人胎肝间充质干细胞体外分离培养和生物学鉴定

目的建立人胎儿肝脏非实质细胞间充质干细胞(nonparenchymalmesenchymalstemcells,NPMSC)的分离、培养方法,观察NPMSC形态学、细胞生物学特性及细胞表型特征,以获得大量人源性间充质干细胞(MSC)。方法采用体外细胞培养技术分离培养人胎儿NPMSC,用显微摄像、MTT和图像分析研究其增殖及生长特征,用流式细胞仪和免疫组化法鉴定其表型。结果每个胎儿肝脏可获得10000±2000个贴壁细胞,可见5～10个高速增生的细胞集落。原代和传代培养均生长缓慢,按1∶3传代,传1代需8～10d,细胞经传代后逐渐纯化,传10代后可达109个细胞。流式细胞仪检测NPMSC不表达CD34,而表达CD166。对传代NPMSC进行人甲胎蛋白和白蛋白免疫组化分析,细胞表达微量甲胎蛋白,不表达白蛋白。结论肝非实质细胞中含有MSC,且量较大,可成为种子细胞。NPMSC在普通培养条件下不向肝细胞分化。     

大鼠肝癌干细胞生物学行为研究

目的研究大鼠肝癌干细胞生物学行为。方法二乙基亚硝胺为诱癌剂制造大鼠肝癌模型，分离培养肿瘤细胞，按照卵圆细胞表面标记物[CD34、c—Kit、Thy—1、甲胎蛋白（AFP）、细胞角蛋白（CK）7、CK8、CK14、CK18、CK19和Y-谷氨酰转肽酶]分选肿瘤细胞，比较各个表面标记物阳性肿瘤细胞亚群与阴性肿瘤细胞亚群移植裸鼠后成瘤能力的差异，找出成瘤能力差异大的亚群细胞组，比较成瘤能力强的细胞与一般细胞的生长特征，并对其细胞周期分布进行研究。结果Thy—1阳性细胞、CK7阴性细胞与AFP阳性细胞成瘤能力明显大于对应细胞亚群（P〈0．01），具备初步的肝癌干细胞特征，这三种细胞比对应亚群细胞具有较低的增殖能力、更长的倍增时间与更小的最大增生倍数，细胞周期检测发现，无论是S期比例，还是（S＋G2／M）期比例，这三种细胞也较对应亚群细胞为低，说明其体外增殖能力较弱。结论CK7阴性、Thy—1阳性和AFP阳性细胞具备大鼠肝癌干细胞的初步特征；大鼠肝癌干细胞可能具备增殖能力较低的生物学行为特征。     

Clinicopathological correlation of radiologic measurement of post-therapy tumor size and tumor volume for pancreatic ductal adenocarcinoma

ObjectivesTumor size measurement is critical for accurate tumor staging in patients with pancreatic ductal adenocarcinoma (PDAC). However, accurate tumor size measurement is challenging in patients who received neoadjuvant therapy before resection, due to treatment-induced fibrosis and tumor invasion beyond the grossly identified tumor area. In this study, we evaluated the correlation between the tumor size and tumor volume measured on post-therapy computed tomography (CT) scans and the pathological measurement. Also, we investigated the correlation between these measurements and clinicopathological parameters and survival.Materials and methodsRetrospectively, we evaluated 343 patients with PDAC who received neoadjuvant therapy, followed by pancreaticoduodenectomy and had pre-operative pancreatic protocol CT imaging. We measured the longest tumor diameter (RadL) and the radiological tumor volume (RadV) on the post-therapy CT scan, then we categorized RadL into four radiologic tumor stages (RTS) based on the current AJCC staging (8th edition) protocol and RadV based on the median. Pearson correlation or Spearman’s coefficient (δ), T-test and ANOVA was used to test the correlation between the radiological and pathological measurement. Chi-square analysis was used to test the correlation with the tumor pathological response, lymph-node metastasis and margin status and Kaplan-Meier and Cox-proportional hazard for survival analysis. P-value < 0.05 was considered significant.ResultsAs a continuous variable, RadL showed a positive linear correlation with the post-therapy pathologic tumor size in the overall patient population (Pearson correlation coefficient: 0.72, P < 0.001) and RadV (δ: 0.63, p < 0.0001). However, there was no correlation between RadL and pathologic tumor size in patients with ypT0 and those with pathologic tumor size of ≤1.0 cm. Post-therapy RTS and RadV group correlated with ypT stage, tumor response grades using either CAP or MDA grading system, distance of superior mesenteric artery margin and tumor recurrence/metastasis.ConclusionAlthough RadL tends to understage ypT in PDAC patients who had no radiologically detectable tumor or small tumors (RTS0 or RTS1), radiologic measurement of post-therapy tumor size may be used as a marker for the pathologic tumor staging and tumor response to neoadjuvant therapy.     

人胎儿骨髓间充质干细胞的分离、培养及生物学特性鉴定

目的 建立人胎儿骨髓间充质干细胞(MMSCs)的分离、培养方法,观察MMSCs形态学、细胞生物学及细胞表型,以获得大量人源性MMSCs。 方法 收集12个人胎儿的骨髓,采用细胞培养技术,分离培养人胎儿MMSCs,用显微摄像、四甲基偶氮唑盐(MTT)和图像分析研究其增殖及生长特征,用流式细胞仪和免疫细胞化学鉴定其表型。 结果 0.5~2 h内尽快分离骨髓细胞,24 h换液后可获得约(300±8.0)个贴壁细胞,5个细胞以上的集落为(15±6)个;培养细胞在种植后1~3d为生长滞留期,第4天达到对数生长期,以后进入平台期;细胞分裂指数曲线的趋势与生长曲线基本类似,在达到对数生长期后,分裂相细胞明显减少。原代及传代培养显示,细胞分裂旺盛,可观察到干细胞特有的不均等分裂,5~7 d可传1代,连续传10代后,每个人胎儿来源MMSCs可扩增达1011-1012个细胞。MMSCs表型为CD166阳性,CD34阴性。对传代MMSCs进行人甲胎蛋白、白蛋白和细胞角蛋白18免疫细胞化学分析,细胞除表达微量甲胎蛋白外,不表达白蛋白和细胞角蛋白18。 结论 人胎儿MMSCs分离成分单纯,容易成功,增殖旺盛,在普通培养条件下不向肝细胞分化。MMSCs作为组织工程重建的种子细胞具有较强的可行性。     

大鼠胰腺导管来源干细胞向胰岛素分泌细胞分化的体外培养

目的:研究在体外条件下从大鼠胰腺导管分离的干细胞向胰岛素分泌细胞分化的产物细胞的形态、表型及功能.方法:采用胶原酶原位消化法消化大鼠胰腺,差异贴壁法培养出胰腺导管来源千细胞(PDSCs),对其进行形态学与表型鉴定.采用无血清培养基,添加Matrigel、exendin-4诱导干细胞向胰岛素分泌细胞分化,鉴定产物细胞的形...     

人胰腺癌细胞系SW1990中肿瘤干细胞样侧群细胞的分离及生物学特性研究

目的 从人胰腺癌细胞系SW1990中分离肿瘤干细胞样侧群(side population,SP)细胞,并进一步研究其生物学特性.方法 细胞常规培养后应用Hoechst 33342和PI染色、荧光激发流式细胞仪检测并分选SP细胞,MTT法检测细胞活性,流式细胞仪分析干细胞标志物CD133的表达,克隆平板测定细胞克隆形成能力,Western blot检测细胞ABC超家族成员G2(ABCG2)蛋白表达.结果 人胰腺癌细胞系SW1990中SP细胞比例为2.70%,可完全被维拉帕米阻断.培养9 d后,SP细胞的A492值为2.1,克隆形成率为(38.7±6.8)%,CD133阳性率为69.63%,均显著高于非SP细胞的0.5,(15.5±2.8)%,16.71%;SP细胞ABCG2表达也较非SP细胞强.结论 胰腺癌细胞系SW1990存在SP细胞.     

放化疗抵抗的胰腺癌细胞中干细胞标志物的表达

目的 观察放化疗抵抗的胰腺癌细胞的Bcl-2、survivin及干细胞标志物Oct-4、ABCG2蛋白表达的变化,探讨这部分肿瘤细胞耐受放化疗的原因.方法 采用同步放化疗干预人胰腺癌细胞系SW1990、BxPC3、pc3、jr305,获得放化疗抵抗的胰腺癌细胞,以不行放化疗处理的细胞作为对照.采用Western blotting检测它们的Bcl-2、survivin、Oct-4和ABCG2蛋白的表达变化.结果 放化疗抵抗的SW1990、BxPC3、pc3、jt305胰腺癌细胞Bcl-2蛋白表达量分别为0.7955±0.0326、0.5718±0.0212、0.6137±0.0382和0.8733±0.0461;survivin蛋白表达量分别为0.8207±0.0490、0.6973±0.021 1、0.7967±0.0346和0.8013±0.0398;Oct-4蛋白表达量分别为0.8728±0.0177、0.7861±0.0139、0.4794±0.0932和0.4216±0.1043;ABCG2蛋白表达量分别为0.7810±0.1370、0.4957±0.1126、0.6102±0.1358和0.4670±0.1274.对照组相应细胞系的4种蛋白表达量分别为0.4723±0.018、0.2954±0.0103、0.3587±0.0201和0.2718±0.0136;0.4717±0.0274、0.3587±0.0113、0.3891±0.0147和0.3326±0.0124;0.6053±0.0142、0.4236±0.0086、0.2385±0.0671和0.1985±0.0582;0.3156±0.0582、0.2360±0.0423、0.2813±0.0512和0.1808±0.0370.放化疗后胰腺癌细胞的4种蛋白表达量均显著增加(P＜0.05).结论 放化疗抵抗的胰腺癌细胞中可能富含肿瘤干细胞.     

MiR-590-3p在胰腺癌干细胞中的表达

目的 应用无血清培养基分离胰腺癌干细胞,检测其miR-590-3p的表达。方法 运用无血清培养基克隆培养ASPC-1、PANC1细胞,检测其单克隆形成、分化及细胞周期、半数抑制浓度(IC50)和表面标记物CD24+、CD44+表达。实时定量PCR法检测细胞miR-590-3p的表达。结果 经无血清培养基培养,(0.94±0.53)％的ASPC-1细胞和(0.57±0.12)％的PANC1细胞能存活,呈克隆球样悬浮生长,并可以在体外连续传代。加入血清后细胞球又重新贴壁生长。ASPC-1细胞球G0/G1期比例和CD24+、C44+、CD24+ CD44+的细胞比例及IC50分别为(75.3±5.4)％、0.96％～2.01％、27.52％～34.47％、0.35％ ～0.44％和(224.37±5.71) μg/ml,均显著高于亲本细胞的(43.7±3.8)％、0.38％～0.42％、17.65％ ～ 18.25％、0.05％～ 0.08％、(11.43±2.10) μg/ml(P值均＜0.05)。PANC1细胞球G0/G1期比例和CD24+、CD44+、CD24 +CD44+的细胞比例及IC50分别为(80.1±4.7)％、5.31％ ～9.84％、72.05％ ～93.06％、4.91％ ～5.21％、(296.58±4.27) μg/ml,均显著高于亲本细胞的(46.1±5.3)％、4.09％～4.97％、47.71％～55.66％、1.48％ ～2.63％、(26.17±3.81) μg/ml(P值均＜0.05)。ASPC-1、PANC1细胞球miR-590-3p表达分别是亲本细胞的4.67和4.52倍。结论 应用无血清培养基可以从ASPC-1、PANC1细胞系中分离出具有干细胞特性的胰腺癌细胞球,其miR-590-3p表达上调,该基因可能是胰腺癌干细胞特性维持的关键基因。     

胚胎干细胞标志物Oct-4在胰腺癌的表达

目的分析胚胎干细胞标志物Oct-4在胰腺癌组织和胰腺癌细胞株中的表达状况。方法应用RT-PCR检测Oct-4mRNA在25例胰腺癌组织以及胰腺癌细胞株SW1990、PC3、JF305和BxPC3中的表达;应用Westernblot法检测胰腺癌组织及胰腺癌细胞株中Oct-4蛋白表达;应用异硫氰荧光素(FITC)标记的流式细胞仪分选法检测Oct-4在胰腺癌细胞株中的表达率。结果4株胰腺癌细胞株均表达Oct-4mRNA和Oct-4蛋白。88%(22/25)胰腺癌组织高表达Oct-4mRNA和Oct-4蛋白,其表达率与胰腺癌的分化程度无关。Oct-4在SW1990的表达率为50.21%、BxPC3为68.53%、PC3为66.27%、JF305为67.61%。结论Oct-4异常表达可能与胰腺癌始发有关,胰腺癌可能来源于胰腺干细胞。     

胰腺癌肿瘤干细胞的悬浮培养法

目的 建立无血清悬浮培养分离人胰腺癌细胞系PANC1干细胞球的方法.方法 采用无血清悬浮法培养PANC1细胞,显微镜下观察干细胞球形成率;流式细胞仪检测细胞CD133表达和细胞周期;以含10%FBS培养基诱导干细胞球细胞分化,荧光显微镜下观察细胞形态及CK18的表达;干细胞球细胞接种NOD/SCID小鼠皮下,观察其成瘤能力.结果 在无血清悬浮培养条件下存活的PANC1细胞形成干细胞球,体外连续传代培养20代始终保持4%0～5%0的干细胞球形成率.干细胞球细胞CD133表达率(5.91±0.7)%,G0/G1期细胞占(80.99±2.60)%,与原代PANC1细胞的(1.44±0.52)%和(69.01±5.03)%相差显著(P＜0.05).将于细胞球细胞置含血清培养基中培养.细胞逐渐恢复原代细胞形态,并表达上皮标志蛋白CK18,2×103的干细胞即在NOD/SCID小鼠皮下成瘤.结论 无血清悬浮法培养PANC1细胞可分离出肿瘤干细胞,其具有自我更新、多向分化和成瘤能力.     

胰腺干细胞的分子标志物

目前,人们已越来越认识到胰腺干细胞的重要性.其中,对胰腺干细胞研究最多的是,用胰岛细胞移植治疗糖尿病,对于胰腺干细胞治疗各种胰腺损伤、胰腺炎、胰腺癌的研究仍在进行当中.对胰腺干细胞分子标志物的掌握是胰腺干细胞治疗研究的基础和重点,但目前缺乏唯一的干细胞标志物和可信的评价指标.此文将介绍干细胞、目前已知的胰腺干细胞分子标志物及其现状,如目前关注较多的PDX1、巢素蛋白、神经元素3、细胞角蛋白、波形蛋白、Notch信号及其他的分子标志物及其现状.     

胰腺癌干细胞的研究进展

胰腺癌是恶性程度极高的消化道肿瘤,是全球癌症相关性死亡的主要原因之一.由于发病机制尚不明确,早期缺乏特征性临床表现及诊断方法,且传统治疗效果欠佳,导致胰腺癌的发病率及死亡率居高不下.近年来,随着干细胞在多个领域研究的不断推进,胰腺癌干细胞(pancreatic cancer stem cells,PCSCs)在胰腺癌发...     

人脂肪间充质干细胞体外分离、培养的方法研究

目的：探讨人脂肪间充质干细胞体外分离及培养方法。方法用消化离心的方法获得人脂肪间充质干细胞,以1×104/cm2接种于体积分数为0．1％胎牛血清的LG-DMEM培养基中,并进行细胞形态学观察,绘制细胞生长曲线,流式细胞仪检测细胞表面抗原,行成脂及成骨诱导检测其多向分化潜能。结果获取的脂肪间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,细胞增殖良好;细胞生长曲线测定表明接种后第4天细胞进入指数增长期,第8天后生长进入平台期,体外能长期培养存活,并保持不分化状态;流式细胞仪检测表明细胞高表达 CD13、CD73,低表达 CD34、CD45及 HLA-DR;能诱导成脂及成骨细胞。结论利用消化离心法在体外能分离得到脂肪间充质干细胞,细胞生长良好,可作为组织工程的种子细胞。     

阻断Hedgehog信号通路对人胰腺癌干细胞自我更新的影响

目的 观察Hedgehog信号通路特异阻断剂环巴明对胰腺癌干细胞自我更新的影响.方法 应用0.5、1、2、5、10 μmol/L环巴明处理胰腺癌PANC1干细胞、PANC1贴壁细胞和永生化胰腺导管上皮H6C7细胞24、48、72 h.采用实时RT-PCR法检测细胞Smo及Gli1 mRNA表达;CCK-8法检测细胞增殖抑制;流式细胞仪检测细胞周期及凋亡.结果 10 μmol/L环巴明处理72 h后,PANC1干细胞、PANC1细胞和H6C7细胞的Smo mRNA表达量分别为1、0.83、2.61;Glil mRNA为57.27、26.35、1;生长抑制率分别为(37.85±13.69)%、(8.53±4.43)%、(43.55±28.98)%.与PANC1细胞比较,PANC1干细胞的Smo、Gli1 mRNA表达显著增加,生长抑制率亦显著增强(P值＜0.05或＜0.01).经10 μmol/L环巴明处理72 h,PANC1干细胞G1期比例从(67.41±6.35)%显著降至(36.53±6.03)%(P＜0.05),细胞凋亡率从(10.95±5.68)%降至(5.73±1.42)%(P＞0.05);PANC1细胞G1期比例从(67.64±6.88)%显著降至(53.13±1.10)%(P＜0.05),细胞凋亡率从(12.08±4.12)%降至(5.66±1.33)%(P＞0.05);而H6C7细胞的G1期比例及凋亡无明显变化.结论 环巴明阻断Hedgehog信号通路可抑制PANC1干细胞增殖,其机制可能与细胞凋亡无关.     

Circulating tumor cells in pancreatic cancer patients: Enrichment and cultivation

AIM:To investigate the feasibility of separation and cultivation of circulating tumor cells(CTCs)in pancreatic cancer(Pa C)using a filtration device.METHODS:In total,24 Pa C patients who were candidates for surgical treatment were enrolled into the study.Peripheral blood samples were collected before an indicated surgery.For each patient,approximately8 m L of venous blood was drawn from the antecubitalveins.A new size-based separation Meta Cell?technology was used for enrichment and cultivation of CTCs in vitro.(Separated CTCs were cultured on a membrane in FBS enriched RPMI media and observed by inverted microscope.The cultured cells were analyzed by means of histochemistry and immunohistochemistry using the specific antibodies to identify the cell origin.RESULTS:CTCs were detected in 16 patients(66.7%)of the 24 evaluable patients.The CTC positivity did not reflect the disease stage,tumor size,or lymph node involvement.The same percentage of CTC positivity was observed in the metastatic and non-metastatic patients(66.7%vs 66.7%).We report a successful isolation of CTCs in Pa C patients capturing proliferating cells.The cells were captured by a capillary action driven size-based filtration approach that enabled cells cultures from the viable CTCs to be unaffected by any antibodies or lysing solutions.The captured cancer cells displayed plasticity which enabled some cells to invade the separating membrane.Further,the cancer cells in the“bottom fraction”,may represent a more invasive CTC-fraction.The CTCs were cultured in vitro for further downstream applications.CONCLUSION:The presented size-based filtration method enables culture of CTCs in vitro for possible downstream applications.     

大鼠胰星状细胞的分离与培养

目的　建立大鼠胰星状细胞 (pancreatic stellate cells,PSCs)的分离培养方法。方法　大鼠胰腺组织经胶原酶和链霉蛋白酶 E消化后 ,用 Nycodenz不连续密度梯度离心法分离 PSC,在 32 8nm紫外光激发下观察细胞的自发荧光现象 ,并以免疫组化技术检测结蛋白 (desmin)、神经胶质原纤维酸性蛋白 (glial fibrillary acidic protein,GFAP)和α-平滑肌动蛋白 (α- sm ooth m uscle actin,α- SMA )的表达 ,同时观察培养细胞的形态和生长特性。结果　新鲜分离的大鼠 PSCs产率、活力和纯度分别约为 2 .5 ×10 6 /g胰腺、95 %和 90 %。培养的 PSCs可自发活化 ,表达 α- SMA,细胞由静止型转化为肌成纤维样细胞表型。原代培养 10天后细胞纯度 >95 % ,传代培养后细胞纯度可达 99%以上。结论　利用 Nycodenz密度梯度离心方法可成功分离大鼠 PSCs,其细胞产率、活力和纯度均可满足体外研究需要。     

Cancer stem cells: Involvement in pancreatic cancer pathogenesis and perspectives on cancer therapeutics

Pancreatic cancer is one of the most aggressive and lethal malignancies. Despite remarkable progress in understanding pancreatic carcinogenesis at the molecular level, as well as progress in new therapeutic approaches, pancreatic cancer remains a disease with a dismal prognosis. Among the mechanisms responsible for drug resistance, the most relevant are changes in individual genes or signaling pathways and the presence of highly resistant cancer stem cells (CSCs). In pancreatic cancer, CSCs represent 0.2%-0.8% of pancreatic cancer cells and are considered to be responsible for tumor growth, invasion, metastasis and recurrence. CSCs have been extensively studied as of late to identify specific surface markers to ensure reliable sorting and for signaling pathways identified to play a pivotal role in CSC self-renewal. Involvement of CSCs in pancreatic cancer pathogenesis has also highlighted these cells as the preferential targets for therapy. The present review is an update of the results in two main fields of research in pancreatic cancer, pathogenesis and therapy, focused on the narrow perspective of CSCs.     

从胎儿胰腺组织建立单克隆胰腺前体细胞的研究

目的 探索一种新的人胰腺干细胞分离纯化体系。方法 从胎儿胰腺中分离获得巢蛋白阳性细胞群体，荧光激活细胞分选(FACS)技术分选单个side population(SP)细胞后进行单克隆细胞培养。应用逆转录-聚合酶链反应(RT-PCR)及放射免疫分析(RIA)方法，研究单克隆SP细胞在体外增殖和向胰岛内分泌细胞分化的能力。结果 胎儿胰腺组织经过分离和体外培养后，可获得巢蛋白阳性细胞，对其进行Htoechst 33342染色，分析显示SP细胞约占0．1％；RT-PCR证实SP细胞有ATP结合盒转运子G2(ABCG2)表达，而非SP细胞则未见表达；SP细胞的克隆形成率约为2．7％，而非SP细胞没有克隆形成。在多种细胞因子和无血清的条件下，单克隆SP细胞经诱导后出现胰岛素、胰升糖素和胰十二指肠同源盒基因-1(PDX-1)mRNA的表达，而巢蛋白、neurogenin3 (Ngn3)和ABCG2 mRNA表达消失；RIA分析可检测到诱导后的细胞内有胰岛素产生。结论 首次报道从胎儿胰腺组织中分离并建立了单克隆胰腺前体细胞。该细胞在体外具有很强的增殖能力，并可分化为胰岛内分泌细胞。     

Functions of the CXC ligand family in the pancreatic tumor microenvironment

Therapeutic resistance is the major contributor to the poor prognosis of and low survival from pancreatic cancer (PC). Cancer progression is a complex process reliant on interactions between the tumor and the tumor microenvironment (TME). Members of the CXCL family of chemokines are present in the pancreatic TME and seem to play a vital role in regulating PC progression. As pancreatic tumors interact with the TME and with PC stem cells (CSCs), determining the roles of specific members of the CXCL family is vital to the development of improved therapies. This review highlights the roles of selected CXCLs in the interactions between pancreatic tumor and its stroma, and in CSC phenotypes, which can be used to identify potential treatment targets.     

EGFR及ADAM9在胰腺癌干细胞与分化细胞中的表达及意义

目的 通过微球体培养富集胰腺癌干细胞,检测其表皮生长因子受体(EGFR)和去整合素-金属蛋白酶9(ADAM9)的表达,并探讨其意义.方法 运用无血清条件培养基悬浮培养胰腺癌PANC1细胞,并连续传代培养.将部分微球体接种于含血清及胶原底物的培养基中进行分化诱导.收集微球体细胞及分化细胞,流式细胞仪检测侧群(SP)细胞比例;实时PCR及蛋白质印迹法检测细胞EGFR、ADAM9 mRNA和蛋白的表达.结果 成功培养出胰腺癌PANC1细胞微球体,并能在体外连续传代.微球体细胞分化诱导后能重新贴壁生长.微球体细胞和分化细胞中SP细胞比例分别为(5.40±0.38)%和(2.80±0.42)%,两者差异具有统计学意义(P＜0.05).微球体细胞与分化细胞相比,EGFR及ADAM9 mRNA表达分别上调约2.5和3.0倍(P＜0.05);微球体细胞EGFR及ADAM9蛋白相对表达量分别为0.90±0.09和0.64±0.07,显著高于分化细胞的0.62±0.11和0.48±0.09(P＜0.05).结论 微球体培养能富集胰腺癌干细胞,ADAM9可能通过EGFR信号通路在胰腺癌的发生、发展中起重要作用.