Kras和Pten在小鼠白血病发生和胚胎造血中的不同作用
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摘要
研究背景及目的
白血病是最常见的儿童恶性肿瘤之一,约占儿童肿瘤的1/3。尽管白血病的发病原因目前尚不清楚,已有研究显示一些产前染色体和基因异常如21三体和TEL-AML重排有增加儿童患白血病风险的倾向,而且有关双胞胎白血病患者的研究显示白血病的发生可以起始于子宫内阶段。Ras是一种膜联小分子GTP酶,在细胞信号转导中发挥重要作用。Rras基因的12、13密码子的突变可引起Ras信号的增强,从而导致Rras蛋白的持续激活,引起细胞增殖分化、恶性转化等改变。Pten是PI3K-Akt信号通路的负性调节蛋白,在细胞周期、系别分化、迁移和造血干细胞、祖细胞的功能中发挥重要作用。目前研究表明儿童白血病中存在癌性Kras的表达和Pten的缺失等基因突变。
因而我们提出一种假设：癌性Kras基因表达和Pten基因的缺失这两种基因突变可能作为早期事件发生在胚胎从而对出生后白血病的发生发挥作用。但目前存在的癌性Kras基因表达和Pten基因的缺失的小鼠模型研究大多应用成鼠的骨髓细胞,而为检验这一假设成立与否,需建立一种于胚胎阶段始可引起造血系统该两种基因突变发生的小鼠模型。
Cre转基因小鼠可敲除特异组织中两侧带有Loxp位点的基因序列,而Vav-iCre (?)、鼠中Cre基因的表达由Vav基因控制。Vav基因主要表达于成鼠的造血系统及胚胎的造血干细胞来源的造血祖细胞,因而Vav-iCre中重组酶主要作用于造血器官的造血细胞,是用来建立胚胎阶段始造血系统特异基因表达或缺失的有效工具。Kras基因在白血病中最常见的突变之一为Kras外显子1的由密码子12编码的甘氨酸突变为天冬氨酸,LSL-KrasG12D小鼠则是在该突变的上游包含有一个两端带有loxp的转录终止密码子,可被Cre重组酶切除,从而导致突变的癌性Kras基因(KrasG12D+)的表达。Pten基因的外显子5为编码磷酸酶的结构域,被认为是Pten基因发挥作用的重要结构域。条件性Pten敲除小鼠(Ptenf/f)则是在小鼠Pten基因外显子5的两端插入loxp序列,可被Cre重组酶敲除,从而导致Pten基因的功能缺失。
本研究旨在利用LSL-KrasG12D小鼠、Ptenf/f和Vav-iCre小鼠平行建立KrasG12D+表达和Pten-/-这两种胚胎阶段始血液系统非病毒致特异基因突变的小鼠模型,获得KrasG12D+或Pten稳定一致表达或缺失的基因型小鼠,观察其不同表型,探索KrasG12D+表达或Pten-/-对胚胎造血及出生后白血病发生的作用,为深入认识儿童白血病的发生机制及可能的防治工作提供动物实验依据。本研究主要分为以下两个部分：
第一部分：Kras G12D+表达对小鼠白血病发生及胚胎造血的影响
方法
1.将LSL-KrasG12D小鼠与Vav-iCre小鼠杂交,在子鼠21天时分笼编号,取部分耳组织提取DNA,通过PCR方法及基因测序方法鉴定子鼠基因型。
2.提前一晚将LSL-KrasG12D母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),分别于E14.5和E15.5取小鼠胚胎,对比观察组、对照组胚胎形态差异,并对正中矢状面切片固定行HE染色,观察对比胎肝组织学变化；取胎肝细胞行流式细胞学检查,检测细胞表面抗体Ter119、CD71的表达,分析观察组、对照组胎肝细胞红系细胞分化的差异。
结果
1. LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后,于48只子鼠中,PCR基因型鉴定结果示Kraswt;Vav-Cre-基因型小鼠17只,占35.24%,LSL-KrasG12D基因型小鼠16只,占33.33%,Kraswt;Vav-Cre+基因型小鼠15只,占31.25%,KrasG12D+基因型小鼠(拟观察组)0只,各基因型发生率不符合孟德尔遗传定律。5只LSL-KrasG12D基因型母鼠及15只Vav-Cre+基因型小鼠的KrasG12D基因突变测序结果示5只LSL-KrasG12D基因型母鼠均含有KrasG12D突变,而Vav-Cre+基因型小鼠不含该突变。
2.从形态学上与Kraswt；Vav-Cre+基因型小鼠的胎肝相比,E14.5的KrasG12D+基因型小鼠胚胎胎肝颜色苍白,并可伴有点状出血点,E15.5的胚胎则出现脑部或弥漫性出血。E15.5胚胎矢状面切片HE染色示KrasG12D+小鼠胎肝肝叶中央部位细胞轻度减少,远端部位有核红细胞增多,伴有严重的细胞坏死。
3.流式细胞学检测E14.5的KrasG12D+基因型小鼠胚胎的新鲜胎肝细胞表面CD71、Ter119的表达结果示Ter119-CD71+细胞群比例为(8.20±4.39)%,较.Kraswt;Vav-Cre+基因型小鼠胚胎的(3.66±0.62)%明显增高,两组相比有显著的统计学意义(P<0.001)。
小结
1.LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后不能获得成活的KrasG12D+基因型小鼠。
2.KrasG12D+基因型小鼠胚胎于E14.5-E15.5因发生脑部出血或弥漫性出血而死亡。
3.KrasG12D+基因型小鼠胚胎胎肝中红系原始细胞及祖细胞比率增高。
第二部分：Pten表达缺失对小鼠白血病发生及胚胎造血的影响
方法
1.将Ptenf/f与Vav-iCre小鼠杂交,获得PtenΔ/+;Vav-Cre+基因型(Pten+/-)小鼠,再次与Ptenf/f小鼠杂交,行基因型鉴定,分析不同基因型表达是否符合孟德尔遗传定律。将小鼠分为Pten-/-(PtenΔ/Δ;Vav-Cre+)基因型(观察组)、Pten+/-(PtenΔ/+;Vav-Cre+)基因型(对照组)和Ptenf/f/Ptenf/+基因型(对照组)三组。每日观察小鼠状态,于50天取小鼠外周血行全血细胞计数,观察细胞分类、比例变化。于观察组小鼠病重时对小鼠行安乐死,取外周血细胞行细胞离心涂片、Giemsa染色,取脾脏、肝脏、胸腺组织固定、HE染色,观察细胞形态,分析肿瘤类型,对骨髓细胞或胸腺细胞行流式细胞学检查,检测其细胞表面抗体CD19、Thy1、CD45、CD4、CD8等表达,进一步分析肿瘤类型,同时对照组小鼠做相同处理,分析观察组小鼠和对照组小鼠生存曲线的差异。
2.小鼠骨髓细胞移植实验：取4×106Pten-／-基因型小鼠的胸腺细胞或骨髓细胞(CD45.2),通过小鼠尾静脉注射入6-8周龄的亚致死量照射(650cGy)的移植受体C57BL/6J (CD45.1/CD45.2)小鼠,每日观察小鼠状态,于小鼠病重时对小鼠行安乐死,提取新鲜的骨髓或脾脏细胞,通过流式细胞学检测CD45.1、 CD45.2及CD3、CD4、CD8表达,CD45.1-CD45.2+细胞群为植入细胞,含有2.5%以上的CD45.1-CD45.2+细胞认为是有效移植。
3.提前一晚将Pten+/-母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),于E14.5取小鼠胚胎,行基因型鉴定,观察胚胎形态有无差异,并对胎肝细胞行流式细胞学检查,检测细胞表面抗体,分析观察组、对照组胎肝造血祖细胞组成成分比例的差异。
结果
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,获得50%Pten+/基因型小鼠,将6只Pten+/-小鼠分别与Vav-iCre小鼠杂交后,于61只子鼠中,PCR基因型鉴定结果示Pten-/-基因型小鼠13只(观察组),占21.31%,Pten+/-基因型小鼠17只,占27.87%, Ptenf/f基因型小鼠15只,占25.00%,Ptenf/+基因型小鼠16只,占26.23%,各基因型发生率符合孟德尔遗传定律。Pten-/-基因型小鼠中位生存时间为82天,而Pten+/-基因型小鼠的中位生存时间大于200天,两组相比其差异具有显著性统计学意义(P<0.001)。
2.于50天取小鼠外周血行全血细胞计数,结果显示Pten-/-基因型小鼠白细胞数量较Pten+/基因型小鼠明显增高,具有显著统计学意义(P<0.05)。
3.从形态学上与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠出现弓背、呼吸抑制及体重减轻,解剖学检查结果示胸腺、脾脏和肝脏体积增大、重量增加(P<0.05)。
4.与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠胸腺HE染色示胸腺出现淋巴母细胞浸润,正常组织结构消失；脾脏、肝脏切片HE染色示脾脏、肝脏出现白血病细胞浸润,正常组织结构破坏；外周血细胞离心涂片Giemsa染色示外周血中出现原始淋巴细胞浸润。
5.应用流式细胞学检测胸腺细胞表面抗体CD19(B细胞)和Thy1(T细胞)的表达,结果示其主要为CD19-Thy+((80.2±2.7)%),而后检测CD45+的胸腺细胞群表面抗体CD4和CD8表达,结果示3只小鼠胸腺肿瘤细胞为CD4+CD8+(1/2),1只为CD8+CD4-(1/6),另外两只为CD4+CD8-(1/3).
6.小鼠的骨髓细胞移植实验结果示4×106的Pten-/-基因型小鼠胸腺细胞或骨髓细胞经静脉注射入移植受体C57BL/6J(CD45.1/CD45.2)小鼠后,于2-16周受体小鼠发病,表现基本同Pten-/-小鼠,流式细胞学检测骨髓细胞中CD45.1-CD45.2+细胞群均>5%((24.4+3.1)%),且该群细胞群表面抗体CD3+。
7.应用流式细胞学检测小鼠胚胎胎肝细胞表面的lin、Scal-1、c-kit、CD34和FcRⅡ/Ⅲ等抗体表达,结果示与Pten++/-基因型小鼠相比,Pten-/-基因型小鼠胚胎胎肝细胞中富含造血干细胞的LSK (lin-Scal-l+c-kit+)细胞群无明显变化,而造血祖细胞(HPC)中的共同髓系祖细胞群(CMP)增高,共同淋系祖细胞群(CLP)减少,两组差异具有显著性统计学意义(P<0.05,P<0.01)。
小结
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,成功获得长期正常生存的Pten+/-基因型小鼠。Pten+/-基因型小鼠与Ptenf/f小鼠杂交后,成功获得可存活的Pten-/-基因型小鼠。Pten-/-基因型小鼠中位生存时间较Pten+/-基因型小鼠的中位生存时间明显缩短。
2. Pten-/基因型小鼠发生肝脾肿大、胸腺增大,细胞形态学及流式细胞学显示急性T淋巴细胞白血病/淋巴瘤(T-ALL/L)特性。
3. Pten-/-基因型小鼠发生的T-ALL/L具有可移植性。
4. Pten-/-基因型小鼠的胚胎胎肝造血祖细胞中CMP增高,而CLP减少。
结论
1.利用Vav-iCre转基因小鼠诱导KrasG12D+在发育阶段表达的KrasG12D+基因型小鼠于E14.5-E15.5死亡,提示该小鼠模型不能用于解释说明胚胎期KrasG12D+表达可导致出生后白血病发生这一问题。
2.胚胎期KrasG12D表达阻碍小鼠胎肝红系祖细胞向成熟红细胞的分化,导致KrasG12D+小鼠胚胎出现贫血。
3.胚胎期始Pten基因的单倍缺失不影响小鼠的表型,而Pten-/-小鼠发生可移植的T-ALL/L。
4.胚胎期Pten表达缺失除了在祖细胞水平引起淋系和髓系的交错变化外,对总的胚胎造血无明显影响。
Background and Objective
Leukemia is the most common children malignancy, accounting for almost30%of children cancer. Although the causes of leukemia are largely unknown, a few prenatal chromosomal and genetic alterations, such as trisomy21(Down syndrome) and TEL-AML1rearrangements have been associated with an increased risk of children leukemia, and studies of twin leukemia have revealed that leukemia can be initiated in utero. Ras are membrane-associated small GTPases and play a critical role in transducing signals, and the mutations at codons12and13cause hyperactive Ras signaling, which leads to constitutive activation of the Ras protein and affect the cell proliferation/differentiation, survival and malignant transformation. Pten (phosphatase and tensin homologue), as a negative regulator of the PI3K-Akt pathway, is involved in cell cycling, lineage commitment, mobilization, and hematopoietic stem/progenitor cell function. Both gain-of-functional Kras mutation(s) and loss of Pten expression have been implicated in children leukemia.
Taken together, we raised the hypothesis that both oncogenic K-ras mutation(s) and Pten deficiency could occur in utero as an early event which contributes to postnatal leukemogenesis. Most existing murine models on these two genetic alterations use adult bone marrow cells and are not appropriate to address this hypothesis.
Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific tissue. In Vav-iCre transgenic mouse, the iCre transgene is expressed under the control of murine Vav gene regulatory elements. The expression of Vav gene is exclusively restricted to hematopoietic cells in adult mice and may be in hematopoietic stem cell (HSC)-derived progenitors in earliest prenatal. Therefore, Vav-iCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs and the Vav-iCre transgene mice are effective tools for constructing fetal-stage-blood-lineage specific nonviral-based genetic modified murine models. In LSL-KrasG12D mice, there is a transcriptional termination codon flanked by loxP sites upstream of a mutation of glycine to aspartic acid encoded by codon12in exonl. Excision of the stop cassette by iCre recombinase allows the expression of oncogenic Kras. The conditional Pten konckout mice (Ptenf/f) were inserted two loxP sequences on either side of the exon5, which encode the phosphatase domain and erased by iCre recombinase leading to disruption of Pten.
In this study, by using LSL-KrasG12D mice, Ptenf/f mice, and Vav-iCre transgene mice, we sought to constructed two fetal-stage-blood-lineage specific nonviral-based genetic modified murine models, and obtain stable and uniformed overexpression or deletion of the gene of interest, to evaluate the effect(s) of deregulated KrasG12D and Pten during fetal hematopoiesis and their potential roles in postnatal leukemogenesis. This study can be divided into two parts:
Part Ⅰ:Effects of oncogenic Kras G12D+in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed LSL-KrasG12D mice and Vav-iCre mice, extract DNA from ear tissues of mice at21days after birth, identified the genetypes of offsprings by PCR and gene sequencing.
2. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5and E15.5, carefully dissect and gather the embryos, HE stain the parasagittal section, observe the differences on gross and histological morphology; collect fresh fetal liver cells and check the expressions of CD71, Ter119by flow cytometric analysis, compare the percentage of primitive progenitor cells and proerythroblasts.
Results
1. Among the first48offsprings, there are17Kraswt;Vav-Cre-mice (35.24%),16LSL-KrasG12D mice (33.33%) and15Kraswt;Vav-Cre+mice (31.25%), but no viable KrasG12D+;Vav-iCre+pups were identified, which is not consistant with the Mendelian inheritance. No KrasG12D mutation was found in15Vav-iCre+littermate mice by sequencing on purified genotyping PCR products.
2. Compared with Kraswt;Vav-Cre+embryos, the E14.5KrasG12D+embryos have pale-colored liver and small hemorrhage foci, the E15.5KrasG12D+embryos appear brain or extensive systemic hemorrhage. In hematoxylin and eosin (HE) stained fetal liver parasagittal section from E15.5KrasG12D+embryos, mild decrease in cellularity in the proximal portion of and increased erythroblasts in the distal portion of the KrasG12D+hepatic lobe were observed. Moreover, severe cell death was observed in the distal portion of the KrasG12D+hepatic lobe.
3. Cells isolated from E14.5Kraswt;Vav-Cre+or KrasG12D+fetal livers were stained with anti-CD71and anti-Ter119for flow cytometric analysis (FCAS). It displayed an increased Ter119-CD71+populations ((8.20±4.39)%) in KrasG12D+fetal livers compared with (3.66±0.62)%in Kraswt;Vav-Cre+fetal livers. The difference is significant (P<0.001)。
Summary
1. No live KrasG12D+; Vav-iCre+pups were detected by crossing LSL-KrasG12Dmice and Vav-iCre mice.
2. KrasG12D+embryos died of brain or extensive systemic hemorrhage between E14.5to E15.5.
3. KrasG12D+embryos fetal livers displayed an increased percentage in primitive progenitor cells and proerythroblasts.
Part Ⅱ:Effects of Pten deletion in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed Ptenf/f mice with Vav-iCre to get Pten+/-mice, then crossed Pten+/-mice with Ptenf/f mice. Identified the genotypes of offsprings, and divided into observation group and control groups. Monitor mice everyday, detecte the complete blood count CBC using peripheral blood (PB) on50day after birth. Euthanize badly sick mice with carbon dioxide, and collect PB to do cytospin and Giemsa stain, fix spleen, liver, thymus tissues and HE stain, isolate bone marrow (BM) cells for FCAS. Meanwhile, dealed with the control groups mice as the same way, and analyse their median survival difference.
2. mice BM cells/thymocytes transplantation:4×106BM cells or thymocytes harvested from Pten-/-mice (CD45.2) were injected intravenously via the tail vein into sub-lethally irradiated (650cGy)6to8-week-old CD45.1/CD45.2mice. Mice were monitored everyday, and were euthanized when badly sick. Fresh cells harvested from BM and spleen were subsequently subjected to FCAS utilizing CD45.1, CD45.2and CD4/CD8antibody. The percentage of CD45.1-CD45.2+was calculated as engraftment cells, and a threshold of2.5%CD45.1-CD45.2+cells was established as a reliable preditor of positive engraftment.
3. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5, carefully dissect and gather the embryos, observe the differences in gross morphology; collect fresh fetal liver cells for FACS, compare the percentage difference of hematopoietic progenitor cells (HPC).
Results
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice occupied50%in offsprings and live normally more than200days. Among the61offsprings by crossing Pten+/-mice with Ptenf/f mice, there are13Pten-/-mice (21.31%),17 Pten+/-mice (27.87%),15Ptenf/f mice (25.00%) and16Ptenf/+mice (26.23%), which is consistant with the Mendelian inheritance. Almost all Pten-/-mice died with a median survival of82days, which was significantly shorter than that of Pten+/-mice(P<0.001).
2. CBC showed the white blood cells (WBC,(36.22±12.46)×109) in Pten-/-mice is signicantly higher than that in Pten+/-mice (7.17±2.01)×109, P<0.05).
3. Compared with Pten-/-mice, the Pten-/-mice showed hunched posture, respiratory distress, ruffled fur and weight loss (incidence100%). Necrospy displayed the presence of a thymic mass accompanied by hepatosplenomegaly (incidence85.7%).
4. Histological examination of the thymus revealed that the normal thymic architecture was effaced and infiltrated by monomorphic lymphoblastic cells with prominent nucleoli and scant cytoplasm. Leukemic cells infiltrated organs such as spleen and liver. Variable leukemic blasts were present in the peripheral blood of diseased mice.
5. Tumor thymocytes were examined for their expression of Thyl and CD19, markers for T cells and B cells respectively. Most of these cells were Thyl+but CD19-((80.2±2.7)%). The CD45+populations were analysed using CD4and CD8, and it showed three (1/2) of the six examined tumors consisted of predominantly CD4+CD8+cells, one (1/6) contained primarily CD4-CD8+cells and the other two (1/3) consisted of CD4+CD8-cells.
6. Mice BM cells/thymocytes transplantation test showed the recipient mice were sick in2-16week after injection. FACS results showed the percentage of CD45.1-CD45.2+populations in BM of recipient mice are more than5%((24.4±3.1)%), and the populations are CD3+.
7. FACS on fetal liver cells revealed that there was no reduction in the hematopoietic stem cell (HSC) enriched lin-sca-1+c-kit+(LSK) population. However, there was an increased percentage of common myeloid progenitor (CMP) population as well as a decreased percentage of common lymphoid progenitor (CLP).
Summary
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice were born and live normally. By crossing Pten+/-mice with Ptenf/f mice, alive Pten-/-mice were born. The median survival of Pten-/-mice was significantly shorter than that of Pten+/-mice.
2. Pten-/-mice developed into T-ALL/L ultimately.
3. The T-ALL/L occurred in Pten-/-mice was transplantable。
4. Pten-/-mice showed an increased percentage of CMP population as well as a decreased percentage of CLP in fetal liver hematopoietic progenitor cells.
Conclution
1. K-rasG12D mutation introduced by Vav-iCre during development leads to fetal lethality, suggesting that this murine model cannot be used to assess the leukemogenic ability of K-ras in utero.
2. Oncogenic KrasG12D+expression during development may block the differention from erythroid progenitor cells to mature erythrocytes, which might be the cause of anemia.
3. None of Pten+/-mice with one copy Pten deletion during fetal stage exhibited T-ALL/L, while Pten-null mice lead to transplantable T-ALL/L.
4. Pten deletion during fetal stage hardly affect the overall fetal hematopoiesis except a skewed lymphoid/myeloid development at the progenitor level。
白血病是最常见的儿童恶性肿瘤之一,约占儿童肿瘤的1/3。尽管白血病的发病原因目前尚不清楚,已有研究显示一些产前染色体和基因异常如21三体和TEL-AML重排有增加儿童患白血病风险的倾向,而且有关双胞胎白血病患者的研究显示白血病的发生可以起始于子宫内阶段。Ras是一种膜联小分子GTP酶,在细胞信号转导中发挥重要作用。Rras基因的12、13密码子的突变可引起Ras信号的增强,从而导致Rras蛋白的持续激活,引起细胞增殖分化、恶性转化等改变。Pten是PI3K-Akt信号通路的负性调节蛋白,在细胞周期、系别分化、迁移和造血干细胞、祖细胞的功能中发挥重要作用。目前研究表明儿童白血病中存在癌性Kras的表达和Pten的缺失等基因突变。
因而我们提出一种假设：癌性Kras基因表达和Pten基因的缺失这两种基因突变可能作为早期事件发生在胚胎从而对出生后白血病的发生发挥作用。但目前存在的癌性Kras基因表达和Pten基因的缺失的小鼠模型研究大多应用成鼠的骨髓细胞,而为检验这一假设成立与否,需建立一种于胚胎阶段始可引起造血系统该两种基因突变发生的小鼠模型。
Cre转基因小鼠可敲除特异组织中两侧带有Loxp位点的基因序列,而Vav-iCre (?)、鼠中Cre基因的表达由Vav基因控制。Vav基因主要表达于成鼠的造血系统及胚胎的造血干细胞来源的造血祖细胞,因而Vav-iCre中重组酶主要作用于造血器官的造血细胞,是用来建立胚胎阶段始造血系统特异基因表达或缺失的有效工具。Kras基因在白血病中最常见的突变之一为Kras外显子1的由密码子12编码的甘氨酸突变为天冬氨酸,LSL-KrasG12D小鼠则是在该突变的上游包含有一个两端带有loxp的转录终止密码子,可被Cre重组酶切除,从而导致突变的癌性Kras基因(KrasG12D+)的表达。Pten基因的外显子5为编码磷酸酶的结构域,被认为是Pten基因发挥作用的重要结构域。条件性Pten敲除小鼠(Ptenf/f)则是在小鼠Pten基因外显子5的两端插入loxp序列,可被Cre重组酶敲除,从而导致Pten基因的功能缺失。
本研究旨在利用LSL-KrasG12D小鼠、Ptenf/f和Vav-iCre小鼠平行建立KrasG12D+表达和Pten-/-这两种胚胎阶段始血液系统非病毒致特异基因突变的小鼠模型,获得KrasG12D+或Pten稳定一致表达或缺失的基因型小鼠,观察其不同表型,探索KrasG12D+表达或Pten-/-对胚胎造血及出生后白血病发生的作用,为深入认识儿童白血病的发生机制及可能的防治工作提供动物实验依据。本研究主要分为以下两个部分：
第一部分：Kras G12D+表达对小鼠白血病发生及胚胎造血的影响
方法
1.将LSL-KrasG12D小鼠与Vav-iCre小鼠杂交,在子鼠21天时分笼编号,取部分耳组织提取DNA,通过PCR方法及基因测序方法鉴定子鼠基因型。
2.提前一晚将LSL-KrasG12D母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),分别于E14.5和E15.5取小鼠胚胎,对比观察组、对照组胚胎形态差异,并对正中矢状面切片固定行HE染色,观察对比胎肝组织学变化；取胎肝细胞行流式细胞学检查,检测细胞表面抗体Ter119、CD71的表达,分析观察组、对照组胎肝细胞红系细胞分化的差异。
结果
1. LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后,于48只子鼠中,PCR基因型鉴定结果示Kraswt;Vav-Cre-基因型小鼠17只,占35.24%,LSL-KrasG12D基因型小鼠16只,占33.33%,Kraswt;Vav-Cre+基因型小鼠15只,占31.25%,KrasG12D+基因型小鼠(拟观察组)0只,各基因型发生率不符合孟德尔遗传定律。5只LSL-KrasG12D基因型母鼠及15只Vav-Cre+基因型小鼠的KrasG12D基因突变测序结果示5只LSL-KrasG12D基因型母鼠均含有KrasG12D突变,而Vav-Cre+基因型小鼠不含该突变。
2.从形态学上与Kraswt；Vav-Cre+基因型小鼠的胎肝相比,E14.5的KrasG12D+基因型小鼠胚胎胎肝颜色苍白,并可伴有点状出血点,E15.5的胚胎则出现脑部或弥漫性出血。E15.5胚胎矢状面切片HE染色示KrasG12D+小鼠胎肝肝叶中央部位细胞轻度减少,远端部位有核红细胞增多,伴有严重的细胞坏死。
3.流式细胞学检测E14.5的KrasG12D+基因型小鼠胚胎的新鲜胎肝细胞表面CD71、Ter119的表达结果示Ter119-CD71+细胞群比例为(8.20±4.39)%,较.Kraswt;Vav-Cre+基因型小鼠胚胎的(3.66±0.62)%明显增高,两组相比有显著的统计学意义(P<0.001)。
小结
1.LSL-KrasG12D小鼠与Vav-iCre小鼠杂交后不能获得成活的KrasG12D+基因型小鼠。
2.KrasG12D+基因型小鼠胚胎于E14.5-E15.5因发生脑部出血或弥漫性出血而死亡。
3.KrasG12D+基因型小鼠胚胎胎肝中红系原始细胞及祖细胞比率增高。
第二部分：Pten表达缺失对小鼠白血病发生及胚胎造血的影响
方法
1.将Ptenf/f与Vav-iCre小鼠杂交,获得PtenΔ/+;Vav-Cre+基因型(Pten+/-)小鼠,再次与Ptenf/f小鼠杂交,行基因型鉴定,分析不同基因型表达是否符合孟德尔遗传定律。将小鼠分为Pten-/-(PtenΔ/Δ;Vav-Cre+)基因型(观察组)、Pten+/-(PtenΔ/+;Vav-Cre+)基因型(对照组)和Ptenf/f/Ptenf/+基因型(对照组)三组。每日观察小鼠状态,于50天取小鼠外周血行全血细胞计数,观察细胞分类、比例变化。于观察组小鼠病重时对小鼠行安乐死,取外周血细胞行细胞离心涂片、Giemsa染色,取脾脏、肝脏、胸腺组织固定、HE染色,观察细胞形态,分析肿瘤类型,对骨髓细胞或胸腺细胞行流式细胞学检查,检测其细胞表面抗体CD19、Thy1、CD45、CD4、CD8等表达,进一步分析肿瘤类型,同时对照组小鼠做相同处理,分析观察组小鼠和对照组小鼠生存曲线的差异。
2.小鼠骨髓细胞移植实验：取4×106Pten-／-基因型小鼠的胸腺细胞或骨髓细胞(CD45.2),通过小鼠尾静脉注射入6-8周龄的亚致死量照射(650cGy)的移植受体C57BL/6J (CD45.1/CD45.2)小鼠,每日观察小鼠状态,于小鼠病重时对小鼠行安乐死,提取新鲜的骨髓或脾脏细胞,通过流式细胞学检测CD45.1、 CD45.2及CD3、CD4、CD8表达,CD45.1-CD45.2+细胞群为植入细胞,含有2.5%以上的CD45.1-CD45.2+细胞认为是有效移植。
3.提前一晚将Pten+/-母鼠与Vav-iCre小鼠放置于一笼拟交配,于发现母鼠有阴道栓的清晨计为胚胎第0.5日(E0.5),于E14.5取小鼠胚胎,行基因型鉴定,观察胚胎形态有无差异,并对胎肝细胞行流式细胞学检查,检测细胞表面抗体,分析观察组、对照组胎肝造血祖细胞组成成分比例的差异。
结果
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,获得50%Pten+/基因型小鼠,将6只Pten+/-小鼠分别与Vav-iCre小鼠杂交后,于61只子鼠中,PCR基因型鉴定结果示Pten-/-基因型小鼠13只(观察组),占21.31%,Pten+/-基因型小鼠17只,占27.87%, Ptenf/f基因型小鼠15只,占25.00%,Ptenf/+基因型小鼠16只,占26.23%,各基因型发生率符合孟德尔遗传定律。Pten-/-基因型小鼠中位生存时间为82天,而Pten+/-基因型小鼠的中位生存时间大于200天,两组相比其差异具有显著性统计学意义(P<0.001)。
2.于50天取小鼠外周血行全血细胞计数,结果显示Pten-/-基因型小鼠白细胞数量较Pten+/基因型小鼠明显增高,具有显著统计学意义(P<0.05)。
3.从形态学上与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠出现弓背、呼吸抑制及体重减轻,解剖学检查结果示胸腺、脾脏和肝脏体积增大、重量增加(P<0.05)。
4.与Pten+/-基因型小鼠相比,Pten-/-基因型小鼠胸腺HE染色示胸腺出现淋巴母细胞浸润,正常组织结构消失；脾脏、肝脏切片HE染色示脾脏、肝脏出现白血病细胞浸润,正常组织结构破坏；外周血细胞离心涂片Giemsa染色示外周血中出现原始淋巴细胞浸润。
5.应用流式细胞学检测胸腺细胞表面抗体CD19(B细胞)和Thy1(T细胞)的表达,结果示其主要为CD19-Thy+((80.2±2.7)%),而后检测CD45+的胸腺细胞群表面抗体CD4和CD8表达,结果示3只小鼠胸腺肿瘤细胞为CD4+CD8+(1/2),1只为CD8+CD4-(1/6),另外两只为CD4+CD8-(1/3).
6.小鼠的骨髓细胞移植实验结果示4×106的Pten-/-基因型小鼠胸腺细胞或骨髓细胞经静脉注射入移植受体C57BL/6J(CD45.1/CD45.2)小鼠后,于2-16周受体小鼠发病,表现基本同Pten-/-小鼠,流式细胞学检测骨髓细胞中CD45.1-CD45.2+细胞群均>5%((24.4+3.1)%),且该群细胞群表面抗体CD3+。
7.应用流式细胞学检测小鼠胚胎胎肝细胞表面的lin、Scal-1、c-kit、CD34和FcRⅡ/Ⅲ等抗体表达,结果示与Pten++/-基因型小鼠相比,Pten-/-基因型小鼠胚胎胎肝细胞中富含造血干细胞的LSK (lin-Scal-l+c-kit+)细胞群无明显变化,而造血祖细胞(HPC)中的共同髓系祖细胞群(CMP)增高,共同淋系祖细胞群(CLP)减少,两组差异具有显著性统计学意义(P<0.05,P<0.01)。
小结
1. Ptenf/f小鼠与Vav-iCre小鼠杂交后,成功获得长期正常生存的Pten+/-基因型小鼠。Pten+/-基因型小鼠与Ptenf/f小鼠杂交后,成功获得可存活的Pten-/-基因型小鼠。Pten-/-基因型小鼠中位生存时间较Pten+/-基因型小鼠的中位生存时间明显缩短。
2. Pten-/基因型小鼠发生肝脾肿大、胸腺增大,细胞形态学及流式细胞学显示急性T淋巴细胞白血病/淋巴瘤(T-ALL/L)特性。
3. Pten-/-基因型小鼠发生的T-ALL/L具有可移植性。
4. Pten-/-基因型小鼠的胚胎胎肝造血祖细胞中CMP增高,而CLP减少。
结论
1.利用Vav-iCre转基因小鼠诱导KrasG12D+在发育阶段表达的KrasG12D+基因型小鼠于E14.5-E15.5死亡,提示该小鼠模型不能用于解释说明胚胎期KrasG12D+表达可导致出生后白血病发生这一问题。
2.胚胎期KrasG12D表达阻碍小鼠胎肝红系祖细胞向成熟红细胞的分化,导致KrasG12D+小鼠胚胎出现贫血。
3.胚胎期始Pten基因的单倍缺失不影响小鼠的表型,而Pten-/-小鼠发生可移植的T-ALL/L。
4.胚胎期Pten表达缺失除了在祖细胞水平引起淋系和髓系的交错变化外,对总的胚胎造血无明显影响。
Background and Objective
Leukemia is the most common children malignancy, accounting for almost30%of children cancer. Although the causes of leukemia are largely unknown, a few prenatal chromosomal and genetic alterations, such as trisomy21(Down syndrome) and TEL-AML1rearrangements have been associated with an increased risk of children leukemia, and studies of twin leukemia have revealed that leukemia can be initiated in utero. Ras are membrane-associated small GTPases and play a critical role in transducing signals, and the mutations at codons12and13cause hyperactive Ras signaling, which leads to constitutive activation of the Ras protein and affect the cell proliferation/differentiation, survival and malignant transformation. Pten (phosphatase and tensin homologue), as a negative regulator of the PI3K-Akt pathway, is involved in cell cycling, lineage commitment, mobilization, and hematopoietic stem/progenitor cell function. Both gain-of-functional Kras mutation(s) and loss of Pten expression have been implicated in children leukemia.
Taken together, we raised the hypothesis that both oncogenic K-ras mutation(s) and Pten deficiency could occur in utero as an early event which contributes to postnatal leukemogenesis. Most existing murine models on these two genetic alterations use adult bone marrow cells and are not appropriate to address this hypothesis.
Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific tissue. In Vav-iCre transgenic mouse, the iCre transgene is expressed under the control of murine Vav gene regulatory elements. The expression of Vav gene is exclusively restricted to hematopoietic cells in adult mice and may be in hematopoietic stem cell (HSC)-derived progenitors in earliest prenatal. Therefore, Vav-iCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs and the Vav-iCre transgene mice are effective tools for constructing fetal-stage-blood-lineage specific nonviral-based genetic modified murine models. In LSL-KrasG12D mice, there is a transcriptional termination codon flanked by loxP sites upstream of a mutation of glycine to aspartic acid encoded by codon12in exonl. Excision of the stop cassette by iCre recombinase allows the expression of oncogenic Kras. The conditional Pten konckout mice (Ptenf/f) were inserted two loxP sequences on either side of the exon5, which encode the phosphatase domain and erased by iCre recombinase leading to disruption of Pten.
In this study, by using LSL-KrasG12D mice, Ptenf/f mice, and Vav-iCre transgene mice, we sought to constructed two fetal-stage-blood-lineage specific nonviral-based genetic modified murine models, and obtain stable and uniformed overexpression or deletion of the gene of interest, to evaluate the effect(s) of deregulated KrasG12D and Pten during fetal hematopoiesis and their potential roles in postnatal leukemogenesis. This study can be divided into two parts:
Part Ⅰ:Effects of oncogenic Kras G12D+in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed LSL-KrasG12D mice and Vav-iCre mice, extract DNA from ear tissues of mice at21days after birth, identified the genetypes of offsprings by PCR and gene sequencing.
2. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5and E15.5, carefully dissect and gather the embryos, HE stain the parasagittal section, observe the differences on gross and histological morphology; collect fresh fetal liver cells and check the expressions of CD71, Ter119by flow cytometric analysis, compare the percentage of primitive progenitor cells and proerythroblasts.
Results
1. Among the first48offsprings, there are17Kraswt;Vav-Cre-mice (35.24%),16LSL-KrasG12D mice (33.33%) and15Kraswt;Vav-Cre+mice (31.25%), but no viable KrasG12D+;Vav-iCre+pups were identified, which is not consistant with the Mendelian inheritance. No KrasG12D mutation was found in15Vav-iCre+littermate mice by sequencing on purified genotyping PCR products.
2. Compared with Kraswt;Vav-Cre+embryos, the E14.5KrasG12D+embryos have pale-colored liver and small hemorrhage foci, the E15.5KrasG12D+embryos appear brain or extensive systemic hemorrhage. In hematoxylin and eosin (HE) stained fetal liver parasagittal section from E15.5KrasG12D+embryos, mild decrease in cellularity in the proximal portion of and increased erythroblasts in the distal portion of the KrasG12D+hepatic lobe were observed. Moreover, severe cell death was observed in the distal portion of the KrasG12D+hepatic lobe.
3. Cells isolated from E14.5Kraswt;Vav-Cre+or KrasG12D+fetal livers were stained with anti-CD71and anti-Ter119for flow cytometric analysis (FCAS). It displayed an increased Ter119-CD71+populations ((8.20±4.39)%) in KrasG12D+fetal livers compared with (3.66±0.62)%in Kraswt;Vav-Cre+fetal livers. The difference is significant (P<0.001)。
Summary
1. No live KrasG12D+; Vav-iCre+pups were detected by crossing LSL-KrasG12Dmice and Vav-iCre mice.
2. KrasG12D+embryos died of brain or extensive systemic hemorrhage between E14.5to E15.5.
3. KrasG12D+embryos fetal livers displayed an increased percentage in primitive progenitor cells and proerythroblasts.
Part Ⅱ:Effects of Pten deletion in murine postnatal leukemogenesis and fetal hematopoiesis
Methods
1. Crossed Ptenf/f mice with Vav-iCre to get Pten+/-mice, then crossed Pten+/-mice with Ptenf/f mice. Identified the genotypes of offsprings, and divided into observation group and control groups. Monitor mice everyday, detecte the complete blood count CBC using peripheral blood (PB) on50day after birth. Euthanize badly sick mice with carbon dioxide, and collect PB to do cytospin and Giemsa stain, fix spleen, liver, thymus tissues and HE stain, isolate bone marrow (BM) cells for FCAS. Meanwhile, dealed with the control groups mice as the same way, and analyse their median survival difference.
2. mice BM cells/thymocytes transplantation:4×106BM cells or thymocytes harvested from Pten-/-mice (CD45.2) were injected intravenously via the tail vein into sub-lethally irradiated (650cGy)6to8-week-old CD45.1/CD45.2mice. Mice were monitored everyday, and were euthanized when badly sick. Fresh cells harvested from BM and spleen were subsequently subjected to FCAS utilizing CD45.1, CD45.2and CD4/CD8antibody. The percentage of CD45.1-CD45.2+was calculated as engraftment cells, and a threshold of2.5%CD45.1-CD45.2+cells was established as a reliable preditor of positive engraftment.
3. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5, carefully dissect and gather the embryos, observe the differences in gross morphology; collect fresh fetal liver cells for FACS, compare the percentage difference of hematopoietic progenitor cells (HPC).
Results
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice occupied50%in offsprings and live normally more than200days. Among the61offsprings by crossing Pten+/-mice with Ptenf/f mice, there are13Pten-/-mice (21.31%),17 Pten+/-mice (27.87%),15Ptenf/f mice (25.00%) and16Ptenf/+mice (26.23%), which is consistant with the Mendelian inheritance. Almost all Pten-/-mice died with a median survival of82days, which was significantly shorter than that of Pten+/-mice(P<0.001).
2. CBC showed the white blood cells (WBC,(36.22±12.46)×109) in Pten-/-mice is signicantly higher than that in Pten+/-mice (7.17±2.01)×109, P<0.05).
3. Compared with Pten-/-mice, the Pten-/-mice showed hunched posture, respiratory distress, ruffled fur and weight loss (incidence100%). Necrospy displayed the presence of a thymic mass accompanied by hepatosplenomegaly (incidence85.7%).
4. Histological examination of the thymus revealed that the normal thymic architecture was effaced and infiltrated by monomorphic lymphoblastic cells with prominent nucleoli and scant cytoplasm. Leukemic cells infiltrated organs such as spleen and liver. Variable leukemic blasts were present in the peripheral blood of diseased mice.
5. Tumor thymocytes were examined for their expression of Thyl and CD19, markers for T cells and B cells respectively. Most of these cells were Thyl+but CD19-((80.2±2.7)%). The CD45+populations were analysed using CD4and CD8, and it showed three (1/2) of the six examined tumors consisted of predominantly CD4+CD8+cells, one (1/6) contained primarily CD4-CD8+cells and the other two (1/3) consisted of CD4+CD8-cells.
6. Mice BM cells/thymocytes transplantation test showed the recipient mice were sick in2-16week after injection. FACS results showed the percentage of CD45.1-CD45.2+populations in BM of recipient mice are more than5%((24.4±3.1)%), and the populations are CD3+.
7. FACS on fetal liver cells revealed that there was no reduction in the hematopoietic stem cell (HSC) enriched lin-sca-1+c-kit+(LSK) population. However, there was an increased percentage of common myeloid progenitor (CMP) population as well as a decreased percentage of common lymphoid progenitor (CLP).
Summary
1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice were born and live normally. By crossing Pten+/-mice with Ptenf/f mice, alive Pten-/-mice were born. The median survival of Pten-/-mice was significantly shorter than that of Pten+/-mice.
2. Pten-/-mice developed into T-ALL/L ultimately.
3. The T-ALL/L occurred in Pten-/-mice was transplantable。
4. Pten-/-mice showed an increased percentage of CMP population as well as a decreased percentage of CLP in fetal liver hematopoietic progenitor cells.
Conclution
1. K-rasG12D mutation introduced by Vav-iCre during development leads to fetal lethality, suggesting that this murine model cannot be used to assess the leukemogenic ability of K-ras in utero.
2. Oncogenic KrasG12D+expression during development may block the differention from erythroid progenitor cells to mature erythrocytes, which might be the cause of anemia.
3. None of Pten+/-mice with one copy Pten deletion during fetal stage exhibited T-ALL/L, while Pten-null mice lead to transplantable T-ALL/L.
4. Pten deletion during fetal stage hardly affect the overall fetal hematopoiesis except a skewed lymphoid/myeloid development at the progenitor level。
引文
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