Telomerase activity in hybrids between telomerase-negative and telomerase-positive immortal human cells is repressed in the different complementation groups but not in the same complementation group of immortality

The expression of telomerase is essential for cells to be immortalized, and most immortal cell lines possessed telomerase activity. Using the cell fusion technique, it has been shown that mortal and telomerase-negative phenotypes of normal cells are dominant over immortal and telomerase-positive phenotypes, suggesting that the normal cells possessed dominant repressor-type activity for telomerase expression. Several telomerase-negative immortal human cell lines were reported, in which telomerase-independent mechanisms was supposed to maintain telomere length. We aimed at seeing whether the telomerase-negative phenotype of these immortal cells is dominant over telomerase-positive phenotype of other immortal cells in correlation with cellular mortality. Results showed that, when telomerase-positive and -negative immortal parental cell lines belonging to the different complementation groups were fused, telomerase-negative mortal hybrid clones arose, i.e. telomerase-negative phenotype was dominant as well as mortal phenotype. However, when immortal hybrid cells arose from telomerase-positive and -negative immortal parents belonging to either the same or different complementation groups, they were all telomerase-positive, i.e. telomerase-negative phenotype appeared to be recessive. Telomerase-negative immortal hybrid was never established from any combinations between telomerase-negative and -positive immortal parental cells.     

中国白裤瑶永生细胞库的建立

目的建立和保存白裤瑶B淋巴母细胞永生细胞株.方法采用EB病毒转化外周血B淋巴细胞,同时加环孢菌素A法.结果成功地建立了含有44株永生细胞的白裤瑶永生细胞库.供血者身体健康,三代均为白裤瑶.结论建立永生细胞库,可永久保存白裤瑶特有的基因组,为民族起源、演进和遗传多样性的研究提供细胞资源.     

EB病毒转化人外周血B淋巴细胞的影响因素研究

目的探讨人类疱疹病毒(EBV)转化外周血B淋巴细胞试验的影响因素。方法用B95-8细胞制备EBV,实时荧光定量-PCR(realtime-PCR)法测定EBV滴度以确保病毒有效性,Ficoll密度梯度离心法分离外周血单个核细胞,研究环孢霉素A(CSA)的浓度、CSA加入时间、淋巴细胞浓度3个因素对EBV转化外周血B淋巴细胞试验的影响,选择最优化条件对三氯乙烯接触工人的B淋巴细胞进行转化。结果制备的病毒滴度分别为5.45×106/ml和1.06×106/ml。不同浓度CSA(0.2、1和2μg/ml)作用以及不同加入时间(同时加入或2h后)B淋巴细胞均成功永生化;正常人B淋巴细胞转化时淋巴细胞浓度不低于2×106/ml时方能成功永生化,而接触三氯乙烯工人的淋巴细胞在1×106/ml浓度下亦转化成功。不同条件下转化成功的B淋巴细胞永生化细胞系在形态上无明显差别,显微镜下可见B淋巴细胞体积增大并聚集成团。结论在建立B淋巴细胞永生化细胞系的过程中,CSA浓度和加入时间对转化结果未见影响,而淋巴细胞与EBV接触时的细胞浓度是影响实验成败的关键因素,接触三氯乙烯工人的B淋巴细胞在1×106/ml浓度下亦转化成功,推测与淋巴细胞致敏状态有关。     

Increasing Diversity of Americans' Faiths Alongside Baby Boomers' Aging: Implications for Chaplain Intervention in Health Settings

Chaplains serving in the health care context provide a ministry to dying patients of inestimable worth as they comfort patients in the last chapter of the journey by being present, listening, and caring. Chaplains also play another important role, helping patients clarify ways in which their beliefs and values might influence health care decisions. This paper reviewed the current trends of spiritual diversity alongside the aging of a large Baby Boomer cohort. Chaplains may be challenged as they participate in the decision-making process, or as they support familes who make decisions about the care of loved ones nearing the end of life. Many of those who seek health care and comfort as the end of life approaches will bring a startling diversity of nonbelief, beliefs, and diverse religious and spiritual practices. This pattern of diversity will profoundly affect patients' decision-making around end-of-life issues. Case studies are used to illustrate possibilities for the chaplain's role at the bedside in the face of such diversity. The dimensional information of a new scale is presented for chaplains to assess diverse afterlife beliefs. As chaplains renew their studies of the worlds living religions, they will be better equipped to serve the needs of this large and spiritually diverse population.     

云南苗族Bardet-Biedl综合征家系外周血B淋巴母细胞系的建立

目的 建立云南苗族巴德-毕氏综合征(Bardet-Biedl syndrome,BBS)家系外周血B淋巴母细胞系,为研究BBS的发病机制、诊断和治疗提供长期的标本来源.方法 取1个BBS核心家系成员新鲜肝素抗凝全血,采用EB病毒转化技术,并加入环孢霉素A(cyclosporine A,CyA)抑制T淋巴细胞,将B淋巴细胞转化成永生化淋巴母细胞系.结果 成功建立了苗族BBS核心家系中12个个体的永生细胞株.结论 EB病毒转化技术可有效保存原来个体完整的基因组,为以后的相关研究提供了长期的DNA资源.

Abstract：

Objective To establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research. Methods With Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree,and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes. Results Twelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully. Conclusion The immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.     

Immune‐related adverse events in urothelial cancer patients: Adjustment for immortal time bias

To investigate the association between the onset, severity, and type of immune‐related adverse events (irAEs) and the efficacy of pembrolizumab in patients with platinum‐pretreated advanced urothelial carcinoma (UC), we retrospectively collected clinical datasets of 755 patients and conducted landmark analysis. Patients who survived for fewer than 3 months were excluded from the evaluation to reduce the immortal time bias. In total, 620 patients were evaluated, of whom 220 patients (35.5%) experienced grade ≥2 irAEs, including 134 patients with grade 2 irAEs and 86 with grade ≥3 irAEs. Propensity score matching extracted 198 patients with and without grade ≥2 irAEs. The onset of grade ≥2 irAEs was associated with longer median progression‐free survival (PFS) (8.3 months vs. 4.5 months, p = 0.003) and overall survival (OS) (20.4 months vs. 14.3 months, p = 0.031) and a higher objective response rate (ORR) (44.8% vs. 30.2%, p = 0.004). Patients with grade 2 irAEs had significantly better oncological outcomes (PFS, OS, and ORR) than grade ≤1 and ≥3 irAEs. Patients with grade ≥3 irAEs had worse outcomes than grade 2 irAEs. Endocrine and skin irAEs were related with better survival outcomes, and the rate of severities was lower in these categories. In conclusion, the occurrence of irAEs, particularly low‐grade irAEs, was predictive of pembrolizumab efficacy in patients with platinum‐pretreated advanced UC.     

建立巴马长寿老人经EBV转染永生细胞株的方法

目的：建立用EBV转化长寿人群外周血淋巴细胞永生细胞株（LCL）的方法。方法：用EBV份百岁以上健康老人，20份89-99岁健康老人及8份66岁以下健康人的血样共36份。采用20％FBS RPM11640培养液，配合环孢菌素A、植物血凝素(PHA)，置37℃5％CO2培养箱内培养。结果：成功地获得24份永生细胞株（其中，百岁老人6份，89-99岁老人12份，66岁以下6份）。结论：采血后24-48h之间接种，转化成功率高。转染后2-3周，大部分标本出现细胞分裂、变形，标志着转化成功。超过5周仍无细胞分裂者即发生细胞凋亡。PHA有缓解细胞凋亡的作用，但不能使细胞分裂。污染是引起转染细胞株凋亡的主要因素。严格的无菌操作、保持培养细胞的适当密度、适时换液、打散较大的细胞克隆团是培养LCL的关键。     

汉族家族性高胆固醇血症患者永生化细胞株的构建及其LDL-R功能的研究

 目的：构建汉族家族性高胆固醇血症（FH）患者永生化细胞株;分析细胞表面低密度脂蛋白受体LDL-R活性。方法：将研究对象20例分为3组：（1）FH纯合组6人，为临床确诊的家族性高胆固醇血症纯合患者;（2）FH杂合组12人,为纯合患者的父母;（3）正常对照组2人，为血脂正常者；采用EB病毒转化外周血淋巴细胞；免疫组化法观察细胞表面LDL-R分布；流式细胞技术观察淋巴细胞LDL-R功能，4 ℃时检测其对LDL结合能力，37 ℃时检测其对LDL内化功能。结果：免疫组织化学法，在光镜下可见淋巴细胞表面散在分布的LDL-R，呈现棕褐色颗粒样；流式细胞技术对3组细胞株检测证实LDL-R功能存在差异，EB病毒转化的细胞株具有永生化特性;正常组LDL-R平均表达量111.35，高于杂合组（97.17）及纯合组（60.62）；纯合组37 ℃内化量228.61，4 ℃ 结合量95.20，结合量为正常人的41.6%，杂合组37 ℃内化量91.28，4 ℃结合量67.42，结合量为正常人的73.8%。结论：采用EB病毒转化外周血淋巴细胞，成功地构建了汉族家族性高胆固醇血症患者的永生化细胞株，此细胞株的功能研究表明其具有稳定的永生化特性，完整地保存了FH患者的细胞种质。     

表达HPA抗原的细胞株稳定性及应用的研究

建立血小板特异性抗原永生化细胞株,为血小板免疫学及血小板交叉配型提供永久的研究材料。采用EB病毒(Ep-stein-Barr virus)转化技术,把携带血小板特异性稀有抗原外周血淋巴细胞转化成永生化淋巴母细胞株。结果:成功地建立了13个稀有血小板特异性稀有抗原建立了永久细胞株,所有的细胞株传代、冻存和复苏的成功率为100%,细胞传至30代没有发现基因突变。由所建的细胞株抽提的DNA样本,分发给第14届国际血小板免疫学研究会做为参比试剂,34个国际实验室基因分型鉴定结果的一致率为97.85%。采用EB病毒转化技术,能成功地血小板特异性抗原外周血淋巴细胞转化成永生化细胞淋巴母细胞株,细胞株稳定传代,并可用作为参比试剂。     

Microenvironmental modulation of asymmetric cell division in human lung cancer cells

Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell–cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133⁺ lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.