恒河猴骨髓间充质干细胞体外培养及诱导分化成骨细胞的实验研究

[目的]体外分离培养非人类灵长类动物恒河猴骨髓MSCs,研究其生长、扩增及诱导分化为成骨细胞的特性.[方法]梯度离心法分离、纯化恒河猴骨髓源MSCs,观察不同接种密度和换液时间对细胞生长的影响;在体外应用成骨添加剂(含地塞米松10-7mol/L、β-甘油磷酸钠10 mmol/L、维生素C 50 mg/L)或rhBMP-2(100 ng/L)定向诱导分化,对分化后的细胞进行免疫组化染色和ALP、BGP定量测定以鉴定成骨细胞,并比较两种定向诱导分化方法的效果.[结果]梯度离心法分离、纯化得到的恒河猴骨髓源MSCs具有分裂和克隆增殖的能力,种植细胞密度为10×104～30×104/cm2,48 h后半量换液,以后每3 d全量换液的方法较合理,MSCs能少量表达ALP活性,分泌的钙量较少;诱导分化后的细胞具有和体内成骨细胞相同的形态学特征,ALP染色阳性,能表达Ⅰ型胶原,不表达Ⅱ型胶原,细胞融合后3 d,ALP和BGP分泌明显增加,融合后14 d细胞ALP、BGP的分泌水平略有升高,但无明显增加;采用成骨添加剂和rhBMP-2诱导分化MSCs后相同时期成骨细胞ALP活性、BGP含量无明显差异.[结论]密度梯度离心法分离得到的非人类灵长类动物骨髓源MSCs,体外培养具有分裂、克隆增殖的能力,在加有成骨细胞诱导剂或rhBMP-2的培养基里能诱导分化为成骨细胞,化学药物在定向诱导分化过程中具有和生长因子相一致的作用.     

骨髓间充质干细胞与胶原三维培养系统的建立

目的建立骨髓间充质干细胞(MSCs)与胶原的三维培养系统。方法 采用贴壁法对(5.8±3.4)×105个原代大鼠骨髓MSCs进行分离培养,通过传代次数的增加对其进行纯化扩增,然后以鼠尾胶原为成份构建胶原膜,用扫描电镜和噻唑蓝(MTT)比色法观察MSCs在胶原膜上的增殖状况。结果MSCs在体外扩增16代后共可获得(4.0±3.3)×1012个细胞,扩增(6.8±1.0)×106倍。扫描电镜结果显示,大鼠MSCs与鼠尾I型胶原有良好的亲和性;MTT结果显示,接种胶原膜后MSCs增殖特性与接种前相比,差异无显著性(P>0.05)。增殖曲线显示MSCs最适接种密度为2×104/cm2。结论大鼠MSCs接种后在胶原膜上增殖状况良好,此三维培养系统适于植入皮肤受损动物模型。     

骨髓间充质干细胞向成骨细胞诱导分化的实验研究

目的:研究体外培养的骨髓间充质干细胞(MSCs)在特定的条件下诱导分化为成骨细胞.方法:从骨髓血中提取MSCs,在含10%胎牛血清,地塞米松、β-甘油磷酸钠和维生素C的条件培养基培养的DMEM培养基中培养,放免法检测骨钙蛋白.比色法检测碱性磷酸酶、von Kossa法钙结节染色.结果:MSCs诱导培养后碱性磷酸酶、钙结节、骨钙蛋白表达明显增强.结论:MSCs在一定条件下能诱导分化为成骨细胞,能作为骨组织工程的种子细胞应用于临床治疗.     

骨髓基质干细胞体外培养及临床应用研究

目的 :通过对人骨髓基质细胞的体外培养、扩增和初步回植的研究 ,探讨MSCs临床应用疗效和前瞻性。方法 :从人髂骨抽取骨髓液 ,体外培养、传代和扩增 ,细胞周期、细胞形态和细胞分裂方式观察 ,并初步对骨科疾病进行回植治疗。结果 :建立一套完整、简单、成熟的体外分离、培养、扩增MSCs的系统方法 ,获得初步良好的临床疗效。结论 :人骨髓基质细胞能在体外大量培养扩增 ,为今后的理论研究和临床应用奠定了基础     

大鼠骨髓间充质干细胞与多孔双相磷酸钙陶瓷支架体外黏附的实验研究

目的研究大鼠骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)诱导培养后在多孔双相磷酸钙(biphasiccalciumphosphate,BCP)陶瓷支架材料上的黏附和生长。方法SD大鼠MSCs经矿化诱导培养、扩增,具有成骨细胞表型后,与多孔BCP陶瓷支架及普通多孔羟基磷灰石(hydroxyapatite,HA)陶瓷支架体外复合培养,扫描电镜观察比较MSCs在两种材料支架表面的黏附数量和形态;同时以0.5、1.0、2.0、3.0和4.0×106/ml浓度细胞悬液接种于多孔BCP支架材料,检测适宜的接种浓度及单位体积支架材料可黏附MSCs数量。结果大鼠MSCs经诱导培养14d后,行矿化沉积茜素红染色、型胶原免疫细胞化学染色及碱性磷酸酶细胞化学染色,结果均为阳性。大鼠MSCs黏附于多孔BCP陶瓷上的细胞数(88.00±6.58)明显高于HA陶瓷组(39.00±3.65),两组比较差异有统计学意义(P<0.01)。当接种浓度为2.0×106/ml时,单位体积的陶瓷支架材料最多可黏附MSCs数量为1.28×107个/cm3,为细胞适宜接种浓度。结论大鼠MSCs在体外经矿化诱导培养可表达成骨细胞表型,细胞浓度为2.0×106/ml时与多孔BCP陶瓷支架材料具有良好的黏附能力。     

不同接种密度对人表皮细胞培养融合的实验研究

目的:研究不同密度接种对人表皮细胞体外培养融合的影响。方法:外科切取无菌皮肤或包皮环切手术标本,经两步酶消化处理后,制备单个表皮细胞悬液。以不同细胞密度接种于含有3T3滋养细胞的培养瓶中,实验室体外原代培养扩增。结果:观察不同接种密度表皮细胞的体外培养增殖,结果表明人表皮细胞具较高的生长、增殖、融合能力。2×106cell/75cm2培养瓶密度接种人表皮细胞于含3T3滋养细胞培养瓶中,表皮细胞贴壁良好,细胞融合时间较短。结论:鼠3T3细胞能促进人表皮细胞贴壁,仍不失为较好的表皮细胞培养滋养层。适宜的表皮细胞接种密度为2×106cell/75cm2培养瓶。     

骨髓间充质干细胞软骨分化生物学的影响因素

间充质干细胞 (MSCs)因具有在适宜的体内或体外条件下分化形成软骨组织的潜能 ,且易于从骨髓中分离和体外大量扩增纯化 ,便于自体移植 ,故被认为是软骨组织工程最有希望的种子细胞来源之一[1] 。然而 ,MSCs在体外培养条件下软骨表型的分化却是一个受多种因素限制的复杂过程。目前其调控机制仍不是很清楚。已知局部环境是影响MSCs向软骨细胞转化的重要因素 ,低氧张力、高细胞密度、局部应用生长因子等均有利于MSCs分化为软骨细胞。1　高细胞密度培养对MSCs软骨分化的影响选用MSCs作为种子细胞时 ,通常先将其诱导成软骨细胞的表现型。…     

人骨髓间充质干细胞体外分离、培养及鉴定的实验研究

目的观察体外培养人骨髓间充质干细胞(MSCs)的形态和生长规律,以证实人MSCs是一种理想的种子细胞,以及为进一步深入研究提供基础理论依据。方法对人骨髓淋巴细胞分离液采用密度梯度离心法和差异贴壁法进行分离、提纯MSCs。观察原代、传代细胞的结构、生长情况,对第2代MSCs表面抗原进行测定。结果MSCs原代培养第14～16d时细胞融合成单层,传代细胞保持原代细胞的形态特征。超微结构显示:第2代MSCs细胞核形态不规则,部分核可见多个核仁,胞质内细胞器形态分化幼稚。细胞的生长曲线显示:传代第3d起呈对数生长,第5d达到高峰,10代后无明显克隆出现。P1代克隆形成率为25.83%±2.93%,P5代为14.67%±1.63%,P10代为4.67%±0.52%。MSCs的表型特征显示细胞均一性较好,MSCs表达CD29,CD44,但不表达CD34,CD45。结论用淋巴细胞分离液密度梯度离心和贴壁筛选法,可分离、纯化人MSCs,方法简单、经济,易应用;MSCs增殖能力强,可在体外大量扩增,能满足组织工程的要求,是理想的种子细胞之一。     

脐血单核细胞的优化培养

目的 优化脐血单核细胞( UCB-MNCs)的培养方法.方法 采用正交实验方法筛选影响UCB-MNCs培养的各因素,优化培养条件.设立优化组、对照组(UCB-MNCs以1.0×106/L接种于含20 μg/L碱性成纤维细胞生长因子(bFGF)、体积分数为0.10胎牛血清、低糖DMEM培养基)培养.其后检测各组培养细胞增殖及形态学变化;流式细胞仪对各组行免疫表型分析.结果 筛选后优化培养条件为:低糖DMEM培养基中含20 mg/L bFGF、50 μg/L干细胞因子(SCF)、10 μg/L重组人粒细胞生长因子(G-CSF)、培养瓶用层粘连蛋白(LN)包被.原代培养的细胞密度优化组细胞( 108.15±6.90)增殖优于对照组(51.48±12.27),差异有统计学意义(P＜0.01).P3代UCB-MNCs中MSCs含量优化组(94.87％)优于对照组(75.35％),差异有统计学意义(P＜0.05).结论 采用正交实验的方法可以筛选出脐血单核细胞的优化培养条件.     

骨髓间充质干细胞软骨分化生物学的影响因素

间充质干细胞(MSCs)因具有在适宜的体内或体外条件下分化形成软骨组织的潜能，且易于从骨髓中分离和体外大量扩增纯化，便于自体移植，故被认为是软骨组织工程最有希望的种子细胞来源之一。然而，MSCs在体外培养条件下软骨表型的分化却是一个受多种因素限制的复杂过程。目前其调控机制仍不是很清楚。已知局部环境是影响MSCs向软骨细胞转化的重要因素，低氧张力、高细胞密度、局部应     

Quantitative study of tissue-engineered cartilage with human bone marrow mesenchymal stem cells

OBJECTIVES: To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. DESIGN: Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. RESULTS: The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 x 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 x 10(6) hMSCs were obtained from the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 x 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. CONCLUSION: Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.     

Bone-like nodules formed by human bone marrow stromal cells: comparative study and characterization

Autologous bone marrow stromal cells have been proposed as an adjuvant in the treatment of bone nonunion. This cell therapy would require the establishment of culture conditions that permit the rapid expansion of these cells ex vivo while retaining their potential for further differentiation. Our aim was to achieve a full differentiation process using human bone marrow aspirates. We first analyzed the effects of mineralization medium (with ascorbic acid and phosphate) and dexamethasone (dex) during the primary culture of human bone marrow stromal (HBMS) cells on the proliferation/differentiation behavior of first-passage cells. The most appropriate schedule was then selected to further characterize this differentiation model. We showed that primary culture of HBMS cells in proliferation medium (DMEM supplemented with 10% fetal calf serum), with a 48-h treatment by mineralization medium and dex resulted in a better osteoblastic differentiation of first-passage cells than primary culture carried out in mineralization medium with or without dex. We showed that culture of HBMS cells under these conditions (primary culture in proliferation medium, followed by subculture in mineralization medium) led to the formation of specifically mineralized bone-like nodules similar to the ones observed with rat bone marrow stromal cells. Our nodules exhibited three distinct cell types, reproducing in vitro a tissue-like structure. This treatment demonstrated an optimal proliferation and expression of osteoblastic markers such as alkaline phosphatase, osteocalcin, and type I collagen. The primary culture allowed the multiplication of the number of adherent progenitor cells at the initial time of plating by a mean factor of 44,000, which was found to be negatively correlated with age. Thus, this differentiation model could provide a new tool to elaborate an autologous cell therapy designed to enhance osteogenesis.     

成人间充质干细胞体外成骨的初步研究

目的 建立一种分离和培养成人骨髓来源的间充质干细胞（MSCs）的方法,观察成人MSCs体外成骨潜能。方法 用Percoll分离液分离出骨髓中的单个核细胞,并在含10％胎牛血清的低糖DMEM培养液中培养。通过传代培养扩增MSCs。为促进成人MSCs体外成骨性分化,第5传代培养时加入成骨性添加剂,培养第4、12天分别用流式细胞仪分析成人MSCs表面分子的表达,并用碱性磷酸酶组化染色和Von Kossa染色。结果 成人MSCs是骨髓黏附细胞中有相应细胞表面蛋白表达和形态均的一细胞群。成骨性添加剂可作用于传代培养的成人MSCs,表现为培养皿表面有相互连接的结节状聚合体、碱酶染色阳性细胞数量增多、Von Kossa染色可见钙化的基质沉积。结论 所建立的成人MSCs的分离和培养条件可分选出骨髓黏附细胞中一组独特的细胞群,成人MSCs具有体外成骨潜能。     

成人骨髓间充质干细胞体外低氧培养及生物学特征

目的建立人骨髓间充质干细胞（human marrow mesenchymal stem cells,hMSCs）体外低氧培养及向成骨细胞分化的生物模型,研究其生物学特征,为骨组织工程提供实验依据。方法采用人淋巴细胞分离液分离健康成人hMSCs,DMEM-LG（含20%胎牛血清）中培养,10～14d后传代培养。取第2代细胞,根据培养氧浓度及培养基类型分为4组：正常氧组（20%O2加DMEM-LG,A组）、低氧组（1%O2加DMEM-LG,B组）、正常氧成骨诱导组（20%O2加条件培养基,C组）及低氧成骨诱导组（1%O2加条件培养基,D组）,通过细胞计数、细胞增殖测定（MTT法）、集落形成检测、RT-PCR检测、碱性磷酸酶（alkaline phosphatase,ALP）活性检测及茜素红染色,研究低氧环境对hMSCs生物学行为的影响。结果与A组相比,B组及D组中的hMSCs有较高的增殖速度,并且不受条件培养基的影响,比较差异有统计学意义（P〈0.01）。B组中的hMSCs生成的集落逐渐增多,A组9d后生成的集落数基本不发生变化。D组培养的hMSCs的ALP活性逐渐增高,但明显低于C组,两者差异有统计学意义（P〈0.01）。定量RT-PCR检测,C组ALP、骨钙素、Ⅰ型胶原蛋白及骨粘连蛋白mRNA表达量明显增加（P〈0.01）。培养4周后,与其他3组相比,C组可见到明显的钙盐沉积和染成红色的钙结节。结论低氧使hMSCs增殖率增加,集落形成能力增强,抑制向成骨细胞分化。     

新生大鼠脑源性神经干细胞培养方法的优化

目的寻求较理想的新生大鼠脑源性神经干细胞(neuralstemcells,NSCs)分离培养方法。方法用不同的接种密度和多种配方的培养基:细胞种植密度采用1×104、1×105、1×106和1×107个/ml4个浓度;培养基除基础成分DMEM/F12外,按照是否添加2%B27、碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)、表皮生长因子(epidermalgrowthfactor,EGF)以及开始培养时胎牛血清浓度不同而分别记为第1～8组。分离培养新生大鼠脑源性NSCs并诱导其分化,应用相差显微镜和免疫细胞化学法对其进行观察和比较。结果分离培养出的细胞聚集成神经球,可在体外大量增殖和长期存活,可诱导分化为神经元和神经胶质,具有神经干细胞特性。开始培养时培养液中加入5%胎牛血清有利于NSCs存活,当血清浓度采用10%时,克隆球迅速分化,干细胞收获量极少;干细胞增生速度在种植密度采用1×106个/ml时优于采用其它种植密度时,适当提高其种植密度可加快增生速度;培养基中加入2%B27、bFGF、EGF是必需的,2%B27、20ng/ml的bFGF和EGF同时加入时NSCs生长最好,bFGF和EGF对加快NSCs的增生具有协同作用。结论成功建立了较理想的新生大鼠脑源性NSCs分离培养方法,为进一步研究和应用奠定了基础。     

人参皂甙Rb1促进大鼠雪旺细胞增殖的实验研究

目的：观察人参皂甙Rb1对体外培养的大鼠坐骨神经经雪旺经细胞增殖能力的影响，探讨其促进神经再生的作用机制。方法：取SD雄性大鼠坐骨骨神经体外培养第2代第5天，加入不同浓度的人参皂甙Rb1，利用MTT比色分析，^3H－胸腺嘧啶核甙测定法检测不同浓度人参皂甙Rb1在不同培养时间对体外培养大鼠旺细胞增殖的影响。结论：人参皂甙Rb1在10μg/ml的浓度对雪旺细胞增殖有明显促进作用，高浓度的人参皂甙Rb1 1mg/ml则显示抑制作用。而200μg/ml人参皂甙Rb1对细胞增殖的促进作用与对照组相近。结论：人参皂甙Rb1在适当浓度范围内可以促进雪旺细胞的增殖，从而为促进活体神经损伤的修复途径提供了一些研究基础。     

同种异体血清在体外培养骨髓间充质干细胞的作用研究

[目的]适当的血清浓度可维持骨髓间充质干细胞(MSCs)的体外培养,血清浓度过低不利于细胞的生长,而高浓度血清则易引起细胞分化。而血清的不同来源对MSCs的培养同样产生影响。传统的培养方法用胎牛血清作为营养支持,临床应用时存在潜在的风险。本研究探讨同种异体血清在体外培养人骨髓间充质干细胞(hBM-SCs)的可行性。[方法]无菌条件下收集人骨髓悬液,分离hBMSCs,分别用含胎牛血清和人血清的培养基培养。分别测定2种方法培养的hBMSCs细胞生长曲线;采用流式细胞仪检测分析培养的hBMSCs表面抗原类型;并将培养的hBMSCs诱导分化为软骨细胞及神经细胞,诱导后的软骨细胞及神经细胞采用免疫组化染色分析。[结果]2种方法培养的间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性,表达强度无显著性差异;人血清培养的hBMSCs细胞生长速度快于胎牛血清培养组,但分化效率低于后者。[结论]通过生长特性、表面抗原表达以及分化潜能等方面的对比研究,人血清培养hBMSCs与胎牛血清培养差别不大,可以作为一种适合临床的安全培养方法。     

The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading

OBJECTIVE: Functional tissue engineering (FTE) of articular cartilage involves the use of physiologically relevant mechanical signals to encourage the growth of engineered constructs. The goal of this study was to determine the utility of deformational loading in enhancing the mechanical properties of chondrocyte-seeded agarose hydrogels, and to investigate the role of initial cell seeding density and nutrient supply in this process. DESIGN: Chondrocyte-seeded agarose hydrogels were cultured in free-swelling conditions or with intermittent deformational loading (10% deformation, 1 Hz, 1 h on/ 1 h off, 3 h per day, five days per week) over a two-month culture period. Disks were seeded at lower (10 million cells/ml) and higher (60 million cells/ml) seeding densities in the context of a greater medium supply than previous studies (decreasing the number of cells/ml feed medium/day) and with an increasing concentration of fetal bovine serum (10 or 20% FBS). RESULTS: Under these more optimal nutrient conditions, at higher seeding densities and high serum concentration (20% FBS), dynamically loaded constructs show >2-fold increases in material properties relative to free-swelling controls. After two months of culture, dynamically loaded constructs achieved a Young's modulus of approximately 185 kPa and a dynamic modulus (at 1 Hz) of approximately 1.6 MPa, with a frequency dependent response similar to that of the native tissue. These values represent approximately 3/4 and approximately 1/4 the values measured for the native tissue, respectively. While significant differences were found in mechanical properties, staining and bulk measurements of both proteoglycan and collagen content of higher seeding density constructs revealed no significant differences between free-swelling and loading groups. This finding indicates that deformational loading may act to increase material properties via differences in the structural organization, the production of small linker ECM molecules, or by modulating the size of macromolecular proteoglycan aggregates. CONCLUSIONS: Taken together, these results point to the utility of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs and the necessity for increasing feed media volume and serum supplementation with increasing cell seeding densities.     

Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 × 10⁴ nucleated cells. After enrichment of the cMSCs by centrifugation on a Percoll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.     

不同年龄人骨髓间充质干细胞体外增殖及成骨分化的研究

目的 :观测年龄对人骨髓间充质干细胞 (humanmesenchymalstemcells ,hMSCs)体外增殖、成骨分化的影响。方法 :使用密度梯度离心法分离不同年龄人骨髓MSCs进行培养 ,保留贴壁细胞传代 ,观察细胞生长情况 ,检测其增殖活性、碱性磷酸酶活性 (ALP)、诱导后骨钙素定量测定。结果 :低龄hMSCs较高龄hMSCs体外生长快、MTT、ALP及骨钙素浓度高。结论 :hMSCs的增殖和成骨分化的能力和活性随着年龄的增加而降低。