are not only involved in the development of the disease, but also related to the recurrence and disease progression, which can be used for risk classification. And targeted therapy.ALL is the most common malignant tumor in children. Children's ALL is sensitive to conventional chemotherapy and has a better prognosis. 5 years of disease free survival can reach 90%. due to the increase of genetic abnormalities with poor prognosis with age, and the sensitivity and treatment effect of adult to chemotherapy is far inferior to that of children, and the prognosis of adult ALL is still poor, and the overall survival of adult is still poor. The rate is only 30%-40%, the incidence of leukemia related complications and the risk of relapse are also relatively high. But the target drugs that can be used for ALL are very limited, and only BCR-ABL1 tyrosine kinase inhibitors are currently available. Therefore, further understanding of the genetic characteristics of ALL and the discovery of new remains that may lead to disease or to promote disease progression and recurrence In the previous work, we have detected the somatic cell acquired JAK1 mutations in 4 adult B-ALL patients by the method of exon sequencing in 4 cases of adult B-ALL, and obtained three new JAK1 heterozygosity mutations in the previous work. Do not define the role of JAK1 mutation in ALL for S646P, A639G and P960S.. It is important for the pathogenesis of ALL and the discovery of new therapeutic targets. The.JAK gene encodes a non receptor tyrosine kinase (JAK, Janus Kinase), including JAK1, JAK2, and four members, conducting downstream signals of cytokines through the pathway and regulating the downstream signals. The expression of target genes and the involvement of.JAKs kinase in cell proliferation, differentiation and immunoregulation also plays an important role in hematopoiesis. The study found that more than half of the Myeloproliferative neoplasms (MPNs) patients have JAK2V617F mutations. The mutation is a key cause of the occurrence of MPNs in vivo and in vivo. The JAK1/2 kinase inhibitor ruxolitinib has been approved by FDA for the use of FDA for the treatment of high-risk myelofibrosis. Other mutations in exons of JAK2 12 are found in the JAK2 V617F negative part of MPNs patients, suggesting that JAK2 abnormalities are an important mechanism for the occurrence of MPNs and an effective therapeutic target. Based on a new discovery in ALL, it is the same as the same Whether the JAK1 kinase of family members can not be ignored also is the focus of this study. In order to clarify whether the newly discovered JAK1 mutation is an important mechanism for the occurrence of ALL, this study focuses on the frequency of the JAK1 mutation in ALL and the correlation with the prognosis, as well as the malignant transformation function in vitro and in vivo. First, we once again collected 152 cases of ALL patients' bone marrow DNA samples. Due to the early detection of JAK1 mutations in 14 and 21 exons, specific primers were designed for the two exons and jak1y652h and n973k mutations were detected in 2 patients with PCR amplification and Sanger sequencing. In 209 patients with all, 5 cases of Jak1 point mutation were detected, 2 were T-ALL, 3 were b-all., y652h and p960s were reported in cosmic. N973k was the reported SNP (singlenucleotidepolymorphism) in the dbSNP database. The residues affected by the present 5 mutations have high conservatism in the evolution process. The incidence of Jak1 mutation in all is about 2.4%, and the incidence of T-ALL is 10.5%, obviously higher than that in B-ALL (P0.05). The clinical features of the mutant positive patients were reviewed, and 4 of the 5 patients (80%) had an early or late recurrence. The Jak1 mutation may be related to the recurrence and resistance of all. Then 3 mutation sites (a639g, s646p and p960s) were selected to perform in vitro functional test, and the known functional mutation jak1v658f was used as the positive control to construct the Lentivirus Expression Vector, which was inserted into the wild type or mutant Jak1 (plv-egfp). 2A) Puro, the plasmid was transferred into hek293v cells by using the transfection reagent, and the effect of Jak1 mutation on the downstream signal pathway was detected by Westernblot. The results showed that the jak1s646p, p960s and v658f mutations were capable of activating the Jak1 itself and the downstream STAT3, the phosphorylation of the erk1/2 signal molecules. The baf3 and Nalm-6 cell lines, which are stable expressing wild or mutant Jak1, are used to detect cell proliferation and growth curves by CCK-8, suggesting that s646p and v658f mutations can lead to baf3 cells not dependent on cytokine growth. Trypan blue staining results suggest that s646p and v658f mutations can resist apoptosis induced by withdrawal IL-3, using flow cytometry. The cell cycle test showed that the proportion of baf3 cells in s646p mutant group and s/g2 phase of Nalm-6 cells increased and promoted cell proliferation. The results of the above cytological test showed that the newly discovered jak1s646p mutation was a functional mutation, similar to jak1v658f, which could lead to the malignant transformation of cells and the over activation of downstream signal pathways. The sensitivity of the Jak1 mutation to the jak1/2 kinase inhibitor ruxolitinib was observed in the cytological experiment. The results showed that ruxolitinib could inhibit the Jak1 itself and the phosphorylation of the STAT3, erk1/2 protein. The stable transfected baf3 cells were treated with different concentrations of ruxolitinib, and the proliferation of the cells was tested CCK-8. The results suggest that ruxolitinib can inhibit the wild. The proliferation of type and mutant baf3 cells has no effect on the K562 cells carrying BCR-ABL1 mutation. The sensitivity of baf3 cells expressing s646p and v658f mutations to ruxolitinib is higher than that of the wild type and other mutation groups, suggesting that the Jak1 gene may be an effective target for treatment. Finally, to study the tumorigenesis of baf3 cells expressing s646p mutation in the body. After 30 days after inoculation, the proportion of white blood cells and GFP positive cells in the peripheral blood of S646P group nude mice was significantly higher than that of the empty and the wild type control group. After the death of the nude mice, the S646P mutation was observed to lead to the liver of nude mice after the death of the nude mice in nude mice. The BaF3 cells of the wild type JAK1 and the S646P mutant were inoculated to the nude mice respectively through the tail vein. The proportion of the spleen increased obviously, the proportion of the original cells in the bone marrow and GFP positive cells increased, suggesting that the S646P mutation, as a kind of functional mutation, resulted in malignant transformation of the cells and promoted the occurrence of malignant tumor like infiltration and invasion in the body. The results confirmed that the newly discovered somatic acquired JAK1 S646P mutation is a kind of acquisition in this experiment. Functional mutations, in vitro and in vivo, have confirmed the existence of malignant transformation ability, which may affect the progression and recurrence of the disease, and may be sensitive to JAK1/2 kinase inhibitors. It may become a new therapeutic target. Based on the results of clinical and basic studies, the extension of the JAK1 gene to ALL patients will contribute to the evaluation of the prognosis. And the development of targeted therapy, especially for relapsed and refractory patients, is likely to be a new and effective treatment for JAK inhibitors.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R733.71

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