辐射损伤导致造血干/祖细胞衰老的机理研究

目的 探讨辐射损伤导致骨髓造血干/祖细胞(HSC/HPC)衰老的可能机制.方法 雄性C57BL/6J小鼠随机分为辐照组和假辐照组,辐照组小鼠经6.5 Gy的X射线全身一次性辐照,假辐照组小鼠处理同辐照组,但不辐照.辐照后24 h免疫磁珠分选法分离并计数两组小鼠的Sca-1+造血干/祖细胞细胞(Sca-1+ HSC/HPC),流式细胞术检测细胞周期,β-半乳糖苷酶(SA-β-Gal)染色检测衰老细胞百分率;混合造血祖细胞集落(CFU-Mix)培养观察分选细胞增殖分化能力,单细胞凝胶电泳(SCGE)检测辐照导致细胞的DNA损伤,RT-PCR检测细胞衰老相关基因p16INK4a、p19Arf、p53、p21 Cipl/wafl mRNA的表达;Western blot法检测p16INK4a、p21Cipl/Wafl蛋白表达.结果 免疫磁性分析法分离纯度的Sca-1+ HSC/HPC可达94％,辐照后小鼠每支股骨的Sca-1+ HSC/HPC数量急剧下降,细胞出现G1期阻滞;形成CFU-Mix集落数量和形成集落的细胞数明显降低;SA-β-Gal染色阳性率显著增高,彗星实验显示细胞拖尾明显延长;p16INK4a、p19Arf、p53、p21cipl/wafl mRNA表达明显增强,p16INK4a、p21Cipl/Wafl蛋白的表达水平上调.结论 辐射导致HSC/HPC DNA的损伤和衰老相关生物学改变,p16INK4a-Rb和p19Arf-p53-p21 Cipl/Wafl信号通路可能起一定作用.     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血于/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述.     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血干/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述。     

人参总皂甙体外诱导CD34+造血干/祖细胞增殖的作用

目的：探讨人参总皂甙(TSPG)协同造血生长因子体外诱导CD34~ 造血干/祖细胞(HSC/HPC)增殖的作用。方法：收集人脐血细胞,采用Stemsep TM干细胞分选系统分离纯化CD34~ HSC/HPC,经不同剂量TSPG加入不同造血生长因子组合进行培养,检测细胞总数、CD34~ 细胞及集落形成细胞总数(CFCs)的变化。结果：TSPG 10、20、50和70μg/ml均可不同程度地提高细胞总数、CFCs和CD34~ 细胞扩增倍数,TSPG 50μg/ml是最佳刺激浓度,可使细胞总数、CFCs和CD34~ 细胞分别增至(2470．50±79．96)倍、(53．96±4．29)倍和(21．86士3．09)倍。结论：合适剂量的TSPG能够促进CD34~ 造血干/祖细胞体外增殖。     

氯化锂激活Wnt/β-catenin信号通路致小鼠造血干/祖细胞衰老及其机制

目的探讨氯化锂(Li Cl)激活Wnt/β-catenin信号通路致小鼠Sca-1+造血干/祖细胞(Sca-1+HSC/HPC)衰老及其机制。方法免疫磁珠法分离纯化小鼠Sca-1+HSC/HPC。纯化后细胞分为:正常对照组,常规培养;Li Cl组,正常对照组基础上,加入Li Cl(终浓度10mmol/L);D-半乳糖致衰组,正常对照组基础上,加入D-半乳糖(终浓度166mmol/L),各组培养48h。造血祖细胞混合集落(CFU-Mix)培养检测Sca-1+HSC/HPC多向分化潜能,细胞计数试剂盒-8(CCK-8)检测Sca-1+HSC/HPC增殖能力,衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老,免疫细胞化学染色和Western blotting检测细胞内β-catenin、糖原合成激酶3β(GSK-3β)、P53、P21蛋白表达,ELISA检测细胞胞质内8羟基脱氧鸟苷(8-OH-d G)含量。结果与正常对照组相比,Li Cl组与D-半乳糖致衰组Sca-1+HSC/HPC增殖能力下降,形成CFU-Mix数量下降,SA-β-Gal染色阳性细胞百分率增加,β-catenin、P53、P21蛋白表达上调,GSK-3β蛋白表达下调,细胞内8-OH-d G水平升高。结论 Li Cl可通过激活Wnt/β-catenin信号通路,使细胞DNA氧化损伤,上调P53/P21途径,这可能是导致小鼠造血干/祖细胞衰老的机制之一。     

去乙酰化酶1在人参皂苷Rg1体内正向调控造血干/祖细胞衰老中的作用

目的:探讨去乙酰化酶1(SIRT1)在人参皂苷Rg1体内正向调控造血干/祖细胞(HSC/HPC)衰老中的作用。方法:免疫磁性分选法分离纯化雄性供体小鼠Sca-1~+HSC/HPC连续移植3代构建HSC/HPC衰老体内模型。~(60)Coγ射线致死剂量辐射雌性受体鼠后分4组,照射对照组、衰老模型组、Rg1治疗衰老组和Rg1预防衰老组。衰老相关β-半乳糖苷酶(SA-β-Gal)染色、造血祖细胞混合集落(CFU-Mix)培养和细胞周期分析检测Rg1体内调控Sca-1~+HSC/HPC衰老的作用。荧光定量PCR及免疫印迹检测衰老调控分子SIRT1、核因子-κB(NF-κB)mRNA及蛋白的表达。结果:连续移植后受体鼠Sca-1~+HSC/HPC出现细胞衰老特征,随移植代数的增加,Sca-1~+HSC/HPC G_0/G_1期细胞比例及SA-β-Gal染色阳性率增高,CFU-Mix数量下降。与同代衰老模型组相比,Rg1治疗衰老组及Rg1预防衰老组受体鼠Sca-1~+HSC/HPC G_0/G_1期细胞比例、SA-β-Gal染色阳性率下降,CFU-Mix数量升高;SIRT6 mRNA及蛋白表达上调,NF-κB mRNA及蛋白表达下调;Rg1预防衰老组各指标变化均较Rg1治疗衰老组明显。结论:Rg1可能通过调控SIRT1/NF-κB信号轴发挥其在连续移植过程中抗Sca-1~+HSC/HPC衰老的作用。     

Ad-hLIF转基因饲养层细胞对CD34+造血干/祖细胞增殖和分化的影响

目的 用腺病毒载体介导的人白血病抑制因子基因(Ad-hLIF)感染WI-38人胚肺成纤维细胞制备饲养层细胞,体外观察转基因细胞对CD34+造血干/柑细胞增殖和分化的影响,体内研究对辐射损伤SCID小鼠造血功能恢复的效果.方法 用RT-PCR和ELISA法鉴定Ad-hLIF转基因饲养层细胞目的 基因的表达后,将经免疫磁珠法分离和流式细胞术检测后的CD34+细胞在饲养层和/或细胞因子培养体系中扩增28 d,检测不同时间点的单个核细胞(MNC)数量及CD34+细胞阳性率;扩增后的MNC经CFDA SE荧光标记后移植入辐射损伤SCID小鼠体内,RT-PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的门巢情况.结果 感染50MOI(multiplicity of infection)Ad-hLIF的饲养层细胞均有绿色荧光,RT-PCR和ELISA法结果 显示hLIF目的 基因能在WI-38饲养层细胞中表达,免疫磁珠法分离的CD34+造血干/祖细胞经流式细胞术检测其纯度可达95.60%±2.58%,MNC在Ad-hLIF转基因饲养层培养体系持续扩增,最高可达356.95±0.87倍,其中CD34+细胞仪在0～14 d能维持较高水平,最高可扩增52.11±1.13倍,以后逐渐降低.将其移植辐射损伤SCID小鼠后,可明显提高小鼠存活率,4周内小鼠骨髓中不仅可观察到CFDA SE荧光标记的细胞,而且经RT-PCR法搭定后.还可检测到表达Alu人源基因的人脐血造血归巢细胞.结论 成功建立的Ad-hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34+造血干/祖细胞,并延缓其分化.扩增的CD34+细胞对辐射损伤SCID小鼠具有造血功能恢复的功能.     

造血干/祖细胞与肿瘤的关系

新近研究发现，骨髓衍生的造血干/祖细胞（bone marrow-derived hematopoietic stem/ progenitor cells）可以不断植入肿瘤，参与肿瘤间质和血管生成;持续存在的炎症环境提供了潜在的致癌性条件，使骨髓衍生干细胞可能成为恶性肿瘤起源细胞。VEGFR1+造血祖细胞(vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cell, VEGFR1+ HPC)在肿瘤转移中可能具有决定性作用，肿瘤细胞先突破原发病灶，进入血流，然后停留在另一个预转移场所，在这个场所中，肿瘤细胞先建立造血干祖细胞簇后生长成瘤。     

Ad-hOSM转基因饲养层细胞对脐血CD34~+造血干/祖细胞增殖及归巢能力的影响

目的:建立转人抑瘤素M(hOSM)基因腺病毒载体的饲养层细胞,观察转基因细胞对脐血CD34+造血干/祖细胞扩增的影响,比较扩增前、后造血干/祖细胞体外迁移能力的变化。方法:建立转hOSM基因腺病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术(FCM)检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,FCM检测各组增殖效果;扩增后的造血干/祖细胞用跨膜迁移实验(Transwell实验)检测自发迁移率和SDF-1诱导迁移率以鉴定体外扩增的造血干/祖细胞的归巢能力。结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实有目的基因表达,免疫磁珠法分离的脐血CD34+造血干/祖细胞纯度可达(96.8±2.28)%,与Ad-hOSM转基因饲养层细胞共培养7 d后CD34+造血干/祖细胞可扩增15.73倍,表面黏附分子CXCR4和CD54表达量仍较高,培养后的细胞用Tran-swell板做体外迁移实验,与转基因饲养层细胞共培养的干细胞,其诱导迁移率为(40.68±1.35)%,明显高于对照组,可以较好的保持其归巢能力。结论:转hOSM基因腺病毒载体的饲养层细胞可有效扩增脐血CD34+造血干/祖细胞,延缓其分化,并且体外扩增后仍保持较高的归巢能力。     

类风湿关节炎患者造血干/祖细胞核因子κB1、κB2基因表达的研究

目的检测类风湿关节炎(rheumatoid arthritis,RA)患者外周血CD34+造血干细胞/祖细胞(hematopoietic stem/progenitor cell,HSC/HPC)的核因子(nuclear factorκB,NFκB)κB1、κB2信号分子的基因表达水平,了解NFκB信号途径分子基因表达的异常与临床指标的关联性。方法收集24例RA患者,9例正常对照组,分别抽取50 ml抗凝外周血,淋巴细胞分离液分离单个核细胞,免疫磁珠分选CD34+HSC/HPC,Trizol法提取总RNA,逆转录为cDNA,实时荧光定量聚合酶反应方法检测患者组和对照组NFκB1、NFκB2 mRNA表达水平的差异,并分析RA患者组甲氨蝶呤(Methotrexate,MTX)治疗组和未治疗组NFκB1、NFκB2mRNA表达水平变化,结合其它临床资料进行相关性分析。结果 RA患者组NFκB1 mRNA表达相对量高于正常对照组[6.57(1.08~76.71),2.14(0.68~7.09),P=0.013];NFκB2 mRNA表达与正常对照组无统计学差异。RA患者外周血CD34+HSC/HPCNFκB1、NFκB2 mRNA表达与有无MTX的治疗无统计学差异,与疾病活动关节评分28(disease activity score 28,DAS28评分)、血沉、C反应蛋白等临床指标无相关性。结论 RA患者外周血CD34+HSC/HPC NFκB1基因表达水平比正常对照增高,可能是RA患者CD34+细胞重要缺陷。     

小鼠p16INK4a基因外显子1α胚胎干细胞条件打靶研究

目的 研究打靶载体的结构与打靶效率的关系，探讨小鼠p16^INK4a基因在活体水平上抑制种瘤发生和发展的功能。方法 利用筛选基因组文库得到的小鼠p16^INK4a基因组DNA片段，设计并构建了针对小鼠p16^INK4a基因外显子1α的条件打靶载体，其短臂为2.0kb EcoR Ⅰ/Xba Ⅰ片段，长臂为5.9kb Spe Ⅰ/Not Ⅰ片段，上游交叉位点（locus of crossing-over,loxP）位于外显子1α起始密码上游240bp处，下游loxP位于外显子1α起始密码下游1633bp处，经重组酶（Cre）介导后可将外显子1α和选择标记Neo基因同时删除。结果 将此条件打靶通过电穿孔转导小鼠胚胎干细胞，获得24个药物抗性克隆，其中1个经Southern杂交证实为正确同源重组克隆。结论 在同源臂两侧各用一个TK基因可以获得较高的正确同源重组率。     

Frequency of P16INK4a and P14ARF Genes Methylation and Its Impact on Bladder Cancer Cases in North Indian Population

Introduction: Amongst the genitourinary cancers, carcinoma of the urinary bladder is one of the leading causes of death in India. Hypermethylation of the CpG islands of gene promoter is one of the earliest and most frequent epigenetic alterations leading to cancer as well as in its development. Several studies have suggested that tumour suppressor genes play a key role in the development of cancer. Methylation in the CDKN2A has been associated with various malignant diseases, but information with respect to urinary bladder cancer is lacking in north Indian population. Materials and methods: We analyzed the methylation of P16INK4a and P14ARF in 80 tissues and matched blood samples of patients suffering from bladder cancer and 80 blood samples of cancer-free individuals by MS-PCR. Results: In tissue and matched blood samples of bladder cancer patients, the incidence of P14ARF hypermethylation significantly increased (OR = 0.31, 95%CI = 0.12–0.8, P = 0.01) and (OR = 0.0, 95%CI=0.0–0.62, P = 0.006) respectively with an increase in age. Clinicopathological analysis revealed that P14ARF hypermethylation in tissue and blood samples was significantly associated with invasive stage (≥ T2) (OR = 0.21, 95%CI = 0.08–0.51, P = 0.0002) and (OR = 0.09, 95%CI = 0.03–0.37, P = 0.00001) respectively. Muscle invasive tumour stage (≥T2) showed significant association with increased risk of P16INK4α promoter hypermethylation in tissue and blood samples of patients (OR = 0.38, 95%CI = 0.17–0.82, P = 0.01) and (OR = 0.13, 95%CI = 0.05–0.36, P = 0.00005) respectively. Conclusion: These results suggest that the CpG island hypermethylation status of the defined panel of genes may be a useful biomarker in patients suffering from bladder cancer.     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

细胞衰老与p16INK4a的转录调控

抑癌基因p16~(INK4a)可特异地抑制CDK4及CDK6,在抑制细胞生长、促进细胞衰老等方面发挥重要的生物学作用。由于p16~(INK4a)功能的重要性,近年来,针对p16~(INK4a)转录调控方面的研究取得了一系列进展,发现了一系列正性和负性调控元件和转录调控因子,如:E47、Id1、Jun B、Bmi-1、RREB等,为进一步认识细胞增殖规律以及衰老进程具有重要的理论意义。     

P16INK4a overexpression and survival in osteosarcoma patients: a meta analysis

Osteosarcoma is one of the most common primary bone malignancies. Although there is a significant improvement of survival on osteosarcoma patients in the past decades, treatment of osteosarcoma is still unsatisfactory for the development of pulmonary metastasis. The potential prognostic value of p16^INK4a in osteosarcoma has been investigated, however, the results from different studies were somewhat controversial. To elucidate whether p16^INK4a is indeed a prognostic factor of osteosarcoma, we conducted a meta-analysis of the published literatures to provide a comprehensive evaluation of the significance of p16^INK4a expression in patients with osteosarcoma. Eight studies with a total of 354 patients with osteosarcoma were examined. The pooled odds ratio (OR) with corresponding 95% confidence interval (95% CI) was calculated to evaluate the effect of p16^INK4a expression on overall survival. Meta-analysis showed that patients with high p16^INK4a expression were significantly associated with favourable overall survival when compared to their counterparts with low or undetectable p16^INK4a expression (OR = 0.270, 95% CI 0.162-0.451, P < 0.001). Sensitivity analysis suggested the pooled OR was stable and not significantly changed when a single study was removed. In conclusion, the results from this meta-analysis highlight that p16^INK4a is an effective biomarker of survival in patients with osteosarcoma.     

Comparison of p16(INK4a) expression with p53 alterations in head and neck cancer by tissue microarray analysis

We investigated whether there is a relationship between loss of p16(INK4a) protein expression and p53 alterations in head and neck squamous cell carcinomas (HNSCCs). For this purpose, immunohistochemistry was performed on tissue microarrays of 664 tumours; this represents the largest HNSCC cohort studied for molecular biomarkers. Loss of p16(INK4a) protein expression was associated with aberrant p53 expression (negative or overexpressed) in the total cohort, and with TP53 mutations in 200 tumours analysed (p < 0.0001 each). Both loss of p16(INK4a) expression and p53 alterations differed significantly across both tumour sites and stages, being more prevalent in the hypopharynx than in the other tumour sites and in advanced tumour stages. As a possible link between p53 status and p16(INK4a) loss, we found that increased DNA methyltransferase 1 protein levels occurred preferentially in tumours with aberrant p53 (p = 0.001) and negative p16(INK4a) expression (p = 0.0004). In the total cohort, there was a borderline significant difference in patient survival across three p16(INK4a) expression levels (negative, positive, high), with loss of p16(INK4a) expression showing shortest survival. It is suggested that loss of p16(INK4a) expression and p53 alterations should be viewed as related events involved in the early carcinogenic process.     

An analysis on the combination expression of HPV L1 capsid protein and p16INK4a in cervical lesions

Human papillomavirus (HPV) infection in cervix is the most important reason for cervical cancer, but only 2% cervical HPV infection will develop into cervical cancer. So how to identify patients at risk of progressive cervical lesions from those infected with HPV to avoid over treatment is a big issue in clinic. The aims of this study were to detect the expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and to investigate the combination expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and its diagnostic efficiency in clinic. Immunochemical method was used to detect the expression of HPV L1 capsid protein and p16^INK4a in 169 cases of abnormal cytology. Histopathologic test was performed to identify cervical lesions of all the cases. χ² test and spearman's rank correlation were used for statistical analysis. The diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), accuracy, and the area under the receive operating characteristic (ROC) curve (denoted by A_Z) were calculated with SPSS 13.0. All the statistical tests were two sided at the 5% level of significance. L1 expression decreased (P < 0.001), but p16^INK4a expression increased (P < 0.001) with histopathologic diagnosis increasing. The expression rates of HPV L1 capsid protein, p16^INK4a, and L1(?)/p16(+) in cervical intraepithelial neoplasia (CIN)2, CIN3, and squamous‐cell carcinoma were statistically different from those in CIN1 (P < 0.001). The expressions of HPV L1 capsid protein, L1(+)/p16(+), L1(+)/p16(?), and L1(?)/p16(?) were negatively correlated with the severity of cervical lesions (P < 0.001), whereas the expressions of p16^INK4a and L1(?)/p16(+) were positively correlated with the severity of cervical lesions (P < 0.001). The specificity and A_Z of combining L1 with p16 ^INK4a were statistically higher than L1 or p16 ^INK4a alone (P < 0.05). L1 and p16^INK4a are useful biomarkers for the early diagnosis of cervical lesions. The combination of L1 and p16^INK4a has a higher diagnostic accuracy than L1 or p16^INK4a alone in diagnosis of cervical lesions. Diagn. Cytopathol. 2010;38:573–578. © 2009 Wiley‐Liss, Inc.     

Mechanisms of inactivation of the p16INK4a gene in leiomyosarcoma of soft tissue: decreased p16 expression correlates with promoter methylation and poor prognosis

The p16INK4a tumour suppressor gene, encoding p16 protein, plays a crucial role in regulation of the G1 cell-cycle phase. To investigate the potential role of p16 in soft tissue leiomyosarcoma (LMS), an immunohistochemical analysis was performed of 77 LMSs for p16 expression. Decreased expression of the p16 protein was identified in 25 of 77 LMSs (32%). Decreased expression of p16 correlated significantly with large tumour size (p=0.0038). In a univariate analysis, large tumour size and decreased expression of p16 were statistically significant adverse prognostic factors (p=0.025 and p=0.0021, respectively). In a multivariate analysis including conventional clinicopathological parameters, decreased expression of p16 protein was revealed as the only independent unfavourable prognostic factor (p=0.012). To elucidate the mechanisms of inactivation of the p16INK4a gene, 49 LMSs for which genomic DNA was available were examined; analysis for homozygous deletion, mutation, and promoter hypermethylation was conducted using differential PCR, PCR-SSCP, and methylation-specific PCR, respectively. Promoter hypermethylation was detected in 11 of 49 LMS cases (22%); homozygous deletion was detected in 3 of 49 cases (6%); and mutation was not recognized in any of the cases studied. Eight of 15 cases (53%) with decreased expression of p16 protein revealed methylation of the p16INK4a gene promoter. Promoter hypermethylation correlated closely with decreased expression and poor prognosis (p=0.0014 and p=0.0088, respectively). These results suggest that decreased expression of p16 protein can be considered as an independent reliable prognostic parameter in patients with soft tissue LMS. Furthermore, promoter methylation was more frequent than either homozygous deletion or mutation in this tumour, and promoter methylation was also shown to have a strong association with inactivation of the p16INK4a gene.