Notch信号和血管内皮生长因子基因对大鼠骨髓间充质干细胞诱导分化后内皮细胞功能的影响

目的 探讨Notch信号和血管内皮生长因子(VEGF165)基因对大鼠骨髓间充质干细胞(MSCs)诱导分化后内皮细胞功能的影响.方法 分离、培养大鼠MSCs,用含VEGF165和碱性成纤维细胞生长因子(bFGF)的细胞培养液培养大鼠MSCs 2周诱导其向内皮细胞分化;用脂质体将携带有VEGF165基因的质粒转染诱导内皮细胞并对转染效果进行鉴定,逆转录-聚合酶链反应(RT-PCR)检测转染前后细胞上Notch信号受体Notch 1和配体Jagged 1的表达变化;用γ-内分泌酶抑制剂L-685458阻断细胞Notch信号通路的转导,划痕实验检测细胞迁移能力;将细胞接种在半固体培养基上,观察其形成毛细血管样结构的能力.结果 转染VEGF165基因的内皮细胞上表达有VEGF165 mRNA,说明实验成功地将VEGF165基因导入诱导后内皮细胞中.转染VEGF165基因后,细胞上Notch信号配体Jagged1 mRNA表达增强(1.08±0.01比1.01±0.02,P＜0.01),Notch1 mRNA表达无明显变化(0.60±0.02比0.59±0.01,P＞0.05);细胞的迁移能力增强(划痕空白处细胞个数:46.45±4.46比41.61±1.42,P＜0.05),形成毛细血管样结构能力无明显变化(细胞分级:3.00±0.89比2.00±0.89,P＞0.05).内皮细胞转染VEGF165基因后,以γ-内分泌酶抑制剂L-685458阻断细胞Notch信号通路的转导,则细胞迁移能力(划痕空白处细胞个数:51.72±3.47比46.45±4.46)和形成毛细血管样结构能力(细胞分级:4.17±0.75比3.00±0.89)均进一步增强(均P＜0.05).结论 转染VEGF165基因可增强大鼠MSCs诱导分化内皮细胞的功能,在此基础上阻断Notch信号通路转导可进一步增强细胞功能.     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

Objective To investigate the role of cardiac myocytes in the differentiation of bone marrow mesenchymal stem cells (MSCs) to cardiac myocytes and the biological properties in the course. Methods The bone marrow of the extremities of the rats was flushed, and bone marrow MSCs were obtained by method of density gradient centrifugation. They were cultured. The second passage of cultured MSCs were labelled with bromodeoxyuridine (BrdU). The cardiac myocytes were obtained from the apex of rat heart with trypsin digestion method, and they were cocultured with labeled MSCs. The developmental changes of bone marrow MSCs were observed under light microscope with immunohistochemical staining for BrdU and a-sarcomeric actin on the 3rd day, anti electron microscopic examination on the 5th day. Results MSCs proliferated fast in primary culture and subculture, positive rate was (90. 34± 2. 31)%, and there was statistical difference when it compared with control group [(4.07±1.35)%, P<0. 01]. The morphology of MSCs changed significantly after coculture. There were new cells nuclei of which were positively stained for BrdU and its cytoplasm positive for actin. Under transmissive electron microscope, sarcomeres like structure and abnormal Z line were observed in cytoplasm. Conclusion Cardiac myocytes can effectively induce bone marrow MSCs to differentiate into cardiac myocytes by coculturing.     

骨髓间充质干细胞防治多脏器衰竭

Objective The patients with lethal irradiation after sucessful hematopoietic stem cells transplan-tation had blood recovery, but did not avoid to died of multiple organ failure(MOF). To overcome the block, the article investigated mechanisms of mesenchymal stem cells (MSCs) protecting lethal radiated mice from multiple organ failure after haploid bone marrow cells transplantation. Method BALB/c mice irradiated with 8Gy60COγ-rays were randomly divided into two groups: MSCs group, infused MSCs labeled with cm-DiI and bone marrow monocytes of CB6F1 mice; Control group, only infused bone marrow monocytes; normal group, mice were infused cm-DiI marked MSCs without irradiation. The distribution of MSCs and the serous densities of Il-2, Il-10 and TNF-α in the recipients were observed after transplantation. Results MSCs collected in the bone marrow and the intes-tine in normal group at 15 d,in MSCs group MSCs enriched the different organs at 3,15 and 30 d. MSCs regulated down the secretion of IL-2 and TNF-α,and up the IL-10 density. Conclusions MSCs protected mice from multiple organ failure through above effects and may be open a new treatment strategy on acute radiation syndrome by stem cells.     

骨髓间充质干细胞防治多脏器衰竭

Objective The patients with lethal irradiation after sucessful hematopoietic stem cells transplan-tation had blood recovery, but did not avoid to died of multiple organ failure(MOF). To overcome the block, the article investigated mechanisms of mesenchymal stem cells (MSCs) protecting lethal radiated mice from multiple organ failure after haploid bone marrow cells transplantation. Method BALB/c mice irradiated with 8Gy60COγ-rays were randomly divided into two groups: MSCs group, infused MSCs labeled with cm-DiI and bone marrow monocytes of CB6F1 mice; Control group, only infused bone marrow monocytes; normal group, mice were infused cm-DiI marked MSCs without irradiation. The distribution of MSCs and the serous densities of Il-2, Il-10 and TNF-α in the recipients were observed after transplantation. Results MSCs collected in the bone marrow and the intes-tine in normal group at 15 d,in MSCs group MSCs enriched the different organs at 3,15 and 30 d. MSCs regulated down the secretion of IL-2 and TNF-α,and up the IL-10 density. Conclusions MSCs protected mice from multiple organ failure through above effects and may be open a new treatment strategy on acute radiation syndrome by stem cells.     

抗CD44单抗IM7对慢性粒细胞白血病CD34+细胞黏附和迁移能力影响的体外研究

Objective To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell(CML-LSC) and its mechanism. Methods CD34+ CD38- CD123+ leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia ( CML) patients BM cells and CD34+ CD38- hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySepTM magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively. Results ①After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60±2.10)% vs. (25.40±1.70)% (P<0.05), respectively. ②The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A570) changing from pre- incubation of (0.62±0.11) to post-incubation of (0.34±0.07), while there was little change of A570 in the HSC group. ③The migration abilityof the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46%～63%, while little change of that in HSC group was detected. ④The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group. Conclusion The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.     

骨髓间充质干细胞移植对烟雾吸入性损伤兔早期肺组织的影响

目的 探讨骨髓间充质干细胞(MSCs)移植对烟雾吸入性损伤兔早期肺组织损伤的影响.方法 采用直接贴壁法体外培养兔MSCs,用流式细胞术鉴定MSCs.将48只兔制备烟雾吸入性损伤模型后按随机数字表法均分成致伤组和MSCs组.MSCs组伤后立即静脉给予含1×107个/ml MSCs的磷酸盐缓冲液(PBS)10 ml;致伤组则给予10 ml PBS.分别于干预后2、6和24 h活杀8只兔,取肺组织行组织病理学观察,同时进行肺损伤评分.结果 流式细胞术检测显示所培养细胞为MSCs.肺大体标本和光镜下均观察到MSCs组肺损伤程度较致伤组明显减轻.虽然MSCs组和致伤组伤后2 h肺损伤评分(分)比较差异无统计学意义(4.0±0.7比4.5±0.6,P＞0.05),但MSCs组伤后6 h和24 h肺损伤评分(分)明显低于致伤组(6 h:6.1±0.9比8.2±0.9,24 h:4.6±0.9比10.4±0.8,均P＜0.01).结论 MSCs移植能明显减轻烟雾吸入性损伤兔肺组织损伤,改善肺损伤评分.

Abstract：

Objective To explore the effect of bone marrow mesenchymal stem cells (MSCs)engraftment on lung tissue at early stage of smoke inhalation injury in rabbits. Methods MSCs were proliferated by the method of whole marrow culture and identified by flow cytometry. Forty-eight rabbits were randomly divided into smoke inhalation group (S group) and MSCs group (M group) after reproduction of rabbit smoke inhalation injury model. 10 ml of phosphate buffer saline (PBS) containing 1 × 107/ml MSCs was intravenously injected in M group, meanwhile 10 ml PBS was injected intravenously in S group. Eight rabbits were sacrificed at 2, 6 and 24 hours after intervention, and the lung tissue was harvested for morphological and pathological observation, and lung injury score was used to evaluate smoke inhalation injury.Results Cultured cells were confirmed to be MSCs with flow cytometry. Lung injury in rabbits of M group was less serious in morphology and histopathology than that in S group. Though there was no significance in lung injury score between M group and S group at 2 hours after injury (4.0±0.7 vs. 4.5±0.6, P＞0.05),the lung injury scores in M group at 6 hours and 24 hours after injury were significantly lower than those in S group (6 hours: 6.1±0.9 vs. 8.2±0.9, 24 hours: 4. 6±0.9 vs. 10.4±0. 8, both P＜0. 01). Conclusion Intravenous engraftment of MSCs could ameliorate lung injury induced by smoke inhalation, and improve lung injury score significantly.     

干细胞因子对内皮细胞增殖、迁移、管状形成能力的影响及对CD133+细胞的趋化效应

目的 探讨干细胞因子(SCF)对脐静脉内皮细胞(HUVEC)增殖、迁移、管状形成能力的影响,以及对CD133+细胞的趋化效应.方法 应用MTT及CCK-8增殖分析法检测HUVEC在不同细胞因子[空白试剂、SCF、血管内皮生长因子(VEGF)、抗人SCF、人IgG]条件下增殖能力的差异性;采用细胞划痕法与Matrigel体外三维成型法分别检测内皮细胞的增殖、迁移和管状形成能力;并应用Transwell技术检测不同细胞因子诱导的CD133+细胞体外趋化效应.结果 MTT及CCK-8增殖分析结果显示SCF无HUVEC增殖刺激活性;SCF可显著提升HUVEC迁移能力;SCF呈剂量依赖性增强HUVEC 管状形成能力,在适宜浓度SCF(100 ng/ml)作用下,HUVEC完整小管形成数量[(30.0 ±3.4)/105HUVEC]显著高于空白试剂组[(5.0±2.6)/105HUVEC,P<0.01];SCF可高效诱导CDl33+细胞体外趋化,SCF组[(118.0±6.5)/104CD133+细胞]Transwell小室跨膜迁移细胞数显著高于空白试剂组[(47.0±4.7)/104CDl33+细胞,P<0.01].结论 SCF可显著增强HUVEC的迁移及管状形成能力,并有效诱导CD133+细胞体外趋化,提示SCF/c-kit信号转导在内皮细胞及其祖细胞的血管新生与血管发生过程中可能发挥重要作用.

Abstract：

Objective To explore the effects of stem cell factor (SCF) on proliferation, transmigration, capillary tube formation of human umbilical vein endothelial cells (HUVEC) and on the chemotaxis of CD133+ cells. Methods In the presence of blank control, SCF, vascular endothelial growth factor ( VEGF) , anti-human SCF (anti-SCF) or human IgG, the difference in proliferation capacity of HUVEC was analyzed by MTT and CCK-8 methods, and wound scratch assay and three-diamensional in vitro Matrigel assay were used for transmigration and capillary tube formation of HUVEC, respectively. In addition, the chemotaxis of CD133 + cells sorted from human umbilical cord blood by flow cytometry was investigated by Transwell migration assay. Results SCF didn't improve the proliferative capacity of HUVEC, but significantly enhanced the transmigration capacity, and increased capillary tube formation in a dose-dependent manner.The number of intact tubules [(30.0 ±3.4)/105 HUVEC] formed by HUVECs in the presence of the optimal concentration of SCF (100 ng/ml) was remarkably higher than that in blank control group [(5.0 ±2.6)/105HUVEC,P <0.01]. SCF also significantly induced a chemotactic response of CD 133+ cells, the transmembrane migration cell number into Transwell lower chamber was significantly higher in SCF group [(118.0 ±6.5)/104 CD133+ cells] than in blank control group [(47. 0 ±4. 7)/104 CD133 + cells,P <0.01]. Conclusions SCF significantly promotes the transmigration and capillary tube formation of HUVEC, and induces a chemotactic response of CD133 + cells. SCF/c-kit signaling possibly plays a critical role in regulating angiogenesis of vascular endothelial cells and vasculogenesis of endothelial progenitor cells.     

Mesenchymal stem cells transplantation improves functional recovery after cardiac arrest: Contribution of necroptosis北大核心CSCD

Objective To explore the effects of bone marrow mesenchymal stem cells (MSCs) transplantation on receptor-interacting protein kinase 1 (RIP1) and RIP3 in rat brain after cardiac arrest (CA). Methods Sprague Dawley (SD) rats were randomly (random number) divided into sham group (n=8), CA group (n=8) and MSCs group (n=8). Animals were subjected to asphyxial cardiac arrest and followed by cardiopulmonary resuscitation (CPR). In MSCs group or CA group, animals received intravenous injection of 1 x 106 MSCs in 0.5 mL phosphate buffer solution (PBS) or 0.5 mL PBS alone at 1 h after successful resuscitation. Neurological deficit scores (NDS) were assessed at 3 d after CPR. Donor MSCs in brain were detected under a fluorescent microscope. HE staining of brain tissue was performed to observe necrotic neurons. Western blot analysis was performed to measure the levels of RIP 1 and RIP3 in brain. Multiple comparisons were made by analysis of variance or Kruskal-Wallis H test. Results At 3 d after CPR, MSCs group demonstrated higher NDS than CA group [72.5(71.5,73.2) vs. 63.0(62.5,64.1), Z=3.376, P=0.001]. DAPI-labeled MSCs were primarily observed in the cerebral cortex. The percentage of necrotic neurons' in MSCs group was significantly lower than that in CA group [(29.6±5.9)% vs. (57.2±6.4)%, t=8.922, P<0.01]. The levels of RIP1 and RIP3 expression in brain in MSCs group were significantly lower than those in CA group [RIP1: 0.227(0.193,0.243) vs. 0.599(0.535,0.629), Z=3.151, P=0.001; RIP3: 0.217(0.203,0.274) vs. 0.543(0.533,0.555), Z=3.361, P=0.001]. Conclusion MSCs transplantation improves neurological function after CPR from CA in rats likely associated with inhibiting necroptosis. © 2018 Chinese Medical Association. All Rights Reserved.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

成人和胎儿骨髓间充质干细胞的比较研究

目的 比较成人和胎儿骨髓间充质干细胞（MSC）的表型和生物学性状差异，为临床选择使用MSC提供实验依据。方法 取正常人和胎儿骨髓单个核细胞，在SF培养基中进行MSC培养，测定生长曲线。电镜观察MSC形态，利用流式细胞仪进行SMC表型测定和细胞周期分析；SA方法测定Ⅰ，Ⅲ型胶原和vWF因子表达。通过碱性磷酸酶染色，苏丹黑染色及骨钙蛋白和脂蛋白酯酶mRNA的表达等来检测细胞向成骨，成脂肪细胞分化情况。结果 从成人和胎儿骨髓中可培养出MSC，并保持多向分化潜能。两者在细胞形态，生长特性，表面抗原表达等方面是相似的。胎儿骨髓MSC的扩增潜力及多向分化能力明显强于成人MSC。成人骨髓MSC的粘附功能则强于胎儿。结论 从成人及胎儿骨髓中可分离培养出MSC，在体外有效扩增且保持其低分化状态和多向分化能力。胎儿MSC较成人MSC更原始，具有更大的多向分化和体外扩增潜能，可作为组织工程的种子细胞；而成人MSC支持造血，促进造血功能恢复和重建造血的功能则强于胎儿，具有更广泛的临床移植应用前景。     

成人骨髓间充质干细胞体外多向分化潜能特性的研究

目的研究体外不同诱导条件下成人骨髓间充质干细胞向成骨细胞、脂肪细胞、神经细胞分化的能力。方法用细胞化学方法对诱导后的骨髓细胞进行脂肪细胞特异油红O染色；Von Kossa及改良的钙钴法进行成骨细胞鉴定；免疫组化方法检测诱导后的骨髓细胞神经元特异性烯醇化酶(NSE)、神经丝蛋白-M(NF—M)、胶质纤维酸性蛋白(GFAP)表达。结果在不同的诱导条件下，诱导后的细胞油红O染色胞浆内可见橙红色脂滴；Von Kossa染色可见细胞间布满黑色颗粒，提示有矿化基质沉积；改良钙钻法碱性磷酸酶染色胞浆呈深棕色或深黑色。免疫组化染色显示向神经细胞方向诱导后细胞NSE( )、NF—M( )、GFAP(-)。结论成人骨髓间充质干细胞在体外具有多向分化的潜能。     

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。     

人血小板裂解液对骨髓间质干细胞成骨成脂分化的影响

目的探讨人血小板裂解液(HPL)体外培养人骨髓间质干细胞(MSCs)及其对MSCs成骨和成脂分化能力的影响。方法采用贴壁法分离培养3株人骨髓来源MSCs,每株细胞在培养时平行分为2个实验组,分别用含10%胎牛血清的低糖培养基和含7.5%HPL的低糖培养基培养,均培养MSCs至第5代,分别用成骨及成脂诱导液对每组MSCs做成骨、成脂分化诱导,诱导成功后分别用茜素红、油红O对成骨和成脂分化结果染色鉴定,并进一步对成骨、成脂诱导结果做定量分析:成骨分化染色后洗脱茜素红在562nm波长下测定OD值并对每个培养孔细胞做蛋白定量,以茜素红OD值与蛋白定量值的比值作为成骨分化的定量值;成脂分化染色后用异丙醇洗脱油红O,在510nm波长下测定OD值,以之作为成脂分化的定量值。对所得的结果做统计学分析。结果对第5代的MSCs做成骨诱导12d后,光镜下可见致密结节形成,茜素红染色及定量鉴定结果显示2种培养液培养的MSCs均能成功向成骨细胞分化,而7.5%HPL培养的MSCs成骨能力比10%FBS培养的MSCs强,定量鉴定结果分别为16.548±4.397、4.151±5.631(P<0.05)。成脂诱导12d后,细胞内有明显脂滴形成,油红O染色及定量分析结果显示MSCs成功向脂肪细胞分化,但7.5%HPL培养的MSCs成脂能力与10%FBS培养的MSCs相比较弱,定量鉴定结果分别为0.239±0.030、0.497±0.105(P<0.05)。结论 HPL可以促进MSCs成骨分化,抑制其成脂分化。     

Notch信号通路对VEGF促大鼠间充质干细胞增殖的作用

本研究旨在探讨Notch信号传导通路在VEGF促大鼠间充质干细胞增殖中的作用.体外培养大鼠间充质干细胞,取对数生长期的细胞用于实验研究.用Notch通路阻断剂DAPT阻断Notch信号传导,观察该通路在VEGF作用下大鼠间充质干细胞的增殖情况.实验分为4组：空白对照组、VEFG组、DAPT组及VEGF＋ DAPT组.应用CCK-8法检测各组细胞增殖情况,RT-PCR法检测各组相关基因（Notch1、Notch2、Flk-1、HES-1）mRNA水平的变化.结果表明,细胞培养3d,各时间点（24h,48 h,72 h） DAPT组及VEGF＋ DAPT组细胞存活率均较低,细胞形态变圆脱落,数目明显减少;VEGF组存活率最高,差异具有统计学意义（P＜0.01）;与对照组相比,VEGF组的Notch1、Notch2和Flk-1的表达水平均上调,而DAPT组及VEGF＋ DAPT组Notch1和Notch2的表达水平均下调,差异均具有统计学意义（P＜0.05）;VEGF组Hes-1基因水平下调,而DAPT组及VEGF＋ DAPT组Hes-1基因水平上调,差异具有统计学意义（P＜0.05）;DAPT组及VEGF＋ DAPT组的Flk-1基因表达水平稍有下调,但差异无统计学意义（P＞0.05）.结论：Notch信号通路在VEGF作用下对大鼠间充质干细胞的增殖具有明显促进作用,而DAPT可抑制这种增殖效应.     

骨髓间充质干细胞分化为神经元样细胞后电生理特性的改变

背景:目前体外实验对骨髓间充质干细胞来源的神经元样细胞的研究多集中于形态学层面和神经标志物方面,对分化后的电生理功能研究较少.目的:观察脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞后电生理特性的变化.设计、时间及地点:细胞学体外培养,对比观察,于2005-06/2007-10在天津市环湖医院细胞室和南开大学生命科学院完成.材料:6周龄雄性Wistar大鼠3只,体质量160g左右.方法:贴壁培养法体外分离纯化间充质干细胞,用脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸联合诱导间充质干细胞向神经元样细胞分化.诱导前和诱导3 d后分别用膜片钳技术检测细胞膜电流.主要观察指标:流式细胞仪检测间充质干细胞表型:倒置显微镜观察诱导分化前后细胞形态变化;免疫细胞化学鉴定神经元特异性烯醇化酶的表达,以及全细胞电流测定结果.结果:①流式细胞仪检测结果显示,CD90阳性率(99±3)%,CD31阳性率(3.4±0.8)%,CD34阳性率(0.3±0.1)%.说明这一细胞群大部分处于未分化的干细胞状态,其纯度可达95%.②光镜下可见未经诱导的间充质干细胞多为扁平形带突起的细胞,似纤维样细胞,诱导3 d后出现神经元样细胞.③免疫细胞化学结果显示,诱导前间充质干细胞的神经元特异性烯醇化酶呈弱阳性,诱导后呈强阳性.诱导72 h时分化率为(24.01±3.76)%.④诱导组神经元样细胞外向电流峰值及最大外向电流密度高于对照组(P＜0.05),但未发现内向钠电流.结论:脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导方法可以诱导间充质干细胞向神经元方向分化,虽未发现具有成熟神经元电生理功能,但有向成熟神经元分化的趋势.     

角化细胞生长因子对骨髓间充质干细胞体外诱导分化为肺泡上皮细胞的实验研究

目的观察角化细胞生长因子(KGF)对体外小鼠骨髓间充质干细胞(MSCs)向肺泡上皮细胞诱导分化的影响。方法建立体外非接触共培养诱导小鼠MSCs向肺泡上皮细胞分化的实验模型,并在培养液中加入KGF(10μl/ml),小鼠分4组(6只/组),实验组A:MSCs(1×105)单独培养组+KGF;实验组B:MSCs+正常肺组织悬液(1×105)+KGF;实验组C:MSCs+损伤肺组织悬液+KGF;实验组D:MSCs+损伤肺组织悬液(1×105)。共同培养8 d,应用激光共聚焦显微镜和RT-PCR检测共培养体系下室MSCs中的SP-C和AQP5的表达情况。结果实验组C于共培养的d 8,较其它实验组的AQP5 mRNA的表达量明显增多(5550.00±244.39vs1650.17±184.02,1600.83±193.00,2890.00±179.09,P均<0.05);于共培养的d 5,与实验组A相比差异也有统计学意义(1743.17±228.41vs1482.00±156.11,P<0.05)。只有实验组C和D表达SP-C,且实验组C各培养时间点的SP-C的mRNA的表达量均较实验组D明显增多(2632.50±231.11vs1599.00±158.95;3976.67±221.81vs2066.83±101.31;5388.33±347.96vs3893.17±178.61,P均<0.01)。结论KGF可以进一步促进体外小鼠MSCs诱导分化为肺泡上皮细胞。     

小鼠骨髓间充质干细胞对胚胎干细胞造血分化的影响

骨髓间充质干细胞作为骨髓基质细胞的前体细胞 ,在体外具有一定的造血支持作用 ,与造血干细胞共移植可促进其植入。本研究旨在初步探讨应用小鼠骨髓间充质干细胞与胚胎干细胞共培养作为一种新的分化体系的可行性。首先分离、培养并鉴定小鼠骨髓间充质干细胞 ,然后利用扩增培养的骨髓间充质干细胞与胚胎干细胞共培养 ,通过造血集落培养和RT PCR观察造血分化的特点。结果表明 ,分离、扩增培养至第四代之后的骨髓间充质干细胞形态均一 ,高表达Sca 1,CD2 9,CD4 4和CD10 5 ,而CD34和CD4 5等造血与内皮细胞特异性表面标志呈阴性 ;特异性诱导体系内传代后 (>4代 )的骨髓间充质干细胞可向脂肪细胞和成骨细胞分化。与悬浮分化体系相比 ,骨髓间充质干细胞共培养体系中初始分化的胚胎干细胞含有显著增加的拟胚体形成细胞而没有造血集落形成细胞。此外 ,RT PCR检测发现 :共培养细胞表达胚胎干细胞特异性转录因子Oct 4 ,而造血标记Flk 1,GATA 1和 β H1为阴性。结论 :间充质干细胞在一定程度上抑制了胚胎干细胞的初始分化 ,但是共培养体系来源的拟胚体产生造血集落的能力显著高于悬浮体系     

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. MATERIAL AND METHODS: Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. RESULTS: FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. CONCLUSIONS: We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

目的 探讨心肌细胞对骨髓间充质干细胞(MSCs)向心肌细胞定向诱导分化的作用及其生物学特性.方法 取大鼠四肢骨髓,用细胞分离液通过密度梯度离心分离MSCs,并将MSCs进行原代培养和传代培养,将传2代MSCs用5-溴脱氧尿核苷(BrdU)进行标记.采用胰蛋白酶消化法取乳鼠心尖部心肌细胞进行培养,并与标记的MSCs混合培养,光镜下观察其动态变化,并于第3日行免疫组化染色,第5日行电镜观察.结果 原代和传代的MSCs增殖迅速,其阳性率达(90.34±2.31)%,与平行对照组[(4.07±1.35)%]比较差异有统计学意义(P<0.01).与心肌细胞混合后,光镜下观察MSCs形态出现明显的变化,第3日BrdU标记阳性的MSCs胞质中肌动蛋白(actin)表达阳性.电镜下观察原始细胞内出现了不规则的肌小节样结构.结论 心肌细胞与MSCs的接触可有效诱导MSCs向心肌细胞定向分化.