大鼠脊髓匀浆上清液对骨髓间充质干细胞的诱导分化作用

目的探讨大鼠脊髓匀浆上清液对骨髓间充质干细胞(MSCs)分化为神经样细胞的作用。方法分离得到的MSCs在体外扩增、传代。用碱性成纤维生长因子(bFGF)预诱导后加入不同时间的臂丛神经损伤大鼠脊髓匀浆上清液诱导。免疫细胞化学染色检测神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达,以鉴定分化细胞的表型特征。结果细胞诱导后具有神经元或胶质细胞样细胞的形态,相应细胞表面标志NSE或GFAP阳性,以损伤后7d脊髓匀浆上清液诱导组NSE阳性和GFAP阳性率最高(P<0.05)。结论臂丛根性撕脱伤大鼠脊髓匀浆上清液在体外能够有效地诱导骨髓MSCs分化为神经样细胞,伤后1w可以作为MSCs移植的最佳时间。     

人骨髓间充质干细胞体外分化为肝细胞样细胞

目的 探讨人骨髓间充质干细胞(MSCs)的体外培养及特异性诱导为肝细胞样细胞的能力。方法 骨髓标本来源于健康志愿者的胸骨,年龄2～35岁,采用淋巴细胞分离液(密度1.077)分离人MSCs,并分别采用HGF、FGF4、HGF FGF4以及无生长因子四种处理因素体外诱导第三代人MSC向肝细胞样细胞分化。通过流式细胞术分析鉴定.MSCs的纯度,于诱导培养的0、7、14、21、28天时留取细胞检测CK18、AFP和白蛋白的表达情况,同时进行糖原染色验证细胞功能。结果 用淋巴细胞分离液分离出的人MSCs纯度可达90％,采用HGF、FGF4及HGF FGF4三种处理因素均可在体外诱导人MSCs特异分化为具有肝细胞样细胞表型和功能的细胞。结论 人MSCs能在体外扩增并定向诱导为肝细胞样细胞。     

三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究

目的探讨三七皂甙对体外定向诱导猪骨髓基质细胞(MSCs)分化为心肌样细胞的影响。方法用穿刺方法抽取猪骨髓,分离并培养骨髓基质细胞,中药三七皂甙定向诱导分化为心肌样细胞。倒置显微镜下观察细胞形态,免疫细胞化学法鉴定心肌细胞特异性抗原标志物肌钙蛋白T,用RT-PCR对心肌肌球蛋白重链(β-MHC)进行鉴定。取培养的猪心肌细胞作为阳性对照,未经三七皂甙诱导正常培养的骨髓基质细胞作为阴性对照。结果猪MSCs经三七皂甙诱导2h后,大部分MSCs可分化为心肌样细胞,免疫细胞化学检测细胞中的肌钙蛋白T呈阳性,RT-PCR显示能表达β-MHC。结论MSCs是骨髓来源的具有多向分化潜能的干细胞,在三七皂甙的定向诱导下,可以向心肌样细胞分化,有望成为心衰及心肌梗死等心肌损伤时干细胞移植治疗的理想细胞材料。     

骨髓间充质干细胞体外诱导分化为神经细胞的实验研究

目的　体外分离培养SD大鼠骨髓间充质干细胞 (rMSC)并诱导分化为神经细胞。方法　取 4周龄健康SD大鼠骨髓 ,以percoll淋巴细胞分离液分离rMSC ,以DMEM +15 %胎牛血清 (FBS)培养 ,以 1mmol Lβ巯基乙醇 (BME)及无血清DMEM培养液处理细胞 ,免疫细胞化学鉴定诱导后细胞。结果　rMSC可以在体外大量培养扩增 ,以BME诱导后大部分rMSC分化为神经元样细胞 ,免疫组织化学染色神经元特异性烯醇化酶 (NSE)呈阳性 ,诱导后细胞可在体外生存 2周。结论　rMSC可在体外诱导分化为神经细胞并在体外存活。     

体外诱导骨髓基质细胞分化为神经元样细胞的实验研究

目的观察大鼠骨髓基质细胞（MSCs）的生长特点及在体外诱导条件下分化为神经元样细胞的能力，并寻找最佳诱导时间。方法无菌条件下，收集大鼠股骨骨髓，分离出MSCs进行培养、扩增、纯化。用神经妥乐平（NT）进行诱导。在不同时间用巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）免疫细胞化学染色鉴定阳性细胞。结果MSCs经诱导后呈神经元样形态。免疫组化鉴定显示Nestin阳性细胞表达在诱导第3天时最多，为48．20％&#177;5．61％，NSE阳性细胞表达在第5天时最多，为31．50％&#177;7．32％。结论MSCs在体外诱导条件下可经神经前体细胞转化为神经元样细胞，且第5天为最佳诱导时间。     

大鼠骨髓间充质干细胞培养、鉴定及神经样细胞分化

目的建立稳定的大鼠骨髓间充质干细胞(BMSCs)体外培养、纯化、扩增的实验体系,并进行细胞表面抗原的鉴定及定向诱导分化检测。方法采用全骨髓贴壁培养法分离、纯化大鼠BMSCs;观察细胞形态,采用细胞免疫化学染色及流式细胞术检测细胞表面CD90、CD29、CD34和CD45表达;分别使用β-巯基乙醇及碱性成纤维细胞生长因子诱导细胞向神经样细胞分化,采用Western印迹法检测诱导后神经标志蛋白[神经巢蛋白(Nestin)、神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)]表达。结果培养至第3代的BMSCs,CD90、CD29表达阳性,CD34和CD45表达阴性;经诱导后,神经标志蛋白表达显著增高(P<0.05)。结论全骨髓贴壁培养法可分离、培养得到高纯度、具备相关生物学特性的BMSCs,且经诱导可定向分化为神经样细胞。     

复方丹参滴丸含药血清诱导大鼠骨髓间充质干细胞分化为心肌样细胞

目的观察复方丹参滴丸含药血清体外诱导大鼠骨髓间充质干细胞(MSCs)分化为心肌样细胞的能力。方法采用全骨髓直接贴壁的方法分离培养大鼠MSCs,流式细胞仪鉴定细胞表面标记;采用复方丹参滴丸的含药血清体外定向诱导第四代MSCs,应用免疫细胞化学法、原位杂交组织化学法、透射电子显微镜鉴定心肌样细胞。结果大鼠MSCs细胞表面抗原CD90、CD106阳性,CD34、CD45、CD31阴性。经复方丹参滴丸含药血清诱导向心肌样细胞分化后,免疫细胞化学法显示α辅肌动蛋白(α-Actinin)、结蛋白(desmin)强阳性,原位杂交组织化学法显示肌球蛋白重链(MHC)强阳性表达。结论复方丹参滴丸含药血清能促使MSCs分化为心肌样细胞,为中药干预MSCs治疗缺血性心脏病提供干细胞实验依据。     

传代和冻存对胎肺间充质干细胞生长及分化的影响

目的 观察传代和冻存对胎肺间充质干细胞（MSCs）生长及分化的影响.方法 以胶原酶消化法自流产胎儿肺组织中分离MSCs,在体外进行传代扩增、冻存、复苏;然后,以相差显微镜观察细胞形态学变化,以流式细胞仪检测表型变化,以神经分化诱导方案研究其向神经元样细胞分化的潜能.结果 经传代和冻存后,人胎肺MSCs的增殖能力、形态和表面标志物的表达均无明显变化,高表达MSCs特异性标志物,不表达造血和内皮细胞标志物,在适宜的神经诱导方案作用下可高效分化为神经元样细胞.结论 传代和冻存对人胎肺MSCs的形态、表型和向神经样细胞的分化潜能无明显影响,胎肺MSCs可作为细胞移植治疗神经系统疾病的备选来源.     

心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究

目的通过向骨髓间充质干细胞(MSCs)培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境,观察MSCs向心肌细胞分化的诱导作用,并与诱导分化剂5-氮杂胞苷(5-aza)比较。方法分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年大鼠骨髓中分离MSCs,用含有心肌细胞裂解液的培养基(A组)、含有5-aza的培养基(B组)、含有5-aza和心肌细胞裂解液的培养基(c组)以及普通培养基(对照组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达α-肌动蛋白、心脏特异性肌钙蛋白T(cTnT)、连接蛋白43及CD31的情况。结果A、B组的MSCs在培养1周后均形成肌细胞形态,并且均表达α-肌动蛋白和cTnT;A组MSCs分化的肌样细胞所含的肌纤维较B组更丰实,细胞生长趋势也优于B组,并且可以表达CD31;B组MSCs分化的肌样细胞不表达CD31;对照组细胞仅表达α-肌动蛋白。结论心肌细胞裂解液是体外诱导MSCs分化为心肌样细胞的理想条件,优于传统的5-aza,在心肌细胞移植技术中可以用于体外模拟心肌细胞微环境。     

犬骨髓间叶干细胞体外定向诱导分化心肌细胞的实验研究

目的:旨在建立骨髓间叶干细胞(MSCs)体外定向诱导分化心肌细胞的方法,为心肌疾患的移值修复治疗提供成体干细胞来源.方法:利用Percoll密度梯度法及MSCs黏附贴壁生长特性进行分离培养与扩增骨髓MSCs,并予以鉴定证实.应用5-氮胞苷对培养早期的骨髓MSCs进行定向诱导分化心肌细胞.通过细胞形态学、细胞免疫组化、透射电镜等技术观察分化细胞的肌管,心肌细胞特异性蛋白与细胞特异性超微结构以鉴定诱导分化的效果.结果:利用Percoll密度梯度法与细胞黏附贴壁生长特性,可分离培养与扩增足量骨髓MSCs.应用化学诱导剂5-氮胞苷10～20 μmol/L孵育早期培养的骨髓MSCs4～5周,可见细胞形成肌管;肌细胞特异性蛋白α-肌动蛋白,肌球蛋白以及心肌细胞特异性分子标志肌钙蛋白I免疫组化染色阳性;透射电镜可见肌丝与房性颗粒.结论:骨髓MSCs可在体外5-氮胞苷的诱导下定向转化为具有典型结构的心肌细胞.     

Endothelial cells modulate osteogenesis in calcifying vascular cells

The potential role of vascular endothelium in atherosclerotic calcification is unknown. Endothelial cells (EC) express bone morphogenetic proteins (BMP), and EC-conditioned medium is osteoinductive in marrow stromal cells. To test whether EC are osteoinductive in vascular cells, we used calcifying vascular cells (CVC) that form nodules and mineralize in vitro. We established a coculture model with EC grown opposite CVC on membranes coated with collagen I or collagen IV, both of which are expressed in atherosclerotic lesions. On collagen I, EC did not alter CVC nodule formation, calcification or expression of the osteogenic marker Cbfa1, the chondrogenic marker collagen IX or smooth muscle cell alpha-actin. However, on collagen IV, EC abolished nodule formation and calcification, and expression of cell markers decreased, suggesting dedifferentiation. Matrix GLA protein (MGP), also expressed in atherosclerotic lesions, was added to CVC in coculture. Unexpectedly, MGP enhanced Cbfa1 expression in CVC on both collagen I and IV. The enhancement was most apparent on collagen IV, where calcification also increased. However, MGP did not restore nodule formation on collagen IV, suggesting that nodule formation and cell differentiation are separate processes. The effect of EC on CVC calcification was suppressed by noggin, an inhibitor of BMP activity, and in part mimicked by replacement of EC by BMP-2. Our results support a role for endothelium in vascular calcification, modulated by collagens and MGP.     

Role of white cells and neoplastic cells in haemostasis

The aim of this review was to assess the role of white cells and neoplastic cells in haemostasis.     

Pooled cells versus individual screening cells in pre-transfusion testing

Pooling of screening red cells used in pre-transfusion testing has been discouraged by many workers since it might lead to the failure to detect alloantibodies. However, there is minimal scientific data to support this view and in the UK a high proportion of blood transfusion laboratories were still using pooled red blood cells for antibody screening in 1982-1983. In order to solve the dilemma of sensitivity of serological tests when using pooled versus individual screening cells, a comparative evaluation of both approaches was carried out. One hundred and five sera known to contain weak, warm-reacting clinically significant alloantibodies were tested by the three same standard sensitive techniques using samples of individual screening cells and a pool of the same cells in parallel. Our results show that when pooled cells were used, 12% of the selected alloantibodies were undetectable and a further 23% were only detectable microscopically. All antibodies examined were easily detectable macroscopically when individual screening cells were used. It is concluded that the sensitivity of pre-transfusion screening tests is reduced when pooled red cells are employed as opposed to individual samples of screening cells and that the use of pooled cells should be discouraged in routine pre-transfusion and antenatal antibody screening.     

Bone marrow-derived cells and stem cells in lung repair

Although it has been many years since publication of the first peer-reviewed studies showing that bone marrow (BM)-derived cells can become mature-appearing epithelial cells, we still know very little regarding the mechanisms, kinetics, cells, and potential clinical utility or pathology associated with this phenomenon. The initial discovery of BM-derived epithelial cells (BMDE) in the liver was published by Petersen and colleagues (Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168-1170). Since that time, BMDE were identified in the skin, eye, GI tract, kidney, and the lung. Surprisingly, once several laboratories started to examine the effects of BM cells after tissue injury, BM-derived cells of different types were found to decrease tissue injury and enhance tissue repair, often without engraftment of marrow-derived epithelial cells. Thus, the potentially beneficial effects of BM-derived cells in some tissue microenvironments may be unrelated to differentiation into nonhematopoietic cell types. Here, I focus on recent findings from my laboratory as well as several other laboratories on the effects of BM cells on lung damage, and BMDE in the lung, including tracheal epithelial cells, bronchiolar epithelial cells, and type II pneumocytes in the alveoli. Potential mechanisms underlying the appearance of marrow-derived epithelial cells, and the role of tissue damage are discussed.     

Embryonic stem cells can form germ cells in vitro

Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering.     

Dedifferentiation of epidermal cells to stem cells in vivo

The effects of growth factors on wound healing have been studied extensively; however epidermal regeneration is not fully understood. We treated eight patients with leg ulcers with recombinant human epidermal growth factor (rhEGF) and compared biopsies of regenerating epidermis with those of controls who did not receive rhEGF. We used immunohistochemistry to identify cells expressing keratin 19 and beta1 integrin in regenerated epidermis from patients and controls. Patients treated with rhEGF had stem cells in the spinous and granular layers of regenerated epidermis. Histological analysis showed that these stem cells had reverted from differentiated to undifferentiated stem cells. Our findings provide evidence for epidermal cell reversion.