Mechanism of in Vitro Differentiation of Bone Marrow Stromal Cells into Neuron-like Cells~

In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cells in vitro.     

大鼠骨髓间充质干细胞向神经细胞分化过程中Notch信号与RhoA/Rho激酶信号的协同作用

目的 探讨Notch信号通路在大鼠骨髓间充质干细胞(MSCs)向神经细胞分化中与RhoA/Rho激酶信号的协同作用.方法 实验分为无血清培养基对照组和盐酸法舒地尔组.采用盐酸法舒地尔诱导大鼠MSCs分化为神经细胞.RT-PCR检测Hes1的表达变化;免疫细胞化学法检测神经元特异性烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化.结果 ①盐酸法舒地尔组MSCs的Hes1 mRNA转录下降(P<0.05).②盐酸法舒地尔可以诱导MSCs向神经细胞分化,NSE、NF-M的阳性表达率显著高于无血清培养基组(P<0.05).结论 盐酸法舒地尔在诱导大鼠MSCs向神经细胞分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经细胞分化. Abstract： Objective To investigate the cross-talk between notch and Rho/Rho GTPase signaling on rat bone marrow mesenchymal stem cells differentiating into neurons. Methods The experiments were divided into serum-free medium control group and fasudil hydrochloride group. Fasudil hydrochloride induces MSCs differentiating into neurons. The expression of Hes1 mRNA was detected by RT-PCR. The expression of neuron-specific enolae(NSE), neurofilament M (NF-M) and glial fibrillary acidic protein (GFAP)was detected by immunocytochemical method. Results ①The Hes1 mRNA expressed by MSCs of fasudil hydrochloride group was significantly decreased(P<0.05).②Fasudil hydrochloride can induce MSCs differentiating into neurons and there was higher expression of neuron-specific enolae(NSE) and neurofilament-M (NF-M) than the serum-free medium control group(P<0.05).Conclusions There may be cross-talk between notch signaling and RhoA/Rho GTPase signaling on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promoted the differentiation of MSCs into neurons.     

Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-αreleased by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-αrelease by RAW264.7 cells evoked by LPS.     

Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells （BMSC） into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC （hBMSC） to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl （NF1）, nestin and neuron-specific enolase （NSE） in rat BMSC （rBMSC）. Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NFI and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S 100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated, rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia milliorrhiza could induce hBMSC to differentiate into neuron-like cells, If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.     

Over-expression of VEGF in marrow stromal cells promotes angiogenesis in rats with cerebral infarction via the synergistic effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group th     

Experimental study on MyoD gene induced differentiation of bone marrow mesenchymal stem cells into myoblasts in vitor

Objective:To explore the possibility of the transfection of MyoD gene induced bone marrow mesenchymal stem cells(MSCs) to differentiate into myoblasts in vitro.Methods:The eukaryotic expression plasmid vector pIRES2-EGRP-MyoD was transfected into MSCs with lipotransfection method,and the positive cells were selected by G418;The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified,purified product was identified by sequencing;The reporter gene enhanced green fluorescence protein(EGFP)was observed in the transfected cells under a fluorescent and a laser confocal microscopes;Immunohistochemical methods was used to examine the expressions of MyoD,myogenin,myosin,myoglobin and desmin in the differentiated,cells.The ultrastructure changes of the cells before and after transfection were observed with electron microscopy.Results:The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified,purified product was as same in sequence as thwat from Genbank;Green fluorescence was observed in the transfected cells under a fluorescent and a laser confocal microscopes;Immunohistochemical methods indicated that MyoD,myogenin,myosin,myoglobin and desmin were expressed in the transfected cells;The transfected cells showed the morphologcal characteristics of matrue cells with filaments in their cytoplasm.Conclusion:MyoD gene can induce cultured MSCs to successfully differentiate into myoblasts,probably providing an experimental foundation for trauma repair.     

Influence of β-catenin small interfering RNA on human osteosarcoma cells

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival,invasion and chemosensitivity of human osteosarcoma cells(U2-OS cells).The siRNA against β-catenin was constructed and transfected into U2-OS cells.The expression of β-catenin was detected by qRT-PCR and Western blotting.Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry,respectively.Cell invasion ability was measured by transwell assay.The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin,suppression of invasion and motility of U2-OS cells,reduced chemosensitivity to doxorubicin in vitro,and little change in cell growth and apoptosis.Additionally,down-regulated MT1-MMP expression was found after transfection.It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression,and the chemosensitivity of osteosarcoma cells against doxorubicin.     

Experimental study on MyoD gene induced differentiation of bone marrow mesen-chymal stem cells into myoblasts in vitro

Objective: To explore the possibility of the transfection of MyoD gene induced bone marrow mesenchymal stem cells ( MSCs) to differentiate into myoblasts in vitro. Methods: The eukaryotic expression plasmid vector pIRES2-EGFP-MyoD was transfected into MSCs with lipotransfection method, and the positive cells were selected by G418; The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was identified by sequencing; The reporter gene enhanced green fluorescence protein ( EFGP) was observed in the transfected cells under a fluorescent and a laser confocal microscopes; Immunohistochemical methods was used to examine the expressions of MyoD, myogenin, myosin, myoglobin and desmin in the differentiated cells. The ultrastructure changes of the cells before and after transfection were observed with electron microscopy. Results: The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was as same in sequence a     

Study on the adoption of Schwann cell phenotype by bone marrow stromal cells in vitro and in vivo

To explore the possibilities of bone marrow stromal cells （MSCs） to adopt Schwann cell phenotype in vitro and in vivo in SD rats. Methods MSCs were obtained from tibia and femur bone marrow and cultured in culture flasks. Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin were added to induce differentiation of MSCs＇. Schwann cell markers, p75, S-100 and GFAP were used to discriminate induced properties of MSCs＇ by immunofluorescent staining. PKH-67-1abelled MSCs were transplanted into the mechanically injured rat sciatic nerve, and laser confocal microscopy was performed to localize the PKH67 labelled MSCs in the injured sciatic nerve two weeks after the operation. Fluorescence PKH67 attenuation rule was evaluated by flow cytometry in vitro. Results MSCs changed morphologically into cells resembling primary cultured Schwann cells after their induction in vitro. In vivo, a large number of MSCs were cumulated within the layer of epineurium around the injured nerve and expressed Schwann cell markers, p75, S- 100, and GFAP. Conclusion MSCs are able to support nerve fiber regeneration and re-myelination by taking on Schwann cell function, and can be potentially used as possible substitutable cells for artificial nerve conduits to promote nerve regeneration.     

Targeted induction of differentiation of human bone mesenchymal stem cells into neuron-like cells

A systematic method of isolating and culturing human bone mesenchymal stem cells （hMSCs）, and inducing them to differentiate into neuron-like cells in vitro was established. The hMSCs were isolated from bone marrow with the lymphocyte-separating medium, cultured and expanded in vitro, and induced after addition of compound neuro-revulsants. The morphological changes of hMSCs were observed, and the expression of surface markers in induced hMSCs was immunocytochemically identified during induction period. The hMSCs could be separated, cultured and expanded in vitro. After induction by compound neuro-revulsants for 48 h, the changes of neuron-like cells, such as cellular shrinkage and neurite growth, were observed in some cells. The immunochemical staining revealed nestin （＋） or NF （＋）, and GFAP （-）. It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.     

人骨髓间充质干细胞向血管内皮细胞诱导的研究

目的 研究人骨髓间充质干细胞(HBMCs)的体外培养和向血管内皮细胞的诱导分化.方法 利用密度梯度离心法将HBMCs从人骨髓中分离出来,体外扩增.将第三代的细胞以含VEGF,bFGF的培养基定向诱导,使其向内皮细胞方向分化.利用免疫细胞化学和流式细胞学检测诱导后的细胞表型.结果 原代未经诱导的骨髓干细胞,培养3周后细胞形态呈梭形,诱导后第7天的细胞呈椭圆形或不规则形,第14天细胞大致呈铺路石样改变.免疫细胞化学显示CD31、CD34、vWF因子呈阳性,流式细胞仪测定CD31、vWF因子阳性率分别为87.5%、82.6%,双阳性率为71.2%.结论 骨髓间充质干细胞在内皮细胞生长因子和碱性成纤维细胞因子的诱导下向血管内皮细胞方向分化. Abstract： Objective To study the culture methods of human bone marrow mesenchymal stem cells(MSCs) and differentiation into vascular endothelial cells in vitro. Methods Bone marrow mesenchymal stem cells were isolated and cultivated by density gradient centrifugation method. Induce the third generation to vascular endothelial cells with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Immunophenotypes of cells were detected by flow cytometry techniques and immunocyte chemistry after the transfection. Results After three weeks of primary culture of MSCs, the cell showed on fusiform shape. After 7 days induction, the cell showed on ellipse and irregular shape. After two weeks transfection, the cell exhibited a Cobblestone-like morphology. The cell expressed CD34,CD31,vWF after transfection by immunocyte chemistry. The positive rates of CD31 vWF were 87.5% and 82.6%, and the double positive rate was 71.2%. Conclusions Bone marrow mesenchymal stem cells can differentiate into vascular endothelial cells by treating with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).     

Pdx-1真核表达载体构建及其在大鼠骨髓间充质干细胞中的表达

目的 构建含有胰腺-十二指肠同源盒-1(Pdx-1)的真核表达载体,并研究其在大鼠骨髓间充质干细胞(MSCs)中的表达情况.方法 克隆Pdx-1基因,构建含Pdx-1的真核表达载体pEGFP-Pdx-1及pcDNA3.1-Pdx-1,转染MSCs,倒置荧光显微镜及逆转录聚合酶链反应(RT-PCR)检测MSCs中Pdx-1的表达.结果 测序结果 表明克隆的Pdx-1基因序列正确,构建的含Pdx-1的真核表达载体能有效的转染MSCs,并检测到该基因mRNA的表达.结论 完成Pdx-1基因克隆,成功构建含有Pdx-1基因真核表达载体,并在转化株中检测到该基因的表达,为糖尿病的细胞替代及基因治疗提供基础. Abstract： Objective To construct eukaryotic expression vector containing the pancreatic-duodenum homeobox-1(Pdx-1)and to study the expression in rat bone marrow-derived mesenchymal stem cells (MSCs). Methods Cloned Pdx-1 gene and constructed containing Pdx-1 eukaryotic expression vector pEGFP-Pdx-1 and pcDNA3.1-Pdx-1, transfected MSCs, detected the Pdx-1 expression in MSCs by invert fluorescence microscope and RT-PCR.Results Sequencing results showed that the cloned Pdx-1 gene sequence was correct, the eukaryotic expression vector containing Pdx-1 could effectively transfected MSCs and detected the gene expression at mRNA level.Conclusions The completion of Pdx-1 gene is cloned successfully and constructed containing Pdx-1 gene eukaryotic expression vector, detected the expression of the gene in the stable transfected MSCs,making preparations for the cell replacement and gene therapy of diabetes.     

骨髓间充质干细胞移植对肺气肿大鼠血浆TNF-α的作用

目的 研究移植骨髓间充质干细胞(MSCs)对肺气肿大鼠血浆肿瘤坏死因子-α(TNF-α)水平的影响.方法 将30只Wistar大鼠随机分为对照组、肺气肿组、肺气肿+MSCs治疗组,每组30只.在气管内注入脂多糖(LPS)并烟雾暴露,建立肺气肿大鼠模型,然后将培养的MSCs经尾静脉注入到肺气肿大鼠体内.观察移植后大鼠肺组织的变化,酶联免疫吸附测定(ELISA)法检测血浆TNF-α含量.结果 肺气肿组、肺气肿+MSCs移植组均出现肺气肿样病理改变,但后者较前者明显减轻.与肺气肿组比较,肺气肿+MSCs组TNF-α含量显著降低.结论 MSCs移植对肺气肿大鼠有保护作用. Abstract： Objective To investigate the effect of bone marrow mesenchymal stem cell (MSCs) transplantation on plasma TNF-α levels in rats of pulmonary emphysema. Methods Thirty wistar rats were randomized into three groupes:normal control group, pulmonary emphysema group, pulmonary emphysema +MSCs transplantation groupe. The pulmonary emphysema model of rats were established by intratracheal instillation of lipopolysaccharide twice and daily exposure to smog. Bone marrow MSCs were infused through tail vein. Morphologic changes of the lung tissues were observed. The level of plasma TNF-α were dectected using enzyme-linked immunosorbent assay. Results Destruction of alveolar walls was observed in rat lungs from pulmonary emphysema group and pulmonary emphysema +MSCs transplantation group. MSCs transplantation group significantly ameliorated the emphysematous changes. Significant differences in the levels of TNF-α between pulmonary emphysema group and pulmonary emphysema +MSCs transplantation group were observed. Conclusions MSCs therapy shows protective effects against pulmonary emphysema.     

骨髓干细胞和伞状支撑骨移植治疗股骨头坏死的动物实验研究报告

目的 探讨骨髓干细胞移植和伞状支撑骨移植治疗股骨头坏死的效果.方法 取6只健康山羊,采用液氮冷冻法制作单侧股骨头坏死动物模型,8周后在坏死侧行骨髓干细胞移植和伞状支撑骨移植术.分别于术后3、6个月做放射学检查,观察股骨头内骨质变化.结果 实验动物一般状况良好.液氮冷冻法8周后达成股骨头坏死模型,模型侧后肢出现跛行.骨髓干细胞移植和伞状支撑骨移植术后3个月X线显示,股骨头外形恢复,股骨头内囊性低密度区消失,可见骨柱影,股骨头内骨质愈合状况良好,原塌陷已修复,股骨头无再塌陷.6个月时,治疗侧股骨头外形正常,骨密度基本均匀,骨柱影已模糊,植骨已融合,股骨头无再塌陷.正常对照侧股骨头无异常变化.结论 骨髓干细胞移植和伞状支撑骨移植术可以有效治疗骨坏死,防治股骨头塌陷,效果良好. Abstract： Objective To study the usefulness to treat the osteonecrosis of the femoral head (ONFH) with bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting. Methods Six goats were established of osteonecrosis of the femoral head models on one side by method of liquid nitrogen frozen, and then they were taken into bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting after 8 weeks. The radiological examination was made at 3 and 6 months after the bong grafting, and observed changes of bone union in the femoral head. Results The general state of the experimental animal was fine after the operation. The model of ONFH was reached on 8 weeks after liquid nitrogen frozen, and limping. After 3 months of bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting, X-ray film showed that low bone density disappeared, the shape of the femoral heads, grafting strut bone and initial bone union were fine in the head, and no repeated collapse of the head. The X-ray film showed that the shape of the femoral heads was normal, grafting strut bone was union in the head, and no repeated collapse of the head after 6 months. There was normal on the control side. Conclusions The ONFH can be treated effectively by bone marrow mesenchymal stem cell transplantation and umbrella strut bone grafting, and the collapse of the femoral head is prevented.     

体外诱导的神经元样细胞对大鼠脑损伤模型修复的影响

目的:探讨大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs) 诱导为神经元样细胞后对大鼠脑损伤模型修复的影响。方法:骨髓贴壁法分离培养大鼠BMSCs并采用流式细胞术进行鉴定,川芎嗪诱导其分化,将诱导24h的细胞直接注射移植到大鼠脑缺血模型海马区组织, 检测分化的神经元样细胞在神经系统微环境中存活及病理变化。结果:细胞移植组病理变化与脑损伤模型组相比, 神经细胞变性、坏死数量明显减少, 间质水肿较轻。结论:骨髓间充质干细胞诱导为神经元样细胞后移植入对大鼠脑缺损伤的修复有一定的作用。     

原因不明的血小板下降在白血病异基因干细胞移植术后早期复发中的预测作用

目的 探讨白血病患者异基因造血干细胞移植(Allo-HSCT)术后原因不明的血小板(PLT)下降与白血病复发之间的关系.方法 51例白血病患者于2006年4月至2009年10月在我科接受了Allo-HSCT术,其中5例患者在术后2年内出现了复发,我们回顾性分析了其复发前外周血PLT计数、骨髓形态学及植入状态的动态变化.结果 5例移植后复发患者移植早期中性粒细胞和PLT造血重建平均时间分别为15.8、25.8 d.当PLT计数降至50×109/L左右时,骨髓巨核细胞进一步减少,骨髓幼稚细胞比率虽较PLT开始下降时有所增多,但均未达到复发的标准,STR检查所有患者从FDC转变为进展性混合型嵌合体(PMC);5例患者在此后的3个月内先后发生形态学复发,复发时骨髓巨核细胞几乎消失,STR检查除1例患者仍为PMC外,其余4例患者均转变为植入失败;统计学分析发现Allo-HSCT术后PLT进行性下降可较出现PMC提前平均75 d、形态学复发提前平均164 d.结论 白血病患者Allo-HSCT术后出现原因不明的血小板进行性下降这一现象,虽然不能用来作为白血病复发的诊断,但对其早期复发有一定的预测作用. Abstract： Objective To explore the relationship between unexplained decline in platelets (PLT) and relapse in leukemia patients after allo-geneic haematopoietic stem cell transplantation(Allo-HSCT). Methods Fifty-one patients with leukemia received Allo-HSCT in our department between April 2006 to October 2009 and 5 of them relapsed within 2 years after transplantation.We retrospectively analyzed the dynamic alteration about the platelets in peripheral blood, bone marrow morphology and the state of implantation in the 5 relapsed patients. Results The average reconstitution time for granulocytes and PLTs was respectively 15.8 days and 25.8 days in the 5 relapsed patients. When PLTs decreased to about 50×109/L, megakaryocytes in bone marrow in all the five patients further reduced. The radio of blast cells in bone marrow increased compared with that when PLT started to decline, but failed to meet the criteria for relapse. STR for all five patients had transformed from FDC to progressive mixed chimerism (PMC). The five patients occurred morphological relapse in the subsequent three months. When the relapse happened, megakaryocytes in bone marrow almost disappeared, STR in 4 patients had transformed to graft failure(GF),except one case was still in PMC. Statistical analysis showed that the progressive decline in PLTs could be an average of 75 days in advance than the appearance of PMC, and an average of 164 days than morphological relapse in the five relapsed patients. Conclusions Although the phenomenon that unexplained progressive decline in PLTs in leukemia patients after Allo-HSCT can not be used as a diagnosis for leukemia relapse, it may be a predict for the early relapse.     

三种补钙方法在预防外周血造血干细胞采集中枸橼酸盐中毒的效果比较

目的 探讨最佳的补钙方法以预防外周血造血干细胞(PBSC)采集中的枸橼酸盐中毒(CI).方法 对141例自体患者或异体供者进行了166例次的PBSC采集,按补钙方法不同将患/供者随机分为三组,即口服组(66例次):采集中每小时服1次10%葡萄糖注射液20 ml+10%葡萄糖酸钙20 ml,整个过程口服3～4次;静注组(52例次):采集中每小时静脉缓慢注射1次10%葡萄糖注射液20 ml+10%葡萄糖酸钙10 ml,整个采集过程应用3～4次;静滴组(50例次):采集中静脉持续滴注10%葡萄糖注射液350 ml+10%葡萄糖酸钙30 ml,速度为25～30滴/min,出现CI症状时稍加快滴速.采集中密切观察并记录CI发生情况.结果 口服组CI发生率为28.1%,其中轻度12例次,中度6例次,无重度中毒者,出现CI的平均时间为开始采集后122 min;静注组CI的发生率为18.2%,其中轻度7例次,中度1例次,无重度中毒者,出现CI的平均时间为开始采集后134 min;静滴组CI的发生率为10.0%,均为轻度中毒者,出现CI的平均时间为开始采集后172 min.三组间的CI发生率(χ2=6.612,P＜0.05)及CI程度(χ2=7.290,P＜0.05)的分布比较差异均有统计学意义,其中以静滴组CI的发生率最低、程度最轻,而CI发生时间差异无统计学意义(χ2=1.427,P＞0.05).结论 静脉滴注葡萄糖酸钙是预防造血干细胞采集中枸橼酸盐中毒的最佳方法,其疗效佳和安全性好,值得临床推广应用. Abstract： Objective To study the best method of calcium supplementation for preventing citrate intoxication(CI)during peripheral blood stem cell(PBSC)harvesting.Methods According to the different methods of administration of calcium gluconate, 166 procedures of PBSC harvesting in 141 patients/donors were randomly divided into three groups - group oral administration, group intravenous injection and group intravenous drip.In group oral administration(n = 66), 10% calcium gluconate 20 ml in 10% GS 20 ml was administrated by oral one time per hour during harvesting.When symptoms of CI appeared, extra administration was given.In group intravenous injection(n = 52), 10% calcium gluconate 10 ml in 10% GS 20 ml was administrated for intravenous injection per hour during harvesting.When symptoms of CI appeared, extra administration was given.In group intravenous drip(n =50), 10% calcium gluconate 30 ml in 10% GS 350 ml was administrated for intravenous drip at the speed of 25 - 30 drops per minute during harvesting.When symptoms of CI appeared, accelerate the intravenous drip.Symptoms of CI were observed and recorded during harvesting.Results The incidences of CI in three groups were 28.1% , 18.2% and 10.0% in which 12 were mild degree and 6 were moderate degree in group oral administration, 7 were mild degree and 1 was moderate degree in group intravenous injection and all 5 were mild degree in group intravenous drip.The mean time of starting symptoms of CI in group oral administration, group intravenous injection and group intravenous drip were 122 minutes, 134 minutes and 172 minutes.There were statistical differences of the incidence(χ2 =6.612, P ＜0.05＝ and the degrees(χ2=7.290, P ＜0.05)of CI among three groups.Both the incidence and degree of CI was the lowest and lightest in group intravenous drip among the three groups.There was no statistical difference of the mean time of starting CI among the three groups(χ2= 1.427, P ＞ 0.05).Conclusions Intravenous drip of calcium gluconate is the best and safe method for effectively preventing the CI during PBSC harvesting.     

Upregulated heme oxygenase-1 expression of mouse mesenchymal stem cells resists to chemotherapy-induced bone marrow suppression

Background Bone marrow hematopoietic function suppression is one of the most common side effects of chemotherapy.After chemotherapy,the bone marrow structure gets destroyed and the cells died,which might cause the hematopoietic function suppression.Heme oxygenase-1 （HO-1） is a key enzyme of antioxidative metabolism that associates with cell proliferation and resistance to apoptosis.The aim of this study was to restore or resist the bone marrow from the damage of chemotherapy by the HO-1 expression of mouse mesenchymal stem cells （mMSCs） homing to the mice which had the chemotherapy-induced bone marrow suppression.Methods One hundred and sixty female Balb/c mice （6-8-weeks old） were randomly divided into four groups.Each group was performed in 40 mice.The control group was intraperitoneally injected for 5 days and tail intravenously injected on the 6th day with normal saline.The chemotherapy-induced bone marrow suppression was established by intraperitoneally injecting cyclophosphamide （CTX） into the mice which performed as the chemotherapy group.The mMSCs were tail intravenously injected into 40 chemotherapically damaged mice which served as the mMSCs group.The difference between the HO-1 group and the mMSCs group was the injected cells.The HO-1 group was tail intravenously injected into the mMSCs that highly expressed HO-1 which was stimulated by hemin.The expression of HO-1 was analyzed by Western blotting and RT-PCR.Cell proliferation was measured using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay.Histopathologic examinations were performed 1 week after injection.Results Compared with the control group,the expression levels of HO-1 mRNA and protein were significantly higher in the HO-1 group （all P ＜0.05）,even obviously than the mMSCs group.CTX treatment induced apoptosis and inhibited proliferation.After injected,the white blood cell （WBC）,red blood cell （RBC） and platelet （PLT） declined fast and down to the bottom at the 7th day.The bone marrow structure was destroyed incomplete.In vitro,the survival rate of cells in chemotherapy group was less than 50％ after 24 hours.In contrast,mMSCs could do a favor to the cellular cleavage and proliferation.They slowed down the cell mortality and more than 50％ cells survived after 24 hours.The effects of blocking apoptosis and bone marrow recovery could be more effective in the HO-1 group.In the HO-1 group,it had observed that the bone marrow structure became complete and the hemogram closed to normal at 7th day.Conclusions HO-1 played an important role in promoting the recovery of CTX-induced hematopoietic damage.We suclgest that HO-1 is able to restore the functions of chemotherapy-induced hematopoietic damage.