游离的gp190胞内功能片段对白血病细胞的生长调节
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摘要
白血病抑制因子(LIF)是一种多效性细胞因子,作用于多种组织细胞发挥不同的生物学作用,广泛调节细胞的生长、增殖和分化。如LIF抑制胚胎干细胞的体外分化;刺激血小板生成,造血细胞增殖;促进成骨细胞增殖;促进神经原形成,肌肉卫星细胞的增殖,参与神经肌肉修复;参与肝的急性期反应,参与炎症的生理病理过程等。这些生物学效应有赖于LIF结合于靶细胞膜上的LIF受体α亚基(gp190),并与LIF受体β亚基(gp130)形成异源性二聚体,激活下游信号转导通路。gp190是LIF的特异性受体,人们已证明,在生理状态下存在可溶性和完整性gp190两种状态。可溶性受体游离于胞外,无跨膜区和细胞内区,对LIF发挥的正常生理效应有拮抗作用。完整性的gp190含有胞外区、跨膜区和胞内区,其中胞内区含有三个功能域,由近膜端至远膜端依次为BOX1、BOX2和BOX3。研究表明,BOX1、2和3各自在细胞增殖分化过程中发挥着不同的作用。gp190胞内区含有3个YXXQ官能体(其中2个位于BOX3中,另一个位于gp190C-末端),它可以与STAT的SH2位点特异性识别,启动信号传递。LIF可以激活信号通路JAK-STAT和Ras-MAPK,人们研究证实单一亚基的单一信号启动位点在离膜状态下完全能够在细胞内启动相应的信号转导通路,LIF的生物多效性可能与受体不同功能域的作用有关。为了明确受体不同功能域在细胞内游离状态时对细胞的生长调节,我们根据gp190细胞内区的不同功能域分别设计了两种小分子——190CT2+3(C-末端199个氨基酸,含有BOX2和BOX3)和190CT3(C-末端119个氨基酸,含有BOX3),初步证明了gp190细胞内区成为游离多肽在细胞内存在时,可以诱导白血病细胞分化。为了进一步验证前期工作,深入探讨它的发生机制,在本课题的实验研究中,我们首先鉴定已构建的190CT2+3和190CT3重组质粒,并进一步对190CT3的YXXQ官能体定点突变,继而研究相关信号转导通路。实验结果证明稳定表达目的基因(190CT2+3和190CT3)的白血病细胞,增殖均受到抑制,细胞向成熟方向分化,信号分子STAT3被激活。当YXXQ突变或PIAS3作用后,190CT3的以上作用被削弱。通过对gp190细胞内区和γ-secretase的研究,说明在生理状态下γ-secretase可能发挥着切割gp190产生游离C-末端的作用。最后我们进行了动物实验,成功建立了白血病裸鼠动物模型。通过对外周血、骨髓和脏器的检测,说明190CT3移植组在裸鼠体内仍然呈现了分化趋势,侵袭转移的恶性程度降低。因此,通过本课题的研究,我们明确了gp190 C-末端不同功能域190CT2+3、190CT3在细胞内游离存在时,促进白血病细胞分化,激活信号分子STAT3;这种游离多肽在生理状态下也可能存在。本研究旨在探索受体细胞内区单一功能域的应用价值,诱导白血病细胞成熟分化。
第一部分、重组人gp190胞内功能片段的白血病细胞株的获得与鉴定
方法:(1)酶切初步鉴定已构建的重组质粒pcDNA3.0-190CT2+3、pcDNA3.0-190CT3,进一步测序;构建重组质粒pcDNA3.0- MUT:通过引物重叠PCR法设计定点突变,在引物中置换突变碱基,将190CT3蛋白序列中三处YXXQ基团的酪氨酸(Y)位点突变为苯丙氨酸(F),将突变后的基因序列插入载体pcDNA3.0。(2)将重组质粒用FuGENE-6脂质体转染人白血病细胞株HL-60和K562,G-418梯度筛选阳性克隆15d,挑选生长旺盛的克隆扩增,设转染pcDNA3.0空质粒载体和野生型HL-60和K562细胞为对照组。鉴定转染后细胞内gp190羧基末端(C-terminal)的表达:用免疫细胞化学,蛋白印迹和RT-PCR的方法进行蛋白水平的半定量检测,确定稳定表达目的基因的细胞株,以备后面实验使用。(3)苏木精染色,显微镜下观察各组细胞形态;细胞计数绘制生长曲线;金黄色葡萄球菌实验检测细胞吞噬功能;流式细胞术检测细胞表面成熟粒单核细胞相关抗原的表达。结果:(1)BamHⅠ、XbaⅠ双酶切重组质粒pcDNA3.0- 190CT2+3、pcDNA3.0- 190CT3,电泳紫外灯下观察可见大小相符的目的片段,测序结果与Genbank核酸序列数据库中LIFR序列(X61615)比对,完全一致。190CT2+3全长597个碱基,190CT3全长357个碱基。对190CT3定点突变,获得339bpDNA片段,经BamHⅠ、XbaⅠ双酶切连接重组入质粒pcDNA3.0,双向测序后与Genbank数据库中LIFR序列(X61615)比对,原190CT3片段的三处编码酪氨酸的cDNA序列TAT正确置换为编码苯丙氨酸的TTC,成功获得重组pcDNA3.0-MUT的真核表达质粒。(2)经G-418筛选得到重组190CT2+3、190CT3、MUT的HL-60和K562细胞和空质粒转染细胞。用免疫印迹法检测LIFR,于20KD、15KD处见目的蛋白条带,选取稳定表达的细胞株进行下一步实验。苏木精染色,光镜下观察细胞形态,结果显示190CT2+3和190CT3诱导后,细胞的杆状核与分叶核增加。统计学分析分叶核比例:190CT3>190CT2+3组,P<0.01,组间差别有意义。(3)细胞计数,绘制生长曲线,190CT2+3和190CT3诱导后,细胞生长速度减慢:190CT3< 190CT2+3< MUT< pcDNA3.0< WT HL-60/K562。流式细胞术检测CD15、CD11b和CD14,结果显示转染190CT2+3和190CT3后标志分子表达增加:190CT3> 190CT2+3> MUT> pcDNA3.0> WT HL-60/K562。结论:(1)将190CT2+3、190CT3和MUT重组质粒转染HL-60/K562细胞,经检测说明重组白血病细胞株已经稳定表达目的基因。(2)190CT2+3、190CT3组分叶核的比例增加,增殖减慢,CD15/CD11b/CD14表达增高,对金葡菌的吞噬功能增强,证明gp190 C-末端功能域对白血病细胞有促进分化,抑制增殖的作用。
第二部分、gp190胞内功能片段信号通路以及与γ-Secretase关系的研究
方法:(1)免疫印迹法检测各组白血病细胞株(HL-60/K562)中磷酸化STAT3和磷酸化MAPK的蛋白表达;免疫荧光双标法检测各组HL-60细胞中STAT3和磷酸化STAT3。(2)构建PIAS3-pEGFP重组质粒:抽提HL-60细胞的总RNA,逆转录得到cDNA,设计四对引物,通过引物重叠延伸PCR法,扩增得到PIAS3编码区全长序列;经XhoⅠ、SacⅡ双酶切连接重组入真核表达载体pEGFP-N1,双向测序后与Genbank数据库中PIAS3序列(NM_006099)比对。通过脂质体瞬时转染190CT3-HL60细胞,通过免疫荧光和免疫印迹法检测转染后不同时间点PIAS3的表达,通过免疫印迹法检测PIAS3作用后,不同时间点STAT3二聚体的表达。细胞计数,绘制转染后的细胞生长曲线。(3)人LIF因子和γ-secretase抑制剂分别作用于HL-60细胞,免疫印迹法检测gp190(全长和C-末端)和PS1(全长和N-末端)的表达。结果:(1)免疫印迹法检测信号分子的蛋白表达,结果显示190CT2+3、190CT3组白血病细胞中(HL-60/K562)磷酸化STAT3明显较对照组增高;磷酸化MAPK的表达情况相反,190CT组表达量较对照组明显降低。激光共聚焦检测的结果显示190CT3组细胞磷酸化STAT3表达增强,STAT3核聚集增多。(2)免疫荧光和免疫印迹法检测证实PIAS3随转染时间的延长,表达增加;在PIAS3的作用下,STAT3二聚体的表达下降,细胞增殖转而加快。(3)LIF作用后,gp190的C-末端约26KD的片段,蛋白表达增加;同时伴随PS1的N-末端(PS1-NTF)蛋白表达逐渐增加,说明gp190胞内区游离多肽增加,γ-secretase分泌酶活性增加。γ-secretase抑制剂的作用下,gp190的C-末端约26KD的片段,蛋白表达减少;同时伴随PS1的N-末端(PS1-NTF)蛋白表达逐渐减少,说明gp190胞内区游离多肽减少,γ-secretase分泌酶活性降低。γ-secretase分泌酶可能切割了gp190产生游离功能域。结论:(1)目的片段190CT2+3与190CT3,即gp190胞内区C-末端功能域在白血病细胞内游离存在时,可以激活信号分子STAT3,同时抑制了信号分子MAPK;C-末端的YXXQ官能体发挥重要作用。(2)PIAS3瞬时作用于190CT3-HL60细胞后,STAT3二聚体减少,细胞增殖加快,说明STAT3在190CT3抑制白血病细胞的增殖过程中发挥关键作用。(3)hLIF刺激引起gp190 C-端片段和PS1 N-端片段表达增加,γ-secretase抑制剂作用使gp190 C-端片段和PS1 N-端片段表达减少,说明生理状态下,γ-secretase可能有切割gp190,产生游离C末端的作用。
第三部分、动物实验
方法:(1)4-5周龄雄性裸鼠由轻到重排列,按照随即配对的分组方法,分成5组(PBS、野生型HL-60、pcDNA3.0-HL60、MUT-HL60和190CT3-HL60),每组7只,尾静脉注射。第一次接种量为2×105个细胞,设立空白对照(注射200μlPBS)。记录接种日期,每天观察小鼠状态及行为表现。30天后第二次接种2×106个细胞。(2)7、14、21、28天分别断尾采集外周血制作血涂片,瑞氏染色,计数外周血中淋巴样细胞和粒细胞百分率;28天取骨髓细胞,流式细胞术检测骨髓细胞中标志分子CD15和HLA-1的表达。第二次接种60天后取出脏器(肝、肾、脾、肺和胃肠)做H-E染色,检测脏器有无病理结构的改变。结果:(1)外周血:CT3组裸鼠粒细胞比例较高,淋巴样细胞比例较低;骨髓:在第28天的检测结果显示,CT3组CD15和HLA-1的表达均升高。(2)组织切片的H-E染色。肝脏:均可见肉芽组织。肺部:野生型HL-60细胞移植组,部分肺泡结构消失,其内有边界不清的肉芽肿,有浆细胞、组织细胞,淋巴样细胞浸润,有出血坏死;pcDNA3.0-HL60移植组,部分肺泡正常结构被破坏,有肉芽肿样结构,部分坏死;MUT-HL60移植组,部分肺泡正常结构被破坏伴坏死、出血;190CT3-HL60移植组,肺部结构正常,肺泡间隔增厚,少量淋巴样细胞聚集,无出血坏死结构。结论:(1)移植后,CT3组细胞在裸鼠体内仍然表现为较成熟的粒细胞形态,维持了较分化的状态。(2)190CT3组HL-60细胞在裸鼠体内恶性程度降低。
小结
1、gp190胞内C-末端功能域在细胞内游离存在时,促进急性髓系白血病细胞向成熟细胞方向分化。
2、gp190胞C-末端功能域在白血病细胞内游离存在时,可以激活STAT3信号通路,同时抑制了信号分子MAPK;C-末端的YXXQ突变或PIAS3转染后,STAT3通路被抑制,细胞增殖加快;γ-secretase可能切割gp190产生细胞内的游离功能域,参与信号传递过程。
3、gp190胞内C-末端功能域作用后的白血病细胞在裸鼠体内恶性程度降低。
Background and objective
LIF induces terminal differentiation of myeloid leukemia cells, also modulates growth/differentiation of many other types of target cells. Other cellular functions influenced by this molecule include: embryogenesis, inflammation, neural development. LIF receptor is a heterodimer consisting of a low affinity binding protein gp190 and a signal transducing unit gp130. gp190 belongs to the hematopoietin receptor family, and is characterized by a cytokine receptor homology (CRH) domain. Cytoplasmic domain of gp190 contains three homologous and functionally important motifs: Box 1, 2 and 3. Gp190 binds to the src-homology 2 (SH2) domain of STAT3 via the YXXQ consensus sequences in the C-terminal. In addition to a membrane form, gp190 also exists in a soluble form that lacks transmembrane domain. Previous reports indicated that certain motif of the cytoplasmic gp190 could activate cell proliferation and differentiation. In the current study, transfecting leukemia cells with vectors containing a sequence encoding the C-terminal fragment (CTF) of gp190 (containing BOX 3 or BOX 2+3 ) inhibited cell proliferation, and induced differentiation. Such effects were significantly attenuated by a mutation in CTF (containing Box 3), as well as by inhibiting STAT3 using a selective inhibitor. STAT3 phosphorylation and nuclear translocation were significantly enhanced in cells expressing the CTF (containg Box 3). The cells expressing the CTF had a slower rate of proliferation upon transplantation into nude mice Analysis of cell morphology and markers for maturity indicated that these cells have higher degree of differentiation. We also found that cytoplasmic region of gp190 could be cleaved byγ-secretase upon LIF stimulation. Taken together, these findings suggest that a gp190 CTF may exist in leukemia cells, and modulate the property of these cells via the STAT3 pathway. It is beneficial to explore the application value about the single motif of cytoplasmic receptor, induce leukemia cells to differentiation.
PartⅠRecombination of human dissociated LIFR (gp190) cytoplasmic receptor in HL-60 cells and identification its expression
Methods: (1) Enzyme BamHⅠand XbaⅠcleaving the recombined plasmid pcDNA3.0-190CT2+3、pcDNA3.0-190CT3, and checking the sequences. Mutant of 190CT3: mutagenic oligonucleotide primers were complementary to the gene sequence in the region to be mutated, except Y-to-F conversion at three YXXQ sequences. The mutagenic oligonucleotide was then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. Desired sequences were amplified using PCR and subcloned into pcDNA3.0. (2) Transfected the eukaryotic expression plasmid pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT into leukemia (HL-60/K562) cells with liposome FuGENE-6. Two control group were setup which were vector pcDNA3.0 tansfected and wild-type HL-60/K562 cells. In the culture processes, we selected the clones with different G-418 concentration. The transfected cells were survival and expanding in presence of neo-gene. We detected the protein expression with western blotting and RT-PCR to identify the leukemia cell lines with stable protein expression. (3) Hematoxylin staining showed the morphous of nuclei. Describing the growth curve. Staphylococcus aureus were added into HL-60 cells. Cell smear was stained using a Wright method, and examined under a light microscope. Percentage of the HL-60 cells containing staphylococcus was used as an index for phagocytosis. The levels of CD15/CD14/CD11b were assayed by flow cytometer. Results: (1)After BamHⅠand XbaⅠcleaving, we observed electrophoretic irradiance strip under the UV light. Sequence detection was consistent with the LIFR sequence in Genbank. After PCR reaction, there is one 300-400bp DNA strip. Blast the MUT sequence and the original LIFR sequence, we found that the original TAT were exactly changed to TTC. The MUT4 colony was expressed the higher MUT by RT-PCR. (2) After subsequent selection by G418, G418-resistent colonies were isolated, namely ,the pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT and pcDNA3.0 leukemia cell lines. What western blotting and RT-PCR analyzed demonstrated that the 190CT had expressed in leukemia cells stably. Cells expressing the gp190 C-terminal fragment were significantly larger than cells transfected with pcDNA3.0. The nuclei of these cells were more leafy than the wild-type as well as the pcDNA3.0 controls. The rate of proliferation was significantly decreased by both 190CT3 and 190CT2+3. Expression of the mature granulocyte marker CD15/CD14/CD11b and the phagocytosis index were significantly increased. Conclusion: 190CT3 and 190CT2+3 could promote the differentiation and inhibit the proliferation of leukemia cells.
PartⅡ: Signaling pathway investigation for human dissociated LIFR (gp190) cytoplasmic receptor
Method: (1)Western blotting analyzed the levels of related signal molecular as P -STAT3(T705), P44/42-MAPK. Immunofluorescence detected the expression of STAT3 and P -STAT3(T705). (2) pEGFP-PIAS3: Total RNA was extracted and reversely transcribed using RT-PCR with the designed primers. PIAS3 coding sequences were then subcloned into the eukaryotic expression vector pEGFP-N1. All constructs were verified with gene sequencing. Western blotting analyzed STAT3 dimer after PIAS3 transfection. (3) After human LIF (20 ng/ml) and theγ-secretase inhibitor dropped in HL-60 cells, detected the expression of gp190-CTF and PS1-NTF. Resultes: (1) Transfection with pcDNA3.0 containing the CTF of the gp190 increased the level of phosphorylated STAT3. Immunofluorescence microscopy revealed that P-STAT3 was significantly increased in both the cytoplasm and nuclei. Cells expressing either 190CT3 or 190CT2+3 had significantly increased level of P–STAT3 in comparison to the wild-type or pcDNA3.0 controls, and decreased leval of P-MAPK. In comparison to cells expressing the gp190 CTF, cell expressing mutant C-terminal had higher rate of proliferatio, lower degree of differentiation. Cells expressing mutant C-terminal gp190 had lower P-STAT3 level and lower P-MAPK. (2) Co-transfection with PIAS3 significantly reduced the level of dimeric STAT3. (3) LIF significantly increased the level of PS1-NTF as well as the amount of the 26-kD cytoplasmic gp190. Theγ-secretase inhibitor significantly decreased the 26-kD cytoplasmic gp190 fragment and PS1-NTF and increased PS1-FL. Conclusion: The gp190 C–terminal fragment could enhance STAT3 phosphorylation and nuclear translocation. After YXXQ mutation or PIAS3 transfection ,activated STAT3 decreased. Gp190 could be cleaved byγ–secretase.
PartⅢBALB/c nude mouse experiment
Method: (1) Animal models: Male BALB/c nude mice (4 weeks of age) were kept under clean condition. At the first time, Mice (7 in each group) received 2×105 HL-60 cells transfected with pcDNA3.0-190CT3 or pcDNA3.0-Mutant in 200μl via the tail vein. A PBS control, a HL-60 wild-type control, and a pcDNA3.0 control were also included. After 30 days, mice were injected 2×106 cells at the second time. (2) Peripheric blood was collected at days 7, 14, 21 and 28. Granulocytes and lymphoid cells were counted using Wright staining under a microscope. Bone marrow was obtained from the femur after sacrifice at days 28. Flow cytometric analysis the CD15 and HLA-1 expression in bone marrow cells. After 60d, the mice were killed and organs were harvest and were in the processing of H.E. staining. Resultes: (1) Lymphoid cell count in the mice receiving HL-60 cells expressing 190CT3 was significantly lower than that in mice receiving the widetype or pcDNA3.0 control. In contrast, granulocyte count was considerably higher in mice receiving cells expressing 190CT3. In mice receiving HL-60 cells expressing mutant 190CT3, count for lymphoid cells and granulocytes was somewhere between that in the wild-type and cells expressing the 190CT3. (2) Cytometric analysis revealed that CD15-positive cells in the bone marrow were significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. CD15-positive cell count in the mice receiving the mutant was higher than the wild-type control, but lower than the 190CT3 group. HLA-1 positive cells in the bone marrow was also significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. (3) Lung pathology: the necrosis configuration was showed in wild-type, pcDNA3.0 and MUT- HL60 transplanted mice, but not in the 190CT3-HL60 mice. Conclusion: gp190 C-terminal fragment trended to differentiation and had low infestation and metastasis in vivo.
Conclusions
In conclusion, this study provided strong, albeit not conclusive evidence that the C-terminal of the gp190 could be released via enzymatic cleavage byγ-secretase. The released fragment could inhibit the proliferation of leukemia cells, induce their differentiation into granulocytes by enhancing STAT3 phosphorylation and nuclear translocation. Results from our in vivo experiments suggest that targeting the gp190 C-terminal and related pathways may be a promising novel approach in the treatment of leukemia. Effects of 190CT3 on the proliferation of HL-60 cells could be partially blocked by eitherboth mutation of the YXXQ sequence in the 190CT3 and or PIAS3 transfection partially blocked the effects of gp190 Box 3 on the proliferation of HL-60 cells. In vivo, 190CT3 in HL-60 had a low malignancy. Gp190 C-terminal fragment prevented tumor growth in animal models of human acute myeloid leukemia.
第一部分、重组人gp190胞内功能片段的白血病细胞株的获得与鉴定
方法:(1)酶切初步鉴定已构建的重组质粒pcDNA3.0-190CT2+3、pcDNA3.0-190CT3,进一步测序;构建重组质粒pcDNA3.0- MUT:通过引物重叠PCR法设计定点突变,在引物中置换突变碱基,将190CT3蛋白序列中三处YXXQ基团的酪氨酸(Y)位点突变为苯丙氨酸(F),将突变后的基因序列插入载体pcDNA3.0。(2)将重组质粒用FuGENE-6脂质体转染人白血病细胞株HL-60和K562,G-418梯度筛选阳性克隆15d,挑选生长旺盛的克隆扩增,设转染pcDNA3.0空质粒载体和野生型HL-60和K562细胞为对照组。鉴定转染后细胞内gp190羧基末端(C-terminal)的表达:用免疫细胞化学,蛋白印迹和RT-PCR的方法进行蛋白水平的半定量检测,确定稳定表达目的基因的细胞株,以备后面实验使用。(3)苏木精染色,显微镜下观察各组细胞形态;细胞计数绘制生长曲线;金黄色葡萄球菌实验检测细胞吞噬功能;流式细胞术检测细胞表面成熟粒单核细胞相关抗原的表达。结果:(1)BamHⅠ、XbaⅠ双酶切重组质粒pcDNA3.0- 190CT2+3、pcDNA3.0- 190CT3,电泳紫外灯下观察可见大小相符的目的片段,测序结果与Genbank核酸序列数据库中LIFR序列(X61615)比对,完全一致。190CT2+3全长597个碱基,190CT3全长357个碱基。对190CT3定点突变,获得339bpDNA片段,经BamHⅠ、XbaⅠ双酶切连接重组入质粒pcDNA3.0,双向测序后与Genbank数据库中LIFR序列(X61615)比对,原190CT3片段的三处编码酪氨酸的cDNA序列TAT正确置换为编码苯丙氨酸的TTC,成功获得重组pcDNA3.0-MUT的真核表达质粒。(2)经G-418筛选得到重组190CT2+3、190CT3、MUT的HL-60和K562细胞和空质粒转染细胞。用免疫印迹法检测LIFR,于20KD、15KD处见目的蛋白条带,选取稳定表达的细胞株进行下一步实验。苏木精染色,光镜下观察细胞形态,结果显示190CT2+3和190CT3诱导后,细胞的杆状核与分叶核增加。统计学分析分叶核比例:190CT3>190CT2+3组,P<0.01,组间差别有意义。(3)细胞计数,绘制生长曲线,190CT2+3和190CT3诱导后,细胞生长速度减慢:190CT3< 190CT2+3< MUT< pcDNA3.0< WT HL-60/K562。流式细胞术检测CD15、CD11b和CD14,结果显示转染190CT2+3和190CT3后标志分子表达增加:190CT3> 190CT2+3> MUT> pcDNA3.0> WT HL-60/K562。结论:(1)将190CT2+3、190CT3和MUT重组质粒转染HL-60/K562细胞,经检测说明重组白血病细胞株已经稳定表达目的基因。(2)190CT2+3、190CT3组分叶核的比例增加,增殖减慢,CD15/CD11b/CD14表达增高,对金葡菌的吞噬功能增强,证明gp190 C-末端功能域对白血病细胞有促进分化,抑制增殖的作用。
第二部分、gp190胞内功能片段信号通路以及与γ-Secretase关系的研究
方法:(1)免疫印迹法检测各组白血病细胞株(HL-60/K562)中磷酸化STAT3和磷酸化MAPK的蛋白表达;免疫荧光双标法检测各组HL-60细胞中STAT3和磷酸化STAT3。(2)构建PIAS3-pEGFP重组质粒:抽提HL-60细胞的总RNA,逆转录得到cDNA,设计四对引物,通过引物重叠延伸PCR法,扩增得到PIAS3编码区全长序列;经XhoⅠ、SacⅡ双酶切连接重组入真核表达载体pEGFP-N1,双向测序后与Genbank数据库中PIAS3序列(NM_006099)比对。通过脂质体瞬时转染190CT3-HL60细胞,通过免疫荧光和免疫印迹法检测转染后不同时间点PIAS3的表达,通过免疫印迹法检测PIAS3作用后,不同时间点STAT3二聚体的表达。细胞计数,绘制转染后的细胞生长曲线。(3)人LIF因子和γ-secretase抑制剂分别作用于HL-60细胞,免疫印迹法检测gp190(全长和C-末端)和PS1(全长和N-末端)的表达。结果:(1)免疫印迹法检测信号分子的蛋白表达,结果显示190CT2+3、190CT3组白血病细胞中(HL-60/K562)磷酸化STAT3明显较对照组增高;磷酸化MAPK的表达情况相反,190CT组表达量较对照组明显降低。激光共聚焦检测的结果显示190CT3组细胞磷酸化STAT3表达增强,STAT3核聚集增多。(2)免疫荧光和免疫印迹法检测证实PIAS3随转染时间的延长,表达增加;在PIAS3的作用下,STAT3二聚体的表达下降,细胞增殖转而加快。(3)LIF作用后,gp190的C-末端约26KD的片段,蛋白表达增加;同时伴随PS1的N-末端(PS1-NTF)蛋白表达逐渐增加,说明gp190胞内区游离多肽增加,γ-secretase分泌酶活性增加。γ-secretase抑制剂的作用下,gp190的C-末端约26KD的片段,蛋白表达减少;同时伴随PS1的N-末端(PS1-NTF)蛋白表达逐渐减少,说明gp190胞内区游离多肽减少,γ-secretase分泌酶活性降低。γ-secretase分泌酶可能切割了gp190产生游离功能域。结论:(1)目的片段190CT2+3与190CT3,即gp190胞内区C-末端功能域在白血病细胞内游离存在时,可以激活信号分子STAT3,同时抑制了信号分子MAPK;C-末端的YXXQ官能体发挥重要作用。(2)PIAS3瞬时作用于190CT3-HL60细胞后,STAT3二聚体减少,细胞增殖加快,说明STAT3在190CT3抑制白血病细胞的增殖过程中发挥关键作用。(3)hLIF刺激引起gp190 C-端片段和PS1 N-端片段表达增加,γ-secretase抑制剂作用使gp190 C-端片段和PS1 N-端片段表达减少,说明生理状态下,γ-secretase可能有切割gp190,产生游离C末端的作用。
第三部分、动物实验
方法:(1)4-5周龄雄性裸鼠由轻到重排列,按照随即配对的分组方法,分成5组(PBS、野生型HL-60、pcDNA3.0-HL60、MUT-HL60和190CT3-HL60),每组7只,尾静脉注射。第一次接种量为2×105个细胞,设立空白对照(注射200μlPBS)。记录接种日期,每天观察小鼠状态及行为表现。30天后第二次接种2×106个细胞。(2)7、14、21、28天分别断尾采集外周血制作血涂片,瑞氏染色,计数外周血中淋巴样细胞和粒细胞百分率;28天取骨髓细胞,流式细胞术检测骨髓细胞中标志分子CD15和HLA-1的表达。第二次接种60天后取出脏器(肝、肾、脾、肺和胃肠)做H-E染色,检测脏器有无病理结构的改变。结果:(1)外周血:CT3组裸鼠粒细胞比例较高,淋巴样细胞比例较低;骨髓:在第28天的检测结果显示,CT3组CD15和HLA-1的表达均升高。(2)组织切片的H-E染色。肝脏:均可见肉芽组织。肺部:野生型HL-60细胞移植组,部分肺泡结构消失,其内有边界不清的肉芽肿,有浆细胞、组织细胞,淋巴样细胞浸润,有出血坏死;pcDNA3.0-HL60移植组,部分肺泡正常结构被破坏,有肉芽肿样结构,部分坏死;MUT-HL60移植组,部分肺泡正常结构被破坏伴坏死、出血;190CT3-HL60移植组,肺部结构正常,肺泡间隔增厚,少量淋巴样细胞聚集,无出血坏死结构。结论:(1)移植后,CT3组细胞在裸鼠体内仍然表现为较成熟的粒细胞形态,维持了较分化的状态。(2)190CT3组HL-60细胞在裸鼠体内恶性程度降低。
小结
1、gp190胞内C-末端功能域在细胞内游离存在时,促进急性髓系白血病细胞向成熟细胞方向分化。
2、gp190胞C-末端功能域在白血病细胞内游离存在时,可以激活STAT3信号通路,同时抑制了信号分子MAPK;C-末端的YXXQ突变或PIAS3转染后,STAT3通路被抑制,细胞增殖加快;γ-secretase可能切割gp190产生细胞内的游离功能域,参与信号传递过程。
3、gp190胞内C-末端功能域作用后的白血病细胞在裸鼠体内恶性程度降低。
Background and objective
LIF induces terminal differentiation of myeloid leukemia cells, also modulates growth/differentiation of many other types of target cells. Other cellular functions influenced by this molecule include: embryogenesis, inflammation, neural development. LIF receptor is a heterodimer consisting of a low affinity binding protein gp190 and a signal transducing unit gp130. gp190 belongs to the hematopoietin receptor family, and is characterized by a cytokine receptor homology (CRH) domain. Cytoplasmic domain of gp190 contains three homologous and functionally important motifs: Box 1, 2 and 3. Gp190 binds to the src-homology 2 (SH2) domain of STAT3 via the YXXQ consensus sequences in the C-terminal. In addition to a membrane form, gp190 also exists in a soluble form that lacks transmembrane domain. Previous reports indicated that certain motif of the cytoplasmic gp190 could activate cell proliferation and differentiation. In the current study, transfecting leukemia cells with vectors containing a sequence encoding the C-terminal fragment (CTF) of gp190 (containing BOX 3 or BOX 2+3 ) inhibited cell proliferation, and induced differentiation. Such effects were significantly attenuated by a mutation in CTF (containing Box 3), as well as by inhibiting STAT3 using a selective inhibitor. STAT3 phosphorylation and nuclear translocation were significantly enhanced in cells expressing the CTF (containg Box 3). The cells expressing the CTF had a slower rate of proliferation upon transplantation into nude mice Analysis of cell morphology and markers for maturity indicated that these cells have higher degree of differentiation. We also found that cytoplasmic region of gp190 could be cleaved byγ-secretase upon LIF stimulation. Taken together, these findings suggest that a gp190 CTF may exist in leukemia cells, and modulate the property of these cells via the STAT3 pathway. It is beneficial to explore the application value about the single motif of cytoplasmic receptor, induce leukemia cells to differentiation.
PartⅠRecombination of human dissociated LIFR (gp190) cytoplasmic receptor in HL-60 cells and identification its expression
Methods: (1) Enzyme BamHⅠand XbaⅠcleaving the recombined plasmid pcDNA3.0-190CT2+3、pcDNA3.0-190CT3, and checking the sequences. Mutant of 190CT3: mutagenic oligonucleotide primers were complementary to the gene sequence in the region to be mutated, except Y-to-F conversion at three YXXQ sequences. The mutagenic oligonucleotide was then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. Desired sequences were amplified using PCR and subcloned into pcDNA3.0. (2) Transfected the eukaryotic expression plasmid pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT into leukemia (HL-60/K562) cells with liposome FuGENE-6. Two control group were setup which were vector pcDNA3.0 tansfected and wild-type HL-60/K562 cells. In the culture processes, we selected the clones with different G-418 concentration. The transfected cells were survival and expanding in presence of neo-gene. We detected the protein expression with western blotting and RT-PCR to identify the leukemia cell lines with stable protein expression. (3) Hematoxylin staining showed the morphous of nuclei. Describing the growth curve. Staphylococcus aureus were added into HL-60 cells. Cell smear was stained using a Wright method, and examined under a light microscope. Percentage of the HL-60 cells containing staphylococcus was used as an index for phagocytosis. The levels of CD15/CD14/CD11b were assayed by flow cytometer. Results: (1)After BamHⅠand XbaⅠcleaving, we observed electrophoretic irradiance strip under the UV light. Sequence detection was consistent with the LIFR sequence in Genbank. After PCR reaction, there is one 300-400bp DNA strip. Blast the MUT sequence and the original LIFR sequence, we found that the original TAT were exactly changed to TTC. The MUT4 colony was expressed the higher MUT by RT-PCR. (2) After subsequent selection by G418, G418-resistent colonies were isolated, namely ,the pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT and pcDNA3.0 leukemia cell lines. What western blotting and RT-PCR analyzed demonstrated that the 190CT had expressed in leukemia cells stably. Cells expressing the gp190 C-terminal fragment were significantly larger than cells transfected with pcDNA3.0. The nuclei of these cells were more leafy than the wild-type as well as the pcDNA3.0 controls. The rate of proliferation was significantly decreased by both 190CT3 and 190CT2+3. Expression of the mature granulocyte marker CD15/CD14/CD11b and the phagocytosis index were significantly increased. Conclusion: 190CT3 and 190CT2+3 could promote the differentiation and inhibit the proliferation of leukemia cells.
PartⅡ: Signaling pathway investigation for human dissociated LIFR (gp190) cytoplasmic receptor
Method: (1)Western blotting analyzed the levels of related signal molecular as P -STAT3(T705), P44/42-MAPK. Immunofluorescence detected the expression of STAT3 and P -STAT3(T705). (2) pEGFP-PIAS3: Total RNA was extracted and reversely transcribed using RT-PCR with the designed primers. PIAS3 coding sequences were then subcloned into the eukaryotic expression vector pEGFP-N1. All constructs were verified with gene sequencing. Western blotting analyzed STAT3 dimer after PIAS3 transfection. (3) After human LIF (20 ng/ml) and theγ-secretase inhibitor dropped in HL-60 cells, detected the expression of gp190-CTF and PS1-NTF. Resultes: (1) Transfection with pcDNA3.0 containing the CTF of the gp190 increased the level of phosphorylated STAT3. Immunofluorescence microscopy revealed that P-STAT3 was significantly increased in both the cytoplasm and nuclei. Cells expressing either 190CT3 or 190CT2+3 had significantly increased level of P–STAT3 in comparison to the wild-type or pcDNA3.0 controls, and decreased leval of P-MAPK. In comparison to cells expressing the gp190 CTF, cell expressing mutant C-terminal had higher rate of proliferatio, lower degree of differentiation. Cells expressing mutant C-terminal gp190 had lower P-STAT3 level and lower P-MAPK. (2) Co-transfection with PIAS3 significantly reduced the level of dimeric STAT3. (3) LIF significantly increased the level of PS1-NTF as well as the amount of the 26-kD cytoplasmic gp190. Theγ-secretase inhibitor significantly decreased the 26-kD cytoplasmic gp190 fragment and PS1-NTF and increased PS1-FL. Conclusion: The gp190 C–terminal fragment could enhance STAT3 phosphorylation and nuclear translocation. After YXXQ mutation or PIAS3 transfection ,activated STAT3 decreased. Gp190 could be cleaved byγ–secretase.
PartⅢBALB/c nude mouse experiment
Method: (1) Animal models: Male BALB/c nude mice (4 weeks of age) were kept under clean condition. At the first time, Mice (7 in each group) received 2×105 HL-60 cells transfected with pcDNA3.0-190CT3 or pcDNA3.0-Mutant in 200μl via the tail vein. A PBS control, a HL-60 wild-type control, and a pcDNA3.0 control were also included. After 30 days, mice were injected 2×106 cells at the second time. (2) Peripheric blood was collected at days 7, 14, 21 and 28. Granulocytes and lymphoid cells were counted using Wright staining under a microscope. Bone marrow was obtained from the femur after sacrifice at days 28. Flow cytometric analysis the CD15 and HLA-1 expression in bone marrow cells. After 60d, the mice were killed and organs were harvest and were in the processing of H.E. staining. Resultes: (1) Lymphoid cell count in the mice receiving HL-60 cells expressing 190CT3 was significantly lower than that in mice receiving the widetype or pcDNA3.0 control. In contrast, granulocyte count was considerably higher in mice receiving cells expressing 190CT3. In mice receiving HL-60 cells expressing mutant 190CT3, count for lymphoid cells and granulocytes was somewhere between that in the wild-type and cells expressing the 190CT3. (2) Cytometric analysis revealed that CD15-positive cells in the bone marrow were significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. CD15-positive cell count in the mice receiving the mutant was higher than the wild-type control, but lower than the 190CT3 group. HLA-1 positive cells in the bone marrow was also significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. (3) Lung pathology: the necrosis configuration was showed in wild-type, pcDNA3.0 and MUT- HL60 transplanted mice, but not in the 190CT3-HL60 mice. Conclusion: gp190 C-terminal fragment trended to differentiation and had low infestation and metastasis in vivo.
Conclusions
In conclusion, this study provided strong, albeit not conclusive evidence that the C-terminal of the gp190 could be released via enzymatic cleavage byγ-secretase. The released fragment could inhibit the proliferation of leukemia cells, induce their differentiation into granulocytes by enhancing STAT3 phosphorylation and nuclear translocation. Results from our in vivo experiments suggest that targeting the gp190 C-terminal and related pathways may be a promising novel approach in the treatment of leukemia. Effects of 190CT3 on the proliferation of HL-60 cells could be partially blocked by eitherboth mutation of the YXXQ sequence in the 190CT3 and or PIAS3 transfection partially blocked the effects of gp190 Box 3 on the proliferation of HL-60 cells. In vivo, 190CT3 in HL-60 had a low malignancy. Gp190 C-terminal fragment prevented tumor growth in animal models of human acute myeloid leukemia.
引文
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