不同浓度兔自体血清对脂肪源性干细胞增殖能力的影响

目的：观察不同浓度兔自体血清对脂肪源性干细胞（adipose-derived stem cells,ADSC）增殖活力的影响,并筛选出最佳血清培养浓度。方法：以大鼠为实验动物,首先制备自体血清,分离获取ADSC;然后选DMEM为基础培养基,采用不同体积分数（分别为5%、10%、20%）自体血清及10%体积分数胎牛血清培养基分别对ADSC进行培养;通过显微镜下细胞形态学观察、活细胞计数及MTT等方法对细胞进行检测,并对检测数据做统计学分析。结果：不同浓度自体血清培养培养条件下ADSC形态正常。与10%胎牛血清组进行比较,5%自体血清组ADSC增殖较慢（P〈0.01）,10%、15%自体血清组增殖较好,但组间无统计学差异（P〉0.05）。结论：兔自体血清培养ADSC是可行的,10%浓度自体血清是最佳培养浓度。     

人血清及神经生长因子对成人鼻黏膜嗅鞘细胞体外增殖的影响

目的:观察人血清及神经生长因子对成人鼻黏膜嗅鞘细胞体外增殖的影响.方法:取成人(自愿者)鼻腔嗅黏膜,制成细胞悬液,分别用DF12培养基(对照组)、10 ng/ml NGF DF12培养基、10%胎牛血清的DF12培养基、10 ng/ml NGF 10%胎牛血清的DF12培养基、10%人血清的DF12培养基和10 ng/mlNGF 10%人血清的DF12培养基培养细胞,并用差时贴壁法纯化嗅鞘细胞.采用MTT法观察上述各种培养液对嗅鞘细胞增殖的影响.结果:加入与不加入NGF培养液组之间的光密度无统计学意义(P＞0.05);胎牛血清组、人血清组与对照组之间的光密度有显著统计学意义(P＜0.01);胎牛血清组和人血清组之间的光密度无统计学意义(P＞0.05).结论:人嗅鞘细胞的体外培养对血清具有依赖性,人自体血清培养可替代胎牛血清用于嗅鞘细胞的体外培养、扩增.本研究结果为人嗅鞘细胞的自体移植治疗脊髓损伤提供了可行性基础.     

自体血清对牛血清适应性人间充质干细胞增殖的影响

目的观察自体血清对牛血清适应性人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)体外增殖的影响.方法用10%的自体血清培养牛血清适应性hMSCs,分别以新生牛血清、胎牛血清及全程自体血清组作为对照,通过相差显微镜、细胞计数、MTT法、CCK-8(cell counting Kit-8)检测观察各组hMSCs的增殖状况.结果各组细胞均呈现正常的形态,细胞生长曲线相似,自体血清促进hMSCs体外增殖的效率与胎牛血清相似,较新生牛血清高;自体血清对牛血清适应性hMSCs的增殖效率有负面影响,但仍比新生牛血清更高.结论10%的自体血清对hMSCs具有与胎牛血清类似的良好促增殖作用,并对牛血清适应性hMSCs的促增殖效率有尚可接受的轻微不利影响.     

不同分离及培养方法对大鼠骨髓间充质干细胞生长增殖和生物学特性的影响

目的 探讨获得大量、高纯度、高活性骨髓间充质干细胞(BMSCs)的有效途径.方法 用密度梯度离心法和贴壁法分离纯化BMSCs,在无血清、含小牛或胎牛血清以及不同体积分数的胎牛血清的培养基中培养,传代扩增,观察各组细胞形态、生长特性,测定生长曲线,免疫细胞化学染色鉴定表面抗原,电镜观察细胞显微结构.结果 密度梯度离心法可获得较纯的原代细胞,但细胞增殖缓慢,容易老化;贴壁法分离的细胞生长增殖迅速,经传代换液,第4代细胞基本纯化;小牛和胎牛血清均能促进BMSCs生长增殖,但在体积分数为0.12的胎牛血清中集落形成率最高(46.50%);细胞表达CD44、CD90,而CD14、CD45阴性;电镜下BMSCs符合干细胞的一般特性.结论 经多次换液、严格控制传代消化时间(2～3 min)和酶浓度(2.5 g/L),采用贴壁纯化法在含体积分数为0.12的胎牛血清L-DMEM培养液中培养可获得大量、高纯度、高活性的BMSCs.     

血清浓度对小鼠骨髓干细胞生物特征的影响

目的：研究不同血清浓度处理对小鼠骨髓干细胞生物特性的影响。方法小鼠全骨髓培养法培养细胞,原代细胞用10%、15%、20%血清体积分数的培养基,观察细胞形态及增殖情况。结果细胞在血清浓度为10%的培养基中增殖速度快,细胞形态以梭型为主;血清浓度为20%时细胞胞体面积增大,且增殖速度减慢。结论体外培养小鼠骨髓干细胞的最佳血清体积分数为10%。     

心肌球培养基血清浓度优化研究

目的 通过比较含有不同胎牛血清浓度的心肌球培养基分离培养大鼠心肌干细胞(CSCs)的效果,探讨最优的心肌干细胞血清培养浓度.方法 选取15只健康新生Wistar大鼠(出生1天龄),无菌条件下取出心脏,经胰酶和Ⅱ型胶原酶反复消化后,种植于培养瓶中,应用含20%胎牛血清的完全培养基培养组织块源性心脏祖细胞(CPCs),将所得的培养外植物源性细胞(EDCs)随机分为A、B、C 3组,各组5瓶细胞(25 mL塑料培养瓶),分别应用含10%、11%、15%不同浓度胎牛血清的心肌球生长培养基培养,培养至第三代CSCs时行细胞表面抗原C-kit染色,鉴定C-kit阳性CSCs占所获得细胞的比率,比较所得的细胞数量,生长周期和生长曲线.结果 从新生大鼠心肌组织中成功分离培养出CSCs,C-kit阳性率87%.培养后所得细胞数量以细胞倍增水平(PDL)表示,各组细胞PDL呈匀速递增,PDL水平和其递增速率(生长曲线所示)比较:B组>C组>A组.应用含11%胎牛血清的心肌球生长培养基培养EDCs获得的CSCs,与其他两组相比,具有生长周期短(P<0.05),生长数量多的特点(P<0.05).结论 含11%胎牛血清浓度是心肌球生长培养基培养原代大鼠心肌干细胞的最优血清培养浓度.     

评估无血清培养子宫内膜间充质干细胞的可行性

目的 使用无血清培养基体外分离扩增子宫内膜间充质干细胞,研究其生物学特性.方法 比较在无血清培养基中和含10%胎牛血清培养基中子宫内膜间充质干细胞细胞形态细胞表型、增殖能力、细胞活率和分化能力等生物学特性.结果 无血清培养基和有血清培养基培养的子宫内膜间充质干细胞形态相似,但是前者呈明显的漩涡状生长,更加细长,立体感更强;无血清和有血清培养的子宫内膜间充质干细胞表面标记物均呈阳性;无血清培养的子宫内膜间充质干细胞细胞活率更高、细胞增殖能力更强.结论 无血清培养基能够在体外扩增子宫内膜间充质干细胞,并使其生物学特性(细胞增殖,细胞活率)优于胎牛血清培养的子宫内膜间充质干细胞,且分化能力无改变,可以取代胎牛血清用于细胞治疗,避免有血清培养的干细胞治疗引起的人畜共患病及免疫原性反应.     

不同血清微环境培养的施万细胞增殖状况比较

目的 将不同浓度大鼠血清、10％胎牛血清培养以及无血清培养的大鼠原代施万细胞的增殖状况进行比较,探讨适合施万细胞生长的血清微环境.方法 将所培养的原代施万细胞分成5％大鼠血清组、10％大鼠血清组、15％大鼠血清组、20％大鼠血清组、10％胎牛血清组、无血清组,另设不含细胞的空白对照组.采用CCK-8细胞增殖试剂,分别于培养后24、48、72、96、120h对各组的细胞增殖情况进行测定.结果 5％大鼠血清组的细胞在96h内的生长状况与10％胎牛血清组相比无显着差异.结论 在96h内,5％的大鼠血清浓度最适合该细胞生长.     

银杏叶提取物对乙醇、鱼油诱导的E47细胞内细胞色素P450 2E1表达的影响

目的:研究银杏叶提取物(GBE)对乙醇、鱼油诱导后的E47细胞内细胞色素P450 2E1(CYP2E1)表达的影响.方法:E47细胞随机分为5组接种培养.对照组:用含体积分数10%胎牛血清的DMEM培养基培养.乙醇组:接种24 h后换含100 mmol/L乙醇、体积分数10%胎牛血清的DMEM培养基培养.鱼油组:接种24 h后换含3 μmol/L鱼油、体积分数10%胎牛血清的DMEM培养基培养.GBE 乙醇组:接种24 h后用150 g/L GBE作用3 h,用PBS洗2次后换用100 mmol/L乙醇、体积分数10%胎牛血清的DMEM培养基培养.GBE 鱼油组:接种24 h后用150 g/L的GBE作用3 h,用PBS洗2次后换用含3 μmol/L鱼油、体积分数10%胎牛血清的DMEM培养基培养.培养24 h后,Western blot法观察E47细胞CYP2E1蛋白的表达,用对硝基苯酚探针测定全细胞CYP2E1的活性,测定丙二醛(MDA)和超氧化物歧化酶(SOD)的含量.结果:与对照组比较,乙醇组、鱼油组细胞CYP2E1蛋白表达量及活性、MDA含量升高,SOD含量降低(P均<0.05).与乙醇组、鱼油组比较,GBE 乙醇组、GBE 鱼油组细胞CYP2E1蛋白表达量及活性、MDA含量降低,SOD含量增高(P均<0.05).结论:GBE能抑制CYP2E1的表达和活性,减轻脂质过氧化反应.     

低抗凝肝素对大鼠肝星状细胞生长的影响

目的:探讨低抗凝肝素对胎牛血清培养的大鼠肝星状细胞生长的影响.方法:肝星状细胞经体积分数为0.4%胎牛血清/DMEM培养液同步化48 h后分为对照组和实验组,对照组以体积分数为10%胎牛血清处理,实验组分别以体积分数为10%胎牛血清和不同质量浓度(8 g/L,40 g/L,200 g/L,1 000 g/L,5 000 g/L)低抗凝肝素处理,应用MTT法测定其对肝星状细胞生长的影响.结果:低抗凝肝素(40～5 000 g/L)对肝星状细胞的增殖有明显抑制作用(P＜0.05).在不同的作用时间,低抗凝肝素(200 g/L)均对肝星状细胞生长有明显抑制作用,且在其作用48 h后肝星状细胞生长速度明显减慢.结论:低抗凝肝素对大鼠肝星状细胞生长具有抑制作用.     

绿色荧光蛋白和酪氨酸羟化酶在骨髓基质细胞中的表达

目的研究反转录病毒介导的绿色荧光蛋白(EGFP)基因和酪氨酸羟化酶(TH)基因在原代培养的骨髓基质细胞(bMSCs)中的表达情况。方法体外分离培养大鼠四肢骨的bMSCs,原代培养8~10 d后,同时利用含EGFP和TH基因的反转录病毒上清依次转染bMSCs并进行抗性筛选,7~10 d可得到稳定表达EGFP的bMSCs,在稳定转染EGFP后10 d进行TH病毒上清的转染,7~10 d可得到稳定表达TH和EGFP的bMSCs。结果原代培养bMSCs 3~4 d后生长至70%~80%汇合,病毒转染EGFP后第2天,细胞呈现弱荧光,经G418筛选2~3 d后出现阳性细胞,约8~10 d生长融合,阳性率60%~70%,第2~5代阳性细胞比率无明显变化。TH阳性率也达60%以上。结论利用反转录病毒载体可有效地将EGFP和TH基因转至bMSCs中,并能在体外稳定表达传代。这种表达EGFP和TH的bMSCs对于研究bMSCs的体内迁移分化及帕金森病(PD)的基因治疗具有重要意义。     

自体骨髓间充质干细胞移植促进交叉韧带重建后腱骨愈合的研究

目的探讨自体骨髓间充质干细胞对促进兔前交叉韧带重建后腱骨愈合的影响。方法采用骨髓穿刺法获取兔骨髓,体外分离、培养、扩增获得骨髓间充质干细胞。18只成年新西兰大白兔随机分为两组,每组9只,每只兔行双侧前交叉韧带重建手术,实验组将骨髓间充质干细胞和生物蛋白胶混合注入兔前交叉韧带重建模型的肌腱和骨道间隙内,对照组只注射生物蛋白胶,分别在2、4、8周各时间点每组处死3只兔子取材。组织学观察腱骨界面的愈合情况。结果对照组,术后8周在腱骨界面之间为瘢痕组织,腱-骨结合较紧密,有类Sharpey纤维成分。实验组,术后2周腱骨结合部位已经出现未成熟的类软骨细胞,术后8周为较成熟的软骨细胞,排列较整齐,从肌腱向骨道逐渐移行,类似于正常前交叉韧带的止点结构。结论前交叉韧带重建后,自体骨髓间充质干细胞移植于腱骨界面,形成类似正常前交叉韧带止点样结构,有利于促进腱骨愈合。     

Autologous serum can induce mesenchymal stem cells into hepatocyte-like cells

Objective： To investigate whether the rabbit serum after radiofrequency ablation to liver tumor can induce mesenchymal stem cells （MSCs） differentiating into hepatocyte-like cells in order to find a new source and culture process for repairing liver injury. Methods： A tumor piece of 1 mm× 1mm×1 mm was transplanted into a tunnel at right liver of rabbits. The model of liver tumor was established after 2-3 weeks. The serum was collected from rabbits 72 h after being subjected to radiofrequency ablation of the liver tumor. Mesenchymal stem cells were isolated from rabbit bone marrow and cultured in DMEM containing autologous rabbit serum. Three kinds of media （L-DMEM） were tested respectively： ① containing 10% fetal calf serum （FCS）; ② containing 30% rabbit autologous serum after radiofrequency ablation of the liver tumor （ASRF）; ③ containing 30% rabbit autologous serum （AS）. MSCs were cultured on 12-well plates until passage 2 and examined under the light and electron microscopy at indicted intervals. The expression of albumin and CKl8 was detected using immunofluorescence to identify the characteristics of differentiated cells. Results： MSCs performed differently in the presence of fetal calf serum, rabbit autologous serum and rabbit autologous serum after radiofrequency ablation of the liver tumor. Induced by the serum after radiofrequency ablation to liver tumor for 7 d, the spindle-shaped MSCs turned into round shaped and resembled hepatocyte-like cells. The reactions were not found in MSCs cultured in FCS and AS groups. After induction for 14 d, slender microvilli, cell-cell junction structure and cholangiole emerged, and the differentiated cells expressed albumin and CKl 8. All those could not been observed in 10% FCS and 30% autologous serum groups. Conclusion： Mesenchymal stem cells differentiate into hepatocyte-like cells in the serum after radiofrequency ablation of liver tumor, providing us a potential cell source and culture process for clinical application in liver     

bMSCs移植对单侧输尿管结扎大鼠肾小管上皮细胞凋亡的影响

目的探讨骨髓间充质干细胞（bMSCs）移植对单侧输尿管结扎（UUO）模型大鼠肾小管上皮细胞凋亡的影响。方法自Wistar大鼠股骨和胫骨分离bMSCs,经体外传代培养、成脂诱导及鉴定后制备细胞悬液。54只Wistar大鼠随机分为假手术对照组（n=18）、bMSC注射组（bMSC组,n=18）和生理盐水注射组（PS组,n=18）。bMSC组和PS组大鼠于UUO模型建立的同时经下腔静脉分别注入bMSCs和等体积生理盐水。于建模后第3、7和14天分批处死大鼠并采集肾脏组织标本,RT—PCR技术检测肾小管细胞FasL mRNA表达,免疫组化染色检测FasL蛋白表达及分布;TUNEL法检测肾小管细胞凋亡,计算凋亡指数。结果与假手术组比较,bMSC组和PS组建模后各时间点FasL mRNA及蛋白表达均明显上调,肾小管细胞凋亡指数显著增加（均P〈0．05）。建模后第3、7天时,bMSC组FasL mRNA和蛋白表达的上调幅度及肾小管细胞凋亡指数的增加程度均明显低于PS组（P〈0．05）;至建模后第14天时,bMSC组与PS组各项检测指标比较,差异均无统计学意义（P〉0．05）。结论静脉注入bMSCs对UUO模型大鼠肾小管上皮细胞凋亡有短期抑制作用。     

An ectopic study of tissue-engineered bone with Nell-1 gene modified rat bone marrow stromal cells in nude mice

Background Tissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 （Nell-1）, a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells （bMSCs） after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique. Methods bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 （Nell-1 group） or Ad-β-galactosidase （AdLacZ, LacZ group） or left untransduced （untransduced group）. The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed, mRNA expressions of osteopontin （OP）, bone sialoprotein （BSP） and osteocalcin （OC） were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase （ALP） activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with β-tricalcium phosphate （β-TCP） at a concentration of 2×10^7 cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. Results Under current transduction conditions, gene transfer efficiency reached （57.9±6.8）%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was （18.1±5.0）%, significantly higher than those of untransduced group （11.3±3.2）% and LacZ group （12.3±3.1）% （P〈0.05）. Conclusions This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.     

海藻酸钠凝胶对骨髓间充质干细胞生物学效应的初步研究

目的：初步探讨海藻酸钠凝胶对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。方法：将在海藻酸钠凝胶上培养的细胞设为实验组，在塑料培养板上的细胞设为对照组。以MTT法检测细胞在两种材料上的增殖；采用甲苯胺蓝染色观察细胞在凝胶上的生长形态；RT-PCR检测bMSCs生长6天后的成骨相关基因。结果：两组的bMSCs均能迅速贴附、铺展进入增殖期；在实验组，细胞成集落样生长较明显，且形态发生改变，伸出较多的伪足，细胞间接触更为紧密；培养至第6天，进行成骨相关基因检测，发现对照组和实验组均有Ⅰ型胶原和碱性磷酸酶表达，但它们在实验组的表达量高于对照组；同时，实验组骨连接蛋白为阳性表达，而对照组为阴性。结论：bMSCs可能具有自发向成骨细胞分化的趋势；海藻酸钠凝胶具有促进干细胞向成骨细胞分化的生物学活性。提示海藻酸钠凝胶可能是比较适合的骨组织工程支架材料。     

Serum- free culture of dendritic cells from patients with chronic myeloid leukemia in vitro and estimation of their cytotoxicity

Objective To establish a serum- free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future.Methods Fetal calf serum, serum- free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony- stimulating- factor, tumor necrosis factor- α and interleukin- 4) and group B (granulocyte/macrophage colony- stimulating- factor, tumor necrosis factor- α and interleukin- 4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T- cell cytotoxicity was measured by (MTT) assay. Results CD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic- phase CML. Group A of serum- free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum- free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class Ⅱ antigen on the surface of DCs was notable (＞50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10%-50%). Although CD1a+/CD14- DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27. 2 fold at DCs∶T cells ratio of 1∶10). At day 12, CD1a+ cells from three patients were studied by displaying G banding and Ph+ cells in these populations were 100%, 98% and 60%, respectively. At an effector∶target ratio of 40∶1, 32% to 45% cytotoxicity was noted with DC- stimulated T cells against autologous leukemia cells. Conclusions A stable serum- free culture system of CML- DCs was established. The expression of CD83 and CD86 on the surface of CML- DCs and DCs’ potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti- leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.     

bFGF基因体外转染兔骨髓间充质干细胞及对其增殖的影响

目的：观察bFGF基因体外转染兔骨髓间充质干细胞（bMSCs）后细胞目的基因的表达及对其增殖的影响。方法：将兔骨髓来源的间充质干细胞分离培养扩增,用PCR扩增法获得绿色荧光蛋白-bFGF基因重组质粒,转染间充质干细胞,用荧光显微镜观察转染后绿色荧光蛋白基因的表达,计算转染率,用MTT检测转染细胞的增殖特性。结果：实验成功地构建了绿色荧光蛋白-bFGF基因重组质粒,倒置荧光显微镜下观察到转染后的bMSCs发出稳定的绿色荧光信号,转染率为（43.32±4.51）%,MTT法显示重组质粒组增殖速度显著高于空白质粒组和未转染细胞组（P〈0.05）。结论：运用转染的方法实现了两种基因在MSCs中的共同表达,为监控MSCs细胞的转染和表达过程提供了一种手段。为后期利用MSCs细胞进行组织工程骨组织制造和移植提供了规范化的监控和程序化生产手段。     

Deltex-1对骨髓间充质干细胞增殖及其向平滑肌细胞分化的影响

目的 探讨Deltex-1对骨髓间充质干细胞(bone marrow mesenchymal stem cells,bMSCs)增殖及其向平滑肌细胞(smooth muscle cells,SMCs)分化的影响.方法 分离培养大鼠骨髓bMSCs,采用流式细胞仪检测鉴定.通过Deltex-1过表达腺病毒载体pAd/Deltex-1感染bMSCs后,采用CCK-8(cell count kit-8)法检测Deltex-1过表达对bMSCs增殖的影响;将未感染、经Deltex-1病毒感染及经空病毒感染的bMSCs与SMCs共培养,采用免疫荧光细胞化学、RT-PCR及Western blot分别检测其bMSCs中SMCs标志物平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SM-MHC)及Notch信号通路相关因子Notch-1的表达,单纯培养的bMSCs、SMCs的相同检测作正常对照.结果 成功分离、培养大鼠bMSCs.CCK-8检测证实:与对照比较,经Deltex-1病毒感染的bMSCs生长减慢,48 h开始显得更为明显(P＜0.05,P＜0.01);免疫荧光细胞化学、RT-PCR及Western blot检测显示:与对照比较,经Deltex-1病毒感染的bMSCs与SMCs共培养后显著表达SMCs的标志物SM-MHC,较弱表达Notch信号通路相关因子Notch-1 (P ＜0.01).结论 Deltex-1过表达可抑制bMSCs的增殖,并促进其向SMCs分化.