【作者】 刘小军；

【导师】 罗建民；

【作者基本信息】 河北医科大学 ， 内科学， 2009， 博士

【摘要】 背景:慢性粒细胞白血病(chronic myelogenous leukemia)是一种获得性多功能造血干细胞突变后克隆性恶性增生性疾病。CML的全球年发病率约为1/10万人口,占全部成人白血病的15%-20%。我国CML的年发病率为0.36/10万人口,仅次于急性髓系白血病和急性淋巴细胞白血病位居第3位。近年来,随着分子生物学及临床医学研究的不断进步,CML的病因学有了很大进展,尤其是Ph染色体,bcr/abl融合基因的发现及其相关研究,为慢性粒细胞性白血病病因诊断及治疗作出了很大贡献。Bcr/abl是一种抑制凋亡的基因,能通过抑制细胞凋亡,而使细胞数量增加,基因组的内在不稳定性增加,使细胞容易发生第二次突变,bcr/abl造成粒细胞恶性增殖的确切机制至今未完全明了。SHIP基因是近年新发现的一种bcr/abl下游底物,该基因仅在造血细胞表达,是PI3K/Akt信号通路的负调控因子。本课题前期研究发现急性白血病患者存在SHIP基因突变,但SHIP基因在CML发病中的作用,尤其是有关临床方面的研究国内外尚无报道。本研究拟检测SHIP基因在慢性粒细胞性白血病患者的表达,探讨SHIP基因在bcr/abl恶性转化中的作用。共分四部分:1、FQ-PCR检测不同时期慢性粒细胞白血病患者SHIP基因表达变化;2、应用干扰RNA技术封闭人白血病细胞系K562中bcr/abl基因后观察SHIP基因的表达变化;3、应用SHIP基因敲除小鼠检测SHIP基因缺失对小鼠造血功能的影响; 4、通过对PI3K/Akt途径中Akt磷酸化水平的变化来了解SHIP在影响细胞凋亡中的机制。方法1实时荧光定量PCR(FQ-PCR)检测CML患者SHIP基因表达情况;2合成bcr/abl基因的特异性siRNA,并转染K562细胞;3构建SHIP-/-转基因骨髓受体小鼠,比较野生型和敲除SHIP基因骨髓小鼠的外周血常规、脾脏细胞学、克隆形成及骨髓细胞迁移能力改变; AnnexV/PI染色比较野生型(SHIP+/+)和SHIP-/-小鼠骨髓细胞的凋亡情况;4 Western blot检测敲除SHIP基因小鼠骨髓细胞Akt磷酸化水平。结果1 SHIP基因在CML患者表达减低FQ-PCR结果显示与正常人相比,CML患者SHIP mRNA表达明显减低,。2 bcr/abl融合基因抑制SHIP基因及蛋白表达2.1封闭bcr/abl融合基因后K562细胞SHIP mRNA表达增加:应用干扰RNA技术封闭K562细胞中bcr/abl融合基因的表达后,基因表达水平较空白对照组及非特异性干扰组减低(P值分别为0.0039,0.0039 ),SHIP基因mRNA的表达水平增加(P =0.037,P =0.016)。分别为:bcr/abl基因:特异性siRNA干扰组(5.63±0.97)×105;空白对照组:(27.14±3.05)×105;非特异siRNA转染组:(22.42±2.15)×105];空白对照组与非特异性转染组间无统计学意义(P=0.055);SHIP基因:特异性siRNA封闭组(3.58±0.37)×105],空白对照组[(1.52±0.37)×105],非特异性siRNA组[(1.53±0.20)×105拷贝],空白对照组非特异性siRNA干扰组表达无统计学意义(P =0.81)。2.2封闭融合基因后K562细胞中SHIP蛋白表达增高:转染特异性siRNA后K562细胞中融合蛋白p210表达明显降低,转染非特异性siRNA组p210表达无显著变化。SHIP蛋白p145在空白对照及非特异性siRNA组中几乎无表达,特异性siRNA封闭p210后SHIP蛋白表达显著增加。3敲除SHIP基因骨髓受体小鼠出现了类似白血病的脏器侵润及相关血液学变化3.1 SHIP-/-小鼠出现类似白血病脏器浸润的表现:移植后3个月后,SHIP基因敲除骨髓受体小鼠部分出现了下肢瘫痪的特殊表现,表现为软瘫,不能支撑体重,对疼痛的反应与对照组小鼠无明显差别,提示仅为运动功能受损,此现象与临床白血病患者中枢神经系统浸润的表现极为相似。另外解剖小鼠发现,基因敲除骨髓受体小鼠肺脏和脾脏体积均较野生型增大。3.2敲除SHIP基因小鼠外周血中性粒细胞比例明显增加:SHIP基因敲除小鼠的外周血中性粒细胞比例明显增加,但早期细胞比例并不高。外周血中未发现有原、早幼粒细胞。血常规检测发现两种小鼠外周血中性粒细胞和淋巴细胞比例有所不同而白细胞总数无显著差异(具体见第3部分表1)。3.3组织学表现为肺脏、脾脏中中性粒细胞及单核细胞高度浸润:与野生型小鼠相比,SHIP -/-小鼠肺脏部分区域明显实变,显微镜下观察实变区有髓系细胞浸润,浸润的细胞主要集中在肺泡区,部分区域还有气管腔内中性粒细胞的浸润。脾脏结构明显受损,正常淋巴细胞生发中心消失红髓白髓界限消失。为大量髓系细胞侵润。3.4细胞学检测发现SHIP基因缺失骨髓受体小鼠脾脏中性粒细胞比例明显增加;外周血中性粒细胞比例亦增高:与正常小鼠脾脏细胞相比, SHIP基因缺失骨髓受体小鼠的脾脏和外周血中性粒细胞比例明显增加,但外周血中未发现幼稚细胞。3.5免疫学检测发现SHIP基因缺失骨髓受体小鼠骨髓、外周血及脾脏的髓系细胞尤其是中性粒细胞比例均显著增加:用髓系细胞表面标志Gr-1和Mac-1进行免疫学标定,进一步证实了上述组织器官中髓系细胞的含量变化,其结果显示SHIP基因缺失小鼠骨髓髓系细胞(Gr1+Mac1+)(40.38±0.87)%比wild-type(WT)小鼠(20.91±1.11)%增加近1倍。脾脏的髓系细胞(14.47±0.67)%增加更明显,为WT(3.49　±0.47)%的4倍左右。外周血髓系细胞比例(60.71±1.72)%也比WT小鼠(23.38±0.62)%明显增多。3.6克隆培养证实敲除SHIP基因导致小鼠骨髓髓系细胞增殖增加:敲除SHIP基因小鼠骨髓单位细胞数形成克隆数量明显多于野生型小鼠,提示该组细胞中的干祖细胞比例高于野生型小鼠。比较两种小鼠克隆形成比例,混合克隆与粒单克隆(CFU-MIX、CFU-GM)的比例在基因敲除小鼠多于野生型小鼠,形成的克隆也比野生型大,提示敲除SHIP基因导致小鼠骨髓细胞增殖能力增强。。3.7 Transwell实验证实敲除SHIP基因使小鼠骨髓细胞运动能力增强:敲除SHIP基因的小鼠骨髓低密度细胞跨膜运动能力明显增强,3组敲除SHIP基因细胞平均跨膜细胞数(0.35x105/2x105)明显多于野生型小鼠(0.07x105/2x105 )(P<0.05)。3.8敲除SHIP基因导致小鼠骨髓细胞对无血清饥饿诱导凋亡耐受力增强:36小时无血清培养后,流式细胞学检测凋亡率发现,SHIP -/-的小鼠骨髓单个核细胞凋亡率(3.86±0.28)明显低于对照组细胞(19.6±1.28)(P<0.05)。提示SHIP基因缺失导致小鼠细胞抗凋亡能力增强。4 SHIP基因缺失细胞内Akt磷酸化水平升高:Western blot检测发现,在SHIP基因缺失的小鼠骨髓细胞中,其Akt的磷酸化水平明显增加,其在未受任何刺激情况下就有明显的p-Akt出现,在受到GM-CSF刺激的情况下表现更为明显,提示SHIP能够抑制Akt的磷酸化。结论1 SHIP基因在CML患者中表达减低甚至表达缺失。2 Bcr/abl融合基因抑制SHIP基因表达。3敲除SHIP基因可促进造血细胞中髓系细胞增殖,抑制细胞凋亡,增强细胞的运动侵袭能力。敲除SHIP基因可使细胞内磷酸化Akt水平增加,并对GM-CSF的反应增强。4 SHIP基因通过调节PI3K/Akt途径发挥其促凋亡作用。更多还原

【Abstract】 Backgroud: Chronic myelogenous leukemia (CML) is a kind of acquired malignancy which is caused by clonal proliferation after mutation in multiple potential stem cells. CML happens at an incident of 1:100,000 globally, composed of 15%-20% of the leukemia in adults. In our county CML ranks as the third most prevelant leukemia at an incidence of 0.36 in 100,000, just behind the acute myelogenous leukemia and acute lymphocytic leukemia.In recent years, with the development of molecular biology and clinical medicine study, great progress has been made in the etiology study of CML. The discovery and study of Ph chromosome and fusion gene of bcr/abl contribute a lot to the diagnosis and therapy of CML. Bcr/abl is a negative regulator of apoptosis, it can increases the cells number by decreasing apoptosis. The increased instability of the genoome makes it easy for secondary mutation to happen in these kind of cells. It is still not very clear why bcr/abl causes malignant proliferation. SHIP protein is a downstream substrate of bcr/abl , it is exclusively expressed in hematopoietic cells, and was found as a negative regulator in PI3K/Akt pathway. Our study group has found out that mutations exist in the acute leukemia patients in our previous study. However, the function of SHIP in the development of CML especially in the clinical study still leaves us with unanswered questions. In our study we examined the expression of the SHIP gene in CML patients and investigated the function of SHIP in the development of CML. The study was carried out in four parts: 1. detect the expression of SHIP gene in CML patients through FQ-PCR; 2. Study the expression change in cell line K562 cells after transfecting the small interfere RNA specific to bcr/abl gene; 3. Use SHIP gene knockout bone marrow receptor mice to study the influence of SHIP gene to the function hematopoietic system; 4. Through examination of the change of p-Akt in PI3K/Akt pathway to study the mechanism of the influence of SHIP in apoptosis.Method:1. real time quantitative PCR was employed to test the expression of SHIP gene in the Chronic myelogenous leukemia.2. synthesis and transfection of siRNA specific bcr/abl fusion gene into K562 cells and then use FQ-PCR and western blot to test the expression of bcr/abl and SHIP at both gene and protein level.3. construct SHIP-/- bone marrow recipient mice to see the influence of SHIP on the hematopoietic system.4. study the p-Akt level change in the SHIP negative bone marrow cells through Western blot.Results:1. SHIP gene expression decreased in the CML patients.Results from the FQ-PCR revealed that the SHIP mRNA expression significantly lower in CML patients compare to the normal controls.2. the fusion gene of bcr/abl inhibit the expression of SHIP.2.1 increased SHIP mRNA was found in K562 cells after blocked the bcr/abl fusion gene.After block the expression of bcr/abl fusion gene in K562 cells with siRNA specific to bcr/abl, the expression of bcr/abl gene significantly blocked comparing to the blank and non-specific interfered controls. (p=0.0039, p=0.0039 respectively) While the expression of SHIP gene increased in the specific interfered group (P =0.037,P =0.016 respectively). The average values are:bcr/abl gene[specific interfered group (5.63±0.97)×105;blank :(27.14±3.05)×105 ; non specific siRNA tranfected group :(22.42±2.15)×105];no significant difference was seen between the later two groups ( P=0.055 ); SHIP gene specific siRNA transfected group (3.58±0.37)×105],blank [(1.52±0.37)×105],non-specific siRNA interfered group [(1.53±0.20)×105copy],no significant difference was seen between the later two groups(P =0.81)。 2.2 the expression of SHIP protein increased in K562 cells after blocking the expression of bcr/ablWestern blot results indicated that the expression of p210 (production of bcr/abl) greatly decreased after transfection with the bcr/abl siRNA in K562 cells compared to the no transfection control and non-functional bcr/abl siRNA group. No significant difference was found between the latter two groups. SHIP protein p145 can hardly be detected in the blank control and the non-functional bcr/abl siRNA group while in the bcr/abl siRNA interfered group the p145 significantly increased.3. infiltration of organs and hematopoietic changes happen in SHIP knockout bone marrow recipients3.1 SHIP -/- bone marrow recipients showed signs of infiltration just as in leukemia patientsThree months after transplantation, some of the SHIP knockout bone marrow recipients showed paralyzed rear legs. They could not bear their body weight, but reaction to pain was not different from the normal controls, which indicate the paralysis may be due to the damage of the motorneuron. This phenomenon mirrors what happens in leukemia patients. After dissection,enlarged lungs and spleens were found in the knockout bone marrow recipients compared to the normal controls.3.2 .SHIP knockout bone marrow recipients showed an increased ratio of neutrophils in peripheral blood.The peripheral blood of the SHIP knockout bone marrow recipients showed increased neutrophils by blood smear. However, no obvious primary cells or blast cells were found in the smears and the complete blood count(CBC). The CBC results showed equal amount of white blood cells, but the components (Neutrophils,Lymphocytes,Eosinophils) of the peripheral blood are significantly different between the two groups.3.3 Histology analysis revealed enlarged lungs and spleens were due to the infiltration of the myeloid cellsCompared with normal controls, SHIP-/- bone marrow recipients show significant pulmonary consolidation in some area of the lungs. Microscopic analysis revealed the consolidation due to the infiltration of the myeloid cells, mainly focused in the alveolus. Some neutrophils appear in the bronchus. The structure of the spleen was significantly damaged. The normal structure of the lymph follicles and the border of red pulp and white pulp disappeared due to the infiltration of vast amounts of myeloid cells.3.4 Morphorlogical study shows neutrophils increased in the SHIP knockout bone marrow recipeints spleen and peripheral blood.Compared with the normal control spleen, increased neutrophils were found in the knockout bone marrow recipients’spleen and peripheral blood but no obvious blast or primary cells are found in the peripheral blood.3.5 Immune analysis indicated the loss of SHIP in the bone marrow increased the percentage of neutrophils in the bone marrow spleen and peripheral blood.Stained the myeloid cells with Gr-1/Mac-1 antibody for flow cytometry analysis, we further analyzed the percentage of the myeloid cells in the previously described organs like bone marrow, spleen and peripheral blood. The results indicated that the percentage of myeloid cells in the SHIP negative bone marrow(Gr1+Mac1+)(40.38±0.87)% is nearly two times of that in the WT (20.91±1.11)% mice bone marrow. In the spleen the difference was even bigger, it was almost four times different from the normal controls.[ (14.47±0.67)% vs(3.49±0.47)%]. In peripheral blood in the SHIP negative bone marrow recipients the average number was (60.71±1.72)% while in the WT recipients it was only (23.38±0.62)%3.6 Colony assays indicate the loss of SHIP gene caused enhanced proliferation in myeloid cells.The colony numbers formed by the SHIP negative bone marrow cells are significantly larger than that formed by the WT bone marrow cells indicating that in the SHIP negative bone marrow there are higher ratios of progenitors or stem cells compared to wild type controls. This was also shown by CFU-MIX and CFU-gm assays. From the size of the colony we can also tell a higher proliferation ability of SHIP negative bone marrow cells.3.7 Migration Assay showed an enhanced migration ability of SHIP negative bone marrow cells.The migration test showed an enhanced moving ability of the SHIP negative bone marrow cells. The number of cells that migrated through the wells in the SHIP negative group(0.35x105/2x105) are significantly higher than those in the WT group(0.07x105/2x105 )(P<0.05).3.8 Cells from the SHIP negative bone marrow show reduced apoptosis induced by serum free starvation.4. Increased level of p-Akt was found in SHIP knockout bone marrow recipients.Results from the Western blot indicated that SHIP can significantly inhibit phosphorylation of Akt. The absence of SHIP causes the significant increase of the p-Akt, in bone marrow cells stimulated with GM-CSF.Conclusion:1. Lower expression of SHIP was found in CML patients2. Bcr/abl fusion gene inhibits the expression of SHIP gene.3. The loss of SHIP gene enhanced the proliferation and migration of hematopoietic cells, but inhibited apoptosis. Higher levels of p-AKT were correlated to increased sensitivity to GM-CSF in the KO cells.4. SHIP gene regulates hematopoiesis through the PI3K/Akt pathway.更多还原