诱导成鼠骨髓源神经干细胞的实验研究

目的：探讨体外诱导成鼠骨髓源神经干细胞的可行性。方法：以含20ng／ml碱性成纤维生长因子(basic fibroblast growth factor，bFGF)与表皮生长因子(epidermal growth factor，EGF)的无血清培养液培养成鼠骨髓细胞，待细胞球形成后进行传代培养与单细胞克隆试验．以含100ng／ml bFGF培养液诱导分化，细胞免疫组化染色进行细胞鉴定。结果：骨髓细胞在诱导条件下形成表达Nestin抗原的细胞球．亦表达造血干细胞标志性抗原CD90。分离成单细胞后很快形成新的细胞球，并可分化出神经元与胶质细胞。结论：骨髓细胞可在体外诱导为能自我增殖的神经干细胞。     

音猬因子体外诱导人骨髓基质干细胞转化为神经元样细胞的研究

目的：研究体外使用音猬因子（sonic hedgehog，SHH）诱导人骨髓基质干细胞（BMSCs）分化为神经元样细胞（神经干细胞、神经细胞、神经胶质细胞）的可行性。方法：从健康志愿者髂骨抽取骨髓5ml．离心、分离BMSCs。接种于含10％胎牛血清（FBS）的DMEM／F12培养液中，BMSCs体外扩增、纯化到第五代后分别种于含有不同浓度诱导液的24孔板中：A组为空白对照；B组加SHH 700ng/ml，碱性成纤维生长因子8（fibroblast growth factor-8，FGF-8）140ng／ml；C组加SHH500ng/ml，FGF-8100ng／ml；D组加SHH250ng/ml，FGF-8 50ng／ml；E组加SHH125ng／ml，FGF-825ng／ml，诱导BMSCs分化。使用免疫组化及免疫荧光鉴定微管相关蛋白（MAP-2）、胶质纤维酸性蛋白（GFAP）、神经元烯醇化酶（NSE）的表达。结果：诱导7d后，部分BMSCs胞体收缩、突起伸出，表现出神经元样细胞的形态。除A组外其余各组免疫组化及免疫荧光染色NSE、MAP-2均为阳性，各组免疫组化及免疫荧光染色均有GFAP阳性细胞存在，且以B组、C组的MAP-2、GFAP、NSE染色阳性细胞数最多。结论：人BMSCs具有较强的自我更新和多向分化潜能，一定浓度的SHH可以在体外有效诱导人BMSCs转化为神经元样细胞。     

骨髓基质干细胞诱导成雪旺细胞的可行性及促轴突生长的体外功能实验

目的 探讨骨髓基质干细胞(BMSCs)向雪旺细胞(SCs)分化的可行性,以及分化成类SCs的表型、分子及功能特征.方法 原代培养F344乳鼠BMSCs,流式细胞仪检测细胞表面特异标记CD29、CD44、CD45的表达;诱导干细胞向成骨细胞和脂肪细胞分化,评价干细胞的生物学特性;采用碱性成纤维细胞生长因子和forskolin等诱导BMSCs向SCs分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定SCs特异性标记物S100和p75的表达;RT-PCR分析诱导前、后SCs相关基因的表达;培养大鼠背根神经节神经元,分别与诱导前后的BMSCs共培养,评价其促轴突生长的功能特性.结果 分离培养的鼠BMSCs CD29、CD44表达呈阳性,CD45表达呈阴性:干细胞诱导的成骨细胞茜素红染色阳性,脂肪细胞油红O染色阳性;BMSCs经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与SCs相似;免疫荧光染色S100和p75阳性;RT-PCR结果S100、CD104均表达增强;与背根神经节神经元共培养,诱导后的BMSCs促进轴突生长的距离为(285.3±36.7)μm,与未诱导组BMSCs的[(113.5±11.5)μm]相比,差异有统计学意义(t=8.966,P=0.001).结论 BMSCs可诱导分化成SCs,其表型、分子及功能特征与SCs相似,诱导分化的BMSCs是一种理想的神经组织工程的种子细胞.     

胎盘间充质干细胞与骨髓间充质干细胞分离培养和生物学特性研究

目的探讨兔胎盘间充质干细胞(placenta-derived mesenchymal stem cells,PMSCs)和兔骨髓间充质干细胞(bone marrow-derived MSCs,BMSCs)体外分离培养、增殖,对其生物学性状进行比较观察。方法取足月待产新西兰大耳白兔1只,采用密度梯度离心法及贴壁培养技术从兔胎盘对PMSCs进行分离、纯化和传代培养。取2周龄新西兰大耳白兔1只,采用直接贴壁法从后肢骨髓中对BMSCs进行分离、纯化和传代培养。用倒置相差显微镜观察两种细胞形态。免疫组织化学染色对第3代细胞表面标志(CD44、CD105、CD34、CD40L)进行鉴定。将BMSCs与PMSCs第2代细胞分别与生物衍生骨进行复合培养5d,每条材料接种(1.0~1.5)×106个细胞,苏木素染色观察细胞与材料复合培养情况。扫描电镜观察两种细胞分别与材料复合培养3d和8d的情况。结果在倒置相差显微镜下观察,两种细胞均为贴壁生长,形态为均一成纤维细胞样。PMSCs增殖力强,细胞的增殖能力随传代次数的增加而有所下降,细胞体外培养10代后,生长速度减慢。两种细胞均表达CD44、CD105,不表达CD34、CD40L。复合培养5d,PMSCs和BMSCs在生物衍生骨表面生长,大量黏附,细胞积聚成团,相互连接成网状,孔隙内也可见细胞生长和增殖,并分泌基质。扫描电镜观察:复合培养3d,可见较多量的细胞在生物衍生骨上黏附,呈梭形或多角形;8d两种细胞均已大量增长,呈层状排列,细胞连接紧密,分泌大量基质,细胞周围有较多的网状胶原形成。结论PMSCs与BMSCs有相似的生物学特性,可作为组织工程的另一成体干细胞来源。     

Lack of telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathway

Objective: To investigate telomerase activity in rabbit bone marrow stromal cells ( BMSCs ) during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor (GDNF) on the expression of telomerase. Methods: BMSCs were acquired from rabbit marrow and divided into control group, GDNF (10 ng/ml) group. Cytokine·NSCs medium (prepared by our lab, Patent No. ZL02134314. 4) supplemented with 10% fetal bovine serum ( FBS) was used to induce BMSCs differentiation along neural pathway. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neuronspecific endase (NSE), and gial fibrillary acidic protein (GFAP). The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activitys were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA). Results; BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomerase activities were undetectable in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomerase expression were observed in the two groups. Conclusions: Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomerase activity of BMSCs.     

犬骨髓间充质干细胞体外分离、诱导成纤维细胞的实验研究

目的探讨体外稳定诱导骨髓间充质干细胞(BMSC)分化为成肌纤维细胞和成纤维细胞的方法,为构建组织工程瓣膜提供种子细胞。方法应用Percoll(密度1·073g/ml)淋巴细胞分离液分离健康犬骨髓标本,取分离的BMSC,在低糖改良Eagles培养液中培养,并用免疫组织化学的方法做细胞表型鉴定,然后用条件培养基对第2、3代细胞进行诱导分化,并用层黏连蛋白单抗对诱导后的细胞做免疫组织化学鉴定。并对诱导后细胞进行冻存,7d后复苏观察细胞的生长、增殖及功能。结果经Percoll液分离的骨髓单核细胞,经贴壁培养后,其表型为波形蛋白阳性、α平滑肌肌动蛋白阳性、CD3-4、层黏连蛋白阴性;诱导后细胞表达层黏连蛋白,阳性细胞数可达(50±3)%;诱导后BMSC经冻存复苏后,细胞复苏率(85±3)%,复苏后细胞生长、增殖旺盛,功能不受影响。结论BMSC体外能定向诱导分化为成肌纤维细胞和成纤维细胞,并符合种子细胞的要求。     

Proliferation and osteoblastic differentiation of bone marrow stem cells: comparison of vertebral body and iliac crest

Bone marrow stem cells (BMSCs) can be obtained from the vertebral body (VB) and iliac crest (IC) for augmenting spinal arthrodesis. However, it is still not evaluated, which of the two sites would have a better BMSCs potential on Proliferation and osteoblastic differentiation is still not evaluated. Fourteen patients (10 men and 4 women) undergoing posterolateral lumbar arthrodesis and pedicle screw instrumentation were involved. The mean age was 54.7 years (range 31–75 years). Bone marrow aspirates were obtained from the vertebral body through the bilateral pedicle and were quantified relative to matched, bilateral aspirates from the iliac crest that were obtained from the same patient and at the same time. The mononuclear cell count and concentration of BMSCs were calculated and compared. Proliferation and osteoblastic differentiation of each of the BMSCs were characterized using biochemical and molecular biology techniques. Concentration (cells/mL) of BMSCs from VB and IC were 3.73 × 10³ and 3.19 × 10³, respectively (P > 0.05). VB and IC exhibited similar proliferation pattern at 3, 5 and 7 days, but BMSCs from the VB exhibited an increased mineralization staining with Alizarin Red S at 14 days. BMSCs from both anatomic sites expressed comparable levels of CD29, CD34, CD44, CD90 and CD105. VB and IC displayed similar levels of expression of ALP, type I collagen and osterix, but VB expressed higher level of osteocalcin and Runx-2, especially at 14 and 21 days. Our studies show that BMSCs from VB have osteogenic differentiation potential similar to IC. Based on these findings, we suggest that BMSCs from VB would be comparable candidates for osseous graft supplementation especially in spinal fusion procedures.     

骨髓间充质干细胞的贴壁培养及内毒素对其增殖活性的影响

目的贴壁法分离培养大鼠骨髓间充质干细胞(BMSCs),并观察不同浓度内毒素对其增殖活性的影响。方法采用全骨髓贴壁培养法培养大鼠BMSCs,倒置显微镜下观察细胞形态,免疫细胞化学方法检测细胞CD44、CD29、CD34的表达,流式细胞术检测细胞周期。加不同浓度的内毒素(0、0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml、100μg/ml)作用24h,用四唑盐比色法(MTT)检测BMSCs的增殖。结果分离培养的细胞三代以后呈均一的成纤维细胞样形态,高表达CD44,但不表达CD34、CD45。80%以上的第三代BMSCs处于G1期。不同浓度的LPS作用24h后各组的平均吸光度值(A)比较有统计学意义(F=3.598,P=0.007);0.01μg/mlLPS组A值与其余各组相比,具有统计学意义(P0.05)。结论全骨髓贴壁培养法可以方便、快捷地获得BMSCs,其在形态学、细胞表面标志物表达和多向分化能力方面具有干细胞生物学特性;0.01μg/mlLPS可明显促进BMSCs的增殖。     

静脉移植5-BrdU标记骨髓间充质干细胞参与创面愈合的作用研究

目的：探讨经尾静脉移植入体内的骨髓间充质干细胞（BMSCs）参与皮肤创面愈合的情况。方法：以SD大鼠为研究对象，分离、培养并鉴定大鼠BMSCs。制作大鼠全层皮肤缺损动物模型；经尾静脉注入经5-BrdU标记的骨髓间充质干细胞，于不同时间点（3、7、14、21、28天）观察移植细胞参与创面愈合的情况。结果：经流式细胞仪鉴定细胞表达CD29、CD90表达阳性；CD45表达呈阴性：经体外标记的5-溴脱氧尿嘧啶标记率〉90％；标记后的干细胞进行移植4周后，实验组大鼠创面局部及边缘可见BrdU标记的阳性细胞聚集，对照组中未发现明显的聚集现象。结论：5-BrdU完全能够满足移植细胞的标记需要。经尾静脉注射移植入体内的BMSCs可能参与皮肤创面愈合。     

Percoll法分离犬骨髓单个核细胞与体外成骨诱导培养的实验研究

目的建立犬骨髓单个核细胞(Bone marrow mononuclear cells,BMNCs)的分离培养、体外扩增和成骨诱导方法,为骨缺损的修复提供理想的骨组织工程种子细胞。方法用1.063g/ml percoll液分离2.5ml Beagle犬骨髓,利用细胞贴壁筛选法进行分离、培养、扩增和成骨诱导。结果采用1.063g/ml percoll液可获得纯度较高的BMNCs,接种细胞生长良好,平均倍增周期为1d,经成骨诱导培养后可定向分化为成骨细胞。结论采用1.063g/ml percoll液能够较好的进行Beagle犬BMNCs的分离培养和体外扩增,分离得到的细胞具备骨髓基质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的特性。     

第三方骨髓间充质干细胞诱导同种异体移植受体免疫耐受机制的研究

目的 初步研究第三方骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)诱导同种异体移植受体免疫耐受的作用机制.方法 40只雌性C57BL/6小鼠作为供体,40只雄性BALB/C小鼠作为受体,建立稳定的同种异体皮肤移植模型,BMSCs取自SD大鼠骨髓.将40只BALB/C小鼠随机分为4组,每组10只.①空白对照组:只进行皮肤移植,未给予其他治疗;②环磷酰胺组(CP组):大剂量环磷酰胺(cyclophosphamide,CP)腹腔注射,200 mg/kg,连用2 d(q.d.);③单纯给予SD-BMSCs移植组(SD-BMSCs组):移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs;④细胞药物联合应用组(CP+SD-BMSCs组):大剂量CP腹腔注射,200 mg/kg,连用2 d(q.d.),并于移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs.检测指标包括:移植皮片存活情况;SD大鼠BMSCs表面抗原CD29、CD34、CD45和CD90鉴定;流式细胞仪检测受体脾脏调节性T细胞(CD4+、CD25+、Foxp3+、Treg细胞)的比例;ELISA检测受体外周血TGF-β、IL-10、IFN-γ的含量;异基因T淋巴细胞与经60Co照射的不同来源BMSCs共培养后,MTT法测定异基因T淋巴细胞增殖的情况.结果 CP+SD-BMSCs组皮肤移植物存活时间为(15.7 ±1.4)d,空白对照组为(6.1±1.1)d,CP组为(12.3±1.5)d,SD-BMSCs组为(12.6±1.8)d,CP+SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P＜0.05).全骨髓贴壁培养的BMSCs表面抗原鉴定:CD29+、CD44+分别为99.7%和96.7%,CD34-、CD45-分别为1.6%和1.3%.流式细胞仪检测Treg含量SD-BMSCs组和CP+SD-BMSCs组明显高于空白对照组和CP组(P＜0.05);ELISA检测受体外局血SD-BMSCs组和CP组TGF-β和IL-10明显高于空白对照组,SD-BMSCs和CP组IFN-γ则明显低于空白对照组(P＜0.05);共培养结果显示:来源于C57小鼠和SD大鼠的BMSCs可以明显抑制T淋巴细胞的增殖反应(P＜0.05),而上述两组组间比较差异则无统计学意义(P＞0.05).结论 第三方BMSCs诱导同种异体移植免疫耐受作用可能与诱导受体Treg细胞增殖和促进免疫耐受因子的表达,抑制免疫排斥因子的表达有关.

Abstract：

Objective To study the immuno-tolerance mechanism of the third-party bone marrowderived mesenchymal stem cells ( BMSCs) in the allogeneic transplantation. Methods Forty female C57BL/ 6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group , with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide ( 200 mg/kg,2 d,q. d. ) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 106 BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+ , CD25+ , Foxp3+ and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA,including TGF-β, IL-10 and IFN-γ. T cells were co-cultured with 60 Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay. Results The skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29+ (99.7% ) ,CD44+ (96.7% ) ,CD34 (1.6% ) ,CD45( 1. 3% ). Compared with the control group and CP group, the ratio of the CD4+ ,CD25+ ,Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P ＜ 0. 05). Analysis of peripheral blood by ELISA showed significant high level of TGF-β, IL-10 and low level of IFN-γ in BMSCs group and CP group, compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased apparently in SD-BMSCs group and C57-BMSCs group (P ＜ 0. 05) , but there was no significant difference between SD-BMSCs group and C57-BMSCs group ( P ＞ 0. 05 ). Conclusions The immunotolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in the allogeneic transplantation might be associated with its effect on the proliferation of Treg cells and increasing expression of TGF-β and IL-10, decreasing expression of IFN-γ.     

骨髓基质干细胞治疗脊髓损伤的体外诱导分化及相关蛋白表达

目的 探讨模拟脊髓细胞环境下骨髓基质干细胞(BMSCs)的分化及蛋白差异表达.方法 分离培养Wistar大鼠骨髓的BMSCs和脊髓的神经细胞,设立BMSCs自然分化组、与神经细胞共培养组和双层培养组,8 d后对各组BMSCs进行神经特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)免疫荧光检测.应用表面增强激光解析离子化-飞行时间质谱技术(SELDI-TOF-MS)筛选双层培养组BMSCs变化明显的蛋白进行分析.结果 BMSCs与神经细胞共培养和双层培养8 d后,BMSCs呈神经细胞形态.NSE和GFAP检测结果 ,BMSCs与神经细胞共培养组明显高于BMSCs与神经细胞双层培养组(P＜0.05),而BMSCs和神经细胞双层培养组又明显高于BMSCs自然分化组(P＜0.05).SELDI-TOF-MS检测到TIP39_RAT和CALC-RAT增加到原来的5.360和2.807倍,INSL6-RAT、PNOC_RAT和PCSK1_RAT减少到原来的38.0%、49.9%和43.8%.结论 在脊髓神经细胞微环境下体外培养BMSCs能诱导其分化成神经细胞,而且接触培养分化率高;BMSCs在向神经细胞分化机制中与TIP39_RAT、CALC_RAT、INSL6_RAT、PNOC_RAT和PCSK1_RAT密切相关.     

Notch信号通路在骨髓间充质干细胞及骨代谢疾病中的功能研究进展

骨髓间充质干细胞是存在于骨髓基质中的一种多能干细胞,具有多向分化的潜能,其天然再生能力对骨骼的生长代谢和骨转换起着至关重要的作用。Notch信号通路是一条在进化中高度保守的信号转导通路,与骨髓间充质干细胞的增殖、分化与凋亡密切相关,影响人体骨骼发育,也是多种骨代谢疾病的重要调节通路。以往对Notch信号通路的研究主要集中在神经干细胞,对骨髓间充质干细胞的研究较少。本文通过查阅文献,阐述不同的影响因素介导Notch信号通路对骨髓间充质干细胞分化的调节,并总结了Notch信号通路在骨代谢疾病如Alagille综合征、Adams Oliver综合征、脊椎肋骨发育不良、HajduCheney综合征、骨折愈合中的研究近况。     

Regulatory effects of dermal papillary pluripotent stem cells on polarization of macrophages from M1 to M2 phenotype in vitro

The M1:M2 macrophage ratio is important for spinal cord injury (SCI) repair. Bone marrow mesenchymal stem cells (BMSCs) can alter macrophage activation, promoting M1 to M2 macrophage conversion and SCI repair; however, clinical BMSC applications have limitations. Previously, we found DPCs to be superior to BMSCs in promoting tissue repair after SCI, which we hypothesized to be mediated by M1 to M2 macrophage conversion. We investigated the regulatory effect of DPCs on M1/M2 macrophage polarization. Dermal papilla cells (DPCs) were isolated from rat vibrissae and characterized. Bone marrow-derived macrophages (BMDMs) were isolated and identified based on specific marker expression, and stimulated to differentiate into M1 macrophages with GM-CSF, IFN-γ, and LPS. These cells were co-cultured with DPCs to evaluate the effect on macrophage differentiation. DPCs expressed dermal papillae-specific markers, including ALP and Sox2, had MSC-expression patterns like those of BMSCs, and were capable of multi-differentiation. BMDMs expressed ANAE and CD68. Three days after induction, differentiated cells exhibited morphology typical of M1-like macrophages and expressed the macrophage marker CD68 and the M1 macrophage markers iNOS, but lacked expression of the M2 macrophage marker CD206. Co-culture with DPCs resulted in a shift to anti-inflammatory M2-like macrophage differentiation, characterized by morphological changes typical of M2 macrophages, downregulation of the characteristic cytokine TNF-α and the proportion of iNOS⁺ cells, and upregulation of the characteristic cytokine IL-10 and the cell-surface marker CD206. The number of CD206-expressing M2 macrophages also increased. These findings demonstrate that DPCs reprogram macrophages to an anti-inflammatory M2 phenotype, which could improve adverse inflammatory microenvironments and promote tissue repair. Thus, DPCs may be an interesting alternative cell source and merit further investigation in applications for SCI therapy.     

双向诱导的骨髓基质干细胞体外成骨与成血管的实验研究

目的 探讨双向诱导的骨髓基质干细胞(BMSCs)形成内皮细胞、成骨细胞与BMSCs共存体系的体外成骨与成血管的能力.方法 采用密度梯度离心法分离培养兔BMSCs.贴壁细胞分为四组:A组(单纯的BMSCs组)、B组(成骨诱导的BMSCs组)、C绀(内皮诱导的BMSCs组)和D组(双向诱导的BMSCs组).通过倒置相差显微镜观察细胞形态学变化,并采用流式细胞仪检测诱导前后的细胞表面抗原,采用茜素红染色观察钙结节,基质胶成血管法观察四组细胞体外成血管的能力.结果 ①经流式细胞学检测,分离培养的BMSCs主要表达CD90、CD105和CD44;BMSCs向内皮细胞诱导1周后,细胞表面抗原CD34和CD133的表达升高,而CD90和CD105的表达减少,相比诱导前差异有统计学意义(P<0.05);BMSCs继续向成骨细胞诱导1周后,细胞表面抗原CD44和HLA-DR升高,相比诱导前差异有统计学意义(P<0.05).②茜素红染色结果显示D组的钙结节大于B组,而A组和C组未见钙结节形成.③体外血管新生试验中,C组的管道数目与面积大于D组,差异有统计学意义(P<0.05),而A组与B组未见管道形成.结论 BMSCs双向诱导形成内皮细胞、成骨细胞与BMSCs共存体系,在体外共培养过程中不仅可以形成钙结节,而且可以形成微血管,有利于骨组织和血管联合构建,是一种良好的构建组织工程骨的种子细胞.     

强直性脊柱炎患者骨髓间充质干细胞的生物学及免疫学特性

目的:探讨强直性脊柱炎(AS)患者骨髓间充质干细胞(BMSCs)的生物学及免疫学特性,为进一步阐明AS的发病机制和寻找新的治疗靶点提供理论依据。方法 :选取37例活动期AS患者(AS组),男34例,女3例,平均年龄(24.3±5.4)岁,HLA-B27均为阳性;49例健康志愿者作为对照组(HD组),男43例,女6例,平均年龄(25.7±4.9)岁;其中HLA-B27阴性44例(HD1组),HLA-B27阳性5例(HD2组)。从每例受检者髂后上棘穿刺采集骨髓组织,分离BMSCs,培养扩增至第3代,以1×104/ml的浓度接种于96孔板中,100μl/孔,从第1天开始每日取3孔进行细胞计数,共计12d,绘制生长曲线;每日取3孔经MTT处理后测定吸光度值,绘制细胞活力曲线,观察BMSCs的生物学特性。使用流式细胞仪检测各受检者BMSCs的细胞表面表型。将第3代BMSCs细胞接种于U形底96孔板,培养4h后使用60Co照射30Gy;取健康志愿者外周血采用密度梯度法分离单个核细胞(PBMCs),加入细胞培养液,计数后按BMSCs∶PBMCs 1∶20、1∶10、1∶5、1∶2、1∶1的比例接种于已接种BMSCs的96孔板,共培养5d,观察双向混合淋巴细胞反应情况;同样获取PBMCs,计数后同样以5个比例接种于96孔板,加入植物血凝素(PHA)4μg/ml,充分接触后共培养5d,观察淋巴细胞增殖反应情况。对组间进行统计学比较。结果:AS组、HD1和HD2组第3代BMSCs体外培养1~12d时的增殖能力、细胞活力无显著性差异(P>0.05);三组细胞表面表型均为高水平表达CD105、CD73和CD90,不表达CD45、CD34、CD14和HLA-DR。HD1组和HD2组BMSCs与不同比例PBMCs共培养的双向混合淋巴细胞反应和PHA刺激的淋巴细胞增殖反应均无显著性差异(P>0.05);AS组与HD组比较有显著性差异(P<0.05)。结论:AS患者BMSCs的生物学特性无明显改变,但其免疫调节功能明显下降,其可能在AS的发病机制中扮演重要角色。     

大鼠骨髓间充质干细胞诱导分化为表皮细胞的实验观察

目的:研究表皮生长因子(Epidermal growth factor,EGF)加条件培养基体外诱导大鼠 MSCs 向表皮细胞定向分化的可行性。方法:采用 Ficoll-Paque 淋巴细胞分离液分离扩增大鼠骨髓 MSCs,免疫细胞化学染色及流式细胞仪进行鉴定。传至第3代的大鼠 MSCs 用表皮生长因子(EGF)、条件培养基等定向诱导 MSCs 分化为表皮细胞;免疫细胞化学对细胞角蛋白 CK5/8、19(Cytokeratin5/8、19)阳性表达细胞进行检测。结果:从大鼠骨髓中分离培养的 MSCs 增殖能力强,细胞表面标志 CD34、CD45阴性,CD29、CD44阳性,流式细胞仪检测显示细胞纯度高,诱导后7d 细胞免疫化学显示角蛋白5/8、19染色阳性,具有表皮细胞特征。结论:从大鼠骨髓中分离培养出的问充质干细胞,具有自我更新和增殖能力强的特点,经诱导可定向分化表达角蛋白。     

兔骨髓间充质干细胞的分离培养及其生物学性状的研究

目的：分离培养兔骨髓间充质干细胞，对其生物学性状进行观察。方法：用密度梯度离心法与贴壁培养法相结合，分离兔骨髓间充质干细胞，进行体外培养扩增，绘制其生长曲线，用倒置显微镜、细胞爬片、扫描电镜、透射电镜观察细胞的生物学性状。结果：密度梯度离心后，活细胞比例在95％以上，贴壁培养的细胞呈纺锤形，细胞增殖力强，平均倍增时问为32h，细胞的增殖能力随传代次数的增加而有所下降。结论：密度梯度离心与贴壁培养相结合可以提高细胞分离效率。体外培养的兔骨髓问充质干细胞生长稳定，增殖力强，可以作为组织工程的种子细胞。     

大鼠骨髓间充质干细胞分离培养及多向分化研究的体外实验

目的 分离培养较高纯度大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）,检测其多向分化潜能.方法 采用密度梯度离心结合差速贴壁法分离培养大鼠BMSCs,观察细胞形态,检测表面标志物CD34、CD44、CD45、CD90表达情况,及其成骨、成脂、成神经诱导后茜素红染色、油红O染色和NSE、GFAP免疫荧光染色情况.结果 细胞呈典型的成纤维细胞样形态,CD44和CD90呈阳性表达,CD34和CD45呈阴性表达,细胞纯度＞98%.成骨、成脂、成神经诱导后,茜素红染色、油红O染色、NSE和GFAP免疫荧光均为阳性.结论 密度梯度离心结合差速贴壁法可分离、培养出高纯度的大鼠BMSCs,该细胞具有成脂、成骨、成神经多向分化潜能,是脊髓损伤修复的一种较理想的种子细胞.     

骨髓间充质干细胞对大鼠慢性胰腺炎中星状细胞的影响

目的 观察骨髓间充质干细胞(BMSCs)对慢性胰腺炎(CP)中星状细胞(PSC)的活化、增殖和分化的影响.方法 将80只SD大鼠随机分为空白组(20只,注射无菌生理盐水)、模型组[20只,尾静脉注射二氯二丁基酯(DBTC)制作大鼠CP模型]、治疗组(20只,于CP模型制作成功后尾静脉注射体外培养的同种异体GFP-MSCs)、假治疗组(20只,于CP模型制作成功后尾静脉注射等量的无菌生理盐水).取大鼠胰腺检测PSC活化表达的Desmin、胶质纤维酸性蛋白(GFAP)、α-平滑肌肌动蛋白(α-SMA)水平,组织中Ⅰ、Ⅲ胶原含量及白细胞介素(IL)-10、肿瘤坏死因子(TNF)-α、转化生长因子(TGF) -β1表达水平.结果 治疗组PSC表达的Desmin、GFAP、α-SMA,组织中Ⅰ (0.135±0.030) ng/g、Ⅲ胶原含量(0.029±0.008) ng/g,TGF-β1 (0.020±0.006) μg/L浓度均低于假治疗组(P＜0.05),但IL-10 (603.799±89.374) g/ml,TNF-α(507.45±90.13) μg/L均高于假治疗组(P＜0.05),后者与模型组之间的差异无统计学意义(P＞0.05),空白组未见明显异常.结论 BMSCs可以通过对细胞因子的调控减少PSC的活化,抑制其增殖及分化,降低细胞外基质的沉积.