血运重建对骨髓间充质干细胞移植后凋亡影响的实验研究

目的 研究血运重建与兔骨髓间充质干细胞移植后凋亡之间的关系.方法 无菌条件下采集兔骨髓间充质干细胞,用荧光标记.直接结扎法建立兔心肌梗死模型,实验动物随机分两组:①1 h后开放阻断的冠脉,将干细胞移植于兔心肌梗死区(20只).②不开放冠脉,1 h后将干细胞移植于兔心肌梗死区(20只).心梗后1 h在心梗区域注人MSCs.2周后用荧光显微镜观察移值细胞是否存活,利用TUNEL法检测植入细胞的凋亡率.结果 移植2周后,用荧光显微镜观察梗死区组织,可见DAPI标记带蓝色荧光的供体细胞核,形态呈椭圆形类似心肌细胞核,并与心肌纤维排列方向一致,证明移植细胞已存活.利用TUNEL法检测,两组移植细胞均出现细胞凋亡,差异无统计学意义.结论干细胞移植入心肌组织后的凋亡与植入区血运重建有关.     

成年犬自体骨髓基质干细胞经冠状动脉移植在心梗后心肌组织内分化

目的　探讨经冠状动脉移植成年犬自体骨髓基质干细胞在心梗后心肌组织内的存活、分化情况。方法　结扎犬冠状动脉前降支制备心肌梗死模型 ;细胞移植组将体外培养、诱导的自体骨髓基质干细胞标记后在心肌梗死后 4周经冠状动脉植入心脏 ;对照组植入等量培养基 ;细胞移植 4周后取犬心脏 ,行组织学检查、免疫组织化学检查及电镜检查。结果　细胞移植组植入骨髓基质干细胞在心肌组织内存活 ;免疫组织化学检查结蛋白、肌钙蛋白I染色阳性 ;电镜示心肌疤痕组织中可见胞浆丰富的异源性细胞 ,圆形或椭圆形 ,细胞体积较大 ,核糖体丰富 ,线粒体较丰富 ,有肌丝样结构 ,单核。对照组心脏标本中可见疤痕组织 ;心肌细胞结蛋白染色阴性 ,肌钙蛋白I染色阳性 ;电镜未发现异源性细胞。结论　成年犬骨髓基质干细胞经冠状动脉移植后在心肌组织内存活并分化为肌源性细胞。     

高压氧对人脐带华通胶间充质干细胞移植心肌梗死大鼠炎性因子的作用

目的探讨高压氧对移植干细胞在梗死心肌组织局部的炎性状况。方法选择SD大鼠65只,随机分为假手术组5只,心肌梗死组15只,干细胞移植组15只,高压氧组15只,高压氧+干细胞移植组(联合组)15只。建立心肌梗死大鼠模型,模型建立后第1天进行高压氧治疗30d。心肌梗死后第7天进行人脐带华通胶间充质干细胞移植,分别于心肌梗死前、心肌梗死后1、7、14、28d采集血清,ELISA法检测血清TNF-a、白细胞介素1β(IL-1β)、IL-6水平,采用EdU染色法,标记人脐带华通胶间充质干细胞,免疫荧光法检测移植细胞存活及增殖率。结果除外联合组14d时IL-6,联合组3、14、28d时TNF-a、IL-6和IL-1β较心肌梗死组、高压氧组、干细胞移植组明显降低(P<0.05);与干细胞移植组比较,联合组干细胞移植后7、21d心肌组织局部干细胞数量及增殖率明显增高(80.6%vs 55.0%,79.1%vs 34.3%,P<0.05)。结论高压氧能够显著降低梗死心肌组织中的炎性因子水平,有效改善移植干细胞生存的微环境,提高移植干细胞在受损心肌局部的生存及增殖率。     

骨髓间充质干细胞移植对心肌梗死后心室肌细胞钾离子通道Ito Kv4.2的影响

目的 研究心肌梗死后移植骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)对心室肌细胞钾离子通道Ito亚单位Kv4.2基因表达的影响方法 40只SD(Sprague-Dawley)大鼠随机分成4组(10只/组):假手术组、心肌梗死组、心肌梗死+干细胞组和心肌梗死+细胞培养基组.开胸结扎冠状动脉前降支建立心肌梗死模型,建模成功后,在梗死周围分别注入BMMSCs和细胞培养基,心肌梗死组仅建立心肌梗死模型,假手术组仅开胸子以前降支穿线但不结扎.2周后行心肌组织HE染色和荧光显微镜观察移植细胞,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测钾离子通道Ito亚单位Kv4.2基因mRNA和蛋白水平.结果 (1)心肌组织免疫荧光检测发现,BMMSCs集中分布于梗死区和梗死心肌周围;(2)心肌梗死组和心肌梗死+细胞培养基组Kv4.2 mRNA量(0.39±0.02,0.41±0.04)和蛋白量(0.47±0.02,0.50±0.05)明显下降,与假手术组(0.76±0.05,0.74±0.06)相比差异有统计学意义(P＜0.01);心肌梗死+干细胞组Kv4.2 mRNA量和蛋白表达量(0.57±0.05,0.64±0.03)较心肌梗死组(0.39±0.02,0.47±0.02)明显升高(P＜0.01).结论 骨髓间充质干细胞移植后心肌梗死大鼠钾离子通道Ito亚单位Kv4.2基因表达上升,可能减少心律失常发生.     

人脂肪间充质干细胞治疗大鼠急性心肌梗死的实验研究

目的:探讨人脂肪间充质干细胞(HADMSCs)移植于大鼠缺血心肌后的增殖分化情况及心功能改善情况。方法:通过结扎法建立心肌梗死模型,1周后将BrdU标记的HADMSCs通过经心外膜注射至心梗移植组梗死心肌,行超声心动图检查。建模后5周行HE染色观察心肌梗死情况,TTC染色计算左室梗死面积百分比,免疫组织化学检测HADMSCs移植后的存活和分布情况及心肌特异性蛋白的表达。结果:超声检查心梗移植组左心室收缩末期内径、舒张末期内径均小于心梗对照组,而射血分数均大于心梗对照组(P0.05);TTC染色结果显示心梗移植组左室梗死面积百分比[(23.6±4.3)%]小于心梗对照组左室梗死面积百分比[(32.4±5.6)%](P0.05)。心肌组织免疫组织化学结果显示带有BrdU标记的移植细胞在心梗周边区域存活,且特异性肌钙蛋白呈阳性表达。结论:HADMSCs能够在大鼠梗死心肌内存活,并分化为心肌样细胞,减小梗死面积,改善心功能。     

同种异体骨髓间充质干细胞转染连接蛋白43基因后移植对心肌梗死大鼠心功能的影响

目的:研究转染真核表达质粒pbudCEA.1_Cx43后的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)移植对心肌梗死大鼠左心功能的影响,明确同种异体干细胞及连接蛋白43(Connexin43,Cx43)基因转染于细胞移植治疗急性心肌梗死的可行性及效果.方法:清洁级SD大鼠60只,随机选出11只作为假手术组,其余大鼠制成心肌梗死模型,再随机分为四组:对照组(n=11);细胞移植组(n=12);基因移植组(n=13);空载体移植组(n=13).另取清洁雄性SD大鼠5只,作为BMSCs供体.心肌梗死各组大鼠结扎前降支后1～3小时心肌内移植入相应的细胞或培养基,移植后6周检测心功能并取心脏检测各种相关指标.结果:同种异体大鼠BMSCs可在梗死心肌组织定居、生存;与对照组比较,假手术组、细胞移植组、基因移植组和空载体移植组左心功能均明显改善(P＜0.05),基因移植组优于细胞移植组和空载体移植组(P＜0.05).结论:BMSCs转染Cx43基因后移植治疗急性心肌梗死可行、有效.     

骨髓间充质干细胞静脉移植对心肌梗死大鼠心功能的影响

目的探讨静脉途径移植骨髓间充质干细胞(MSCs)对急性心肌梗死(AMI)大鼠心功能的影响。方法雄性SD大鼠20只,分为干细胞移植组(10只)和对照组(10只)。结扎大鼠左冠状动脉建立心肌梗死模型。同种异体大鼠MSCs体外分离、纯化、扩增,4’,6-二脒-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)标记,并通过尾静脉于心肌梗死后1周移植入AMI大鼠体内,对照组则注射等量培养液。4周后测定心功能,并行免疫组织化学检测。结果心肌梗死4周后,干细胞移植组心肌组织冷冻切片中可以观察到DAPI标记细胞存在,免疫组织化学显示阳性标记细胞a-Actin肌动蛋白检测阳性。干细胞移植组心功能较对照组有明显改善(P<0.05)。结论MSCs经静脉移植可以归巢至梗死心肌处,并能改善受损的心功能。     

同种异体骨髓间充质干细胞移植治疗急性心肌梗死的实验研究

目的探讨同种异体骨髓间充质干细胞(BMMSCs)移植对急性心肌梗死(AMI)大鼠心脏结构和功能的影响及其作用机制。方法采用Wistar大鼠30只,随机分成BMMSCs移植组、培养液注射组(开胸后在梗死区周围注射等量细胞培养液);体外分离纯化、扩增BMMSCs,4,6二脒基-2-苯吲哚(DAPI)标记。BMMSCs移植组于造模后1周经心肌直接注射MSCs,培养液注射组注射同等剂量的细胞培养液。造模后4周测定心功能并行免疫组织化学检测。结果造模后4周,与培养液注射组比较,BMMSCs移植组左室射血分数明显增加,左室收缩末期内径和舒张末期内径显著降低(P均<0.05);心肌梗死区毛细血管密度明显增高(P<0.05);BMMSCs移植组大鼠梗死心肌中可见DAPI标记阳性细胞;与培养液注射组比较,BMMSCs移植组可以显著减少非梗死区心肌细胞的凋亡(P<0.05)。结论 BMMSCs移植能够提高AMI梗死区毛细血管密度,改善大鼠的心功能。     

骨髓间质干细胞静脉移植治疗大鼠急性心肌梗死的实验研究

目的:观察多次静脉移植骨髓间质干细胞治疗心肌梗死的可行性及对心肌梗死后心功能的影响。方法:骨髓间质干细胞培养传至5代后,制备成终浓度为4×109个/L的悬液备用。将40只SD大鼠均结扎冠状动脉左前降支,建立急性心肌梗死模型后,随机分为3组:①对照组(10只):只静脉注射0.85%氯化钠溶液;②静脉移植组(15只):心肌梗死模型制备后24h,从尾静脉注射骨髓间质干细胞0.5ml(含2×109个/L)连续7d;③心外膜移植组(15只):心肌梗死模型制备后1~3h,于梗死周边区(心肌颜色苍白区周边)分6点注射骨髓间质干细胞,每点50μl。5周后,观察3组的生存情况及心脏的结构功能。结果:心肌梗死对照组有3只大鼠死亡,其他组无死亡。与对照组相比,静脉移植组、心外膜移植组的左室结构与功能明显改善(P<0·05),静脉移植组与心外膜移植组差异无统计学意义(P>0·05)。结论:骨髓间质干细胞可以在体外大量扩增,异体输入无不良反应,静脉注射与直接心肌注射无明显差别,方法可行。     

IGF-1预处理对骨髓间充质干细胞心肌再生及抗炎效果的影响

目的:探讨胰岛素样生长因子1(IGF-1)预处理对骨髓间充质干细胞(MSCs)心肌再生及抗炎效果的影响。方法:10μg/L的IGF-1预处理MSCs,流式细胞仪分析趋化因子受体4的表达;建立大鼠心肌梗死模型,尾静脉分别注射不含MSCs的100μl培养液(对照组),含经IGF-1预处理的MSCs(IGF-1-MSCs组)和无预处理的MSCs(MSCs组),每组8只大鼠;MSCs移植4周后观察心肌组织炎性因子(肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-6)在mRNA和蛋白水平的表达,分析移植细胞的归巢率,连接蛋白43(CX43)的表达,心肌特异性蛋白肌钙蛋白T的表达。结果:流式细胞术显示MSCs经IGF-1预处理48h后的趋化因子受体4显著增高。IGF-1-MSCs组MSCs移植4周后:①更多的MSCs归巢到心肌梗死周边区(P<0.05);②显著增加梗死周边区肌钙蛋白T阳性细胞(P<0.05)和CX43密度(P<0.05);③降低了心肌梗死后心肌炎性因子的表达。结论:IGF-1预处理能够显著提高MSCs的心肌再生能力,并提高其在梗死心肌中的抗炎效果。     

Adult stem cells and cancer stem cells: tie in or tear apart?

Stem cell research is one of the new frontiers of medical science. Because of the unique self-renewable ability and powerful potential to differentiate, stem cells can be viewed as the mother of all cells in the body and have been investigated as a possible tool for reversing the degeneration and damage on organs. Recently, successful isolating cancerous stem cells from leukemia, breast and brain cancers provide a new target for eliminate cancer; however, it hints an increasing caution in using adult stem cells for organ repair. Cancerous stem cells share the same properties of self-renewal and differentiation with normal stem cells, with the addition of similar phenotype of adult stem cells isolated from the same tissue. Some believe that cancerous stem cells are derived from mutation of the normal stem cells, whereas others suspect it to be from different origins. Further investigation of the intrinsic factor underlying the behavior of adult stem cells and cancerous stem cells will shed light on both the fields of tissue engineering and cancer therapy. In this review, recent progresses in the studies of adult stem cells and cancerous stem cells are summarized to facilitate a better understanding and elicit much attention in this field.     

干细胞治疗基础与临床——国内外最新动态

近年来,干细胞治疗作为一项治疗人类多种疾病的崭新技术,相关的基础研究与临床试验呈现出快速增长的趋势,该文对胚胎干细胞,成体干细胞及诱导性多能干细胞当前的进展情况进行了综述,干细胞临床治疗拥有广阔的发展前景。     

Defined factors induce reprogramming of gastrointestinal cancer cells

Although cancer is a disease with genetic and epigenetic origins, the possible effects of reprogramming by defined factors remain to be fully understood. We studied the effects of the induction or inhibition of cancer-related genes and immature status-related genes whose alterations have been reported in gastrointestinal cancer cells. Retroviral-mediated introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins, including Nanog, Ssea4, Tra-1-60, and Tra-1-80 in esophageal, stomach, colorectal, liver, pancreatic, and cholangiocellular cancer cells. Induced cells, but not parental cells, possessed the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, which was supported by epigenetic studies, indicating methylation of DNA strands and the histone H3 protein at lysine 4 in promoter regions of pluripotency-associated genes such as NANOG. In in vitro analysis induced cells showed slow proliferation and were sensitized to differentiation-inducing treatment, and in vivo tumorigenesis was reduced in NOD/SCID mice. This study demonstrated that pluripotency was manifested in induced cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation-inducing treatment. Retroviral-mediated introduction of iPC cells confers higher sensitivity to chemotherapeutic agents and differentiation-inducing treatment.     

Erythroid Differentiation of Fetal,Newborn and Adult Haemopoietic Stem Cells

Erythroid regeneration was studied in lethally irradiated mice given transplants containing equivalent numbers of haemopoietic stem cells (i.e. CFU) from fetal liver, neonatal marrow or adult marrow. Adult marrow was taken from normal control mice, whose CFU for the most part were not in active cell cycle, as well as from phenylhydrazine-treated groups whose CFU were in similar state of proliferation (i.e. ?40–50% in DNA synthesis) as those derived from fetal liver and neonatal marrow. Splenic and femoral radioiron (⁵⁹Fe) incorporation were measured at intervals after transplantation and were found to begin earliest in mice given fetal liver, then in animals given neonatal marrow and latest in recipients of adult marrow. Peripheral reticulocytes showed a similar pattern of recovery. The data reported herein suggest that the differences in erythroid regeneration evoked by transplants of fetal liver, neonatal marrow or adult marrow, are not solely attributed to the degree of proliferation in the pluripotential stem cell compartment. These data may, however, suggest a shorter doubling time for cells comprising the fetal and newborn committed erythroid compartments.     

Glioma stem cells: turpis omen in nomen? (the evil in the name?)

High‐grade gliomas remain incurable and lethal. Through the availability of the stem‐like cells responsible for glioblastoma (GB) formation, expansion, resilience and recurrence, the discovery of glioma cancer stem cells (GCSCs) is revolutionizing this field. GCSCs provide an unprecedented opportunity to reproduce and study GB pathophysiology more accurately. This critically emphasizes our ability to unambiguously identify, isolate and investigate cells that do qualify as GCSCs, to use them as a potential model that is truly predictive of GBs and of their regulation and response to therapeutic agents. We review this concept against the background of key findings on somatic, neural and solid tumour stem cells (SCs), also taking into account the emerging phenomenon of phenotypic SC plasticity. We suggest that basic approaches in these areas can be imported into the GCSC field, so that the same functional method used to identify normal somatic SCs becomes the most appropriate to define GCSCs. This, combined with knowledge of the cellular and molecular basis of normal adult neurogenesis, promises to improve the identification of GCSCs and of selective markers, as well as the development of innovative, more specific and efficacious antiglioma strategies.     

自体骨髓间充质干细胞联合造血干细胞共移植治疗恶性血液病五例疗效观察

目的 研究自体骨髓间充质干细胞(MSCs)与造血干细胞共移植治疗恶性血液病的安全性和可行性,及其对移植后造血重建的影响.方法 从无骨髓浸润的恶性血液病患者本人骨髓中分离、培养间充质干细胞,经放化疗等预处理后,与造血干细胞共移植治疗恶性血液病患者5例,其中恶性淋巴瘤4例,粒细胞肉瘤1例,并观察其对移植后造血重建的影响.结果 MSCs联合造血干细胞共移植治疗恶性血液病患者5例,MSCs输注过程顺利,未见明显不良反应.移植后造血恢复过程中,中性粒细胞≥0.5×109/L的中位时间为9.4(8～11)d、血小板≥20×109/L的中位时间为12.2(10～14)d.结论 MSCs联合造血干细胞共移植治疗恶性血液病安全性好,未见明显副作用.结果 提示输注MSCs可促进造血恢复,但其远期疗效仍有待于进一步观察.     

Stem cell concepts in myelodysplastic syndromes: lessons and challenges

According to the cancer stem cell (CSC) hypothesis, CSCs are the only cancer cells that can give rise to and sustain all cells that constitute a cancer as they possess inherent or acquired self-renewal potential, and their elimination is required and potentially sufficient to achieve a cure. Whilst establishing CSC identity remains challenging in most cancers, studies of low-intermediate risk myelodysplastic syndromes (MDS), other chronic myeloid malignancies and clonal haematopoiesis of indeterminant potential (CHIP) strongly support that the primary target cell usually resides in the rare haematopoietic stem cell (HSC) compartment. This probably reflects the unique self-renewal potential of HSCs in normal human haematopoiesis, combined with the somatic initiating genomic driver lesion not conferring extensive self-renewal potential to downstream progenitor cells. Mutational ‘fate mapping’ further supports that HSCs are the only disease-propagating cells in low-intermediate risk MDS, but that MDS-propagating potential might be extended to progenitors upon disease progression. The clinical importance of MDS stem cells has been highlighted through the demonstration of selective persistence of MDS stem cells in patients at complete remission in response to therapy. This implies that MDS stem cells might possess unique resistance mechanisms responsible for relapses following otherwise efficient treatments. Specific surveillance of MDS stem cells should be considered to assess the efficiency of therapies and as an early indicator of emerging relapses in patients in clinical remission. Moreover, further molecular characterization of purified MDS stem cells should facilitate identification and validation of improved and more stem cell-specific therapies for MDS.