成年鼠骨髓间充质干细胞体外诱导分化为心肌样细胞

目的探讨成年大鼠骨髓间充质干细胞(MSC)体外培养条件及定向分化为心肌样细胞的可行性。方法通过取成年SD大鼠股骨干骨髓,进行体外培养和传代,用10μmol/L 5-氮杂胞苷(5-aza)分别对原代、一代和三代MSC进行体外定向诱导,用免疫组化方法分析检测分化后细胞的心肌特异性抗原(肌丝蛋白、肌钙蛋白)并通过RT-PCR鉴定心肌细胞特异因子基因(ANP、GATA-4)的表达。结果原代、一代MSC在体外经5-aza诱导4周后,细胞形态无明显变化,而经5-aza诱导4周后的三代MSC形态变大,呈杆形、球形,诱导细胞相互连接,连接细胞出现肌管样结构。免疫组化检测经10μmol/L 5-aza诱导4周的原代、一代MSC未见肌丝蛋白、肌钙蛋白T阳性细胞,而诱导三代MSC肌丝蛋白阳性比例近20%,肌钙蛋白阳性比例约8%。RT-PCR显示诱导三代MSC 4周后有ANP和GATA-4心肌细胞特异基因表达。结论MSC能够在体外经5-aza诱导分化为心肌样细胞,具有向心肌细胞转化的潜力,是自体心肌细胞移植的一种良好供体。     

Radiation protocols determine acute graft-versus-host disease incidence after allogeneic bone marrow transplantation in murine models

PURPOSE: Effects of radiation sources used for total body irradiation (TBI) on Graft-versus-Host Disease (GvHD) induction were examined. MATERIALS AND METHODS: In a T cell receptor (TCR) transgenic mouse model, single fraction TBI was performed with different radiation devices ((60)Cobalt; (137)Cesium; 6 MV linear accelerator), dose rates (0.85; 1.5; 2.9; 5 Gy/min) and total doses before allogeneic bone marrow transplantation (BMT). Recipients were observed for 120 days. Different tissues were examined histologically. RESULTS: Acute GvHD was induced by a dose rate of 0.85 Gy/min ((60)Cobalt) and a total dose of 9 Gy and injection of 5 x 10(5) lymph node cells plus 5 x 10(6) bone marrow cells. Similar results were obtained using 6 MV linear accelerator- (linac-) photons with a dose rate of 1.5 Gy/min and 0.85 Gy/min, a total dose of 9.5 Gy and injection of same cell numbers. TBI with (137)Cesium (dose rate: 2.5 Gy/min) did not lead reproducibly to lethal acute GvHD. CONCLUSIONS: Experimental TBI in murine models may induce different immunological responses, depending on total energy, total single dose and dose rate. GvHD might also be induced by TBI with low dose rates.     

Radiation protocols determine acute graft-versus-host disease incidence after allogeneic bone marrow transplantation in murine models

Purpose:?Effects of radiation sources used for total body irradiation (TBI) on Graft-versus-Host Disease (GvHD) induction were examined.

Materials and methods:?In a T cell receptor (TCR) transgenic mouse model, single fraction TBI was performed with different radiation devices (⁶⁰Cobalt; ¹³⁷Cesium; 6 MV linear accelerator), dose rates (0.85; 1.5; 2.9; 5 Gy/min) and total doses before allogeneic bone marrow transplantation (BMT). Recipients were observed for 120 days. Different tissues were examined histologically.

Results:?Acute GvHD was induced by a dose rate of 0.85 Gy/min (⁶⁰Cobalt) and a total dose of 9 Gy and injection of 5×10⁵ lymph node cells plus 5×10⁶ bone marrow cells. Similar results were obtained using 6 MV linear accelerator- (linac-) photons with a dose rate of 1.5 Gy/min and 0.85 Gy/min, a total dose of 9.5 Gy and injection of same cell numbers. TBI with ¹³⁷Cesium (dose rate: 2.5 Gy/min) did not lead reproducibly to lethal acute GvHD.

Conclusions:?Experimental TBI in murine models may induce different immunological responses, depending on total energy, total single dose and dose rate. GvHD might also be induced by TBI with low dose rates.     

Response of human bone marrow progenitor cells to X-rays in vitro

In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23 degrees C, was characterized by a two-term exponential relationship, with D0 values of 0.42 +/- 0.19 Gy and 1.38 +/- 0.37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D0 = 1.18 +/- 0.15 Gy. The biphasic response at 23 degrees C became a single exponential response when the temperature at irradiation was 30 or 37 degrees C.     

Dominant lethals following acute spermatogonial X-irradiation in the rat.

Spermatogonial X-irradiation with 0, 400 or 600 R in adult rats and 200 R in fetal rats was carried out to assess genetic damage by the test of induced dominant lethality. From postimplantation embryonic losses observed, dominant lethal rates were established to be significantly raised by a dose of 400 or 600 R. With 200 R given either to adult or to fetal rats, no excess was detected in embryonic death rates.     

MR imaging characteristics of cranial bone marrow in adult patients with underlying systemic disorders compared with healthy control subjects

BACKGROUND AND PURPOSE: In young adults, hematopoietic bone marrow has usually converted to fatty marrow. Fat hyperintensity on T1-weighted MR images facilitates the evaluation of marrow abnormalities. Our purpose was to compare cranial marrow signal intensity patterns in adults with systemic disorders and in healthy subjects. METHODS: MR images in 25 adults with underlying systemic disorders (chronic anemia, lymphoma, leukemia, or other infiltrative processes) and 44 healthy aged-matched individuals were retrospectively reviewed. Calvarial and clival marrow signal intensity on sagittal T1-weighted images was graded relative to that of orbital fat, white matter (WM), and gray matter (GM). Marrow was classified as homogeneous (uniformly isointense), diffusely heterogeneous (mottled), or focally heterogeneous (generally isointense with a focal lesion). RESULTS: In 84% of the control subjects, bone marrow was iso- or hyperintense relative to WM. Patients had abnormal diploic (n = 22) or clival (n = 17) marrow; 22 had calvarial marrow that was hypointense relative to WM compared with that in seven healthy subjects (P <.001). Marrow hypointensity relative to WM was a sensitive (93%) and specific (86%) marker of pathologic abnormality. Although marrow hypointensity relative to GM was specific (96%), it was not sensitive (67%). Calvarial and clival marrow patterns, respectively, were homogeneous in 81% and 64% of control subjects and 76% and 60% of patients. Clival marrow intensity varied more than did calvarial intensity; therefore, clival criteria were less sensitive and accurate in systemic disease detection. CONCLUSION: Homogeneous diploic marrow hypointense relative to WM on non-contrast-enhanced T1-weighted images suggests an underlying systemic or hematologic disorder and requires appropriate clinical correlation and evaluation.     

The effect of 5-fluorouracil on the radiosensitivity of granulocytopoietic stem cells in the bone marrow and the blood

The radiation tolerance of haematopoietic stem-cells, originating from blood and bone marrow, after exposure to 5-Fluorouracil (5-FU) was investigated. The CFU-C Colony Formation Test was used to determine the surviving fraction of cryopreserved mononuclear bone marrow and blood cells, after incubation in culture medium containing 1 ng/ml 5-FU for 24 hours. With the same assay system we determined the radiation tolerance of peripheral CFU-C from three patients before and after 5-FU radio-chemotherapy (1000 mg/m2/24 h per day for four days), while the influence of the small radiotherapeutic treatment volume was neglected. Neither in the in vitro experiments nor in the patients we found a significant variation of the dose-survival curves with or without 5-FU. So it is concluded, that the enhanced myelodepression of 5-FU combined with radiation may be due to an additive effect or by effects on the microenvironment or on the pluripotent stem cell system.     

骨髓基质细胞的体外培养与临床应用

目的　验证骨髓基质细胞 (bonemarrowstromalcells,BMSCs)向成骨细胞转化的能力及其临床应用价值。方法　取健康成年大白兔股骨大转子红骨髓 ,在DMEM标准培养液中原代培养骨髓基质细胞 ,传代细胞分别用DMEM标准培养液和加入地塞米松、β 甘油磷酸钠、L 抗坏血酸的条件培养液培养 ,倒置显微镜观察不同培养条件下细胞的形态学特征 ,进行酶学测定 ,计数传代细胞中成骨样细胞转化率 ,免疫组化法检测Ι型胶原表达。同样方法培养人BMSCs,与同种异体骨基质明胶充分混合 ,应用于临床修复创伤所致的骨缺损、骨不连或骨折延迟愈合。结果　体外培养条件下 ,原代细胞呈长梭形 ,具有成纤维细胞的生长特性 ,条件培养的传代BMSCs变为三角形或锥形 ,呈多层生长 ,无接触抑制现象 ,具有与成骨细胞相似的形态和生长特点。ALP染色阳性率可达 85 %以上 ,Ι型胶原免疫组化染色呈强阳性 ,具有与成骨细胞类似的功能性表现。临床应用 4 2例病人 ,随访 8～ 2 6个月 ,取得了满意疗效。结论　BMSCs能够向成骨细胞分化和增殖 ,是骨组织工程学种子细胞的理想来源 ,并有广阔的临床应用前景     

人骨髓基质细胞体外扩增方法的研究

 目的 为提高人骨髓成纤维细胞集落(CFU-F)的产率,缩短培养时间.方法 在以往的人骨髓基质细胞体外扩增方法基础上进行了改进,每10ml培养体系中,含1×10<'7>个单个核细胞,以30%人AB血清代替小牛血清,增加了碱性成纤维细胞生长因子(bFGF),再加入氢化可的松和适量的IMDM培养液,培养第9d观察结果.结果 在一定范围内人AB血清、bFGF均能促进CFU-F增殖,提高其产率,并缩短培养时间3～5 d.结论 可应用到人骨髓基质细胞体外扩增及传代中,对于研究CFU-F的性质及在造血疾病中的应用具有重要意义.     

大鼠骨髓多能成体祖细胞转化为皮肤组织细胞的在体研究

目的 观察大鼠骨髓多能成体祖细胞(mulipotent adult progenitor cells,MAPCs)是否可在体向皮肤组织细胞分化.方法 采用免疫磁性分选技术(MACS)获取MAPCs;通过皮肤受损的C57BL/6小鼠与免疫缺陷性裸鼠的尾静脉注入MAPCs,免疫组织化学技术检测愈合皮肤中大鼠MHC I类抗原的表达.结果 MHC I抗原在C57BL/6小鼠愈合皮肤处的毛囊皮脂腺周围及毛囊外根鞘与皮脂腺交界处均有阳性表达细胞;裸鼠实验组愈合皮肤内出现毛囊样结构,在表皮基底部及部分毛囊样结构中有MHC I抗原阳性表达的细胞.结论 在皮肤组织损伤的条件下,MAPCs可迁移至受损皮肤表面及皮肤附属器毛囊周围,并通过转化为表皮细胞参与受损皮肤的愈合.     

胎肝及骨髓来源造血干/祖细胞电离辐射敏感性的比较研究

目的研究电离辐射对胎肝与骨髓来源造血干/祖细胞(hematopoietic stem and progenitor cells, HSPCs)的影响差异。方法取14.5 d小鼠胎肝及8周龄小鼠骨髓, 分选c-Kit+细胞体外接受60Co单次5、10 Gy照射, 检测HSPCs中细胞凋亡、线粒体活性氧(ROS)水平、集落形成能力、DNA损伤情况。将12只CD45.1背景的C57BL/6J小鼠按随机数表法分成骨髓组(BM)和胎肝组(FL), 60Co全身照射, 第1次4.5 Gy照射, 间隔30 min后再进行第2次5 Gy剂量照射, 6 h后进行移植, 12周后检测嵌合率、谱系分化以及细胞周期。分选胎肝及骨髓的c-Kit+细胞检测线粒体压力。结果与骨髓HSPCs相比, 照射后胎肝HSPCs的细胞凋亡比例显著升高(t=16.21、12.27, P<0.05), ROS水平显著增加(t=68.72、18.89, P<0.05);集落形成能力显著降低, 在5 Gy时胎肝HSPCs成集落能力显著降低, 照射剂量为10 Gy时完全不能形成集落(t=12.41、15.67、9.46, P...     

Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro

Purpose

Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10^?7, 10^?6 and 10^?5 M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro.

Methods

BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue.

Results

The cell viability was not significantly impaired until up to a concentration of 10^?5 M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10^?6 M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10^?5 M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10^?7 and 10^?6 M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10^?7–10^?5 M nicotine (P < 0.05).

Conclusion

It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.     

Hematopoietic bone marrow in the adult knee: spin-echo and opposed-phase gradient-echo MR imaging

Hematopoietic bone marrow in the distal femur of the adult may be mistaken for a pathologic marrow process in magnetic resonance imaging of the knee. We investigated the incidence of hematopoietic marrow in the distal femur in a series of 51 adult patients and compared spin-echo (TR/TE in ms: 500/35, 2000/80) and opposed-phase gradient-echo (0.35 T, TR/TE in ms: 1000/30, = 75°) magnetic resonance images. Zones with intermediate to low signal intensity on T1-weighted spinecho and opposed-phase gradient-echo sequences representing hematopoietic marrow within high signal intensity fatty marrow were observed in 18 of the 51 patients. Five patterns of marrow signal reduction were identified; type 0: uniform high signal, i.e., no signal change; type I, focal signal loss; type II, multifocal signal loss without confluence; type III, confluent signal loss; and type IV, complete homogeneous reduction in marrow signal. Opposed-phase gradient-echo sequences demonstrated markedly greater red-yellow marrow contrast than conventional spin-echo sequences. Follow-up studies in three patients using a gradient-echo sequence with TE varying from 10 to 21 ms at 1-ms increments showed a cyclic increase and decrease in red and yellow marrow signal intensity depending on the TE. The contribution of intravoxel chemical shift effects on red-yellow marrow contrast in opposed-phase gradient-echo images was verified by almost complete cancellation of the TE-dependent marrow signal oscillation with use of a chemically selective pulse presaturating the water protons.Hematopoietic marrow in the adult distal femur in the absence of hematologic abnormalities is found primarily in women of menstruating age. It may be residual and may represent a biologic variation in the normal adult pattern of red-yellow marrow distribution. Reconverted red marrow appears to be related to increased erythrocyte demand. Residual and reconverted red marrow should not be mistaken for bone marrow malignancy. Opposed-phase gradient-echo imaging is easily implemented and appears ideally suited to monitor the distribution of hematopoietic marrow as a function of age and erythrocyte demand in vivo.     

电离辐射对小鼠骨髓细胞p53、Wafl、gadd45基因转录水平的影响

目的研究X射线全身照射对小鼠骨髓细胞p53、wafl、gadd45基因转录水平的影响,以探讨电离辐射诱导小鼠骨髓细胞G1阻滞的分子机理.方法采用Northern blot和半定量RT-PCR方法分别检测p53、wafl、gadd45基因转录水平变化.结果2GyX射线全身照射后2～72 hp53基因转录水平均有不同程度的升高,wafl基因在照射后2 h开始升高,4 h达峰值,一直持续到照后48 h开始恢复,gadd45转录水平除在照射后2 h一过性升高外,从8 h即开始不同程度下降,照射后72 h开始恢复.分别以0.5～6.0 Gy X射线全身照射后12 h取小鼠骨髓细胞作量效研究表明,p53的改变在照射后各组虽有不同程度的升高,但未见明显的剂量依赖,wafl基因转录呈明显的剂量依赖性升高,6.0 Gy X射线照射后达对照组的3.41倍,gadd45未见升高趋势.结论在电离辐射诱导的小鼠骨髓细胞周期G1阻滞中p53-wafl通路起重要作用,而gadd45可能不参与该过程调控.     

骨髓间充质干细胞成骨定向分化的研究进展

就骨髓间充质干细胞的形态和表面分子特征、体外成骨定向分化的特性及影响因素等方面的研究进展作一综述 ,并初步探讨了骨髓间充质干细胞在骨组织工程中的应用前景。