MPL A497-L498ins4一个新突变基因对BaF3细胞增殖的作用研究

发布时间：2018-05-01 11:22

本文选题：骨髓增殖性疾病 + MPL　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:骨髓增殖性肿瘤(MPN)是一类以一系或多系髓系细胞包括红系、粒系和巨核系增殖为主要特征的克隆性造血干细胞疾病。其中经典的三类MPN疾病包括真性红细胞增多症(PV)、原发性血小板增多症(ET)、原发性骨髓纤维化(PMF),其临床表现较为相似且可以相互转化。目前发现JAK2 V617F、JAK2 exon12、MPL exon10以及CALR exon9为MPN中PV、ET、PMF三种疾病的主要分子病因。我们前期通过对771例确诊的MPN病人进行上述基因筛查时发现其中2例患者同时存在MPL基因的一种新突变类型(命名为MPL A497-L498ins4)。MPL基因编码血小板生成素受体(TPOR)蛋白分子,TPOR的近跨膜区aa514-518无配体结合时可阻止受体二聚体相互靠近,进而激活其下游如JAK2-STAT信号通路引起细胞增殖。以往研究报道的MPL基因突变类型主要为MPL W515点突变,并曾证实该突变可导致细胞产生自主促增殖能力,其机制可能是由于该突变引起TPOR的aa514-518区域结构异常进而导致细胞自主促增殖活性。MPL A497-L498ins4为插入4个氨基酸的突变,但并不在TPOR的aa514-518区域,那么该突变是否为引起患者MPN发生的驱动基因呢?为此本课题组拟通过慢病毒基因转导技术将该基因突变以及对照基因转导小鼠原B淋巴细胞(Ba F3)中,从细胞学水平探讨该突变具有自主促细胞的作用。方法:1.构建MPL A497-L498ins4及对照基因的慢病毒表达载体用RT-PCR法获取MPL A497-L498ins4及对照基因MPL W515L、MPL WT的CDS区全长,将PCR产物及慢病毒表达载体pCDH-MCS-T2A-copGFP-MSCV(简写为mscv)通过双酶切、酶切产物回收、连接反应将上述目的基因插入到慢病毒表达载体中,并分别命名为mpla497-l498ins4-mscv(目的)、mplw515l-mscv(阳性对照)、mplwt-mscv(野生型对照)重组质粒,测序鉴定。2.建立可稳定表达mpla497-l498ins4及各对照基因的baf3细胞株采用脂质体转染法将各目的基因及包装质粒转导至293t包装细胞中,获得慢病毒颗粒后按最适比例感染baf3细胞,用流式细胞仪分选gfp阳性细胞,对分选后的细胞扩大培养,获得可稳定表达目的基因及各对照基因的baf3细胞株。3.比较分析mpla497-l498ins4在无il-3培养条件下对baf3细胞的增殖作用正常情况下,baf3细胞需要依赖鼠il-3因子才能维持正常生长。突变型mpl可使baf3细胞不依赖il-3自主增殖。因此本研究通过il-3撤退实验观察mpla497-l498ins4对baf3细胞增殖的影响,以此证实该突变体是否具有自主促细胞增殖的作用。设立mplw515l-baf3细胞、mplwt-baf3细胞、mscv-baf3细胞及baf3细胞分别作为本实验的阳性对照、野生型对照、载体对照及空白对照,所有各组均加无il-3培养基进行培养,收集0h、24h、48h、72h各时间点各组细胞,采用cck-8法测定各实验组中baf3细胞增殖情况并进行统计分析。4.比较分析mpla497-l498ins4对其配体tpo的敏感性mpl基因编码血小板生成素受体(tpor),其配体为tpo。mpl基因突变可致tpor不依赖配体tpo自主结构性活化,因此突变型mpl在缺乏或低浓度tpo的情况下依然可引起细胞自我增殖,而野生型mpl则必须依赖一定浓度的tpo刺激才能引起细胞增殖。因此本研究可通过检测tpo敏感性实验再次证实mpla497-l498ins4突变是否具有自主促细胞增殖活性。分组同上,设立6个tpo不同浓度梯度(2ng/ml、1ng/ml、0.1ng/ml、0.01ng/m、0.001ng/ml、0ng/ml)培养96h,采用cck-8法测定各实验组中baf3细胞增殖情况并进行统计分析。结果:1.各基因慢病毒表达载体的构建与鉴定各基因mpla497-l498ins4、mplw515l、mplwt成功连接到慢病毒表达载体pcdh-mcs-t2a-copgfp-mscv中,经一代测序法分别对插入载体的各基因进行序列鉴定,Blast分析结果显示各组载体插入基因序列均正确。2.各基因慢病毒转导BaF3细胞模型的建立与鉴定利用已构建的表达载体,制备MPL A497-L498ins4及对照(阳性对照MPL W515L、野生型MPL WT、空载体MSCV)各基因慢病毒悬液并感染Ba F3细胞株,流式检测各组感染率依次为:82%、77%、70.5%、95.7%。应用流式分选仪对小于90%GFP阳性率的细胞进行分选,最终使各组GFP阳性率均呈90%以上。应用RT-PCR方法及CD110-PE流式检测方法分别鉴定各组细胞转导基因的mRNA与蛋白表达情况:结果显示除空载体MSCV组外其它各组均有目的基因表达,且各蛋白分子均定位于BaF3细胞膜上。所有结果提示成功建立稳定表达各基因的Ba F3细胞模型。3.MPL A497-L498ins4在无IL-3培养条件下对Ba F3细胞的增殖能力分析应用CCK-8检测不同时间点各组BaF3细胞不依赖IL-3生长情况,结果显示:MPL WT组、MSCV组及BaF3组在各时间点细胞增殖无显著变化,且各组之间无统计学差异(P0.05)。MPL A497-L498ins4组与MPL W515L组在48h、72h细胞均出现明显增殖,与MPL WT、MSCV、BaF3三组相比,均有统计学差异(P0.01),但MPL A497-L498ins4组与MPL W515L组细胞增殖情况无统计学差异(P0.05)。4.MPL A497-L498ins4突变对其配体TPO敏感性分析应用CCK-8检测各组BaF3细胞在TPO不同浓度下的增殖情况,结果显示:在2ng/ml及1ng/ml TPO浓度时,MPL A497-L498ins4、MPL W515L、MPL WT三组细胞均有明显增殖,与MSCV、Ba F3两组比较有统计学差异(P0.01);在其余TPO浓度时,MPL WT、MSCV、BaF3三组无明显增殖,而MPL A497-L498ins4、MPL W515L组细胞增殖较为显著,且与其余三组相比,均有统计学差异(P0.01)。结论:MPL A497-L498ins4突变体可致Ba F3细胞不依赖IL-3生长,同时该突变体对低浓度TPO较野生型MPL敏感,提示该突变体具有与MPL W515L突变体同样的自主促细胞增殖活性。
[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.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3

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