【作者】 邹剑峰；

【导师】 侯健；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 多发性骨髓瘤(multiple myeloma, MM)是一种浆细胞恶性肿瘤,以骨髓中异常浆细胞恶性增殖、分泌单克隆免疫球蛋白、正常免疫球蛋白受到抑制以及溶骨病变为特征。发生率占血液系统肿瘤的10%,占所有肿瘤的1%,死亡率占所有肿瘤的2%。美国癌症学会统计表明,2009年美国MM的新发病例约为20,580,包括男性患者11,680例,女性患者8900例,死亡约10,580例,MM发病率仅次于非霍奇金淋巴瘤,成为血液系统的第二位常见的恶性肿瘤。在我国也呈逐年升高的趋势。MM的治疗主要包括传统化疗,造血干细胞移植以及新药治疗。目前,新的靶向治疗药物已经成为治疗MM的重要手段,但就目前而言,骨髓瘤仍是不可治愈的疾病。维甲酸在诱导分化治疗急性早幼粒细胞白血病领域取得的巨大成功,鼓舞人们进行深入研究肿瘤的分化治疗策略。但在诱导骨髓瘤细胞分化方面其作用机制尚未完全阐明。因此,需要对其分子生物学机制进一步研究,以便为临床新药的开发与使用提供理论依据,进而制定新的联合用药方案,提高临床疗效。内质网(endoplasmic reticulum, ER)是真核细胞中负责分泌性蛋白折叠和组装的细胞器。当细胞在病原感染、化学损伤、遗传突变、营养剥夺,以及正常分化(如B细胞分化)等各种内外源因素刺激下,ER中未折叠或错误折叠的蛋白增多,出现ER应激(ER stress),应激信号能通过ER膜传递到细胞核中,继而引起一系列特定的靶基因转录上调和蛋白质翻译水平下调,以使细胞能继续存活,这种反应就是未折叠蛋白反应(unfolded protein response, UPR)。MM细胞是浆细胞克隆增生性肿瘤,同正常浆细胞一样,骨髓瘤细胞具有高度发达和扩张的粗面ER,每秒钟能产生成千上万个免疫球蛋白分子,大量的免疫球蛋白在分泌之前必须经过ER的正确折叠和组装。如果这些蛋白不能被正确折叠,就会出现ER应激,诱导UPR的产生。浆细胞通过UPR减少免疫球蛋白分子的翻译,增加ER伴侣分子GRP78(glucose regulating protein 78)和GRP94(glucose regulating protein 94)以及折叠酶的表达,增强ER相关蛋白质降解途径(ER-associated degradation, ERAD)活性,提高细胞处理未折叠蛋白的能力,使之存活。目前研究显示ER膜上有3种跨膜蛋白感应ER应激：PERK(PKR-like ER kinase)、ATF6(activating transcription factor-6)以及具有激酶和核酸内切酶活性的IRE1(inositol-requiring enzyme 1)。活化后的IRE1具有核酸内切酶活性,对XBP-1 mRNA进行剪接形成376aa的XBP-1剪接蛋白(XBP-1s);未经剪接的XBP-1 mRNA则形成261aa的蛋白(XBP-1u)。XBP-1是具有b-zip结构的转录因子,大量证据表明XBP-1对B细胞分化成熟为浆细胞,并分泌免疫球蛋白是至关重要的,为浆细胞分化所必需。XBP-1-/-的B淋巴细胞胞浆中可以表达免疫球蛋白,但不能大量的分泌免疫球蛋白,也不能完成B淋巴细胞的最终分化过程；在XBP-1-/-的B淋巴细胞中经基因转染后重新表达XBP-1 mRNA,可以有效地恢复免疫球蛋白的分泌。因此,XBP-1在UPR时表达上调,是UPR的关键靶基因,也对浆细胞的成熟、分化具有重要意义。目前研究显示UPR在正常B细胞向浆细胞的分化过程中起重要的作用,没有UPR,浆细胞不能完成最终的分化和分泌免疫球蛋白。XBP-1在UPR活化时表达上调,并增加UPR靶基因伴侣分子GRP78和GRP94的表达,调节UPR。XBP-1也是UPR反应元件中与浆细胞分化相关的唯一转录因子。当前研究国际上对诱导分化治疗MM研究甚少,尤其通过修饰未折叠蛋白反应诱导MM分化还是空白。因此,我们设想通过UPR的经典诱导剂二硫苏糖醇(DTT)或衣霉素(tunicamycin)诱导MM细胞发生UPR,观察对MM细胞分化的影响,研究XBP-1u、XBP-1s以及下游的伴侣分子GRP78、GRP94等的表达,然后采用RNAi等技术,影响XBP-1u、XBP-1s等的表达修饰UPR,最终达到明确诱导MM细胞分化的机制。本实验研究包括以下三个部分：第一部分小剂量UPR诱导剂诱导骨髓瘤细胞分化的实验研究目的探讨小剂量UPR诱导剂对骨髓瘤细胞的分化作用。方法用小剂量UPR诱导剂(TM, DTT)处理MM细胞株U266, RPMI-8226。Annix V检测细胞的凋亡率。经细胞涂片,瑞氏染色后,显微镜下观察细胞形态；流式检测检测细胞表面CD49e的表达率；用人λ轻链ELISA试剂盒检测细胞培养上清液中轻链蛋白的浓度。结果UPR诱导剂TM 0.4μmol/L, DTT 0.5mmol/L处理骨髓瘤细胞株U266,RPMI-8226 72小时后,Annix V检测细胞的凋亡率均低于5%。细胞形态学观察与对照组相比即可见到细胞胞核缩小,胞浆丰富,核浆比例下降,核仁减少或消失,核染色质变粗、变密。有些细胞具有典型的碱性胞浆,胞浆丰富,出现胞核偏心,核染色质粗糙,凝聚成块状,着色深,排列成车轮状,,呈现成熟浆细胞特有的形态改变,有别于细胞凋亡的早期形态学改变。U266细胞CD49e的阳性率在对照组8.61±0.82%,TM处理72h后,U266细胞CD49e的阳性率为43.15±3.78%,有统计学差异(p<0.01),DTT处理72h后CD49e的阳性率为38.08±2.9%,有统计学差异(p<0.01)。RPMI-8226细胞CD49e的阳性率对照4.89±0.37%,TM处理72h后,CD49e的阳性率为25.54±2.67%,有统计学差异(p<0.05)；DTT处理72h后CD49e的阳性率为11.83±1.38%(p<0.05)。U266细胞的培养上清液中轻链蛋白的浓度,对照组456.25±19.07ng/ml, TM处理72h组,692.35±45.4ng/ml,有统计学差异(p<0.01),DTT处理72h后,轻链蛋白的浓度为614.42±22.67ng/ml,有统计学差异(p<0.05)。RPMI-8226细胞的培养上清液中轻链蛋白的浓度,对照组292.24±23.59ng/ml,TM处理72h组,407.16±18.09,有统计学差异(p<0.01),DTT处理72h后,轻链蛋白的浓度为478.35±15.37g／ml,有统计学差异(p<0.05)。在原代细胞经UPR诱导剂处理72小时后,也得到有类似的结果,细胞具有形态学分化的表现,CD49e细胞的阳性率增加,培养上清液中轻链蛋白的浓度增加。结论小剂量UPR诱导剂可以诱导骨髓瘤细胞株和原代细胞进一步的分化。第二部分UPR诱导剂诱导骨髓瘤细胞分化过程中相关基因表达情况的研究目的研究小剂量UPR诱导剂(TM, DTT)在诱导骨髓瘤细胞分化过程中,未折蛋白反应相关基因GRP78,GRP94的转录水平变化,与UPR和B细胞分化密切相关的XBP-1u, XBP-1s在转录和蛋白翻译水平的变化。方法小剂量UPR诱导剂(TM, DTT)处理骨髓瘤细胞株U266,RPMI-8226,荧光Realtime PCR检测基因GRP78, GRP94, XBP-1u,, XBP-1s的转录水平变化,Western Blot检测XBP-1u, XBP-1s蛋白翻译水平的变化。结果小剂量UPR诱导剂(TM, DTT)处理骨髓瘤细胞株U266,RPMI-8226后与对照组相比,荧光Realtime PCR检测基因GRP78, GRP94, XBP-1u,的转录水平是明显上调的,上调水平约1.5～3.12倍,而XBP-1s的转录水平是下降的。Western Blot检测XBP-1u的翻译水平是相对增加的,而XBP-1s的蛋白翻译水平下调。结论在骨髓瘤细胞的诱导分化过程中发生了未折叠蛋白反应,XBP-1u的转录和翻译水平是相对增加的。第三部分调控基因XBP-1u/XBP-1s表达对骨髓瘤细胞分化的影响目的研究基因XBP-1u/XBP-1s表达水平的差异对骨髓瘤细胞分化的影响。方法分别采用siRNA沉默XBP-1u的表达,构建过表达质粒转染骨髓瘤细胞过表达基因XBP-1u和XBP-1s后,通过形态学观察,流式检测细胞表面CD49e的表达率,和ELISA试剂盒检测培养上清中轻链蛋白的含量。结果采用siRNA沉默XBP-1u的表达后,骨髓瘤细胞的进一步分化被阻断；过表达XBP-1u可以促进骨髓瘤细胞的分化,表现为形态学上分化成熟表现,CD49e的阳性率增加,细胞上清液中轻链含量是增加的；而过表达XBP-1s则不能促进骨髓瘤细胞的分化。结论XBP-1的表达水平与骨髓瘤细胞的分化密切相关,其中XBP-1u的上调在其中起着重要作用。更多还原

【Abstract】 Multiple myeloma (MM) is a neoplasm of malignant plasma cells, characterized by accumulation of malignant plasma cells in the bone marrow, production of a monoclonal protein, reduction in uninvolved immunoglobulin and lytic bone lesions. It accounted for 10% of hematological malignancies and 1% of all cancers. It represents approximately 2% of all cancer deaths.The American Cancer Society has estimated 20,580 new cancer cases of MM in the United States in 2009, including 11,680 cases in men and 8900 cases in women, with an estimated 10,580 deaths. MM is the second most prevalent blood cancer after non-Hodgkin’s lymphoma. Its incidence also appears to be increasing in our country. The treatments of MM include conventional chemotherapy, hematopoietic stem cell transplantation and new drugs. And multiple myeloma is still incurable nowadays. As to all-trans retinoic acid (ATRA) in inducing differentiation therapy took great effect in acute promyelocytic leukemia. People try to test new strategies to cancer therapy. The mechanism in induction of myeloma cells’differentiation is unclear. Therefore, further study on their molecular biology mechanism is needed. Further understanding of the mechanism will be useful to a new agent development and its clinical application, make sense to design new combinations of therapies to raise clinical response.Endoplasmic reticulum (ER) is an organelle of eukaryotic cells, and it is major in r secretary protein folding and assembly of the secreting protein. When the cells in pathogen infection, chemical injury, genetic mutations, nutritional deprivation, as well as normal differentiation (such as B cell differentiation), and other internal and external source of stimulation factors, unfolded or misfolded proteins accumulated in ER, and induce cell stress occurs (ER stress), stress signals pass through the ER membrane to the nucleus, and then cause a series of specific target gene transcription regulated and protein translation level down-regulated, so that cells can survive, and this reaction is called unfolded protein response (UPR). MM are the plasma cell clone proliferate tumors, the same as normal plasma cells, myeloma cells have highly developed and expansion of rough ER, can produce tens of thousands of immunoglobulin molecules per second, a large quantity of immunoglobulin must be the correct folding and assembly in the ER before secretion. If these proteins can’t be correctly folded, there will be ER stress, and induced UPR. Plasma cells through UPR reducing the translation of immunoglobulin molecules, increasing ER Chaperone GRP78 (glucose regulating protein 78) and GRP94 (glucose regulating protein 94), as well as expression of folding enzymes in the ER associated protein degradation pathway increased (ER-associated degradation, ERAD) activity, to increase processing capacity of unfolded proteins to make cell survival.The current study shows that there are three kinds of transmembrane protein of ER stress sensors in ER membrane:PERK (PKR-like ER kinase), ATF6 (activating transcription factor-6), as well as IRE1 (inositol-requiring enzyme 1).with the kinase and endonuclease activity. IRE1 with endonuclease activity after the activation, carried out on XBP-1 splicing. XBP-1 mRNA splicing form translation of 376aa protein (XBP-1s); and the unspliced XBP-1 mRNA will be translated to 261aa protein (XBP-1u). XBP-1 is a transcription factors with b-zip structure. Some studies revealed that XBP-1 is critical for plasma cell differentiation and maturation, and is necessary for secreting immunoglobulin. XBP-1(?) B-lymphocytes can express cytoplasm immunoglobulin, but can not secretary a large amount of immunoglobulin, it can not complete the final differentiation of B lymphocytes; in XBP-1(?) B-lymphocytes the gene re-expression of transfected XBP-1 mRNA in B lymphocytes, can effectively restore the secretion of immunoglobulin. Therefore, during the UPR, the XBP-1 expression increases, and XBP-1 is a key UPR target genes, is important to the plasma cells maturation and differentiation.The current studies have shown that UPR played a key role in the process of normal B-cell differentiation to plasma cells. Without UPR, plasma cells can not complete the final differentiation and secretion of immunoglobulin. Expression of XBP-1 up-regulated during the UPR activation and increased UPR target genes chaperone GRP78, GRP94 expression, regulating UPR. XBP-1 is the only one transcription factor of UPR-responsive element which related to the plasma cell differentiation.Currently, little International Research studied on the MM-induced differentiation therapies. Particularly it is still blank through the modification of unfolded protein response to induce the differentiation of MM. Therefore, we tested the adoption of the classic UPR inducer dithiothreitol (DTT) or tunicamycin (tunicamycin) to induce UPR and cell differentiation on MM cells. Observe the impact on MM cells’ differentiation. To study the XBP-lu, XBP-1s, as well as the expression of downstream Chaperone GRP78/GRP94, and then using RNAi technologies interfere the XBP-1u, XBP-1s expression to modify UPR, and finally identify the mechanism of mycelia cells induced further differentiation. Experimental studies include the following three parts:PartⅠStudy on Low-Dose UPR Inducing Agents Induced Myeloma Cell DifferentiationObjective To investigate the effect of low-dose UPR inducers in myeloma cell differentiation.Methods MM cell lines U266, RPMI-8226 were treated with a small dose of UPR inducer (TM, DTT). cell apoptosis detection by Annexin V. observed he cell morphology changes under the microscope after the cell smear, Wright stain; expression of CD49e on the cell surface was detect by flow cytometry; concentrations of light chain protein in cell culture supernatant were detected byλlight chain ELISA detection kitResults Myeloma cell lines U266, RPMI-8226 were treated by UPR inducers TM 0.4μmol/L, DTT 0.5mmol/L after treatment 72 hours, apoptosis rate were evaluated by Annexin V positive rate was less than 5%. Compared with the control group, Cell morphology changed to mature feature, the cell nucleus narrowed, rich in cytoplasm, the ratio of nucleus and cytoplasm declined nucleolus disappearance, or reduction of nuclear staining. Some cells have a typical alkaline cytoplasm, rich in cytoplasm. With eccentric nuclei, coarse nuclear chromatin, massive form, arranged in the wheel-shaped, the morphological changes were quite different from the morphological changes of cell early apoptosis. The CD49e positive rate of U266 cells, in control group were 8.61±0.82%, after TM treatment 72h, CD49e positive rate was 43.15±3.78%, a statistically significant difference (p<0.01), after DTT treatment 72h CD49e positive rate was 38.08±2.9%, there is significant difference (p<0.01). RPMI-8226 cells, CD49e positive rate of the control group were 4.89±0.37%, after TM, treatment 72h, CD49e positive rate were 25.54±2.67%, a statistically significant difference (p<0.05); after DTT treatment 72h, CD49e positive rate were 11.83±1.38%(p<0.05). concentration of light chain protein of the cell culture supernatant in the U266 control group 456.25±19.07ng/ml, after TM treatment 72h were 692.35±45.4ng/ml, a statistically significant difference (p<0.01), after DTT treatment 72h concentration of light chain protein were 614.42±22.67ng/ml, a statistically significant difference (p<0.05).RPMI-8226 cell lines in the control group were 292.24±23.59ng/ml, after TM treatment 72h were 407.16±18.09ng/ml, a statistic significant difference (p<0.01), after DTT treatment 72h Light chain protein concentration were 478.35±15.37ng/ml, a statistically significant difference (p<0.05).In primary cells treated UPR inducers by the 72 hours later, also had similar results, the with the mature morphological feature, CD49e positive cells increased, concentration of light chain protein in the supernatant up-regulated.Conclusion Low-dose UPR inducing agents tunicamycin and DTT can induce myeloma cell lines and primary cells further differentiation.PartⅡStudy on Related Gene Expression in UPR Inducing Agents Induced Myeloma Cell DifferentiationObjective To study the unfolded protein response related genes GRP78, GRP94 changes of transcription level in B cell differentiation with the low dose UPR inducers (TM, DTT) closely related to myeloma cell induction of and differentiation process, changes of XBP-1u, XBP-1s in the mRNA transcription and protein translation level.Methods Human myeloma cell lines U266, RPMI-8226 were treated with low-dose UPR inducer (TM, DTT), gene GRP78, GRP94, XBP-1u,, XBP-1s transcription level changes were detected by fluorescence Realtime PCR, XBP-1u, XBP-1s protein translation level changes. By Western Blot DetectionResults after the treatment of low-dose UPR inducer (TM, DTT) compared with the control group the gene GRP78, GRP94, XBP-1u, transcription levels were significantly up-regulated, and increases the level were about 1.5 to 3.12 folds, while the transcription of XBP-1s levels down-regulated. XBP-1u had a relative up-regulated in the level of translation, while XBP-1s protein translation levels were down-regulated by Western BlotConclusion UPR was activated during the induction of myeloma cell differentiation, XBP-1u was relative up-regulated both in transcription and translation levels.PartⅢRegulation of XBP-1u/XBP-1s Gene Expression on Myeloma Cells DifferentiationObjective To identify the different expression level of gene XBP-1u/XBP-1s on myeloma cell differentiation.Methods siRNA interfere the expression of XBP-1u, and construction plasmid and transfected myeloma cells to over express gene XBP-1u and XBP-1s, through morphological observation, flow cytometry detection expression of CD49e in cell surface, and ELIS A kit test light chain protein in the cell culture supernatant.Results after silencing the expression of XBP-1u, further differentiation of myeloma were blocked; over-expression of XBP-1u can promote the myeloma cells differentiation, showing the differentiation and maturation morphological changes, up-regulated the CD49e positive rate, and the concentration of light chain protein in the cell culture supernatant.Conclusion XBP-1 is related to myeloma cell induction differentiation, in which up-regulation of XBP-1u played a key role in the differentiation.更多还原