本文選題：骨髓增殖性疾病 + MPL��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:骨髓增殖性腫瘤(MPN)是一類以一系或多系髓系細(xì)胞包括紅系、粒系和巨核系增殖為主要特征的克隆性造血干細(xì)胞疾病。其中經(jīng)典的三類MPN疾病包括真性紅細(xì)胞增多癥(PV)、原發(fā)性血小板增多癥(ET)、原發(fā)性骨髓纖維化(PMF),其臨床表現(xiàn)較為相似且可以相互轉(zhuǎn)化。目前發(fā)現(xiàn)JAK2 V617F、JAK2 exon12、MPL exon10以及CALR exon9為MPN中PV、ET、PMF三種疾病的主要分子病因。我們前期通過對771例確診的MPN病人進(jìn)行上述基因篩查時發(fā)現(xiàn)其中2例患者同時存在MPL基因的一種新突變類型(命名為MPL A497-L498ins4)。MPL基因編碼血小板生成素受體(TPOR)蛋白分子,TPOR的近跨膜區(qū)aa514-518無配體結(jié)合時可阻止受體二聚體相互靠近,進(jìn)而激活其下游如JAK2-STAT信號通路引起細(xì)胞增殖。以往研究報道的MPL基因突變類型主要為MPL W515點突變,并曾證實該突變可導(dǎo)致細(xì)胞產(chǎn)生自主促增殖能力,其機(jī)制可能是由于該突變引起TPOR的aa514-518區(qū)域結(jié)構(gòu)異常進(jìn)而導(dǎo)致細(xì)胞自主促增殖活性。MPL A497-L498ins4為插入4個氨基酸的突變,但并不在TPOR的aa514-518區(qū)域,那么該突變是否為引起患者M(jìn)PN發(fā)生的驅(qū)動基因呢?為此本課題組擬通過慢病毒基因轉(zhuǎn)導(dǎo)技術(shù)將該基因突變以及對照基因轉(zhuǎn)導(dǎo)小鼠原B淋巴細(xì)胞(Ba F3)中,從細(xì)胞學(xué)水平探討該突變具有自主促細(xì)胞的作用。方法:1.構(gòu)建MPL A497-L498ins4及對照基因的慢病毒表達(dá)載體用RT-PCR法獲取MPL A497-L498ins4及對照基因MPL W515L、MPL WT的CDS區(qū)全長,將PCR產(chǎn)物及慢病毒表達(dá)載體pCDH-MCS-T2A-copGFP-MSCV(簡寫為mscv)通過雙酶切、酶切產(chǎn)物回收、連接反應(yīng)將上述目的基因插入到慢病毒表達(dá)載體中,并分別命名為mpla497-l498ins4-mscv(目的)、mplw515l-mscv(陽性對照)、mplwt-mscv(野生型對照)重組質(zhì)粒,測序鑒定。2.建立可穩(wěn)定表達(dá)mpla497-l498ins4及各對照基因的baf3細(xì)胞株采用脂質(zhì)體轉(zhuǎn)染法將各目的基因及包裝質(zhì)粒轉(zhuǎn)導(dǎo)至293t包裝細(xì)胞中,獲得慢病毒顆粒后按最適比例感染baf3細(xì)胞,用流式細(xì)胞儀分選gfp陽性細(xì)胞,對分選后的細(xì)胞擴(kuò)大培養(yǎng),獲得可穩(wěn)定表達(dá)目的基因及各對照基因的baf3細(xì)胞株。3.比較分析mpla497-l498ins4在無il-3培養(yǎng)條件下對baf3細(xì)胞的增殖作用正常情況下,baf3細(xì)胞需要依賴鼠il-3因子才能維持正常生長。突變型mpl可使baf3細(xì)胞不依賴il-3自主增殖。因此本研究通過il-3撤退實驗觀察mpla497-l498ins4對baf3細(xì)胞增殖的影響,以此證實該突變體是否具有自主促細(xì)胞增殖的作用。設(shè)立mplw515l-baf3細(xì)胞、mplwt-baf3細(xì)胞、mscv-baf3細(xì)胞及baf3細(xì)胞分別作為本實驗的陽性對照、野生型對照、載體對照及空白對照,所有各組均加無il-3培養(yǎng)基進(jìn)行培養(yǎng),收集0h、24h、48h、72h各時間點各組細(xì)胞,采用cck-8法測定各實驗組中baf3細(xì)胞增殖情況并進(jìn)行統(tǒng)計分析。4.比較分析mpla497-l498ins4對其配體tpo的敏感性mpl基因編碼血小板生成素受體(tpor),其配體為tpo。mpl基因突變可致tpor不依賴配體tpo自主結(jié)構(gòu)性活化,因此突變型mpl在缺乏或低濃度tpo的情況下依然可引起細(xì)胞自我增殖,而野生型mpl則必須依賴一定濃度的tpo刺激才能引起細(xì)胞增殖。因此本研究可通過檢測tpo敏感性實驗再次證實mpla497-l498ins4突變是否具有自主促細(xì)胞增殖活性。分組同上,設(shè)立6個tpo不同濃度梯度(2ng/ml、1ng/ml、0.1ng/ml、0.01ng/m、0.001ng/ml、0ng/ml)培養(yǎng)96h,采用cck-8法測定各實驗組中baf3細(xì)胞增殖情況并進(jìn)行統(tǒng)計分析。結(jié)果:1.各基因慢病毒表達(dá)載體的構(gòu)建與鑒定各基因mpla497-l498ins4、mplw515l、mplwt成功連接到慢病毒表達(dá)載體pcdh-mcs-t2a-copgfp-mscv中,經(jīng)一代測序法分別對插入載體的各基因進(jìn)行序列鑒定,Blast分析結(jié)果顯示各組載體插入基因序列均正確。2.各基因慢病毒轉(zhuǎn)導(dǎo)BaF3細(xì)胞模型的建立與鑒定利用已構(gòu)建的表達(dá)載體,制備MPL A497-L498ins4及對照(陽性對照MPL W515L、野生型MPL WT、空載體MSCV)各基因慢病毒懸液并感染Ba F3細(xì)胞株,流式檢測各組感染率依次為:82%、77%、70.5%、95.7%。應(yīng)用流式分選儀對小于90%GFP陽性率的細(xì)胞進(jìn)行分選,最終使各組GFP陽性率均呈90%以上。應(yīng)用RT-PCR方法及CD110-PE流式檢測方法分別鑒定各組細(xì)胞轉(zhuǎn)導(dǎo)基因的mRNA與蛋白表達(dá)情況:結(jié)果顯示除空載體MSCV組外其它各組均有目的基因表達(dá),且各蛋白分子均定位于BaF3細(xì)胞膜上。所有結(jié)果提示成功建立穩(wěn)定表達(dá)各基因的Ba F3細(xì)胞模型。3.MPL A497-L498ins4在無IL-3培養(yǎng)條件下對Ba F3細(xì)胞的增殖能力分析應(yīng)用CCK-8檢測不同時間點各組BaF3細(xì)胞不依賴IL-3生長情況,結(jié)果顯示:MPL WT組、MSCV組及BaF3組在各時間點細(xì)胞增殖無顯著變化,且各組之間無統(tǒng)計學(xué)差異(P0.05)。MPL A497-L498ins4組與MPL W515L組在48h、72h細(xì)胞均出現(xiàn)明顯增殖,與MPL WT、MSCV、BaF3三組相比,均有統(tǒng)計學(xué)差異(P0.01),但MPL A497-L498ins4組與MPL W515L組細(xì)胞增殖情況無統(tǒng)計學(xué)差異(P0.05)。4.MPL A497-L498ins4突變對其配體TPO敏感性分析應(yīng)用CCK-8檢測各組BaF3細(xì)胞在TPO不同濃度下的增殖情況,結(jié)果顯示:在2ng/ml及1ng/ml TPO濃度時,MPL A497-L498ins4、MPL W515L、MPL WT三組細(xì)胞均有明顯增殖,與MSCV、Ba F3兩組比較有統(tǒng)計學(xué)差異(P0.01);在其余TPO濃度時,MPL WT、MSCV、BaF3三組無明顯增殖,而MPL A497-L498ins4、MPL W515L組細(xì)胞增殖較為顯著,且與其余三組相比,均有統(tǒng)計學(xué)差異(P0.01)。結(jié)論:MPL A497-L498ins4突變體可致Ba F3細(xì)胞不依賴IL-3生長,同時該突變體對低濃度TPO較野生型MPL敏感,提示該突變體具有與MPL W515L突變體同樣的自主促細(xì)胞增殖活性。
[Abstract]:Objective: myeloproliferative tumor (MPN) is a class of cloned hematopoietic stem cell diseases characterized by one or multiple myeloid cells, including red, granulated and megakaryocytes. The classic three types of MPN diseases include true erythrocytosis (PV), primary thrombocytopenia (ET), primary myelofibrosis (PMF), and its clinical table Now we have found that JAK2 V617F, JAK2 exon12, MPL exon10, and CALR exon9 are the main molecular causes of PV, ET, PMF, and PMF three diseases in MPN. We found a new type of mutation in 2 of these patients at the same time. MPL A497-L498ins4).MPL gene encodes a thrombopoietin receptor (TPOR) protein molecule. When TPOR's near transmembrane region aa514-518 has no ligand binding, it can prevent receptor two polymer from close to each other, and then activate its downstream, such as JAK2-STAT signaling pathway, to induce cell proliferation. The previous study reported that the type of MPL gene mutation was mainly MPL W515 point mutation, and had been used in the previous study. It is confirmed that the mutation can lead to the ability of the cells to produce autonomous proliferation. The mechanism may be that the mutation causes the abnormal aa514-518 regional structure of TPOR and lead to the cell independent proliferation activity.MPL A497-L498ins4 as the insertion of 4 amino acids, but not in the aa514-518 region of TPOR, then whether the mutation is caused by the MPN hair of the patient. What about the driving genes of birth? To this end, we intend to use the gene transduction technique of lentivirus gene transduction to transduce the gene mutation and the control gene into the primary B lymphocyte (Ba F3) of mice, and explore the role of the mutation to promote cell growth from the cytological level. Method: 1. the RT-PCR method for the construction of MPL A497-L498ins4 and the control gene of the lentivirus expression vector is used. The whole length of MPL A497-L498ins4 and the control gene MPL W515L, MPL WT CDS region, PCR product and Lentivirus Expression Vector pCDH-MCS-T2A-copGFP-MSCV (short for MSCV) were cut through double enzyme, the enzyme cut product was reclaimed, and the above target gene was inserted into the Lentivirus Expression Vector, and named mpla497-l498ins4-mscv (purpose), mplw. 515l-mscv (positive control), mplwt-mscv (wild type control) recombinant plasmid, sequencing and identification of.2. to establish a stable expression of mpla497-l498ins4 and the baf3 cells of each control gene transduced into 293T packaging cells by liposome transfection, and acquired the lentivirus particles and infected baf3 cells according to the optimum proportion and used flow. GFP positive cells were selected by cytometer, and the cells were cultured and cultured, and the baf3 cell line.3., which could express the target gene and each control gene, was compared and analyzed. Under the normal condition of the proliferation of baf3 cells without IL-3 culture, baf3 cells need to rely on the mouse IL-3 factor to maintain normal growth. Variant MPL can make baf3 cells independent of IL-3 proliferation. Therefore, the effect of mpla497-l498ins4 on the proliferation of baf3 cells was observed by IL-3 withdrawal test in this study to confirm whether the mutant had the role of autonomic cell proliferation. The establishment of mplw515l-baf3 cells, mplwt-baf3 cells, mscv-baf3 cells and baf3 cells was used as this experiment, respectively. Positive control, wild type control, carrier control and blank control, all groups were cultured with no IL-3 medium and collected 0h, 24h, 48h, 72h each time point cells. CCK-8 method was used to determine the proliferation of baf3 cells in each experiment group and to analyze.4. comparison and analysis of mpla497-l498ins4 sensitivity MPL gene to its ligand TPO. The encoding of the thrombopoietin receptor (tpor), whose ligand is tpo.mpl gene mutation, can cause tpor independent structural activation of the ligand TPO, so the mutant MPL can still cause cell self proliferation in the absence or low concentration of TPO, and the wild type MPL must rely on a certain concentration of TPO stimulation to induce cell proliferation. The TPO sensitivity test can be used to confirm whether the mpla497-l498ins4 mutation has the active cell proliferation activity again. In the same group, 6 TPO different concentration gradients (2ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/m, 0.001ng/ml, 0ng/ml) are trained for 96h, and CCK-8 method is used to determine the proliferation of the baf3 cells in the experimental groups and to carry out statistical analysis. Fruit: 1. gene Lentivirus Expression Vector Construction and identification of each gene mpla497-l498ins4, mplw515l, mplwt were successfully connected to the Lentivirus Expression Vector pcdh-mcs-t2a-copgfp-mscv. The genes of the inserted vectors were sequenced by one generation sequencing method. The Blast analysis results showed that each carrier gene sequence was correct.2. base sequence. Due to the establishment and identification of the BaF3 cell model of lentivirus transduction, MPL A497-L498ins4 and control (positive control MPL W515L, wild type MPL WT, and airborne MSCV) were prepared and infected with the lentivirus suspension and infected Ba F3 cell lines. The rate of flow detection in each group was 82%, 77%, 70.5%, and 95.7%. applied flow sorting apparatus. The cells with the positive rate of less than 90%GFP were selected, and the positive rates of GFP were all above 90%. The mRNA and protein expression of the cell transduction genes were identified by RT-PCR and CD110-PE flow detection. The results showed that all the other groups except the MSCV group had the target gene expression, and all the protein molecules were located. On the BaF3 cell membrane, all the results suggested that the Ba F3 cell model,.3.MPL A497-L498ins4, which was stable to express each gene, was used to analyze the proliferation ability of Ba F3 cells without IL-3 culture. The BaF3 cells were not dependent on IL-3 growth in the CCK-8 detection at different time points. The results showed that the MPL group, the group and the group at all time points were shown. There was no significant difference in cell proliferation, and there was no significant difference between each group (P0.05).MPL A497-L498ins4 group and MPL W515L group in 48h, 72h cell proliferation, compared with MPL WT, MSCV, BaF3 three groups, there were statistical differences (P0.01), but there was no statistical difference between the group and the cell proliferation. The sensitivity analysis of 8ins4 mutation to its ligand TPO was used to detect the proliferation of BaF3 cells at different concentrations of TPO in each group. The results showed that in the concentration of 2ng/ml and 1ng/ml TPO, MPL A497-L498ins4, MPL W515L, there were significant proliferation in the three groups. The proliferation of T, MSCV, BaF3 three groups was not obvious, but the proliferation of MPL A497-L498ins4 and MPL W515L groups was more significant, and compared with the other three groups, there were statistical differences (P0.01). Conclusion: MPL A497-L498ins4 mutant can cause Ba F3 cells to not depend on the growth of Ba F3 cells, and the mutant is sensitive to the wild type. The L W515L mutant has the same autonomous cell proliferation activity.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.3

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