体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

Wnt3a 转基因基质细胞的建立及其对脐血CD34+ 造血干/祖细胞扩增作用的研究

Wnt 信号通路在造血干/祖细胞自我更新的过程中发挥至关重要的作用 . 纯化的 Wnt3a 蛋白可以实现造血干/祖细胞的扩增 . 通过病毒转染原代小鼠骨髓基质细胞,建立转基因滋养层细胞 . 通过共培养对转基因滋养层细胞扩增 CD34+ 造血干/祖细胞的作用进行了研究 . 实验结果显示 , 与普通滋养层加细胞因子组相比,经转基因滋养层加细胞因子组培养的 CD34+造血干/祖细胞集落形成能力 (CFC) 是其 (1.55±0.06) 倍;混合集落形成能力是其 (1.95±0.26) 倍;高增殖潜能集落形成能力 (HPP-CFC) 是其 (1.45±0.40) 倍; LTC-IC 活性是其 (3.83±0.86) 倍 . 结果表明,转基因滋养层细胞通过分泌具有天然活性的 Wnt3a 蛋白能在体外有效地扩增造血干/祖细胞的数量 .     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34⁺富集细胞在SCF+IL-3+IL-6+FL+Tpo细胞因子组合下的体外扩增特性,发现：CD34⁺富集细胞具有很高的扩增潜力,在本实验条件下其总细胞持续扩增了8周,扩增倍数达31270.9±8640.5倍;而MNC在培养至第4周扩增就已呈现下降趋势,最大仅扩增了53.3±6.2倍。对比集落和CD34⁺细胞的扩增发现,MNC的集落密度和CD34⁺细胞含量由第0天至第7天有一个上升的过程,而CD34⁺富集细胞在培养过程中,集落密度和CD34⁺细胞含量却始终呈下降趋势。在体外培养过程中,CD34⁺富集细胞的CFU-GM和CD34⁺细胞最大分别扩增了185.7±14.1和191.7±188.8倍,明显高于MNC的12.4±3.2和50.6±33.2倍;而CD34⁺富集细胞和MNC的BFU-E则只实现了少量扩增,分别为7.2±5.2和10.1±3.4倍。结果显示,从CD34⁺富集细胞出发扩增造血干/祖细胞,可以得到更多的CD34⁺细胞和CFU-GM集落形成细胞。      

低氧环境中微囊化成骨细胞支持造血干/祖细胞扩增

在模拟骨髓造血壁龛(hematopoietic niche)的氧分压条件下,探讨微囊化成骨细胞(osteoblasts,OB)对脐血造血干/祖细胞(HSPC)体外扩增的支持和调控机理．分离培养人髂骨OB,采用聚电解质络合法将第3代的OB以密度为8×105 ml包埋在直径为0.5 mm的明胶-海藻酸钠-壳聚糖(GAC)微胶珠中．将微珠+造血干/祖细胞(A′组)、造血干/祖细胞(B′组)及微珠(C′组)置于6孔板,在5%氧分压下进行培养．同时在20%常氧条件下设置同样分组培养作为对照(A,B,C)．通过流式细胞分析和半固体细胞集落培养,观察比较各培养体系中造血干/祖细胞的扩增,并检测体系内白血病抑制因子(LIF)和白介素-6(IL-6)的含量变化以探讨作用机理．经过倒置相差显微镜观察,人成骨细胞在微珠中分散均匀,生长状态良好．微珠内部有丰富的孔道供营养物质传递,有大量造血干/祖细胞弱黏附于微珠表面．经过7天的培养,A′、B′、A、B四组造血细胞的扩增倍数分别为(49.0 ± 4.6),(3.3 ± 0.5),(17.7 ± 1.2)和(1.9 ± 0.2)．A′、B′、A 组的CD34+细胞分别扩增了(87.6 ± 8.3), (2.2 ± 0.3)和(14.9 ± 1.0)倍,B组则出现下降．A′、B′、A、B四组CFU-Cs集落扩增倍数分别为(9.8 ± 0.8),(3.5 ± 0.4), (6.9 ± 0.7)和(2.6 ± 0.2)．低氧共培养体系比常氧共培养体系和非共培养体系对造血干/祖细胞的扩增有更大的促进作用．A′、B′、C′中IL-6和LIF含量明显高于对应的A、B、C组,与扩增倍数的差异相对应．微囊化成骨细胞对造血干/祖细胞扩增有明显的促进作用,5%氧分压接近体内造血壁龛氧环境,在此环境中成骨细胞分泌细胞因子量增多并通过其对造血干/祖细胞的扩增进行调节．     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增。在该生物反应器内, 采用SCF+TPO+Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34+细胞的效果。培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34+细胞扩增倍数、培养物中CD34+细胞含量均相近, 无显著性差异; 而CD34+CD38-细胞扩增倍数以及培养物中CD34+CD38?细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养。可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增。     

BCL-2反义硫代磷酸寡脱氧核苷酸对原代急性白血病细胞选择性抑制作用

为探讨BCL-2反义硫代磷酸寡脱氧核苷酸(AS-PS-ODN,ASPO)对急性原代白血病细胞和正常或良性血液病骨髓细胞的作用是否存在差别。应用台盼蓝拒染试验测定细胞存活力;用造血祖细胞集落培养:粒—单系祖细胞集落(CFU-GM),多向祖细胞集落(CFU-Mix),后红系祖细胞集落(CFU-E)、前红系祖细胞集落(BFU-E)和白血病祖细胞集落(CFU-AML,CFU-ALL)培养检测细胞增殖能力;免疫细胞化学染色检测细胞BCL-2蛋白表达变化。结果发现(1)正常或良性血液病骨髓细胞经10μmol/L ASPO处理一周,同对照组比较:细胞生长数、CFU-GM、CFU-Mix、CFU-E、BFU-E及BCL-2蛋白表达均无显著差别(P<0.05)。(2)急性原代白血病细胞经5μmol/L ASPO处理一周,同对照组比较:细胞生长数显著减低;CFU-AML或CFU-ALL显著降低(P < 0.05),而CFU-GM明显增高(P< 0.05);对照组BCL-2蛋白表达率为77.92％±22.50％,ASPO组于培养的第3天为54.05％±20.20％(P<0.01)和第6天为55.35％±22.74％(P<0.05)均显著低于对照组。因此认为BCL-2ASPO具有选择性地抑制白血病细胞的增殖和BCL-2蛋白的表达的作用。     

遗传性红细胞增多症相关HIF2α基因点突变对人CD34+造血干祖细胞体外红系分化的影响

该文旨在研究与遗传性红细胞增多症相关的低氧诱导因子HIF2α基因点突变对人造血干祖细胞红系分化的影响。通过构建人HIF2α基因编码区、携带疾病相关的两个点突变体(M535V和G537R)以及文献中报道的HIF2α基因阳性对照突变(P531A)慢病毒表达载体,分别包装病毒并感染人脐血来源的CD34+造血干祖细胞,并进行常氧及5%低氧条件下的红系定向诱导分化培养。利用FACS流式分析比较红系分化进程特征分子CD71和CD235a的表达变化,结合荧光定量PCR检测HIF2α调控的红系分化相关的靶基因表达水平。结果显示,构建的HIF2α及突变体的慢病毒载体经病毒包装后感染K562细胞可在RNA和蛋白水平实现过表达;与对照组相比,感染表达HIF2α基因或其突变体病毒后的脐血CD34+HSPC在常氧及5%O_2条件下诱导红系分化培养的细胞CD71和CD235a的表达动态均无明显改变,但HIF调控的红系分化相关基因EPOR和VEGFA的表达水平有一定升高。综上,在体外红系分化培养体系中,慢病毒介导的HIF2α及突变体的过表达不直接影响造血干祖细胞的红系分化进程,提示疾病相关的HIF2α基因突变造成的红系分化异常增多的细胞内外调控机制需要更进一步的深入研究。     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增.在该生物反应器内, 采用SCF TPO Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34 细胞的效果.培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34 细胞扩增倍数、培养物中CD34 细胞含量均相近, 无显著性差异; 而CD34 CD38-细胞扩增倍数以及培养物中CD34 CD38-细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养.可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增.     

血小板生成素诱导胎肝CD34~ 造血干/祖细胞增殖分化与周期蛋白表达的关系

为了解胚胎时期巨核细胞增殖分化特有的内在机制 ,本研究观察了在体外培养体系中 ,胎肝源CD3 4 造血干 /祖细胞在血小板生成素 (thrombopoietin ,TPO)作用下增殖分化特征与相关周期蛋白B1、D1和D3表达及细胞内水平变化的关系。结果发现 :( 1)经 12d培养后 ,TPO使胎肝源CD3 4 细胞数从 1× 0 5个细胞 /ml增加到 13 12± 4 0 6× 10 5个细胞 /ml,CD4 1 细胞增加到了 95 % ,CD3 4 细胞下降到了 3 % ,大部分细胞的DNA倍性为 2N ,少数为 4N ,无大于 4N巨核细胞 ,TPO对MegaCultTm C胶原半固体培养体系中胎肝源CD3 4 细胞形成CFU Mk集落产率的影响呈明显的剂量效应关系 ;( 2 )在整个培养期间 ,周期蛋白B1表达逐渐增加 ,并保持在一个高水平上 ,培养后期 ,高水平的周期蛋白B1出现在G1期细胞上 ;( 3 )周期蛋白D1和D3表达先增加 ,培养后期细胞内水平下降 ,且以G2期细胞为主。该结果提示 :( 1)TPO通过上调周期蛋白B1和在所有细胞周期时限上调周期蛋白D1和D3表达 ,促进巨核细胞祖细胞的增殖分化 ;( 2 )周期蛋白B1在G2 M期的持续高水平和周期蛋白D1和D3在G2 M期的水平下降 ,可能导致胎肝源巨核细胞核内有丝分裂延迟或阻滞。     

人卵黄囊造血的探讨

采用卵黄囊组织切片、涂片的形态学、细胞化学染色、造血干/祖细胞体外培养及CD_(34)单克隆抗体免疫荧光检测等方法研究表明:人卵黄囊中存在造血岛,造血岛内由于造血微环境的特点致使此期造血主要向红系分化。血岛中检测出CD_(34)~ 细胞,比例高于胎肝及成人骨髓,干/祖细胞于体外培养形成红系集落。结论:人胚胎期造血源于卵黄囊。     

牛胚胎生殖细胞的分离与培养

胚胎生殖细胞 (Embryonicgermcells,EG)是由生殖嵴原始生殖细胞 (Primordialgermcells,PGCs)中分离得到的一种未分化而多潜能的干细胞。牛EG细胞的研究在EG细胞核移植、转基因及建立生物反应器方面具有广阔的应用前景。本研究从 2 9- 70日龄牛胎儿PGCs分离得到EG细胞 ,经过抑制分化培养 ,其中一个细胞系传至 6代。所分离得到的EG细胞具有典型的EG细胞形态 ,AP及PAS染色呈阳性 ,核型正常 ,同时观察到这些细胞在体外进行自发性分化 ,可形成类胚体、成纤维样细胞及神经样细胞     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

Resistance and augmentation of innate immunity in mice exposed to starvation

Mice were exposed to starvation for 3 days. Body temperature and various parameters were examined. By starvation, body temperature, blood glucose and ACTH decreased, especially on days 2 and 3. The level of corticosterone increased at this time. On the other hand, the number of lymphocytes yielded by the liver, spleen and thymus decreased from day 1 to 3. The change of the distribution of lymphocyte subsets was unique because NK, NKT and extrathymic T cells were stress-resistant in the liver. Conventional T and B cells were stress-sensitive. Reflecting the increased proportion of NK and NKT cells, NK and NKT activities were augmented. The increased proportion of NKT cells produced both IFNγ and IL-4 (Th0-type profile). The proportion and some functions of granulocytes and macrophages increased on Day 1 after starvation. These results suggest that starvation has a potential to increase the functions of unconventional lymphocytes and myeloid cells.     

Comparative immunocytochemical demonstration of ACTH-, LH and FSH-containing cells in the pituitary of neonatal,immature and adult rats

Summary By means of immunocytochemistry, the development of ACTH-, LH- and FSH cells was examined in the anterior pituitary of 5-day-old neonatal, 15-day-old immature and adult rats. ACTH-positive cells are angular and the periphery of these cells is strongly reactive with anti-ACTH serum. In contrast, LH- and FSH-immunopositive cells are ovoid elements, ranging in cell size and intensity of staining. Angular cells, in which only the cell periphery reacted with anti-LH serum, were observed in neonatal and immature rats; however, these cells were not stained with either anti-FSH serum or anti-ACTH serum. Observation of serial semithin sections revealed that ACTH-immunopositive cells do not react with either anti-LH or anti-FSH serum. Finally, it was observed that ACTH cells and LH cells are both functionally differentiated already in 5-day-old neonatal rats.     

Cell cycle, morphology and pluripotency of octaploid embryonic stem cells in comparison with those of tetraploid and diploid cells

To examine the alteration of cellular characteristics on ploidy transition of embryonic stem (ES) cells, octaploid cells (8H1 cells) were established from tetraploid H-1 (ES) cells, and compared with tetraploid and diploid H-1 (ES) cells (4H1 and 2H1 cells, respectively). The duration of G₁, S, and G₂/M phases were essentially the same among 2H1, 4H1, and 8H1 cells, suggesting that cell cycle progression is conserved. The ratio of cell volume of 2H1, 4H1, and 8H1 cells was about 1 : 2 : 4, indicating that these polyploid cells were generated through cell cycle progression without cell division. The morphology of 8H1 cells was flagstone-like and flatter than that of 4H1 cells, and differed from the spindle-like shape of 2H1 cells, suggesting that transformation occurred during the ploidy transitions. Alkaline phosphatase activity was expressed equivalently in 2H1, 4H1, and 8H1 cells, and solid tumors that contained endodermal, mesodermal, and ectodermal cells were formed by 2H1, 4H1 or 8H1 cells after interperitoneal injection into the mouse abdomen, suggesting that pluripotency was preserved in the ploidy transition.     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.     

Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding

Epidemiological, experimental and clinical data indicate that cadmium and lead are osteotoxins in man and other species. The relative sensitivities of a clonal human osteosarcoma cell line (HOS TE 85) and a clonal rat osteosarcoma cell line (ROS 17.28) to the cytotoxic effects of cadmium and lead were tested in serum-free media without added growth factors. The rat osteosarcoma cells were more sensitive to cadmium with cytotoxicity and inhibition of proliferation at 0.25 versus 0.75 and 1.0 mol l^– cadmium, respectively, for human osteosarcoma cell lines. The lower sensitivity to cadmium of human osteosarcoma cells is attributed, at least partly, to induction of metallothionein synthesis by cadmium and zinc in this cell line; in the rat osteosarcoma cell line, they do not induce metallothionein synthesis. Human osteosarcoma cells were more sensitive than rat osteosarcoma cells to lead with inhibition (IC₅₀) of proliferation at 4 mol l^– lead and cytotoxicity at 20 versus 6 and over 20 moll^– lead, respectively, for these variables in rat osteosarcoma cells. Both cells lines attained the highest lead concentration in the 15 000 × g (mitochondrial) fraction. The lead in the mitochondrial, microsomal, nuclear and cytosolic fractions of the human cell line did not decrease during 24 h post-washout. Binding of lead was much less stable in the less sensitive rat cells, with 50–100% loss of mitochondrial, microsomal and nuclear lead during 24 h post-washout.     

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (∼2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (∼12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (∼0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharyngx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.     

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.