兔骨髓间充质干细胞向尿路上皮分化后构建膀胱组织工程化移植物

背景：尿路上皮细胞是泌尿系组织工程领域重要的种子细胞,但是难以体外大量扩增;有研究表明骨髓间充质干细胞可以向尿路上皮细胞分化,但关于分化后细胞在植入动物体内后上皮生成情况,以及分化后细胞在组织工程领域的具体应用研究尚不多。目的：分离、扩增兔骨髓间充质干细胞,诱导骨髓间充质干细胞向尿路上皮细胞分化并与兔膀胱脱细胞基质构建组织工程化移植物,了解分化后细胞作为种子细胞的效果。方法：12只8周龄雄性新西兰大白兔胫骨穿刺,抽取骨髓,密度梯度离心法分离骨髓间充质干细胞,第4或5代细胞以条件培养基培养2周,进行分化后细胞的鉴定。随后将分化后细胞种植在膀胱脱细胞基质上构建组织工程化移植物,进行膀胱修补;另12只动物作为对照组以尿路上皮细胞与膀胱脱细胞基质构建复合物行膀胱修补。结果与结论：骨髓间充质干细胞培养成功并在体外扩增,由条件培养基培养诱导分化后,PCR检测提示分化后细胞干细胞标志物CD44表达降低,而上皮细胞标志物UP1a表达升高,行免疫荧光检测发现分化后细胞能表达尿路上皮特异性标志物UP1a,而骨髓间充质干细胞无表达。分化后细胞构建的组织工程化移植物在膀胱修补2周后可形成稳定连续的上皮覆盖,上皮层厚度与尿路上皮细胞构建的组织工程化移植物类似。提示骨髓间充质干细胞向尿路上皮诱导分化后可作为泌尿系组织工程的种子细胞,可能是尿路上皮细胞之外的又一新选择。     

Continuous flow cell separator use for bone marrow processing

Many techniques have been described for processing bone marrow prior to transplantation, purging or cryopreservation. Effective techniques incorporate centrifugation and, or, density separation to produce an ideal marrow concentrate. We report on the use of a continuous flow cell separator (COBE Spectra) for marrow processing. Preliminary results indicate that the improved technology incorporated in this machine together with the new algorithm control of its collection functions allows for rapid collection of an ideal marrow concentrate. The addition of an inert sedimenting agent prior to processing enhances differential mononuclear cell collection and elimination of red blood cells and granulocytes. By this technique a volume depletion of 87% was achieved with recovery of 76.4% mononuclear cells and 86.5% CFU-GM progenitor cells. Marrow processed in this manner has been successfully transplanted; patients receiving such marrow show no delay in engraftment and their grafts have been sustained.     

Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations

BACKGROUND: The performance of cultures to assess possible bacterial contamination of bone marrow and peripheral blood progenitor cell preparations is required by the standards of the American Association of Blood Banks. STUDY DESIGN AND METHODS: Consecutive (n = 893) bone marrow and peripheral blood progenitor cell preparations were cultured for assessment of possible contamination by microorganisms. RESULTS: Consecutive bone marrow and peripheral blood progenitor cell preparations (n = 893) were cultured; the overall positive rate detected was 2.5 percent (22/893). The isolates predominantly were skin contaminants (gram-positive cocci) and so-called water-borne organisms (gram-negative rods). The 6.0-percent rate of positivity in 317 bone marrow preparations was higher than the 0.5-percent rate in 576 peripheral blood progenitor cell preparations (p < 10(-6)). Culture- positive preparations were transfused to 16 patients at this institution; however, none of these transfusions led to documented sepsis with the contaminating organism. CONCLUSION: The culture method described here complies with the standards of the American Association of Blood Banks. Contamination can be detected in both bone marrow and peripheral blood progenitor cell preparations. When contaminated preparations are transfused, there are few complications that can be attributed to the contamination.     

Influence of preharvest tumor cell contamination in bone marrow or blood does not predict resultant tumor cell contamination of granulocyte colony-stimulating factor mobilized stem cells

Tumor cell contamination of stem cell collections harvested from breast cancer patients is a common phenomenon described by several investigators but with findings that vary among reports. Although so-called co-mobilization of these cells has been hypothesized, the origin of tumor cell contamination in stem cells is still unknown. A total of 47 G-CSF mobilized stem cell grafts from patients with nodal-positive (n = 30), chemosensitive metastatic (n = 11), and 5 women with inflammatory breast cancer were evaluated for cancer cells by immunocytochemistry. Additionally, 40 bone marrow aspirations and 23 peripheral blood samples collected prior to apheresis and after one to two cycles of conventional chemotherapy were available for examination. Tumor cell contamination of leukapheresis correlated best with preharvest blood state. This was valid when the nominal (positive/negative) presence of tumor cells in blood was compared to the nominal presence of tumor cells in apheresis samples and when the it was correlated to the tumor cell load of apheresis samples (TCL = tumor cells per 10(6) nucleated cells investigated). The correlation between blood and stem cells was better (nominal and quantitative) than that between marrow and stem cells, despite the larger sample size of marrow aspirations. The presence or absence of cancer cells in apheresis samples could not be safely predicted by the presence or absence of tumor cells in marrow or blood alone. Diagnostic specificity seems to improve from a combination of results from marrow and blood analysis. No correlation was found in quantitative analysis of tumor cell contamination between marrow and blood. In conclusion, the results suggest that blood and bone marrow represent different compartments for epithelial cancer cells and that contaminating tumor cells in stem cell harvests may be derived from the blood and/or marrow compartment. The tumor cell contamination of a stem cell harvest cannot be safely predicted by a preceding blood or marrow analysis.     

Human bone marrow lymphocytes. I. Distribution of lymphocyte subpopulations in the bone marrow of normal individuals.

This study was undertaken to determine the proportions and in vitro immune capacities of lymphocyte populations in the bone marrows of normal humans. Relatively pure mononuclear cell suspensions were obtained from bone marrow aspirates by linear sucrose gradient centrifugations. Simultaneous peripheral blood and bone marrow specimens from each individual were assayed for lymphocyte surface markers and mitogen responsiveness. Maximal possible contamination of bone marrow aspirates by peripheral blood was determined by performing aspirates on individuals who had received 51chromium-labeled autologous erythrocytes. Rhymus-derived (T) lymphocytes, as determined by the sheep red blood cell (E) rosette assay, comprised 8.6-(plus or minus 1.6)% of the total bone marrow lymphocyte pool. Bone marrow-derived (B) lymphocytes, as determined by the presence of a complement receptor, made up 15.4-(plus or minus 1.9)% of the lymphocyte pool whereas 74.6 (plus or minus 2.4)% of mononuclear cells lacked easily detectable surface markers. These findings could not be explained by contamination with peripheral blood lymphocytes since contamination was corrected for in the calculations. Lymphocyte-enriched suspensions of bone marrow cells responded to stimulation with phytohemagglutinin, concanalin A, and particularly pokeweed mitogen. In vitro incubations of bone marrow and peripheral blood lymphocytes with tritiated thymidine followed by determinations of E and erythrocyte antibody complement (EAC) rosettes were performed. Simultaneous rosetteradioautographs demonstrated that the proliferative potential of bone marrow B lymphocytes was greater than peripheral blood B lymphocytes (P less than 0.01). On the other hand, the proliferative potential of bone marrow T lymphocytes was the same as that of peripheral blood T lymphocytes. These findings demonstrate that in addition to containing B lymphocytes the normal bone marrow contains a small fraction of T lymphocytes similar to the mature T lymphocyte pool found in the peripheral blood. These T cells most probably enter the bone marrow parenchyma as part of the normal recirculating lymphocyte pool.     

ABO血型不合的异基因骨髓移植

目的 探讨ＨＬＡ 相合,ＡＢＯ 血型不合的异基因骨髓移植（ａｌｌｏＢＭＴ） 中存在的免疫及造血问题。方法 对本所３８ 例ＡＢＯ 血型主要不合,２３ 例次要不合的ＨＬＡ 相合的ａｌｌｏＢＭＴ 受者进行分析,并选用同期ＡＢＯ 血型相合的ａｌｌｏＢＭＴ 患者作配对比较。结果ＡＢＯ 血型不合的ａｌｌｏＢＭＴ患者,输注骨髓后无一例发生急性溶血。经配对ｔ 检验及χ２ 检验,ＡＢＯ 血型不合对骨髓植活、血小板恢复,ＧＶＨＤ 及５ 年无病生存率均无影响; 在ＡＢＯ 血型主要不合组, 红系开始恢复时间明显延迟,使红细胞输用量明显增多;其中５ 例患者发生纯红细胞再生障碍（ 纯红再障） ,持续约７ ～２４ 个月。发生纯红再障者均为“Ｏ”型血受者,红系恢复与血型抗体滴度具有相关性。结论ＡＢＯ 血型不合可以进行ａｌｌｏＢＭＴ,但对发生纯红再障高危的患者宜慎重。     

Purging of leukemia-contaminated bone marrow grafts using suicide adenoviral vectors: an in vivo murine experimental model

Autologous bone marrow transplantation is an alternative therapeutic option for acute myeloid leukemia patients lacking a compatible donor. However, bone marrow from these patients may contain residual leukemic cells that should be ideally eliminated prior to the infusion of the graft. With the aim of developing more efficient protocols of graft purging, adenoviral-mediated gene transfer protocols have been conducted. We studied whether suicide adenoviral vectors expressing the cytosine deaminase gene (AdCD) could be used for selectively killing leukemic WEHI-3B cells. The AdCD transduction followed by the 5-fluorocytosine exposure abrogated the growth of WEHI-3B cells in vitro, with a minimal effect on normal hematopietic progenitors. To test the efficacy of the purging protocol in vivo, bone marrow cells were mixed with syngenic WEHI-3B cells and this chimeric cell population was transduced with AdCD vectors. Infected cells were injected into myeloablated Balb-c mice, which then received a 5-fluorocytosine treatment for 4 days. All mice transplanted with unpurged bone marrow developed leukemia and died. However, 90% of recipients receiving the purging treatment were healthy up to 9 months post-transplantation and had a perfectly re-established hematopoietic system, without any signal of leukemic cell presence. In conclusion, suicide adenoviral vectors are proposed as a tool for the purging of Adenoviral-susceptible myeloid leukemia cells contaminating autologous bone marrow grafts.     

Bone deficit in ovariectomized rats. Functional contribution of the marrow stromal cell population and the effect of oral dihydrotachysterol treatment.

This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions.     

Transplantation of gene-modified human bone marrow stromal cells into mouse-human bone chimeras

Transplantation of BM stromal cells engineered to secrete therapeutic factors could represent a treatment for a large array of hematologic disorders. The aim of this study was to evaluate the susceptibility of human BM stromal cell precursors to retroviral gene transfer, then the ability of those to be transplanted in vivo. We have transduced a recombinant retrovirus encoding the mouse CD2 antigen into STRO-1+ cells selected from adult and fetal BM. Gene-modified stromal cells were injected intravenously into NOD-SCID mice engrafted previously with pieces of human fetal hematopoietic bone. Using nested PCR, transgenic human cells were detected both in the marrow of human bone grafts and in the BM, liver, and spleen of host mice 7 weeks after grafting. These data indicate that BM stromal progenitor cells are targets for retrovirus-mediated gene transfer and can home to hematopoietic tissues on engraftment through the bloodstream of nonconditioned hosts.     

Correlation of CD34+ cell counts with volume of bone marrow collected for allogeneic bone marrow harvests

The quantity of bone marrow collected for allogeneic bone marrow transplantation is based on collecting 10 to 15 cc of bone marrow/kg of recipient weight. We hypothesized that the percentage of CD34+ cells collected during a bone marrow harvest decreased at the end of the harvest because of increasing amounts of peripheral blood contamination. We performed a prospective, blinded study in which we measured CD34+ percentages and cell counts at 200-cc intervals during bone marrow harvests from 11 consecutive human leukocyte antigen (HLA)-matched sibling bone marrow donors. We observed that the percentage of CD34+ cells in aspirated bone marrow did not vary significantly from the start to the end of the bone marrow harvest, and the total number of CD34+ cells/kg increased in a linear fashion, thus disproving our original hypothesis. In conclusion, the percentage of CD34+ cells in aspirated bone marrow will remain constant throughout a bone marrow harvest.     

ABO incompatibility as an adverse risk factor for survival after allogeneic bone marrow transplantation

BACKGROUND: Graft ABO incompatibility has not been thought to affect patient survival after allogeneic bone marrow transplantation, although it may be associated with prolonged erythroid aplasia and immediate or delayed hemolysis. STUDY DESIGN AND METHODS: A retrospective analysis of a cohort of 292 allogeneic transplant recipients measured survival in a subgroup of ABO-incompatible bone marrow graft recipients. RESULTS: Patients with acute myelogenous leukemia or myelodysplastic syndrome receiving non-T-cell-depleted bone marrow grafts had an 85-percent greater risk of death within 100 days of transplant (relative risk, 1.85, 95% CI, 1.33-2.58; p = 0.003) than comparable patients receiving ABO-compatible grafts. Both ABO major- and minor-mismatched graft recipients were at risk.The increased mortality rate was not due to an increase in graft failure or acute graft-versus-host disease; rather, patients died of multiple-organ failure and sepsis, which is consistent with regimen-related toxicity. This effect was not seen in a larger group of 112 chronic myelogenous leukemia patients undergoing similar treatment. CONCLUSION: ABO incompatibility may be a significant prognostic risk factor after allogeneic bone marrow transplantation in susceptible subgroups of recipients. Care is necessary to design hematopoietic stem and progenitor cell-processing and -transfusion policies to minimize this risk.     

全骨髓贴壁法分离培养大鼠骨髓间充质干细胞的生物学特性及优势

背景：骨髓来源的间充质干细胞含量极低,体外纯化、扩增活性好、分化潜能高的骨髓间充质干细胞,对进一步研究至关重要。目的：进一步验证全骨髓贴壁法体外分离、培养、纯化骨髓间充质干细胞的生物学特性、表型及多向分化潜能。方法：通过全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,进行形态学观察,流式细胞仪检测细胞标记物表达,分别进行成骨、成脂诱导分化。结果与结论：成功纯化、扩增了高细胞活性、高分化潜能的骨髓问充质干细胞。所得细胞呈成纤维细胞样;表达CD29、CD90,不表达CD45;成骨、成脂诱导分化后,茜素红染色、油红O染色阳性。证实全骨髓贴壁法操作简单、对细胞活性损伤小,可以得到高纯度、高活性、高分化潜能、生物学形态和特征稳定的骨髓间充质干细胞。     

混合骨髓移植的实验研究

目的：探讨在异基因骨髓移植治疗白血病中既控制移植物抗宿主病（ＧＶＨＤ）又同时保留移植物抗白血病（ＧＶＬ）作用的移植方案。方法：采用近交系小鼠移植模型，在异基因骨髓、脾细胞移植物中混合一定比例的同基因脾细胞，观察移植后受鼠ＧＶＨＤ死亡率、脾结节数、移植后不同时间骨髓造血重建细胞来源以及受鼠脾脏淋巴细胞对供、受鼠双方来源的刺激细胞增殖反应；采用Ｌ６１５小鼠白血病模型观察混合移植对荷瘤小鼠生存时间及死亡原因的影响。结果：混合移植可以有效减轻ＧＶＨＤ，异基因造血免疫细胞在受鼠体内一过性存留并发挥移植物抗宿主反应，使荷瘤小鼠生存时间明显延长。结论：异基因骨髓、脾细胞移植物中混合一定比例的同基因脾细胞使移植受鼠体内既具有主要组织相容性抗原复合物（ＭＨＣ）不合的免疫活性细胞发挥一过性ＧＶＬ作用，同时减轻了异基因骨髓移植的严重ＧＶＨＤ。     

Cryopreserved primate bone marrow cells can be used for retroviral-mediated gene transfer.

Retroviral-mediated gene transfer into Rhesus monkey bone marrow cells, which were cryopreserved, stored, and then transduced at the time of thawing, was studied for potential application in gene therapy protocols. Albumin density gradient fractionation was used to define subpopulations of cryopreserved cells transduced by a murine retroviral vector. The transfer of the bacterial neomycin phosphotransferase gene into Rhesus monkey bone marrow that was cryopreserved and thawed was found to be preferential in a light-density population enriched for granulocyte-macrophage colony-forming units (CFU-GM). These results are similar to results obtained with freshly harvested bone marrow in which this population is enriched for CD34 antigen-positive cells, as well as for cells that were undergoing cell division. This method may be useful in enriching for transduced precursors in future gene transfer experiments in primates.     

Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.     

自体骨髓采集术病人的护理

[目的]探讨自体骨髓采集术的护理方法并采集足够细胞数量的骨髓液,降低术中及术后不良反应。保证自体骨髓移植术的成功。[方法]对6例自体骨髓采集术病人采取全方位的护理措施。[结果]经过采用一系列全方位的护理措施,6例病人均采集足够细胞数量的骨髓液,采集术中、术后无不良反应发生,使骨髓移植过程顺利完成。[结论]为自体骨髓采集术病人提供全方位的护理,有利于骨髓液的采集成功,更为骨髓移植的成功奠定基础。     

肝硬化干细胞移植治疗中自体骨髓采集临床研究

目的探讨肝硬化患者自体骨髓采集过程的临床表现。方法 122例行自体骨髓采集的肝硬化患者,比较采集前5min、局部麻醉、穿刺术中及术后15min患者心率、收缩压、舒张压变化;比较前3管骨髓采集中患者疼痛评分及每管抽取时间。结果以双侧髂后上嵴为骨髓采集部位,局部麻醉、穿刺及抽取骨髓过程中患者心率均高于术前（P〈0.05）,收缩压、舒张压与术前比较差异无统计学意义（P〉0.05）,术后15min收缩压、舒张压低于术前（P〈0.05）、但在正常范围,心率与术前比较差异无统计学意义（P〉0.05）;双侧局部麻醉时疼痛程度评分差异无统计学意义（P〉0.05）,右侧穿刺时疼痛程度评分高于左侧（P〈0.05）,抽取双侧第1管骨髓时疼痛程度评分高于第2,3管（P〈0.05）。结论正确掌握穿刺方法及抽取速度,可采集较高数量和质量的自体骨髓干细胞。     

Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses

The use of flow cytometric enumeration of blasts in bone marrow aspirates has been of limited value in situations where blood contamination of the specimen is present. Assessment of sequential pulls of bone marrow aspirates from the same patient show decreasing proportions of blasts that are detected in later specimens. To address this problem, the intensity of CD16 on maturing neutrophils was compared for bone marrow biopsies, peripheral blood, and bone marrow aspirates. A comparison between bone marrow biopsy and aspirate specimens from the same individuals showed similar proportions of neutrophils with mature phenotype in most, but not all pairs. Other cell populations (total mature lymphocytes, monocytes, neutrophils, and blasts) were also similar between the two specimen types, with one exception of a patient with myelodysplasia, exhibiting a unique blast population in the biopsy that was not evident in the aspirate. The proportion of mature myeloid cells expressing a mature neutrophil phenotype (high levels of CD16) was found to be 17% (±6.7, n=47) in trephine marrow biopsy specimens. In contrast, marrow aspirates contained more of the mature neutrophil phenotype (38%±16, n=33) with about 1/3 of the aspirates indistinguishable from biopsies. Using a simple formula to normalize the aspirate specimens to the average neutrophil composition of marrow biopsies, it was possible to correct for the dilutional effect of added blood to both normal bone marrow aspirates and aspirates with elevated blast counts. These results suggest three alternative means of circumventing the problem of blood dilution of marrow aspirate specimens. (1) Blast counts by flow cytometry can be obtained from disaggregated biopsy specimens. (2) A bone marrow aspirate can be assessed for the proportion of mature neutrophils present and only those with low proportions (<30%) of phenotypic mature neutrophils are considered adequate for blast counting. (3) The aspirates with high proportions of mature neutrophils may be normalized based on the proportion of dim CD16 maturing myeloid cells to a level observed in bone marrow biopsies (based on an average mature neutrophil composition). Such an approach for identifying the amount of hemodilution in each specimen may enhance the utility of flow cytometry in enumeration of blasts in bone marrow, especially in cases where myeloblast count is crucial for prognosis, such as myelodysplastic syndromes (MDS).     

Effects on donors of second bone marrow collections

Second bone marrow transplants have been used successfully to treat marrow graft failure or relapse following the original marrow transplantation. Hospital records of 16 related two-time bone marrow donors were reviewed to determine what risks a person faces in making a second marrow donation. One donor suffered a minor complication following the first collection, but no donors suffered complications during the second donation. The volume of marrow collected for the first and second donations (1253 +/- 504 vs. 1261 +/- 471 mL) was similar. However, more marrow donors received transfusions of homologous red cells during the second collection than during the first collection (5/16 compared to 1/16; p = 0.06). To determine what factors might contribute to the high incidence of homologous red cell transfusion during second donations, the experiences of donors who received homologous blood during the second donation were compared to the experiences of donors who received only autologous blood. Persons receiving homologous blood during the second collection received more red cell units than persons receiving autologous blood (2.0 +/- 0.7 units compared to 0.9 +/- 0.5 units; p less than 0.01). Their precollection hemoglobin levels were lower (12.3 +/- 1.2 vs. 14.4 +/- 1.4 g/dL [123 +/- 12 vs. 144 +/- 14 g/L]; p less than 0.01), and the time between the first and second collections was significantly less (45 +/- 8 days vs. 342 +/- 432 days; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

猪骨髓间质干细胞的分离培养及分化潜能的鉴定

目的：建立猪骨髓间质干细胞(MSCs)的体外分离培养和鉴定的方法，探讨体外培养的间充质干细胞的一些生物学特点，为利用猪的实验研究提供实验基础。方法：猪的髂嵴穿刺吸取骨髓，经密度梯度离心得到骨髓单个核细胞，接种后形成单层贴壁的成纤维样的细胞。检测细胞周期，多向诱导分化鉴定分离的细胞。结果：体外培养的原代MSCs12～14d达到融合，传代后仍具有分化成骨的能力，细胞周期显示有80％的细胞处于GO／G1期。结论：体外培养猪的MSCs具有分化成骨的潜能，生长稳定，传代后仍保持未分化状态．猪骨髓间充质干细胞分离培养体系的建立为基础研究和组织工程提供了一个有价值的动物模型。