肿瘤坏死因子α仪对骨髓瘤患者骨髓间充质干细胞TAZ表达和成骨潜能的影响

目的 探讨肿瘤坏死因子α(TNF-α)、具有PDZ结合域的转录共刺激因子TAZ和骨髓瘤骨病的关系.方法 分离培养多发性骨髓瘤(MM)患者和正常人的骨髓间充质干细胞(MSCs),鉴定其生物学特性;酶联免疫吸附试验(ELISA)检测MM患者和正常人骨髓血清中TNF-α的浓度.MM患者骨髓CD138+细胞与正常人MSCs行Transwell成骨共培养后,采用实时定量PCR检测MSCs成骨指标和TAZ mRNA表达量,von Kossa染色法鉴定钙质沉积度.采用实时定量PCR和Westernblot检测MSCs中TAZ mRNA和蛋白质的表达量.结果 MM患者的MSCs成骨潜能和TAZ表达量较正常人降低.MM患者骨髓血清中的TNF-α浓度为(355.4±49.1)pg/ml,正常人为(92.3±17.2)pg/ml.CD138+骨髓瘤细胞可以抑制正常人MSCs向成骨细胞分化,TNF-α单克隆抗体可以部分逆转此抑制效应.随着培养体系中TNF-α(0、0.1、1、10 ng/mL)浓度梯度增加,MSCs中TAZ表达量逐渐降低.结论 MM患者的骨髓MSCs成骨潜能较正常人明显降低.骨髓瘤细胞可能是通过TNF-α抑制TAZ的表达,抑制MSCs的成骨作用.     

骨髓增生异常综合征患者骨髓来源间充质干细胞的成骨分化特点及其临床意义

目的：探讨骨髓增生异常综合征（myelodysplastic syndrome,MDS)患者骨髓来源间充质干细胞( mesenchymal stemcell, MSC)成骨分化特点及其临床意义。方法: 骨髓标本取自河北大学附属医院血液内科30 例未经治疗的新诊断的MDS患者。分离培养MDS患者及正常人的MSC,体外培养并观察MSC的形态学特点。在适当条件下诱导MSC向成骨细胞和成脂细胞分化,茜素红染色观察成骨诱导分化第14 天钙结节形成情况,实时荧光定量PCR法检测未分化MSC中成骨分化转录因子Ostefix、RUNX2 以及参与造血细胞向白血病细胞转化的Jagged-1 的mRNA表达水平。结果：MDS患者的骨髓来源的MSC表现为细胞体积增大、分化潜能下降。与对照组相比,成骨分化转录因子Osterix 和RUNX2 表达水平明显下降(均P<0.05);茜素红染色结果显示,MDS组钙结节含量也明显少于正常对照组,而Jagged-1 的表达水平明显升高(均P<0.05)。结论：MDS骨髓来源的MSC细胞体积增大、成骨分化能力明显减弱以及Jagged-1 的表达水平升高,可能在MDS造血功能衰竭和向急性髓系白血病转化的过程中发挥了重要作用。     

间充质干细胞的历史沿革、生物学特性与应用前景

上世纪70年代,首次有报道骨髓中少部分塑性贴附细胞能够分化形成类似骨或软骨的集落,这些细胞最终可以分化为间质系统细胞,被定义为“间质干细胞（mesenchymal sten cells,MSCs）”。MSC能表达多种表面抗原,但不具有特定的特征。MSCs具有自身更新能力和多向分化潜能,向骨、脂肪、软骨、骨骼肌细胞、甚至肝细胞、神经细胞分化,近年还发现MSC易于外源基因的转染和表达,被认为是很好的基因载体。MSC支持造血的作用已得到公认。目前的MSC的移植实验多为先在体外诱导分化为相应组织细胞后再植入体内,诱导分化操作易改变细胞特性,运用到临床治疗上,会发生种种无法预测的可能。     

间充质干细胞的历史沿革、生物学特性与应用前景

上世纪70年代,首次有报道骨髓中少部分塑性贴附细胞能够分化形成类似骨或软骨的集落,这些细胞最终可以分化为间质系统细胞,被定义为"间质干细胞(mesenchymal sten cells,MSCs)".MSC能表达多种表面抗原,但不具有特定的特征.MSCs具有自身更新能力和多向分化潜能,向骨、脂肪、软骨、骨骼肌细胞、甚至肝细胞、神经细胞分化,近年还发现MSC易于外源基因的转染和表达,被认为是很好的基因载体.MSC支持造血的作用已得到公认.目前的MSC的移植实验多为先在体外诱导分化为相应组织细胞后再植入体内,诱导分化操作易改变细胞特性,运用到临床治疗上,会发生种种无法预测的可能.     

TGF-β1和bFGF对骨髓基质干细胞增殖、分化的影响

目的探索体外培养条件下转化生长因子-β1(TGF-β1)和碱性成纤维生长因子(bFGF)对大鼠骨髓间充质干细胞(MSCs)增殖与分化的影响。方法用DMEM冲洗骨髓腔,收集骨髓细胞悬液,接种、体外培养、扩增并分别加入不同浓度TGF-β和bFGF,通过MTT法检测不同生长因子对MSCs增殖的影响,通过RT-PCR技术测定碱性磷酸酶(ALP)、骨钙素(Osteocalcin,OCN)和骨桥蛋白(Osteopontin,OPN)的表达了解生长因子对MSCs分化的影响。结果低浓度TGF-β和bFGF促进MSCs增殖,而高浓度TGF-β和bFGF抑制MSCs增殖;TGF-β和bFGF促进MSCs的ALP、OCN和OPN表达。结论低浓度TGF-β和bFGF可以作为大鼠MSCs较理想的诱导因子,可促进其增殖和分化,为体外培养组织工程化骨提供较好的种子细胞。     

骨髓间充质干细胞在异基因骨髓移植中的研究进展

骨髓间充质干细胞（MSCs）是骨髓基质细胞的前体细胞,分泌多种造血相关因子,表达多种黏附因子,同时具有免疫调节功能,在造血微环境发挥重要作用。因此,利用MSCs做滋养层,体外扩增造血干细胞（HSC）,临床联合移植MSCs和HSC,提高造血重建的能力,减少或减轻移植物抗宿主病（GVHD）的发生,具有很广阔的研究前景。     

骨髓间充质干细胞在异基因骨髓移植中的研究进展

骨髓间充质干细胞(MSCs)是骨髓基质细胞的前体细胞,分泌多种造血相关因子,表达多种黏附因子,同时具有免疫调节功能,在造血微环境发挥重要作用.因此,利用MSCs做滋养层,体外扩增造血干细胞(HSC),临床联合移植MSCs和HSC,提高造血重建的能力,减少或减轻移植物抗宿主病(GVHD)的发生,具有很广阔的研究前景.     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的：深入研究骨髓间充质干细胞（mesenchymal stem cells,MSCs）多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法：大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子（KGF）、胰岛素-转铁蛋白-硒（ITS）、尼克酰胺的无血清DMEM／F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果：培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论：MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的:深入研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法:大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子(KGF)、胰岛素-转铁蛋白-硒(ITS)、尼克酰胺的无血清DMEM/F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果:培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论:MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

CIK的体外增殖及体内外杀瘤活性的实验研究

目的:从人骨髓造血前体细胞体外培养扩增树突状细胞(dendritic cells,DCs),测定其表型及T细胞刺激活性.方法:采用Mini-MACS分离技术,从正常人骨髓、脐血分离CD34~ 造血干细胞,体外以重组hGM-CSF,hTNF-α,hIL-3诱导培养2周,流式细胞术检测扩增细胞的表面表型及细胞内IL-12的表达,体外同种混合淋巴细胞反应检测扩增DCs的T细胞刺激活性.结果:从正常人骨髓、脐血分离得到高纯度(>90%)的CD34~ 造血干细胞,经重组hGM-CSF,hTNF-α的共同诱导培养,扩增得到大量DCs,加人hIL-3可以进一步增加DCs产量;FACS检测表明,扩增的DCs表达HLA-DR,CD40,CD54,CD80,CD86分子,细胞内有hIL-12的P35,P40亚基的表达;与外周血单核细胞培养生成的DCs相比,由CD34~ 干细胞扩增的DCs具有更强的激发同种T细胞增殖的能力.结论:人CD34~ 干细胞体外经诱导培养,可以生成大量功能成熟的DCs,从而为进一步开展DCs的基础及临床研究打下了基础.     

Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with multiple myeloma.

Multiple myeloma (MM) is a B-cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM). The role of the BM microenvironment in the pathogenesis of the disease has been demonstrated, especially for the survival and growth of the myeloma plasma cells. Functional characterization of the major component of the BM microenvironment, namely the recently characterized mesenchymal stem cells (MSCs), was never performed in MM. Based on a series of 61 consecutive patients, we evaluated the ability of MSCs derived from myeloma patients to differentiate into adipocytes and osteocytes, inhibit T-cell functions, and support normal hematopoiesis. MSCs phenotypic characterization and quantification of interleukin-6 (IL-6) secretion were also performed. As compared to normal MSCs, MSCs from MM patients exhibited normal phenotype, differentiation capacity and long-term hematopoietic support, but showed reduced efficiency to inhibit T-cell proliferation and produced abnormally high amounts of IL-6. Importantly, these characteristics were observed in the absence of any detectable tumor plasma cell. Chromosomal analysis revealed that MM patients MSCs were devoid of chromosomal clonal markers identified in plasma cells. MM MSCs present abnormal features that may participate in the pathogenesis of MM.     

Antigenic open reading frames from HHV-8 are present in multiple myeloma patients and normal individuals at similar frequency

It has been proposed that the absence of a humoral response to human herpes virus 8 (HHV-8) in patients with multiple myeloma (MM) reflects strain variation or the mutation, or absence, of the antigenic regions of HHV-8 recognized in ELISA screening tests. We therefore assessed DNA sequence of three antigenic regions (ORF65, ORF73 and ORFK8.1) and the transforming hypervariable K1 ORF of HHV-8 in fresh bone marrow cells, bone marrow derived dendritic cells (DCs) and bone marrow stromal cells (BMSCs) from 12 patients with MM and 8 normal individuals. HHV-8 ORFs were detectable by nested PCR in MM patients (ORF65: 67% ORF73: 22% and K8.1: 58%), but were also surprisingly frequent in normal individuals (ORF65: 37%, ORF73: 12.5% and K8.1: 62%). HHV-8 sequences were more frequently detected in cells from BMSC and DC culture than from fresh bone marrow in MM. In contrast no HHV-8 sequences were detected in BMSC from normal individuals. Sequence analysis of ORF65 failed to demonstrate productive mutations in any MM sample. K1 genomic sequences were detected in 42% of MM and 37% of normals and exhibited 98% homology with the K1-A1 HHV-8 strain. In conclusion, our data do not support the presence of a K1-C3 strain of HHV-8 with ORF65 expression deficiency in MM patients. HHV-8 infection appears to be common in the general population when sensitive PCR is employed and multiple samples are analyzed.     

A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth

Mesenchymal stem cells (MSCs) have emerged as excellent candidates for clinical application because of their capabilities of immunomodulatory and supporting hematopoiesis. Nevertheless, it is unclear whether the characteristics of MSCs are altered in diseased states. In this study, we obtained and expanded MSCs from bone marrow of patients with myelodysplastic syndromes (MDS). Our results showed that MSCs derived from MDS (MDS-MSCs) were similar to normal adult bone marrow derived MSCs in morphology, growth property, surface epitopes, and differentiation ability in vitro. In addition, MDS-MSCs had normal karyotype and ultrastructure. However, MDS-MSCs appeared to be impaired in immunomodulatory and supporting hematopoiesis function. Although, MDS-MSCs could express hematopoietic cytokines and support hematopoiesis in long term culture, Real time quantitative polymerase chain reaction showed that the expression of hematopoietic cytokines in MDS-MSCs was much lower than that of normal adult derived MSCs. Moreover, MDS-MSCs showed reduced hematopoiesis support function, as compared to their normal counterparts. Lastly, the capacity of MDS-MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. These results indicate that MDS-MSCs have impaired immunomodulatory and hematopoiesis support functions, suggesting their critical role in the pathogenesis of MDS.     

与骨髓MSC共孵育的T淋巴细胞对白血病细胞的杀伤作用

目的 探讨经MSC诱导免疫耐受的T淋巴细胞对与MSC同源的肿瘤细胞的杀伤活性,以证实MSC诱导免疫耐受的同时,是否也削弱了移植物抗肿瘤的作用。方法 初治的白血病患者留取原代肿瘤细胞,液氮保存,诱导缓解后培养同一患者的骨髓MSC,取异体T淋巴细胞与MSC共孵育,比较与MSC共孵育的T淋巴细胞对MSC同源的肿瘤细胞的杀伤作用是否减弱。结果 与MSC共孵育后,T淋巴细胞对MSC同源的肿瘤细胞仍有杀伤作用,但与未共孵育者相比。杀伤活性减弱。结论 MSC在诱导免疫耐受的同时也削弱了GVI,的作用,提示在以MSC诱导免疫耐受预防GVHD的方案中,应考虑同时加强GVL作用,以防肿瘤复发。     

慢性粒细胞白血病骨髓来源的间充质干细胞免疫调节功能的研究

目的:研究慢性粒细胞白血病骨髓来源的间充质干细胞的免疫学特征,比较其与正常人来源的间充质干细胞是否存在免疫功能的差异。方法:分离正常人和慢性粒细胞白血病患者的骨髓间充质干细胞,分别检测它们对T 细胞周期、活化、抑制和增殖的作用。结果:CML 和正常志愿者骨髓来源的MSC 的细胞形态和表型没有差异,CML 患者来源的MSC 抑制T 细胞增殖的作用减弱,抑制T 细胞周期的作用减弱,抑制T 细胞活化的能力减弱,抑制T 细胞凋亡的作用增强。结论:CML 患者骨髓来源的MSC存在明显的免疫调节功能缺陷,使用CML 患者自体的MSC 移植治疗可能不是一种很好的选择,对于CML 患者最好选用异基因的MSC 移植治疗。      

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34⁺ Lin⁻ cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.     

The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.