人小涎腺成纤维样单克隆细胞的成骨分化

目的：探索人小涎腺成纤维样单克隆细胞的体外培养、扩增方法,鉴定其性质,研究向成骨细胞分化的潜能。方法：组织块贴壁法原代培养人小涎腺细胞,收集成纤维样细胞,用96孔板稀释铺板法克隆培养,免疫荧光和RT-PCR检测细胞表型,并进行成骨诱导分化,通过骨钙素、一型胶原免疫细胞化学染色及碱性磷酸酶、茜素红钙结节染色鉴定成骨分化。结果：成功的在体外扩增培养出人小涎腺成纤维样单克隆细胞,免疫荧光证实细胞表达CD13、CD29、CD106和CD44,RT-PCR显示CK18、α-SMA、vimentin、CD106、nestin基因表达与人骨髓间充质干细胞相似。成骨诱导8天后,碱性磷酸酶表达阳性率100%,19天后骨钙素和一型胶原大量表达,并形成钙结节。结论：所建立的分离培养体系能够获得大量人小涎腺成纤维样单克隆细胞,免疫表型类似于间充质干细胞。在成骨诱导培养条件下具有良好的向成骨细胞分化的潜能。     

人成骨细胞体外培养、鉴定及护骨素表达的研究

目的：用酶消化法从正常人松质骨中分离成骨细胞，进行体外培养和功能鉴定，观察用17β－雌二醇（17β-E2）和雌激素受体拮抗剂ICI182780处理后，成骨细胞中护骨素（OPG）的表达变化。方法：用胰酶－胶原酶消化法从正常人松骨骨中分离培养成骨细胞，Ⅰ型胶原染色用VanGie-son法，钙化结节用茜素红染色，用钙钴法显示成骨细胞中碱性磷酸酶的表达，骨钙素和OPG基因的表达用RT－PCR检测，OPG蛋白用W esternBlot检测，结果：用酶消化法分离的人成骨细胞，在体外培养时可维持合成Ⅰ型胶原，碱性磷酸酶、骨钙素，并形成钙化结节等功能，体外培养的人成骨细胞表达OPG，17β-E2上调其表达，并能被雌激素受体拮抗剂ICI182780阻断。结论：用酶消化法进行人成骨细胞培养，方便易行，可培养大量高纯度人成骨细胞供研究使用；17β-E2上调OPC表达，而绝经后雌激素缺乏时，OPG的表达相应减少，可能是骨质疏松的发病因素之一。     

成人成骨细胞和MG-63细胞株孕激素受体的表达

目的 观察成人成骨细胞和MG-63细胞株孕激素受体(progesterone receptor，PR)亚型表达的情况。方法 用改良胶原酶消化法从正常成人松质骨中分离成骨细胞，观察细胞形态，钙钴法行碱性磷酸酶染色，Van Gieson法行Ⅰ型胶原染色，茜素红行矿化结节染色；半定量RT-PCR检测骨钙素和PR亚型mRNA表达；Western blot测定PR蛋白质表达。结果 所分离培养的细胞具有成骨细胞的形态特征，保持了其在体内的功能；人成骨细胞和MG-63细胞株均表达PRA、PRB mRNA和蛋白质。结论 人成骨细胞和MG-63细胞株可能受孕激素的影响。     

人骨髓间充质干细胞定向诱导分化为成骨细胞及其鉴定

目的探讨将成人骨髓间充质干细胞(MSCs)定向诱导分化为成骨细胞的方法,并对所诱导细胞的成骨特性进行鉴定. 方法分离人骨髓,梯度离心并结合全骨髓法进行培养,培养基中添加成骨诱导剂地塞米松、β-甘油磷酸钠及抗坏血酸.贴壁细胞传代,取第3代细胞鉴定其成骨特性;在倒置相差显微镜下观察细胞形态,钙-钴法染色检测碱性磷酸酶(ALP)表达,免疫组织化学检测Ⅰ型胶原表达,原位杂交检测骨连接素(ON)、骨桥素(OP)表达,Von Kossa 染色检测钙结节形成. 结果第3代人MSCs呈典型的成骨细胞形态,可连续传代10次;ALP染色阳性率达85%;Ⅰ型胶原、ON和OP表达阳性;Von Kossa 染色可见钙结节形成. 结论成功地将人MSCs诱导分化为成骨细胞,所诱导的细胞具有典型的成骨细胞特性.     

接骨丹介导成骨细胞增殖促进骨折愈合的观察

[目的]观察接骨丹对新西兰乳兔成骨细胞增殖的影响,探讨接骨丹促进骨折愈合的机制。[方法]普通级新西兰大白乳兔,耳缘静脉空气栓塞处死,无菌条件下取颅盖骨,Ⅰ型胶原酶消化法获取成骨细胞并进行体外培养、鉴定及传代。将鉴定后的第二代成骨细胞随机分为正常对照组及实验组,正常对照组应用正常兔血清培养,实验组应用接骨丹含药血清培养,MTT法观测两组细胞的生长曲线;不同血清干预48 h后,倒置显微镜观测接骨丹对成骨细胞碱性磷酸酶及Ⅰ型胶原表达的影响,Elisa法观测接骨丹对成骨细胞培养上清液中骨钙素表达的影响。[结果]Ⅰ型胶原酶消化法成功获取并建立成骨细胞体外培养体系,MTT观测显示实验组能够显著促进成骨细胞的增殖,倒置显微镜观测实验组中碱性磷酸酶及Ⅰ型胶原的表达要优于正常对照组,并能够促进成骨细胞分泌骨钙素。[结论]接骨丹能够促进骨折的愈合,其机制可能与促进成骨细胞中碱性磷酸酶、Ⅰ型胶原及骨钙素的表达进而促进成骨细胞增殖和分化有关。     

青少年特发性脊柱侧凸患者成骨细胞中核因子κB受体活化子配体和骨保护蛋白的表达

目的:从成骨细胞(osteoblast,OB)水平探讨核因子KB受体活化子配体(receptor activator of NFκBligand,RANKL)、骨保护蛋白(osteopmtegerin,OPG)与青少年特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)患者骨量降低的关系.方法:AIS患者(AIS组)20例,男5例,女15例,年龄11～19岁,平均14.75岁,Cobb角40°～96°,平均59.65°;同年龄非脊柱畸形患者(对照组)8例,男6例,女2例,年龄10～19岁,平均15.25岁.两组均采用双能X线吸收测量仪(dual-energy X-ray absorptiometry,DEXA)测量骨密度(bone mineral density,BMD),测量部位包括非优势侧股骨近端及腰椎.所有受试者术中取适量髂前上嵴的松质骨,运用植块法培养成骨细胞.培养过程中观察细胞形态学变化,并用碱性磷酸酶染色法、钙结节染色法和RT-PCR检测骨钙素表达,并进行表型鉴定.取P2代细胞应用RT-PCR和Western blotting检测RANKL和OPG的mRNA及蛋白表达情况.结果:植块法培养可以获得较多的原代细胞.碱性磷酸酶染色、钙结节染色及RT-PCR检测骨钙素表达证实所培养的细胞表现成骨细胞特性.AIS组腰椎(L2～L4)及股骨近端的BMD值明显低于对照组;RANKL的核酸及蛋白水平的表达量均较对照组高(P<0.01):OPG的核酸及蛋白水平的表达量与对照组比较无统计学差异(P>0.05);RANKL/OPG比值明显高于对照组(P<0.01).结论:成骨细胞中RANKL及OPG在核酸及蛋白水平的表达异常可能与AIS患者骨量降低的分子机制相关.     

hBMP-2基因转染兔骨髓间充质干细胞的表达及生物学效应的观察

目的:观察人骨形态发生蛋白-2(humanbonemorphogeneticprotein-2,hBMP-2)基因转染兔骨髓间充质干细胞(bonemarrowmesenchymalstemcells,BMSCs)后的表达及表达产物对BMSCs增殖和向成骨细胞分化的影响。方法:利用腺病毒表达载体Adeno-XTM将hBMP-2基因转染兔BMSCs,用免疫组化染色。检测细胞内BMP-2的表达。然后通过MTT法分析其对细胞增殖的影响,并分别通过体外检测Ⅰ型胶原合成和表达情况、碱性磷酸酶染色和钙结节VonKossa染色,观察腺病毒介导hBMP-2基因转染兔BMSCs的成骨分化能力。结果:转基因细胞6周时仍能表达外源性基因。基因表达产物hBMP-2能明显促进BMSCs的增殖以及I型胶原的合成,转染后第14天碱性磷酸酶染色多数细胞为阳性,第21天出现钙结节。结论:hBMP-2基因转染BMSCs后可获得稳定表达,且基因表达产物能促进BMSCs增殖,并诱导其向成骨细胞分化。     

低氧环境下共培养的骨膜细胞和髓核细胞骨向分化能力的研究

[目的]探讨低氧环境对体外共培养的骨膜细胞、髓核细胞骨向分化能力的影响。[方法]采用组织块法分离兔骨膜细胞,胰酶、胶原酶消化法获取髓核细胞,传至3代进行共培养实验,实验分为2组:正常氧组(20%O2)、低氧组(5%O2),共培养后用CCK-8检测细胞增殖情况,用AKP试剂盒、BCA试剂盒检测碱性磷酸酶活性,RT-PCR检测骨钙素、Ⅰ型胶原、RUNX2以及HIF-1a mRNA的表达,免疫组化试剂盒检测Ι型胶原的表达,茜素红染色检测钙盐沉积或钙结节。[结果]骨膜细胞和髓核细胞在体外成功分离和培养,共培养后保持了较高的增殖率,经过成骨诱导培养后成功诱导为成骨细胞,细胞增殖曲线为"S"型,两组在1、3、5、7、9 d的光吸收值(OD)比较差异有统计学意义(P0.05);低氧组的骨钙素、Ⅰ型胶原、RUNX2以及HIF-1a mRNA表达水平高于常氧组(P0.05),碱性磷酸酶(ALP)活性高于常氧组(P0.05),细胞成骨染色(茜素红染色、免疫组化)结果显示低氧组较常氧组表达增多。[结论]低氧条件下体外共培养的骨膜细胞和髓核细胞经成骨诱导后向成骨细胞分化的能力更强。     

转染核结合因子a1/成骨细胞特异性转录因子2基因在兔皮肤成纤维细胞成骨表达中的作用

目的探讨外源性小鼠核结合因子a1(Cbfa1)／成骨细胞特异性转录因子2(Osf2)基因在兔皮肤成纤维细胞中的瞬时表达对该细胞成骨表型表达的作用。方法在阳离子脂质体介导下,将含外源性小鼠Cbfa1／Osf2基因的真核表达载体pSG5一Cbfa1／Osf2导入新西兰兔皮肤成纤维细胞。用RT—PCR方法检测Cbfa1、骨钙素、碱性磷酸酶及Ⅰ型胶原前肽基因表达,以Western—Blot方法检测Cbfa1蛋白表达,以对硝基苯二磷酸底物法及放射免疫方法分别检测碱性磷酸酶活性及骨钙素分泌情况,并通过茜素红染色及扫描电子显微镜检测转染细胞形成骨性结节的能力。结果转染pSG5-Cbfa1／Osf2真核表达质粒的兔皮肤成纤维细胞内可见Cbfa1 mRNA及Cbfa1蛋白瞬时表达,骨钙素mRNA、碱性磷酸酶mRNA及Ⅰ型胶原前肽mRNA大量表达,碱性磷酸酶活性增强,骨钙素大量分泌,细胞表面形成骨性结节。结论转染．pSG5-Cbfa1／Osf2真核表达质粒的兔皮肤成纤维细胞能表达成骨相关基因及蛋白质,并在其表面形成骨性结节。     

人羊膜间充质细胞向成骨细胞诱导分化的实验研究

目的 观察人羊膜间充质细胞(human amnion mesenchymal cells,hAMCs)体外诱导向成骨细胞分化,为骨组织工程提供种子细胞。方法 从剖宫产后废弃的人羊膜组织分离培养hAMCs,经成骨细胞诱导条件培养基诱导后,对细胞形态特征、碱性磷酸酶、骨桥素、骨钙素表达以及I型胶原分泌进行观察和检测。结果 原代培养的hAMCs形态呈长梭形或不规则形,呈均匀分布生长,传代后细胞体积略变大,约5～7d传代1次。经成骨细胞诱导培养15d后,hAMCs碱性磷酸酶、骨钙素、骨桥素的表达呈阳性,并且检测有I型胶原分泌。结论 hAMCs易于体外分离培养及扩增,体外成骨细胞定向诱导的hAMCs具有典型的成骨细胞的形态和功能性特征,是良好的骨组织工程种子细胞。     

Long-term culture, low-temperature culture, and hyperoxic culture do not prolong fish-to-mouse islet xenograft survival

Abstract: Macroscopic, anatomically discrete islets called Brockmann bodies (BB) were harvested from tilapia with microscissors and then cultured under various conditions known to prolong islet allograft survival. BBs were cultured in 95% air/5% CO₂ at 37°C either overnight (groups 1 and 2) or for 1 week (group 3); in 95% O₂/5% CO₂ at 37°C for 1 week (group 4), or in 95% air/5% CO₂ at 24°C for 1 week (groups 5 and 6). Viability and function of cultured islets was confirmed by fluorescein diacetate/ethidium bromide staining and by transplantation under the left renal capsules of streptozotocin-diabetic balb/c mice. Recipient mice in groups 2 and 6 received CsA (50 mg/kg/d p.o.) × 4 days. All recipient mice became normoglycemic posttransplant. Mean graft survival times (± S.D.) for groups 1, 2, 3, 4, 5, and 6 were 7.6 (± 1.1), 6.2 (± 1.5), 6.4 (± 0.79), 8.2 (± 2.3), 7.0 (± 0.71), and 6.2 (± 0.45) days, respectively. Rejection (i.e., nonfasting plasma glucose levels > 200 mg/dl) was confirmed histologically in all instances; rejection was characterized by infiltrates composed of mononuclear cells, plasma cells, and eosinophils. In our study, neither short-term CsA nor any of the culture protocols significantly prolonged discordant fish-to-mouse islet xenograft survival.     

Renal fibroblast culture

The interstitial cells in the kidney are not a homogeneous cell population but consist of different cell types like fibroblasts, dendritic cells or lymphocyte-like cells. Fibroblasts are the most abundant interstitial cell type. They are regarded as the most important cells for the production and degradation of extracellular matrix and are assumed to play a pivotal role in renal interstitial fibrosis, which correlates directly with the decrease in excretory renal function. Renal fibroblasts also have endocrine activity: cortical fibroblasts are supposed to synthesize erythropoeitin, and inner medullary fibroblasts are involved in the regulation of water and electrolyte homeostasis. A powerful tool for the further elucidation of fibroblast function are studies on cultured cells. Different techniques for the isolation of fibroblasts have been reported, including the cultivation of fibroblasts from outgrowths of minced tissue and the selective removal of contaminating epithelial cells by various methods. Several aspects have to be considered while culturing fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their site of origin, state of differentiation and culture conditions. Their identification in culture exclusively by morphological criteria is therefore critical especially in mixed cultures with other cell types. Unfortunately, a constitutively expressed, specific marker for all fibroblasts is still not available. Since myofibroblast formation is considered as a key event in renal interstitial fibrosis, the transformation of fibroblasts to myofibroblasts is of special interest. Studies on cultured fibroblasts provide an effective tool to examine factors that affect this transformation and regulate the production and degradation of extracellular matrix. In addition, this technique can be used for further characterization of the endocrine activity of cultured fibroblasts. A better understanding of the biology of fibroblasts is essential to develop therapeutic strategies for the treatment of renal tubulointerstitial fibrosis, the pathologic equivalent of progressive renal failure.     

Murine autogeneic mixed lymphocyte culture. Role of culture conditions

In an attempt to determine whether culture conditions significantly influence autoreactivity, we tested the effects of fetal calf, syngeneic, and autogeneic serum with and without 2-mercaptoethanol on the murine self-reactive mixed lymphocyte culture. We used both unfractionated and T-enriched lymph node cells as responders and unfractionated and B-enriched irradiated spleen cells as stimulators in a primary mixed lymphocyte culture (MLC). Using unfractionated cells as responders to various stimulator cell populations, we found excellent allogeneic reactions in all media tested but minimal or no self-reactivity in syngeneic of autogeneic serum. When using T-enriched lymph node cells as responders, allogeneic reactivity was excellent but self-reactivity was present only in the cultures supplemented with fetal calf serum and 2-mercaptoethanol. The possibility that the substances in fetal calf serum and/or 2-mercaptoethanol may be needed to enhance minimal, but biologically relevant, self-reactivity is discussed, as well as the possibility that the culture supplements may be inducing "nonspecific" self-reactivity.     

Fetal skin organ culture

The fetal response to cutaneous injury has been investigated in a variety of models; most have studied the differences between fetal and adult healing mechanisms in vivo and in cell culture. Further disclosure of the cellular and biochemical events requires a model that can be manipulated to study single factors influencing fetal tissue repair without the complex interactions that occur in vivo, but in a system that more closely approximates normal skin than cell culture models. This paper presents a method for the organ culture of fetal skin and its advantages as a model to help elucidate fetal healing mechanisms. Skin sections (1 x 1 cm) excised from the backs of fetuses of New Zealand white rabbits on day 27 gestation (term = 31 days) were placed eccentrically in 65-mm culture dishes and fed daily with 2.5 mL of DMEM containing 10% fetal calf serum, antibiotics, and 10 mM ascorbic acid. A separate group, treated similarly, received 4-mm punch wounds to assess the in vitro response to wounding. The specimens were incubated at 37 degrees C in humidified room air on a rocker platform to provide alternate exposure of the skin to air and medium. Gross observation at 3 weeks showed cells extending into the central wound, indicating that viable cells were proliferating and/or migrating from the tissue. Skin was examined histologically and was viable over the 3-week period studied. Organ culture, by maintaining tissue in the natural extracellular matrix, allows cell-to-cell contact and communication to be maintained while allowing controlled environmental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)