大黄素对体外大鼠骨髓基质细胞向成骨细胞方向分化的影响

目的 :观察大黄素对骨髓基质细胞向成骨细胞方向分化的影响 ,探讨大黄素对干细胞分化方向的调节作用。方法 :用密度梯度离心的方法和传代贴壁筛选的方法相结合 ,分离纯化骨髓基质细胞。对硝基苯磷酸盐法判断是否成功诱导骨髓基质细胞向成骨细胞方向分化 ,碱性磷酸酶染色法、放射免疫测定法观察大黄素对骨髓基质细胞向成骨细胞方向分化的影响。结果 :结合密度梯度离心的方法和传代贴壁筛选的方法 ,分离纯化后可得到成分较为均一的骨髓基质细胞 ,其间存在有间质干细胞 ,经诱导后向成骨细胞方向分化。大黄素可增加大鼠骨髓基质细胞成骨诱导后细胞碱性磷酸酶的含量 ,其中10 -6mol·L-1浓度组作用 7d时效果明显。大黄素在 2 .5× 10 -7～ 10 -5mol·L-1浓度范围内未能增加细胞上清液和细胞裂解液中骨钙素含量。结论 :大黄素能促进骨髓基质细胞向成骨细胞方向分化 ,这一作用主要体现在骨形成过程的早期 ,对矿化期没有促进作用。     

抗骨质疏松药物作用靶位分子水平研究进展

1成骨细胞上的药物作用靶位多功能骨髓基质干细胞或多功能骨髓间充质干细胞(p luripotent mesenchymal stem cell,MSC)分化形成纤维样系统细胞,包括前成骨细胞、成骨细胞、成软骨细胞、成纤维细胞和网织细胞。来源于MSC的骨髓基质成纤维样系统细胞的前体细胞,在成年的哺乳动物体内分为2种:定向成骨前体细胞和可诱导成骨前体细胞。不论是MSC,还是已形成的前体细胞都可在一定条件下继续分化或被诱导分化为成熟的成骨细胞。     

IRAK-4对成骨细胞分化BMP-Smad通道的影响

骨形成是一个复杂现象,当骨折发生时,附近骨髓中的间充质干细胞在细胞外基质和损伤部位的各种细胞因子刺激激活下发育分化成为成骨细胞[1]。之后,经过成骨细胞增殖、细胞外基质成熟和矿化等三个阶段,最终完成骨形成过程。这些过程易受多种因素的影响。其中成骨细胞的作用为合成骨基质,由间充质干细胞分化发育形成[2]。破骨细胞的作用为降解骨基质,由骨髓单核巨噬细胞分化演变而成。各种病     

骨髓来源成骨细胞体外成骨表达的研究

目的探索在体外诱导兔骨髓基质干细胞(bone marrow stem cells,BMSCs)向成骨细胞分化及成骨表达的特性。方法抽取兔骨髓,通过离心法获取单个核细胞,在体外经条件培养基培养21d,通过倒置显微镜及激光共聚焦显微镜观察成骨细胞的形态学特点;应用MTT法测定成骨细胞活性;并检测成骨细胞分泌碱性磷酸酶活性及形成矿化结节情况。结果体外BMSCs可在成骨条件培养液下诱导培养后可向成骨细胞分化,经倒置显微镜和激光共聚焦显微镜观察证实,诱导后BMSCs由长梭形转变为短胖形的成骨细胞,MTT检测成骨细胞活性良好,分泌碱性磷酸酶并形成矿化结节。结论骨髓基质干细胞在体外可定向诱导为成骨细胞,并具有成骨细胞功能,有希望成为理想的种子细胞应用于临床。     

骨髓基质细胞体外培养成骨的研究进展

近年来,随着组织工程的兴起,作为组织工程化组织关键的种子细胞来源越来越引起人们的重视。骨髓基质细胞(Marrow stem cells,MSCs)是骨髓中存在的具有潜在成骨活性的一组干细胞。通过体外培养骨髓基质细咆,证实了骨髓成骨的细胞学基础是骨髓基质细胞中含有向成骨细胞系转化的骨祖细胞及基质干细胞,现对这一领域的研究进展综述如     

盐酸四环素缓释微球对大鼠成骨细胞活性的影响

目的 观察盐酸四环素缓释微球对体外培养的成骨细胞活性的影响.方法 体外分离培养SD大鼠成骨细胞并通过形态学观察、碱性磷酸酶(ALP)钙-钴法染色鉴定其细胞生物学特征;将盐酸四环素聚乳酸-聚羟基乙酸共聚物(PLGA)微球、盐酸四环素分别与成骨细胞共培养,采用四甲基偶氮唑盐(MTT)法检测各实验组成骨细胞的增殖情况,通过碱性磷酸酶活性测定法检测各实验组成骨细胞中ALP的活性,免疫组化Ⅰ型胶原蛋白(Col Ⅰ type)染色观察各实验组成骨细胞中Ⅰ型胶原的表达情况.结果 盐酸四环素-PLGA微球能显著促进SD大鼠成骨细胞的增殖,同时增强了ALP的表达,其效应持续时间高于盐酸四环素组和空白对照组,Ⅰ型胶原在3组细胞中的表达无显著差异.结论 制备的盐酸四环素-PLGA微球具有缓释促进成骨细胞增殖和增加ALP活性的作用,在骨创伤的修复重建治疗中具有潜在的应用价值.     

二甲胺四环素增强博安霉素的抗肿瘤转移作用

博安霉素对小鼠Lewis肺癌的肺转移有显著抑制作用;用等毒性剂量进行比较,博安霉素的肺转移抑制率高于丝裂霉素。单独给博安霉素(5mg·kg^-1)对肺转移抑制率为67%,对其中大转移瘤结(直径>2mm)的抑制率为85%;博安霉素与二甲胺四环素(5mg·kg^-1)联合使用时,对肺转移瘤以及时大转移瘤结的抑制率分别为88%和100%,两药相互作用指数CDI<0.5(P<0.01),表明二甲胺四环素能明显增强博安霉素的抗转移作用。酶联免疫测定证明,二甲胺四环素能降低肺巨细胞癌PG细胞IV型胶原酶的表达,Fura-2/AM荧光法测定,二甲胺四环素能显著降低PG细胞内游离Ca²⁺的水平。本研究结果提示博安霉素与二甲胺四环索联合用药可能有利于控制肿瘤转移,二甲胺四环素的增效机制可能与抑制IV型胶原酶的表达、干扰肿瘤侵袭与转移的过程有关。     

二甲胺四环素的反相—高效液相色谱分析

本文报道了用反相高效液相色谱分析二甲胺四环素的方法,采用Waters 10C184.6mm I.D.×250mm的色谱柱,以30％二甲基甲酰胺-70％0.02mol/L柠檬酸水溶液力流动相,流速1.0ml/min,UV350nm检测,可将二甲胺四环素与表二甲胺四环素、去甲基四环素、去甲基金霉素完全分离。二甲胺四环素的峰面积与进样量呈很好的线性相关关系(r=0.9999),回收率99.16％,变异系数CV％<2％。本方法可用于二甲胺四环素产品及胶囊的含量分析。     

CXCR4抑制剂与血液系统肿瘤的治疗

基质细胞衍生因子(SDF-1)又名CXCL12或前B细胞刺激因子(pre-B cell stimulatory factor,PBSF),是主要由骨髓基质细胞和不成熟的成骨细胞产生的趋化因子。SDF-1表达于骨髓、淋巴结、肺、大脑、心脏、肝脏、胸腺、脾脏和肾脏等组织和器官。SDF-1受体之一的CXCR4是一个具有7个跨膜受体的G蛋白偶联受体,定位于第4号染色体,具有类似于白介素(IL)-8受体的结构,表达于造血干/祖细胞、血液和骨髓的多种细胞。     

bFGF对骨髓基质细胞的增殖作用及其临床意义

目的探讨成纤维细胞生长因子(bFGF)对骨髓前体细胞的增殖作用及其临床意义。方法分离骨髓前体细胞体外培养,用碱性磷酸酶化学染色及图像分析测定细胞克隆数;采用免疫组化(ABC法)检测ALP、骨钙素及钙,研究bFGF对NASP的促增殖作用。结果骨髓细胞中许多基质前体细胞以非贴壁细胞形式存在,bFGF不仅能促进NASP细胞增殖,而且能诱导NASP细胞向成骨细胞分化。结论bFGF主要作用于非贴壁生长的基质前体细胞,bFGF能增加骨细胞的数量,促进骨样组织形成,为临床用bFGF治疗骨折、骨不连提供理论依据。     

白细胞介素1对体外培养小鼠骨髓基质细胞增殖及细胞周期的影响

目的研究白细胞介素１对小鼠骨髓基质细胞增殖的影响。方法取出小鼠骨髓基质细胞作原代培养，用［３Ｈ］ＴｄＲ参入法及ＭＴＴ法观察其增殖情况，利用流式细胞仪观察其细胞周期的变化。结果白细胞介素１在２５０×１０３～１０００×１０３ＵＬ－１范围内可促进小鼠骨髓基质细胞的增殖，并引起Ｓ期细胞的增多。结论白细胞介素１可促进体外培养的小鼠骨髓基质细胞增殖。     

Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields.

As leukemia is one of the health hazards that is sometimes associated with exposure to extreme low frequency fields, we studied the in vitro effects of ELF fields on haemopoietic cell proliferation. First, the cytotoxic effect of 80 microT, 50 Hz magnetic fields on 3T3 cell proliferation was investigated using the neutral red test. Many chemicals are believed to cause damage because they interfere with basal or "housekeeping" cell functions. The basal cell functions are present in every cell. Non-specialized, actively dividing cells are suitable for measuring cytotoxic effects. Cytotoxic doses can be identified by exposing actively dividing cells in vitro and measuring growth inhibition caused by interference with these basal cell functions. 80 microT, 50 Hz magnetic fields caused no cytotoxicity: we were not able to demonstrate any interference with essential cell functions in the non-differentiated 3T3 cell line. Furthermore, the in vitro effects of ELF fields on murine haemopoietic and stromal stem cell proliferation were studied. Haemopoiesis is a continuous process, where mature blood cells are replaced by the proliferation and differentiation of more primitive progenitor and stem cells. Blood formation is tightly regulated by the stromal micro-environment. Exposure of murine bone marrow cells, from male and female mice, to 80 microT (50 Hz) magnetic fields showed a reduction in the proliferation and differentiation of the granulocyte-macrophage progenitor (CFU-GM) compared to non-exposed bone marrow cells. The results on the effect of the ELF-field on stromal stem cell proliferation (CFU-f) are somewhat equivocal at the moment. CFU-f from female mice showed a reduction while CFU-f from male mice were not decreased.     

Effects of neopterin on the hematopoietic microenvironment of senescence-accelerated mice (SAM)

The pteridine neopterin (NP) is produced by monocytes and is known to be a useful marker of immunological activation, although, it remains elusive whether neopterin itself exhibits biological functions. Recently, we found that NP stimulates hematopoietic cell proliferation and differentiation by activating bone marrow stromal cell function. In order to elucidate the biological effect of NP on stromal cells, its effects on hematopoiesis was determined in the mouse model of age-related stromal impairment, senescence-accelerated mice (SAMs). An intraperitoneal administration of NP increased the number of peripheral leukocytes and CFU-GM in the bone marrow and spleen of young SAMs, however, no increase of CFU-GM in old SAMs (stromal impairment) was observed when compared with young SAMs. NP also increased the CFU-GM colony formation of bone marrow and spleen cells from young SAMs in a soft agar culture system, but it did not enhance CFU-GM colony formation of cells from old SAMs cultured in this system. Treatment with NP induced the production of hematopoietic stimulating factors, including IL-6 and GM-CSF, by bone marrow stromal cells from young SAMs but stromal cells from old SAMs did not respond to NP stimulation. Further studies will be required to clarify the mechanism by which NP stimulates the production of hematopoietic growth factors from stromal cells, the results of this study indicate that NP is a potent hematopoietic regulatory factor by activating stromal cell function(s).     

Macrophage inflammatory protein-3alpha influences growth of K562 leukemia cells in co-culture with anticancer drug-pretreated HS-5 stromal cells

Stromal cell monolayers have been an important means of studying the regulation of hematopoiesis, because they produce cytokines. Cytosine arabinoside, vincristine, daunorubicin, and doxorubicin are common drugs for hematological cancer therapy, and they may have some effects on bone marrow stroma during chemotherapy. The aim of this study was to elucidate interactions between the bone marrow stromal microenvironment and leukemic cells after drug treatment. We tested the hypothesis that human HS-5 stromal cells, pretreated with anticancer drugs, affected the growth of leukemic K562 cells by changing the cytokines in the culture microenvironment. Thereafter, proliferation of K562 cells increased nearly 2.5-fold compared the co-cultivation with drugs-pretreated HS-5 stromal cells and drugs-untreated HS-5 stromal cells. The results indicated that co-cultivation with HS-5 stromal cells pretreated with drugs caused significant K562 cell proliferation. Cytokines in the microenvironment were detected via the RayBio((R))Human Cytokine Antibody Array Membrane. The levels of the cytokines CKbeta, IL-12, IL-13, IGFBP-2, MCP-1, MCP-3, MCP-4, MDC, MIP-1beta and MIP-1delta were decreased, with a particularly marked decrease in MIP-3alpha. In co-culture medium, there was a 20-fold decrease in MIP-3alpha in daunorubicin-pretreated HS-5 cells and at least a 3-fold decrease in Ara-C-pretreated cells. This indicated a significant effect of anticancer drugs on the stromal cell line. Using phosphorylated Erk and pRb proteins as cell proliferation markers, we found that phosphorylation of these markers in K562 cells was inhibited during co-cultivation with drug-pretreated stromal cells in MIP-3alpha-supplemented medium and restored by MIP-3alpha antibody supplement. In conclusion, anticancer drug pretreatment suppresses the negative control exerted by HS-5 cells on leukemic cell proliferation, via modulation of cytokines in the microenvironment, especially at the level of MIP-3alpha.     

A new paradigm for stem cell therapy: Substance-P as a stem cell-stimulating agent

Bone marrow is a reservoir for hematopoietic stem cells, endothelial precursor cells, and bone marrow stromal cells (also generally called mesenchymal stem cells), whose positive role in tissue repair is highly anticipated. In this report, we introduce a novel function of substance-P (SP), an 11-amino-acid peptide, as an injury-inducible messenger to mobilize bone marrow stem cells to the blood and finally to engage in tissue repair. This new drug may substitute for ex vivo cell culture of therapeutic cells by stimulating cell proliferation in the bone marrow in vivo and mobilizing those therapeutic cells to the patient’s own blood stream. Again, the additional role of SP in mitigating inflammation-mediated tissue damage can further rationalize the clinical development of SPpeptide as a stem cell stimulant.     

降脂壮骨方剂含药血清对大鼠骨髓基质细胞骨形态发生蛋白-2表达的影响

目的研究高胆固醇血清以及降脂壮骨方剂含药血清干预下大鼠骨髓基质细胞骨形态发生蛋白-2（BMP-2）表达的变化。方法取成年大鼠股骨,用密度梯度离心方法分离大鼠骨髓基质细胞,原代培养细胞至第三代,并用流式细胞仪进行细胞鉴定。选取第三代细胞,随机分为三组。A组正常血清对照组：2％空白血清（体积比）;B组高胆固醇血清损伤组：终浓度为4mmol／L高胆固醇血清;C组中药血清预处理＋高胆固醇血清损伤组：2％中药血清（体积比）预处理2h后＋终浓度为4mmol／L高胆固醇血清。回收细胞提取总RNA,检测BMP-2表达变化,并进行统计学分析。结果与空白血清对照组相比,高胆固醇血清损伤组骨髓基质细胞增殖受抑制;BMP-2的mRNA表达水平降低（P〈0．01）。与高胆固醇血清损伤组相比,中药血清预处理＋高胆固醇血清损伤组骨髓基质细胞生长状态明显改善,BMP-2的mRNA表达水平增高（P〈0．01）。结论降脂壮骨方剂含药血清可以增强高胆固醇血清环境下骨髓基质细胞BMP-2、雌激素受体（ER）,明显改善骨髓基质细胞生长状态,有利于骨质疏松症的治疗。     

骨髓基质细胞与可注射聚丙交酯多孔微载体共培养的体外实验研究

目的体外共培养骨髓基质细胞与聚丙交酯多孔微载体,研究微载体对骨髓基质细胞的增殖、凋亡等生理特性的影响,为细胞移植治疗提供一种安全可靠的载体。方法密度梯度离心法获取大鼠骨髓基质细胞,与可注射聚丙交酯多孔微载体在体外共培养,倒置显微镜及扫描电镜下观察细胞的生长及黏附在微载体的情况,并应用MTT法、流式细胞仪比较共培养、单独培养以及黏附在微载体的细胞的增殖及凋亡等生理特性。结果两者共培养时,骨髓基质细胞可长期存活、增殖,并可以黏附于微载体的表面及内部孔洞中,共培养时微载体对骨髓基质细胞的增殖、凋亡等特性无明显影响。结论可注射聚丙交酯多孔微载体是一种安全、可靠的细胞载体。     

Drugs toxic to the bone marrow that target the stromal cells

Drugs that cause toxicity to the bone marrow are a heterogeneous group of compounds that act by various mechanisms. The etiology of this pathology is poorly understood but the highly proliferative nature of the hematopoietic cells is assumed to make the bone marrow more sensitive to toxicity. Recent evidence suggests that drugs can also affect specific aspects of stromal cells and the extracellular matrix that they establish. The data support the view that characteristics other than a high proliferation rate could confer susceptibility of the bone marrow to the toxic effects of drugs. This article discusses those drugs that have been shown to have direct effects on the bone marrow stromal cells.     

碱性成纤维细胞生长因子对非贴壁骨髓基质前体细胞增殖及分化作用的研究

目的　探讨碱性成纤维细胞生长因子 (bFGF)对非贴壁骨髓前体细胞的增殖及分化作用。方法　分离骨髓前体细胞体外培养 ,用碱性磷酸酶化学染色及图像分析仪测定细胞克隆数 ;采用免疫组化 (ABC法 )检测PCNA、Ⅰ型胶原、钙及骨钙素。研究bFGF对非贴壁前体细胞的促增殖及分化作用。结果　骨髓细胞中许多基质前体细胞以非贴壁前体细胞 (NASP)形式存在 ,bFGF不仅能促进其增殖 ,而且能诱导其向成骨细胞分化。结论　bFGF主要作用于非贴壁生长的基质前体细胞 :能增加骨细胞的数量 ,促进骨样组织形成 ,为临床用bFGF治疗骨折、骨不连提供理论依据。     

In vitro myelotoxicity of environmental contaminants

Myelotoxicity of pesticides and algal toxins was detected in vitro by using the granulocyte-macrophage colony forming unit assay (CFU-GM), and the MTT test with SR-4987 cells, an established stromal cell line derived from a long term murine bone marrow culture, which may represent a suitable in vitro model for studying haematotoxicity. Comparison of the IC50s and NOELs obtained with the CFU-GM assay and those determined by testing the established stromal cells in the MTT cytotoxicity test indicate that inhibition of the proliferation of SR-4987 stromal cells is a sensitive in vitro endpoint for measuring myelotoxicity. It is suggested that this assay could be used as rapid and easy screening test for determining the haematotoxicity of environmental toxins. A comparison with results obtained with the MTT test on a non-differentiated cell line, 3T3-L1, was carried out to distinguish between non-specific interference with cell proliferation and specific toxicity on haemopoietic cells.