自体骨髓单个核细胞移植治疗大鼠后肢缺血

目的　探讨自体骨髓单个核细胞（BM-MNC）移植用于大鼠缺血后肢的治疗后实现血管再生的能力。方法　建立大鼠后肢缺血动物模型,将取于自体的BM-MNC制成细胞悬液注射于缺血部位,分别在移植后2和30d时行动脉造影,用免疫组化方法检测内皮祖细胞（EPC）,毛细血管密度以及测定血管内皮生长因子(VEGF)的表达。结果　缺血肌组织中的EPC含量增高（P<0.01）。BM-MNC移植组在移植早期VEGF表达显著增高（P<0.01）。细胞移植后30dBM-MNC移植组毛细血管密度明显高于其他组（P<0.01）,血管造影可见侧支循环建立。结论　自体骨髓单个核细胞移植于大鼠后肢缺血区能促进血管新生,改善侧支循环,可望成为一种简单有效的治疗下肢缺血的方法。     

骨髓单个核细胞移植治疗脑出血

BACKGROUND:It has been proved that bone marrow mononuclear cell transplantation can obviously improve neurological function of rats with cerebral hemorrhage. OBJECTIVE:To investigate the effects of transplanted bone marrow mononuclear cells on the neurological function and apoptosis in perihematomal brain tissues following cerebral hemorrhage in a rat model. METHODS:Twenty-four Sprague-Dawley rats were given stereotaxical injection of collagenase IV into the caudate nucleus to establish cerebral hemorrhage models in transplantation group (n=12) and model group (n=12), and then at 6 hours after cerebral hemorrhage, rats in these two groups were administrated 3x10¹⁰/L allograft bone marrow mononuclear cells and the same amount of PBS, respectively. Another 12 rats were given no interventions as control group. Neurological functions of rats were assessed at 1, 4, 8, 16 days after cerebral hemorrhage; pathological changes of the injury sites were observed at 16 days after transplantation; neuronal apoptosis rates in the perihematomal brain tissue were detected by flow cytometry at 2 and 4 days after transplantation. RESULTS AND CONCLUSION:The modified neurologic severity scores in the transplantation group were significantly lower than those in the model group at 8 and 16 days after cerebral hemorrhage (P < 0.05). In the control group, cells in each layer arranged closely with complete structure, and neurons and glial cells were in good shape; in the model group, perihematomal brain tissues were loose with intercellular gap, in which most neurons and glial cells became necrotic; in the transplantation group, cells in each layer arranged closely and regularly, and glial cell proliferation occurred. Besides, compared with the model group, the neuronal apoptosis rate in the transplantation group was significantly lower (P < 0.05). To conclude, bone marrow mononuclear cells can significantly enhance the neurological function recovery and reduce neuronal apoptosis in the brain of cerebral hemorrhage rats.     

同种异体骨髓单个核细胞移植治疗大鼠急性心肌梗死实验研究

目的探讨大鼠同种异体骨髓单个核细胞（bone marrow mononuclear cells，BM—MNCs）在急性心肌梗死区分化增殖潜能及其修复重建心肌作用。方法健康雄性Wistar大鼠24只，用结扎冠状动脉左前降支的方法建立大鼠心肌梗死模型，随机分为对照组（AMI＋培养基，12只），移植组（AMI＋BM—MNCs，12只）；分别将制备的培养基和BM—MNCs悬液心外膜下植入梗死心肌周围。移植术后4周，观察心肌梗死区及其周边区组织形态学特点、心肌梗死面积变化。结果实验组与对照组相比心肌梗死面积明显缩小（P〈0．01）；实验组心肌梗死区内有BrdU标记阳性的BM—MNCs移植细胞存活，向心肌源性细胞分化并且诱导了大量的新生毛细血管。结论同种异体BM—MNCs移植在细胞水平完成了对心肌梗死区再心肌化和再血管化过程，可以改善急性心肌梗死后的心脏功能。     

骨髓单个核细胞移植对肾缺血再灌注后肾小管坏死及细胞凋亡的影响

目的：观察和分析外源性骨髓单个核细胞（BMMNCs）对肾缺血再灌注诱发的肾小管坏死及凋亡的影响。方法：密度梯度离心法分离获取BMMNCs并行DAPI标记。制作SD大鼠肾缺血再灌注损伤模型，通过下腔静脉进行BMMNCs移植。分别于缺血再灌注后不同时相获取肾脏标本，荧光显微镜下观察DAPI的标记情况，HE染色做肾组织学检测，TUNEL法检测肾组织凋亡细胞，免疫组织化学染色观察增殖细胞核抗原（PCNA）的表达。结果：BMMNCs移植组大鼠肾脏组织中可见DAPI荧光细胞，部分存在于肾小管上皮组织中，未见明显细胞坏死及变性征象，其凋亡细胞计数显著减少，而PCNA阳性细胞数增多。结论：外源性BMMNCs移植可减少缺血再灌注诱发的肾小管上皮细胞的变性、坏死和凋亡，促进缺血再灌注损伤后肾小管上皮细胞的增殖，从而提示BMMNCs移植有助于缺血再灌注损伤后肾小管的修复及其结构完整性的维持。     

自体骨髓单个核细胞移植治疗糖尿病性下肢缺血83例

背景：自体骨髓单个核细胞移植治疗糖尿病周围血管病变是目前广泛开展的一项技术,对缺血性病变有较好的效果,有个案报道对周围神经损伤所致临床症状有改善作用。 目的：观察自体骨髓单个核细胞移植前后糖尿病足病患者缺血性及神经性病变指标的变化。 方法：行自体骨髓单个核细胞移植治疗的糖尿病足病患者83例153条肢体。每例患者抽取自体骨髓250~400 mL,经密度梯度法提取单个核细胞,提取的单个核细胞数量为1.01×10⁸~4.96×10⁹个,平均数量为(2.04±0.53)×10⁸个,稀释后双下肢肌肉内注射,每点1~3 mL,间隔约3 cm。移植后3,6个月进行缺血性指标和神经病变指标检测。 结果与结论：自体骨髓单个核细胞移植后增加了双下肢血流量和外周血管数量,延长了行走距离,提高了皮温,促进了溃疡的愈合。治疗3个月及6个月后均有明显效果,差异有显著性意义。治疗6个月后,还可以改善糖尿病周围神经的自觉症状,增加运动及感觉神经传导速度,差异有显著性意义。     

骨髓单个核细胞移植后心肌梗死区的血管生成及细胞因子分泌

背景：干细胞移植为治疗心肌梗死带来新的希望,但研究结果不一致,存在争议。细胞移植能否长期持久地改善心脏功能、改善缺血心功能的机制这些问题均不明确。 目的：观察经冠状动脉自体骨髓单个核细胞移植对急性心肌梗死犬心功能、血管生成和细胞因子分泌的影响。 方法：杂种犬分为骨髓单个核细胞组(n=10)和生理盐水组(n=6),前上嵴或髂后上棘穿刺分离得到骨髓单个核细胞,结扎冠状动脉前降支建立急性心肌梗死模型,于心肌梗死后2 h分别经冠状动脉内移植骨髓单个核细胞和生理盐水,心肌梗死后2 h及6周时分别测定超声心动图指标,骨髓单个核细胞移植后6周vWF免疫组化染色检测心肌组织的毛细血管密度,RT-PCR检测血管内皮生长因子(血管内皮生长因子188、血管内皮生长因子164)、碱性成纤维细胞生长因子、基质金属蛋白酶9 mRNA的表达。 结果与结论：经冠状动脉自体骨髓单个核细胞移植后6周,超声心动图显示,骨髓单个核细胞组射血分数和每搏输出量比生理盐水组显著升高;梗死边缘区新生血管数量明显高于生理盐水组。骨髓单个核细胞组梗死区血管内皮生长因子188、血管内皮生长因子164和碱性成纤维细胞生长因子mRNA表达水平显著高于生理盐水组。骨髓单个核细胞组梗死区基质金属蛋白酶9 mRNA 表达水平显著低于生理盐水组。结果说明自体骨髓单个核细胞经冠状动脉内注射移植,改善急性心肌梗死后心功能,促进梗死边缘区血管生成,提高促血管生长因子血管内皮生长因子188、血管内皮生长因子164和碱性成纤维细胞生长因子的mRNA表达水平,减少基质金属蛋白酶9 mRNA表达水平。     

自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变

背景：有研究表明移植骨髓单个核细胞治疗糖尿病下肢神经病变动物模型,通过在组织内能促进血管再生和增加血管生成因子及神经营养因子能改善临床症状。                             目的：观察自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变的临床效果。 方法：30例糖尿病下肢闭塞症患者60条下肢,按治疗方式的不同分为2组：自体骨髓单个核细胞移植的治疗组和对侧下肢非自体骨髓单个核细胞移植的对照组,各30条下肢。 结果与结论：移植4周后,治疗组总有效率高于对照组(P < 0.05)。两组治疗后神经病变自主症状问卷神经病变主觉症状问卷评分均较治疗前明显降低,治疗组评分降低更明显(P < 0.01),治疗组胫神经和腓总神经感觉和运动神经传导速度均较对照组快(P < 0.01),患者未出现并发症和不良反应。说明自体骨髓单个核细胞移植治疗糖尿病下肢周围神经病变的临床效果较好。     

自体骨髓单个核细胞肝内移植治疗失代偿期肝硬化

背景：大量研究发现,骨髓间充质干细胞是肝干细胞的主要肝外来源,骨髓间充质干细胞在特定环境下可分化为肝组织和肝细胞从而参加肝脏的修复与重建。 目的：观察自体骨髓单个核细胞移植治疗失代偿期肝硬化的效果及安全性。 方法：选择肝硬化失代偿期患者20例,在无菌条件下由髂后上棘抽取骨髓200 mL,在体外分离纯化骨髓单个核细胞并制成10 mL细胞悬液,经肝动脉将细胞悬液移植入肝脏,分别在移植后第2、4、8、12周复查肝脏功能,观察实验室指标,临床症状及不良反应。 结果与结论：移植后2周内所有血清学指标均无显著改变,移植后4周白蛋白由(27.05±5.23) g/L升到(30.02±5.02) g/L,纤维蛋白原由(1.55±0.53) g/L升到(2.55±0.53) g/L,凝血酶原时间由(24.05±5.23) s降到 (17.05±5.13) s,胆碱酯酶和甲胎蛋白也有不同程度上升,氨基转移酶、总胆红素水平在移植前后无显著性变化。单个核细胞移植后食欲改善、体力好转10例,腹胀减轻6例,腹水减少7例,下肢浮肿减轻4例。全部患者在移植中未发生严重并发症,移植近期无不良反应出现。     

不同途径移植骨髓单个核细胞修复创伤性脑损伤

背景：干细胞移植存在多种途径,目前还无法确定哪一种是最佳途径。而对于不同的脑损伤个体,所移植的细胞种类、移植途径和移植时间都将会影响到治疗效果。目的：探讨不同途径移植骨髓单个核细胞对脑损伤大鼠神经功能的影响。方法：Ficoll淋巴细胞分离液梯度离心分离大鼠骨髓单个核细胞,CFDA-SE体外标记后备用;自由落体法制备大鼠创伤性脑损伤模型,模型制作成功后,立即通过损伤区、侧脑室和颈内动脉移植CFDA-SE标记的骨髓单个核细胞,每一种移植途径都设对照组(以等体积的DMEM代替骨髓单个核细胞)。治疗后不同时间点进行mNSS评分,最后一次行为学评分完毕取脑组织,荧光倒置显微镜下观察骨髓单个核细胞在损伤区域的存活和迁移情况。结果与结论：治疗后7,10,14 d,对照组和移植组mNSS评分均较1 d和3 d时降低(P < 0.05);治疗后7 d和10 d,颈内动脉移植组和对照组相比mNSS评分降低(P < 0.05);治疗后14 d,颈内动脉移植组荧光细胞数量较其他组多(P < 0.05),且分布广。结果表明经颈内动脉移植骨髓单个核细胞能明显改善大鼠神经功能,且移植细胞能够在损伤区大量存活和迁移。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

经冠脉移植骨髓单个核细胞对猪梗死心肌的修复作用

目的观察经冠脉移植骨髓单个核细胞的定植、分化和对心功能的影响。方法小型猪冠状动脉前降支结扎90min后再灌注制备心肌梗死模型,分为骨髓细胞移植组(BM-MNCs组,n=6)和对照组(n=5),分别经冠脉移植PKH26标记的骨髓单个核细胞和培养基,术后6周进行病理学检查并行血流动力学和超声心动图检查心功能变化。结果在BM-MNCs组,心肌梗死后6周在缺血心肌内可找到移植细胞,其Ⅷ因子和desmin免疫组化染色均为阳性;血流动力学指标显示左室等容舒张期压力最大变化率和心排量与心肌梗死后90min比较明显改善(P<0.05);超声心动图显示每搏输出量和心排量比对照组明显改善(P<0.05);心肌梗死边缘区的小血管数目明显多于对照组(P<0.05)。结论经冠脉移植骨髓单个核细胞可定植于梗死区心肌,并可分化为肌源性细胞和血管内皮细胞,可促进小血管再生,有改善心功能的潜能。     

自体骨髓干细胞原位移植治疗急性心肌梗死的临床研究

目的:用粒细胞集落刺激因子(G-CSF)动员自体骨髓干细胞进入心肌梗死部位原位移植以修复梗死心肌组织,探讨其对急性心肌梗死患者心肌梗死面积及心功能的影响。方法:25例初次急性心肌梗死患者在入院后随机分为干细胞原位移植组(n=12)和对照组(n=13)。干细胞原位移植组于入院后在常规治疗基础上加用G-CSF动员自体骨髓干细胞进行心肌梗死原位移植;对照组按急性心肌梗死常规方法治疗。于入院第(1、28d)描记常规12导联心电图,采用Wagner的QRS波群记分法评价心功能,于入院(7、28d)行核素心肌灌注断层显像,测量心肌梗死面积。结果:G-CSF治疗4周,干细胞原位移植组QRS记分值明显降低,显著低于对照组(P＜0.05);心肌梗死面积由36.0%±8.3%下降至18.0%±5.8%,显著小于对照组(P＜0.05)。结论:用G-CSF动员自体骨髓干细胞来修复坏死心肌组织的"干细胞原位移植"疗法,能减小心肌梗死的范围,改善心功能。     

Cardiac contractility after transplantation of autologous mononuclear bone marrow cells in patients with myocardial infarction

Autologous bone marrow mononuclear cells were transplanted by intracoronary infusion to patients with myocardial infarction after recovery of coronary perfusion. Controls received traditional therapy alone. Echocardiography was carried out before and 3 and 6 months after cell therapy. Cell transplantation did not appreciably improved left-ventricular contractility in comparison with the control group. In none patient cell therapy provoked malignant ventricular arrhythmias. Intracoronary infusion of bone marrow mononuclear cells in patients with myocardial infarction did not improve cardiac contractility and did not aggravate the course of the disease. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 15–20, January, 2006     

Morphological and molecular analysis of angiogenesis after intramyocardial transplantation of autologous bone marrow mononuclear cells

We studied the peculiarities of angiogenesis in the postinfarction period after transmyocardial laser revascularization and intramyocardial implantation of mononuclear bone marrow cells into the pericicatrical zone of the left ventricular myocardium in dogs. Morphological manifestation of angiogenesis in the myocardium after application of laser and cell technologies are angiomatosis, formation of large thin-wall vessels and sinusoids. The angiogenic effect of implanted mononuclear bone marrow cells is determined by high content (43-47%) of CD31⁺ cells in both adherent and nonadherent fractions. More pronounced angiogenic potential of nonadherent cells is determined by intensive expression of cytokine VEGF-B and D mRNA essential for arterial vessels growth. Immunohistochemical studies showed that about 90% cells of the nonadherent fraction are endothelial precursors expressing endothelial cell markers isolectin B₄ and VEGF-R2. It was found that the use of adherent mononuclear bone marrow cells during the postinfarction period induces ossification of the epicardium and subepicardial myocardium layer, formation of cartilage plates, and focal calcification. Implantation of nonadherent mononuclear bone marrow cells into transmyocardial laser channels did not induce ectopic ossification of the myocardium.     

自体骨髓单个核细胞移植治疗缺血性心脏病临床疗效观察

目的 研究经冠状动脉内移植骨髓单个核细胞治疗缺血性心力衰竭的可行性和疗效。方法 缺血性心力衰竭病人20例，抽取骨髓用密度梯度离心法分离出骨髓单个核细胞，经外周动脉穿刺插管，将自体骨髓单个核细胞注入冠脉。比较病人的临床症状，NYHY分级和治疗后由于心血管事件造成的再住院情况。结果 病人自觉症状改善，心功能提高，ECT检查显示在缺血区有血管新生。结论 自体骨髓单个核细胞移植是安全有效的。     

骨髓间充质干细胞治疗急性心肌梗塞的实验研究

目的研究自体骨髓间充质干细胞(MSC)经冠脉内注射治疗急性心肌梗死(AMI)的有效性和可行性。方法球囊阻塞法制成AMI模型。经冠脉注入标记的MSC,4周后核素心肌断层显像检测相对心肌梗死面积、心肌灌注评分和射血分数,经导管检测左室收缩压(LVSP)、左室舒张末压(LVEDP)、压力升高最大速率( dP/dt)和压力下降最大速率(-dP/dt)。免疫荧光法分析MSC的植入和分化。结果与对照组相比,细胞治疗组射血分数显著增加(P<0.05),心肌灌注评分和相对心肌梗死面积显著降低(P<0.01,P<0.05),LVSP、 dP/dt和-dP/dt显著增加(P<0.05,P<0.01,P<0.01),LVEDP显著降低(P<0.05)。标记的MSC在心肌中阳性表达肌凝蛋白重链、连接蛋白43、平滑肌肌动蛋白和Ⅷ因子相关抗原。结论MSC在体内分化为心肌和血管组织,可改善AMI猪的心功能。     

Reparation of the myocardium after transplantation of mononuclear bone marrow cells

Safety and efficiency of intracoronary transventricular transplantation of autologous mononuclear bone marrow cells in rats with postinfarction cardiosclerosis were studied. The cells migrated to the damaged area and were detected only in the cicatricial tissue; they have fibroblast-like phenotype and some of them were stained with Fapα (marker of reactive fibroblasts). More active proliferation of non-muscular cells and formation and maturation of collagen fibers in the cicatricial tissue were observed after transplantation of mononuclear cells. This led to thickening of the cicatricial wall, but the size of the scar and index of dilatation of the left ventricle remained unchanged. The number and volume density of newly formed blood vessels in the damaged area increased after transplantation, but no labeled cells were seen in the vascular walls. It can be hypothesized that stimulation of neoangiogenesis is mediated by paracrine mechanisms, which also explains improvement of global contractility of the left ventricle (increased contractility index in functional tests). Thus, transplantation of mononuclear bone marrow cells leads to thickening and strengthening of the cicatricial wall, stimulates angiogenesis, and improves global myocardial contractility. However, no morphological signs of reverse remodeling of the left-ventricular myocardium were revealed.     

Colony-forming cells in rat myocardium after destructive exposure and intramyocardial transplantation of bone marrow cells

The content of colony-forming cells in myocardial cell culture from the perinecrotic zone of rat heart was evaluated on day 40 after cryodestruction. The mean cellularity after cryodestruction was 12-fold lower than in intact animals. Intramyocardial transplantation of bone marrow cells (mononuclears, mesenchymal stem cells, and mesenchymal stem cells treated with 5-azacitidine) into the perinecrotic zone increased the content of colony-forming cells. After transplantation of mesenchymal stem cells and mesenchymal stem cells treated with 5-azacitidine, the number of colonies reached 33±7 and 11±3, the mean cellularity being 2975±80 and 1105±42 cells/cm², respectively. Hence, intramyocardial transplantation of mesenchymal stem cells created an appreciable pool of colony-forming cells in the myocardial perinecrotic zone. Treatment with 5-azacitidine reduced survival of mesenchymal stem cells after intramyocardial transplantation. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 29–33, January, 2007