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.     

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Bonemarrowmesenchymalstemcells, alsocalledbonemarrowstromalcells (BMSCs), areisolatedfrombonemarrow, andtheycanmultiplyinvitroanddifferentiateintoosteogeniccells,chondrocytes, adipocytes, musclecellsandneuralcells.1 5 RecentstudieshavedemonstratedthatBMSCsfromadultratscoulddifferentiateintoSchwann likecellsinspecificconditions.6, 7 BMSCsmayhavepotentialapplicationforautologousneuraltransplantation. Inthisstudy, wetrytoinvestigatethedifferentiativecapabilityofadulthumanBMSCsintoSchwann l…     

家兔作为胎龄期骨髓间充质干细胞动物模型的实验研究

目的建立以家兔为对象的胎龄期BMSC研究动物模型。方法通过人工授精方法,获得孕期3周的孕兔4只,行剖腹产术取出胎兔20只,冲取胎兔骨髓,以贴壁培养法进行体外扩增培养。测量第3代胎兔BMSC的生长曲线,克隆形成率,并进行成骨、成脂及成软骨诱导分化。另外,将原代细胞冻存30 d后复苏,测量其第3代生长曲线,对冻存前后增殖能力的变化进行观察。扫描电镜观察胎兔BMSC与β-TCP形成细胞材料复合物的体外形态。将BMSCs-β-TCP复合物植入裸鼠皮下,于术后1、3、6个月分别取材,行HE、VG、Masson染色观察。结果胎兔来源的BMSC于倒置相差显微镜下观察,细胞饱满均匀,呈梭形或倒三角形状;传代后各代细胞形态未发生明显变化,生长曲线相差不大;成骨、成脂以及成软骨诱导观察到钙结节、脂肪空泡、黏多糖。冻存30 d后复苏,其第3代生长曲线与冻存前相比未见明显变化。电镜下观察,与β-TCP复合7 d后,细胞能均匀紧密贴合于材料上,伸展良好分布均匀。植入裸鼠皮不同时间点取材行HE、VG、Masson染色,均显示有新生骨组织生成。结论以家兔为研究动物模型,可以在胎龄期获取并分离得到BMSC,并可在异位构建组织工程骨,是间充质干细胞动物研究的新选择。     

胶质细胞源性神经营养因子(GDNF)在大鼠骨髓基质干细胞中的表达

目的构建表达外源性胶质细胞源性神经营养因子(GDNF)的转基因大鼠骨髓基质干细胞(BMSCs),拟应用于脊髓损伤的基因治疗研究。方法提取新生大鼠肾组织mRNA,用逆转录PCR法扩增GDNF全长cDNA,构建其真核表达载体PEG-GFP-GDNF,酶切及测序鉴定;然后转染原代培养的大鼠BMSCs,荧光免疫细胞化学及细胞形态学检测GDNF的表达。结果扩增到全长687bp序列准确的GDNF编码片段,并在GDNF转基因原代大鼠BMSCs中检测到重组GDNF的表达。结论体外可构建成功表达GDNF的转基因BMSCs,有望应用于脊髓损伤等中枢神经系统疾病的治疗研究。     

端粒酶在大鼠胚胎神经干细胞的表达及其生物学特性

目的观察Wistar大鼠胚胎神经干细胞及其分化细胞的端粒酶活性，探讨神经干细胞维持其生物学特性的分子生物学基础及神经干细胞分化调控的内在机制。方法显微镜下分离大鼠胚胎室管膜下区细胞，在含生长因子的神经干细胞完全培养液中培养形成神经干细胞，然后在不含生长因子而含10％血清培养液中分化14～16d，应用改良的端粒重复序列扩增法(TRAP)研究神经干细胞及其分化细胞的端粒酶活性。结果Wistar大鼠胚胎神经干细胞表达较强的端粒酶活性，而分化后的细胞则不再表达端粒酶活性。结论端粒酶活性的改变可能是胚胎神经干细胞诱导分化的机制。随着端粒酶活性的逐渐下降，胚胎神经干细胞随之分化为具有一定生物学特性的细胞。     

成骨诱导的人骨髓基质干细胞生物安全性评价

目的分析人骨髓基质干细胞在体外成骨诱导培养条件下，其染色体核型及端粒酶活性是否发生异常的改变，细胞是否产生致肿瘤性，为验证人骨髓基质干细胞作为骨组织工程种子细胞的安全性提供实验依据。方法采用常规细胞染色体G显带处理方法对3例成骨诱导培养的人骨髓基质干细胞样本进行核型分析，采用端粒酶活性PCRELISA检测法分析细胞的端粒酶活性，并通过裸鼠皮下致肿瘤试验对细胞的致肿瘤性进行研究。结果人骨髓基质于细胞体外成骨诱导培养至第7代，未发现染色体核型异常改变，相应的端粒酶活性也未出现异常增高。致肿瘤试验，在观察期内均未见结节形成或可疑病灶产生，符合围家医疗产品生物学评价标准的要求。结论人骨髓基质干细胞在体外长期成骨诱导培养条件下，染色体核型及端粒酶活性均未出现异常改变，且无致肿瘤性，符合作为骨组织工程种子细胞的生物安全性应用要求。     

人GDNF基因的腺病毒载体构建及其在人神经干细胞中的表达

目的：观察构建的含人胶质细胞源性生长因子（GDNF）基因腺病毒载体对人神经干细胞的感染及其基因表达情况,为脊髓损伤的基因及神经干细胞治疗提供前期实验依据。方法：从12周龄流产人新鲜胚胎中提取人神经干细胞并进行培养,行Nestin免疫荧光染色进行检验。将全长558bp编码人GDNF的cDNA克隆到重组腺病毒载体质粒,并在人胚肾细胞（HEK293细胞）中包装出含有目的基因hGDNF的腺病毒,然后用该腺病毒感染人神经干细胞,应用荧光显微镜和Western-blot检测病毒感染及外源基因的表达情况,并进行GFAP和Tubulin免疫荧光染色检测神经干细胞向神经细胞分化情况。结果：Nestin免疫荧光染色显示所培养细胞为阳性染色红色,表明其具有神经干细胞性状;感染48h后观察到神经干细胞中有大量的增强绿色荧光蛋白（EGFP）表达,以及hGDNF蛋白高表达;感染后的神经干细胞有长的伪足伸出,呈GFAP和Tubulin染色阳性,表明促进了神经干细胞向神经元的分化。结论：腺病毒对神经干细胞具有较高感染效率,可作为一种良好的基因导入载体,实现外源基因hGDNF在神经干细胞内的有效表达,并可为神经干细胞分化为神经元提供更有利条件。     

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。     

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.     

Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects

We compared bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) of adult rabbits under identical conditions in terms of their culture characteristics, proliferation capacity, osteogenic differentiation potentials induced by adenovirus-containing bone morphogenetic protein 4 (Ad-BMP4) in vitro, and capacity to repair calvarial defects in the rabbit model by autologous transplantation ex vivo. According to the results of growth curve, cell cycle, and telomerase activity analysis, ADSCs possess a higher proliferation potential. Both of the Ad-BMP4 transduced MSCs expressed BMP4 mRNA and protein and underwent osteogenic differentiation. Up-regulated mRNA expression of all osteogenic genes was observed in differentiated BMSCs and ADSCs, but with different patterns confirmed by real-time RT-PCR. Deposition of calcified extracellular matrix was significantly greater in differentiated ADSCs compared with differentiated BMSCs. X-ray and histological examination indicated significant bone regeneration in the calvarial defects transplanted with Ad-BMP4 transduced autologous MSCs compared to the control groups. There was no significant difference in new bone formation in Ad-BMP4 transduced MSCs based on quantitative digital analysis of histological sections. The use of ADSCs often resulted in the growth of fat tissue structures in the control groups, and the fat tissue structures were not seen with BMSC cells. Our data demonstrate that BMP4 can be potently osteoinductive in vivo, resulting in bone repair. ADSCs may be an attractive alternative to BMSCs for bone tissue engineering under appropriate stimuli. But the easy adipogenic differentiation needs to be considered when choosing adipose tissue for specific clinical application.     

大鼠骨髓间充质干细胞体外成脂分化能力的实验研究

目的:研究大鼠骨髓间充质干细胞体外成脂分化能力与传代次数的关系。方法:用全骨髓培养法,分离骨髓间充质干细胞,加含有地塞米松、胰岛素、3-异丁基-1甲基黄嘌呤、吲哚美辛诱导剂的培养液进行诱导,诱导第10天进行细胞形态学观察和油红O染色鉴定,并进行光密度值测定。结果:随着传代次数增加,油红O染色细胞计数值与光密度值均下降。结论:大鼠MSCs成脂能力随传代次数增加而降低。     

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.     

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。     

人骨髓基质干细胞染色体核型与端粒酶活性的检测分析

目的分析人骨髓基质干细胞作为组织工程种子细胞,在长期体外扩增及大规模培养条件下,其染色体核型及端粒酶活性是否发生异常的改变。方法采用常规细胞染色体G显带处理方法及端粒酶活性PCR-ELISA检测法分析3例人骨髓基质干细胞样本。结果人骨髓基质干细胞体外培养至P10代,未发现染色体核型异常改变,相应的端粒酶活性也未出现异常增高。结论人骨髓基质干细胞在体外长期培养扩增的条件下,染色体核型及端粒酶活性未出现异常改变,符合种子细胞安全性的应用要求。     

Expression of glial cell line-derived neurotrophic factor family members and their receptors in pancreatic cancers

BACKGROUND: The glial cell line-derived neurotrophic factor (GDNF) is a member of neurotrophic polypeptide family, which promotes survival and rescue of various neural cells in the central and peripheral nerve systems. We previously reported that GDNF promotes tumor cell invasion in pancreatic cancer cell lines. The purpose of this study was to investigate GDNF family expression and the status of related receptors in actual cancer tissues, and assess correlations with clinicopathologic behavior. METHODS: Immunohistochemical assessment of GDNF, neurturin, persephin, artemin, GDNF family receptor alpha-1 and alpha-2, and RET was performed for 51 cases of surgically resected pancreatic cancer. RESULTS: In all intrapancreatic nerves, GDNF and artermin were expressed strongly. In pancreatic cancer tissues. The expression of RET was stronger than that seen in normal ductal cells and was significantly related to the survival rate after resection (P = .026) and lymphatic invasion (P = .014). Intrapancreatic neural invasion was significantly related to the expression of GDNF (P = .047). CONCLUSIONS: We conclude that the expression of RET in pancreatic cancer tissues may be a useful prognostic marker and GDNF may play an important role in neural invasion.     

Effects of androgens on telomerase activity in normal and malignant prostate cells in vitro

BACKGROUND: Recent studies have shown that sex hormones regulate telomerase activity in endometrium and breast tissues. The present study was designed to clarify the effects of androgen on telomerase activity in normal and malignant prostate cells. METHODS: Androgen-sensitive (LNCaP) and -independent (TSU-Pr1 and DU145) prostate cancer cell lines and normal prostate cells including basal cells were cultured in the presence or absence of 5alpha-dihydrotestosterone (DHT). RESULTS: Prostate cancer cell lines exhibited high telomerase activity, and normal prostate cells showed low activity. Short or prolonged androgen-deprivation reduced telomerase activity in LNCaP cells, and DHT induced telomerase activity at the G(1) phase of the cell cycle. DHT did not modulate telomerase activity in TSU-Pr1, DU145, and normal cells. CONCLUSIONS: LNCaP cells had an androgen-dependent pathway to activate telomerase, whereas TSU-Pr1 and DU145 cells as well as normal prostate cells had an androgen-independent pathway. These findings suggest that the regulatory mechanism of telomerase varies during the progression of prostate cancers.     

Qtracker体外标记兔成骨诱导分化后细胞的初步研究

目的 探讨Qtracker体外标记兔成骨诱导分化后细胞的特点及可行性.方法 抽取3个月龄健康新西兰大白兔骨髓,贴壁培养骨髓基质干细胞(BMSCs),传至第3代后向成骨细胞诱导,并做鉴定.Qtracker分别以1、2、4、8、16和32 nmol/10~6细胞的浓度标记成骨诱导分化后细胞,分别记为A、B、C、D、E、F组;未标记的细胞作为空白对照组(G组).分别利用荧光显微镜计数和流式细胞术两种方法枪测标记阳性率,台盼蓝拒染法检测标记后细胞存活率,甲基噻唑基四唑(MTT)法观察Qtracker染料对细胞增殖的影响.结果 兔BMSCs经诱导后能向成骨细胞诱导分化.经Qtracker标记后,荧光显微镜下胞浆呈绿色荧光.随着标记浓度的增加,A、B、C、D、E、F组细胞标记阳性率逐渐增高,于8 nmol/10~6细胞的浓度标记时,在荧光显微镜下计数,标记率可达到(93.58±2.08)%;通过流式细胞仪检测,其标记率为(95.24±1.31)%,经两种方法测定,D、E、F组间标记率差异均无统计学意义(P>0.05),且与其他各组比较差异均有统计学意义(P<0.05);G组各时间点标记阳性率均为0.以不同浓度标记后各组细胞存活率均在96%以上,且各组之间差异无统计学意义(P>0.05).标记Qtracker后对细胞的增殖无影响(P>0.05).结论 Qtracker可用于兔成骨诱导分化后细胞的体外标记,在浓度为8 nmol/10~6细胞下可得到最佳的标记率,且其对成骨诱导分化后细胞的增殖无明显影响.     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

补骨脂素诱导BMSCs成骨分化中的LncRNA表达谱分析

目的观察补骨脂诱导骨髓间充质干细胞(BMSCs)成骨分化过程中长链非编码RNA(lncRNA)的表达谱及相关靶基因和信号通路。方法取P2代BMSCs分为三组:空白组(含10%FBS的L-DMEM)、成骨诱导组(含10%FBS及成骨诱导剂的L-DMEM)、补骨脂素组(含10%FBS的L-DMEM及10μmol/L补骨脂素),同一环境不同诱导条件干预21 d;利用Agilent lncRNA芯片筛选出成骨诱导分化过程中表达差异2倍以上的lncRNA,并通过荧光定量PCR对芯片结果进行验证。选取筛选结果中表达差异较大的lncRNA进行GO和KEGG分析。结果成骨诱导分化21 d后持续表达超过2倍的lncRNA共有446个差异表达的lncRNAs。在BMSCs成骨分化过程中,共筛选出5个lncRNA显著上调,4个lncRNA显著下调。其中XR009483上调最显著,而XR007366下调最显著,经PCR验证与芯片结果相符。经GO分析富集度较高的为骨及软骨发育、干细胞分化等生物进程,以及胶原合成、骨化和钙化等生物功能。KEGG分析主要富集于15个生物学通路,其中富集评分较高的是TGF-beta、Wnt、NF-kappa B及Calcium等生物学通路。结论补骨脂素诱导BMSCs成骨分化过程中lncRNA表达谱发生显著变化,提示差异表达的lncRNA可能与BMSCs成骨分化密切相关,为研究补骨脂素促BMSCs成骨分化机制奠定了一定基础。