单细胞克隆肝癌干细胞向间充质样细胞的分化潜能

探讨单细胞克隆肝癌干细胞（liver cancer stem cell,LCSCs）向间充质样细胞分化的潜能。方法：通过有限稀释法获得单个细胞来源的LCSC克隆,采用RT-PCR法鉴定干细胞标志物;将该单细胞克隆分别用成骨、软骨和脂肪诱导分化培养基培养3周后,采用Real-time PCR及特殊染色技术比较诱导前后LCSC表达成骨、软骨及脂肪细胞特异标志物的差异。结果：单细胞克隆的LCSC表达多种干细胞标志物、干细胞因子（stem cell factor,SCF）、干细胞因子受体、C-kit、巢蛋白（nestin）、CD34、三磷酸腺苷结合转运蛋白G超家族成员2（ATP-binding cassette sub-family G member 2,ABCG2）、CD133。分化诱导培养3周后,成骨方向诱导的细胞茜素红染色呈现橘红色钙结节形成,软骨方向诱导的细胞阿尔新蓝染色显示蓝色蛋白多糖沉积,脂肪方向诱导的细胞油红O染色显示大量脂滴形成。Real-time PCR结果显示,诱导后成骨细胞特异标志物骨钙素和Ⅰ型胶原、软骨细胞特异标志物蛋白聚糖和Ⅱ型胶原、脂肪细胞特异标志物脂联素和过氧化物酶体增殖物激活受体γ的mRNA表达均较对照组显著上调（Ⅰ型及Ⅱ型胶原间为P<0.05,其他指标间均为P<0.01）。结论：LCSC具有可塑性,在特定的微环境下具有向间充质样细胞分化的潜能。     

体外诱导骨髓间充质干细胞向神经细胞分化的研究进展

骨髓间充质干细胞因其具有多向分化的能力,被广泛应用于临床中,特别是用于治疗神经退行性疾病。体外诱导分化骨髓间充质干细胞的方法很多,也有很多中药作为诱导剂参与其的诱导分化。目前诱导分化的方法主要分为两大类:化学诱导法及共培养法。诱导分化后细胞的鉴定主要是通过观察细胞的形态、分析细胞的增殖能力、检测细胞表面标志物等方法。不同诱导方法的诱导机制不一样,诱导分化的结果也不一样。本文对骨髓间充质干细胞体外诱导分化为神经元细胞的方法及研究进展进行综述。     

成骨诱导自体脂肪源性干细胞用于修复兔颅骨缺损

目的探讨脂肪源性干细胞作为骨组织工程的种子细胞在修复骨缺损中的应用前景。方法提取兔脂肪源性干细胞,成骨诱导,将成骨诱导前后的脂肪源性干细胞种植到自制松质骨支架上,将细胞-支架复合物和单纯支架材料,分别移植至兔颅骨缺损部位。术后14周,处死取材,Micro-CT扫描。结果与未诱导的脂肪源性干细胞复合支架、单纯支架以及空白处理对比,成骨诱导后的脂肪源性干细胞复合支架成骨量显著增高,差异具有统计学意义。结论脂肪源性千细胞可作为骨组织工程的种子细胞,其成骨诱导后与自制松质骨复合植入体内,能显著促进骨缺损的修复。     

骨髓间充质干细胞定向诱导成骨分化的蛋白质组学分析

目的通过对人骨髓间充质干细胞（hBMSCs）定向诱导成骨细胞分化过程中不同分化阶段的比较蛋白质组学分析,寻找与干细胞分化的启动、进展及成熟有关的功能蛋白质,为研究干细胞的调控分化提供线索。方法采用贴壁培养法从人骨髓中分离获得hBMSCs,体外扩大培养,以成骨诱导培养体系诱导BMSCs定向成骨细胞分化,选取诱导的1、7、14、21d,分别提取未诱导细胞及诱导组细胞的总蛋白,采用荧光差异凝胶电泳技术分离各组总蛋白,图像分析检测差异表达的蛋白质点,经酶切后行蛋白肽质量指纹谱和数据库检索,鉴定差异蛋白表达。结果贴壁细胞于3d左右开始伸出突起,2～6d时细胞增殖较缓慢,逐渐长为梭形,7d开始细胞增殖迅速,细胞呈漩涡状生长,15d左右逐渐达到完全融合。碱性磷酸酶染色鉴定BMSCs向成骨诱导分化,BMSCs染色阴性,成骨细胞阳性。BMSCs与成骨细胞凝胶图像存在多个蛋白差异点,选取45个差异点,切胶作质谱分析。经质谱分析和数据库检索后鉴定到23个差异表达蛋白质点,大多数差异点蛋白的功能涉及细胞骨架形成、能量代谢、信号转导等过程。结论应用蛋白质组学方法,从功能蛋白质组学的层面阐述BMSCs定向诱导成骨分化相关的重要蛋白的变化,可以为更深入揭示BMSCs定向分化的分子机制提供依据。     

脐带来源的华通氏胶间充质干细胞的分离、特性及应用前景

间充质干细胞(MSCs)的来源非常广泛。虽然各种组织来源的MSCs生物学特性不尽相同，但在标准培养条件下该类细胞均可以贴壁生长；细胞群体中有 95%以上的细胞表达典型的间充质标记分子，如 CD73、CD90 和 CD105，但缺乏造血标记分子CD14、CD19、CD34、CD45和CD79a的表达；特别是均具有分化为成脂细胞、成骨细胞和成软骨细胞的能力。这些性质是国际细胞治疗协会给出的定义该类细胞的最低标准。脐带间充质干细胞是指来源于新生儿脐带组织中的多能干细胞。目前来看，从脐带不同解剖学部位(区室)获得的MSCs的生物学性质也存在差异，其中从脐带华通氏胶中分离获得的华通氏胶间充质干细胞，在增殖能力、分化潜能、免疫调节能力等方面均明显优于其他区室来源的间充质干细胞，是用于组织器官损伤修复及免疫调节的一种理想的“种子”细胞，被认为具有更好的临床应用前景。本文围绕近年来华通氏胶间充质干细胞的分离建系、增殖特性、分化潜能以及临床应用方面的研究进展进行了综述，其中重点介绍了化学诱导分化方案的建立和优化，认为与通过遗传改变诱导重编程方法相比，化学诱导分化方法具有更好的临床应用潜力。     

人骨髓间充质干细胞多向分化的诱导与鉴定

目的探讨人骨髓间充质干细胞(hBMSCs)体外多向分化的潜能,为体内移植实验提供种子细胞。方法采用全骨髓贴壁培养法,分离、自制无血清培养基培养hBMSCs;取生长良好的P4代hBMSCs,免疫荧光法检测其表面抗原CD34、CD44和CD105的表达;应用含不同诱导剂的培养基进行诱导培养,油红O染色检测成脂诱导分化,茜素红染色鉴定成骨细胞诱导分化,阿利新蓝染色检测成软骨诱导情况。结果 hBMSCs表面抗原CD44、CD105呈阳性,CD34呈阴性;经过诱导培养,油红O染色、茜素红染色以及阿利新蓝染色均呈阳性。结论分离培养的hBMSCs能够分化为成脂细胞、成骨细胞和软骨细胞,说明hBMSCs具有多向分化的潜能,可以作为体内移植实验较为理想的种子细胞。     

人脑胶质瘤组织培养的干细胞样细胞具有体外高侵袭能力

目的:分离培养人脑胶质瘤干细胞样细胞(glioma stem-like cell,GSLC),研究其体外侵袭力。方法:选取中国医科大学第一医院神经外科2008年10月至2009年1月间住院患者手术切除的脑胶质瘤组织8例,以无血清成球培养法培养胶质瘤细胞球;免疫细胞化学实验检测其CD133的表达;荧光免疫显微镜观察其分化后胶质细胞标志物GFAP和神经元标志物TU-20的表达;matrigel侵袭实验检测其侵袭力,并与原代脑胶质瘤细胞进行比较。结果:成功分离培养出人脑胶质瘤细胞球细胞,该细胞表达干细胞标志物CD133;能自我更新与增殖;诱导分化后,GFAP和TU-20均为阳性表达,提示其为GSLC。胶质瘤细胞球细胞侵袭细胞数显著多于原代胶质瘤细胞[(261.23±87.20)vs(116.08±63.88)个,P<0.01];此外,胶质瘤细胞球细胞穿过matrigel胶后可再次聚集成球状生长。结论:成功分离培养人脑胶质瘤组织中的GSLC,其体外具有较高的侵袭力,可能参与脑胶质瘤的侵袭和转移。     

多发性骨髓瘤患者骨髓间充质干细胞的生物学特性研究

目的研究多发性骨髓瘤(MM)患者骨髓间充质干细胞(MSCs)的生物学特性及其体外支持造血的能力。方法取15例MM患者的骨髓MSCs,并在体外培养和扩增。观察细胞形态,应用流式细胞术检测MSCs免疫表型;体外诱导MSCs向骨和脂肪分化;RT-PCR检测MSCs造血因子表达情况;将MM骨髓MSCs作为滋养层,应用甲基纤维素半固体培养,检测其支持造血能力。结果 MM来源的MSCs形态为典型的纤维细胞样,该细胞群体表达CD29、CD44、CD105,而CD14、CD31、CD34、CD45、HLA—DR均表达阴性。油红O染色和VonKossa染色检测显示MM骨髓MSCs可以向骨和脂肪分化。MM骨髓MSCs表达造血相关因子,在体外可以维持和扩增造血干/祖细胞。结论 MM患者骨髓中存在具有多向分化能力的MSCs,其具有体外支持造血的功能。     

Pellet培养与纤维蛋白凝胶支架体外成软骨能力的比较

目的比较Pellet培养和纤维蛋白凝胶支架两种培养方式诱导脂肪干细胞向软骨细胞分化及合成细胞外基质的差异。方法人脂肪组织酶消化分离脂肪干细胞,培养基为DMEM含10%胎牛血清,利用Pellet培养和纤维蛋白凝胶支架两种培养体系将P3代脂肪干细胞诱导成软骨后21天取材进行苏木精-伊红染色,番红O染色,DMMB法测定胞外基质中GAG含量,荧光定量PCR测定Ⅱ型胶原基因表达。结果苏木精-伊红染色与番红-O染色显示,纤维蛋白凝胶实验组软骨细胞外基质大量分泌,并融合成片,说明纤维蛋白凝胶支架可更好促进干细胞成软骨分化、维持软骨细胞表型。GAG/DNA结果显示,纤维蛋白凝胶组促GAG分泌的能力优于其余各组(P0.05),其Ⅱ型胶原m RNA表达的能力也最强(P0.05)。结论利用纤维蛋白凝胶支架诱导人脂肪干细胞成软骨分化的能力优于无支架的Pellet三维培养体系。     

过表达miR-145的脐带间充质干细胞外泌体改善肾细胞癌细胞786-0对舒尼替尼的药物敏感性

目的:探究过表达miR-145的脐带间充质干细胞外泌体对肾透明细胞癌细胞786-0舒尼替尼药物敏感性的影响.方法:分离纯化人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs),流式细胞术鉴定其细胞表型,茜素红及红油O染色观察其成骨与成脂分化能力;差...     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的:深入研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法:大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子(KGF)、胰岛素-转铁蛋白-硒(ITS)、尼克酰胺的无血清DMEM/F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果:培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论:MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

体外条件下TGF-β3、BMP-2和DEX诱导兔滑膜间充质干细胞向软骨细胞谱系分化的研究

目的：本实验拟将分离纯化得到的滑膜间充质干细胞（ synovial-derived mesenchymal stem cells,SMSCs ）在体外培养条件下进行成软骨刺激诱导,并从转录和翻译两个水平寻找进入软骨细胞分化谱系的证据,进而判断转化生长因子β3（ transforming growth factor-β3,TGF-β3）、骨形态发生蛋白（ bone morphogenetic protein-2,BMP-2）和地塞米松（ dexamethasone,DEX ）诱导 SMSCs 进入软骨细胞分化谱系。方法贴壁法分离纯化得到 SMSCs,在体外培养条件下用含500 ng / ml BMP-2、10 ng / ml TGF-β3、10-7 M DEX 的高糖 DMEM 培养基进行刺激诱导,并在倒置相差显微镜下观察分化过程中其形态学的变化,以 RT-PCR检测I、II型胶原及软骨特异性Aggrecan （ AGN ）的mRNA表达,细胞免疫荧光化学染色的方法检测细胞分化过程中I、II型胶原的表达,碱性甲苯胺蓝细胞化学染色检测软骨特异性GAG的表达,证实SMSCs的诱导成软骨作用。结果 SMSCs在前述诱导条件下,诱导后14天细胞逐渐由小梭形变为多角形、类软骨细胞样形态,RT-PCR可以检测到I、II型胶原及AGN基因的表达,细胞免疫荧光化学染色I、II型胶原、碱性甲苯胺蓝细胞化学染色结果呈阳性。而未经诱导的SMSCs形态基本保持梭形,基因表达和染色呈阴性,两组间差异显著。细胞免疫荧光化学染色分析SMSCs在软骨诱导培养基中诱导后14天表达I、II型胶原;未经诱导的SMSCs不表达I、II型胶原。说明SMSCs在软骨诱导培养基中诱导后14天进入软骨细胞分化谱系,可作为种子细胞在同样的诱导条件下向软骨分化。结论 SMSCs作为新的MSCs家族成员,显示出与BMSCs相似的多向分化潜能,500 ng/ml BMP-2、10 ng/ml TGF-β3、10-7 M DEX的高糖DMEM培养基中培养后14天,SMSCs已进入软骨细胞分化谱系。SMSCs可作为半月板组织工程的种子细胞。     

Hyperthermia‐treated mesenchymal stem cells exert antitumor effects on human carcinoma cell line

BACKGROUND:

Mesenchymal stem cells (MSCs) possess the potential for differentiation into multilineages. MSCs have been reported to play a role as precursors for tumor stroma in providing a favorable environment for tumor progression. Hyperthermia destroys cancer cells by raising the temperature of tumor‐loaded tissue to 40°C to 43°C and causes indirect sensitizing effects when combined with chemo‐ and/or radiotherapy. However, how hyperthermia affects the tumor‐supportive stroma is unknown. Here, the authors investigated the effects of hyperthermia‐treated MSCs, from different sources, on the human ovarian cancer cell line SK‐OV‐3.

METHODS:

MSCs from adipose tissue and amniotic fluid were untreated or heat‐treated (HS‐MSCs). The culture supernatant of each treatment group was collected and transferred to the SK‐OV‐3 cells.

RESULTS:

The morphological analysis and cell proliferation assay showed a reduced viability of the tumor cells in the conditioned medium with the HS‐MSCs. Further investigations revealed that the conditioned medium of the HS‐MSCs induced a higher nuclear condensation and a greater number of sub‐G1 cells among the tumor cells. Analysis of the mRNA expression demonstrated that the conditioned medium of the HS‐MSCs induced up‐regulation or down‐regulation of several tumor‐associated molecules. Finally, the cytokine array of each conditioned medium showed that angiogenin, insulin‐like growth factor binding protein 4, neurotrophin 3, and chemokine (C‐C motif) ligand 18 are involved as main factors.

CONCLUSIONS:

This study showed that the conditioned medium of the HS‐MSCs exerted a suppressive effect on tumor progression and malignancy, suggesting that hyperthermia enables tumor stromal cells to provide a sensitizing environment for tumor cells to undergo cell death. Cancer 2009. © 2009 American Cancer Society.     

Screening for induction of differentiation and toxicity to blast cells by chemotherapeutic compounds in human myeloid leukemia

Bone marrow cells from 9 patients with acute myeloid leukemia and 1 patient with a blast crisis of chronic myeloid leukemia were cultured to determine their ability to be induced to differentiate by different chemotherapeutic compounds. Five of these 10 patients showed differentiation to granulocytic and/or monocytic cells by culture with medium containing the myeloid cell differentiation-inducing protein MGI-2. Actinomycin D induced differentiation in cells from 2 of the patients who did not show differentiation with MGI-2 containing medium. In these 7 patients there was an increase in the ratio of differentiated myeloid cells to blasts. None of these 10 patients showed induction of differentiation by cytosine arabinoside, adriamycin, or daunomycin, but treatment with these compounds showed in some patients an increase in the ratio of differentiated myeloid cells to blasts. The results indicate that this ratio can be increased by differentiation and also in some patients by toxicity to blast cells. With dexamethasone or vinblastine there was no induction of differentiation and no increase in this ratio in any of the 10 patients tested. After in vivo chemotherapy with low dose cytosine arabinoside, cells from one patient showed a similar response in culture to actinomycin D as cells before chemotherapy, whereas in another patient the cells had acquired the ability to respond to actinomycin D. In contrast, after high-dose in vivo chemotherapy with cytosine arabinoside and daunomycin, cells from a third patient seemed to have lost the ability to differentiate in vitro by MGI-2 containing medium or actinomycin D. The results indicate that pre-screening for differentiation-inducing compounds and compounds that show toxicity to blast cells should be useful to select the appropriate compounds to be used for therapy, and that it is advisable to screen the cells both before and after initiation of therapy.     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的：深入研究骨髓间充质干细胞（mesenchymal stem cells,MSCs）多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法：大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子（KGF）、胰岛素-转铁蛋白-硒（ITS）、尼克酰胺的无血清DMEM／F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果：培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论：MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

与骨髓MSC共孵育的T淋巴细胞对白血病细胞的杀伤作用

目的 探讨经MSC诱导免疫耐受的T淋巴细胞对与MSC同源的肿瘤细胞的杀伤活性,以证实MSC诱导免疫耐受的同时,是否也削弱了移植物抗肿瘤的作用。方法 初治的白血病患者留取原代肿瘤细胞,液氮保存,诱导缓解后培养同一患者的骨髓MSC,取异体T淋巴细胞与MSC共孵育,比较与MSC共孵育的T淋巴细胞对MSC同源的肿瘤细胞的杀伤作用是否减弱。结果 与MSC共孵育后,T淋巴细胞对MSC同源的肿瘤细胞仍有杀伤作用,但与未共孵育者相比。杀伤活性减弱。结论 MSC在诱导免疫耐受的同时也削弱了GVI,的作用,提示在以MSC诱导免疫耐受预防GVHD的方案中,应考虑同时加强GVL作用,以防肿瘤复发。     

过氧化物酶体增殖物激活受体-γ在再生障碍性贫血发病机制中的作用

目的 检测再生障碍性贫血（AA）患者骨髓间充质于细胞（BMSC）过氧化物酶体增殖物激活受体-γ（PPAR γ）的表达及其对BMSC成脂的影响,探讨AA患者骨髓脂肪化的发生机制.方法 分离培养16例AA患者、20例白细胞减少症患者（对照组）BMSC,显微镜下观察其形态;用Westemblot检测AA患者BMSC内PPARγ的表达;将对照组BMSC分为三组：单纯诱导组（脂肪诱导培养液）、吡格列酮组（脂肪诱导培养液＋ PPARγ配体吡格列酮）及GW9662组（脂肪诱导培养液＋吡格列酮＋ PPARγ拮抗剂GW9662）,诱导成脂21 d后,油红O染色计数脂肪细胞分化率,并用酶联免疫吸附（ELISA）法检测细胞培养上清肿瘤坏死因子α（TNF-α）含量.结果 连续传至第5代AA组BMSC出现脂肪细胞分化,而对照组BMSC传至第8代仍未出现脂肪细胞分化.AA患者BMSC内PPAR γ蛋白相对表达量为1.46±0.10,高于对照组的0.86±0.06（P＜ 0.05）.对照组BMSC诱导成脂中吡格列酮组脂肪细胞分化率为（87.42±0.67）％,高于单纯诱导组的（44.69±2.61）％及GW9662组的（39.29±1.59）％（P＜0.05）,后两组差异无统计学意义（P＞0.05）;吡格列酮组细胞培养上清中TNF-α表达量为（95.04±3.41） pg/ml,高于单纯诱导组的（30.84±3.48） pg/ml及GW9662组的（31.43±3.51）pg/ml（均P＜ 0.05）,后两组间差异无统计学意义（P＞0.05）.结论 AA患者BMSC出现脂肪化明显,PPARγ在AA患者BMSC中表达增高,其拮抗剂GW9662可抑制BMSC向脂肪细胞分化,提示PPARγ参与了AA患者骨髓脂肪化的病理改变.     

IL-1β produced by aggressive breast cancer cells is one of the factors that dictate their interactions with mesenchymal stem cells through chemokine production

The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-κB signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an IκB dominant negative mutant, nuclear translocation of p65 and induction of NF-κB signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1β, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1β secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1β, which increases the production of chemokines by MSCs.     

Hyperthermia on mesenchymal stem cells (MSCs) can sensitize tumor cells to undergo cell death†

Hyperthermia, the procedure of raising the temperature of tumor-loaded tissue to 40°–43°C, has been applied to various established cancer treatments. Although the mechanism of hyperthermia in cancer treatment is well-known, there are few or no studies regarding the effect of hyperthermia on the tumor-supportive stroma. Mesenchymal stem cells (MSCs) display the potential for differentiation into various tissues. MSCs are also reported to play a role as potential precursors for tumor stroma in providing a favorable environment for tumor progression. Here, we investigated the effects of hyperthermia-treated MSCs on the viability and growth of cancer cells. Culture supernatants from non-shocked or heat-shocked MSCs (NS-MSCs or HS-MSCs) were added to MCF7 cells. Morphological analysis and cell proliferation assay showed the reduced viability and growth of MCF7 cells by addition of culture medium conditioned by HS-MSCs. Additionally, exposure to the conditioned medium by HS-MSCs induced cell cycle arrest at G2/M phase, increased MHC class I, Fas receptor, and TNF-R expressions, and decreased MDR1 expression in the MCF7 cells. In particular, the conditioned medium of HS-MSCs accelerated the inhibition of tumor cell growth by several chemotherapeutic drugs. These data present new aspects of hyperthermia in cancer treatment, suggesting that hyperthermia can enable tumor stroma provide a sensitizing environment for tumor cells to undergo cell death.