乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

目的 通过乳腺癌细胞系及其干细胞的培养,化疗药干预和流式细胞仪筛选鉴定,探讨不同乳腺癌细胞系中CD44⁺CD24^-/low细胞比例,及富集乳腺癌干细胞相关亚群的方法。方法 通过细胞培养乳腺癌细胞系MCF-7、MDA-MB-231,观察生长曲线,比较化疗药物干预下生长情况;利用流式细胞仪检测两种乳腺癌细胞系中CD44⁺CD24^-/low细胞比例;无血清悬浮培养,化疗药(多西紫杉醇、表阿霉素)干预这两种乳腺癌细胞系,观察其是否形成细胞球。结果 (1)MDA-MB-231细胞系倍增时间短,生长速率高于MCF-7细胞系;(2)MCF-7细胞系中可能存在较大比例肿瘤干细胞,其对化疗抵制,能自我更新;(3)化疗敏感性用两独立样本t检验,MCF-7细胞,差异没有统计学意义;MDA-MB-231细胞,差异有统计学意义,提示MDA-MB-231细胞系对该方案化疗较敏感;(4)无血清悬浮培养,MDA-MB-231细胞系未发现明显细胞球;MCF-7细胞系初次无血清培养约6天出现细胞球。加入化疗药筛选后两种细胞,见大部分肿瘤细胞逐渐死亡,未发现明显细胞球;(5)流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中主要是CD44⁺CD24⁺亚群和CD44^-CD24⁺亚群,CD44⁺CD24^-/low细胞比例分别2.07%和0.20%。结论 (1)MDA-MB-231细胞系增值较快,恶性度相对较高,其对TA联合化疗药物较敏感;MCF-7细胞系中可能存在少量肿瘤干细胞,对化疗抵制,能自我更新;(2)无血清培养液培养MCF-7细胞系能形成悬浮细胞球;流式细胞仪检测两种细胞系中CD44⁺CD24^-/low细胞比例小;(3)CD44⁺CD24^-/low表型可能不是乳腺癌干细胞唯一特异性的表面标志。     

CD44和CD24标记的宫颈癌细胞亚群特性

目的 考察Siha细胞系中的CD44^+/CD24⁺能否富集宫颈癌干细胞。方法 用流式细胞仪分选出CD44⁺/CD24⁺Siha细胞,用无血清悬浮培养观察成球能力、裸鼠移植瘤实验观察成瘤能力、透射电子显微镜观察辐射前后两组细胞形态变化,并通过Transwell侵袭实验比较细胞侵袭能力的差异。结果 耐放疗细胞中CD44^+/CD24⁺Siha细胞比例明显高于其在亲代Siha细胞中的比例;无血清培养CD44⁺/CD24⁺Siha细胞组可以形成致密且体积较大的细胞球,CD44^+/CD24⁺Siha细胞组致瘤时间早,成瘤率高;辐射后CD44⁺/CD24⁺Siha细胞较亲代Siha细胞更抗凋亡;CD44⁺/CD24⁺Siha细胞组的迁移细胞数明显高于亲代Siha细胞组,所有数据均具有统计学意义。结论 CD44⁺/CD24⁺Siha具备部分干细胞特性,CD44⁺/CD24⁺可能成为宫颈癌干细胞特异性表面标志物。     

CD44+CD24-/low乳腺癌干细胞分选鉴定及其多药耐药性研究

目的:观察MACS免疫磁珠法分选CD44+CD24-/low乳腺癌干细胞活性,并检测其与多药耐药的关系。方法:运用MACS免疫磁珠法从多药耐药乳腺癌细胞株MCF-7/ADR中分选CD44+CD24-/low乳腺癌干细胞,流式细胞术测定分选前后CD44+CD24-/low细胞比例,微球体培养法检测分选细胞自我更新能力,流式检测CD44+CD24-/low细胞表面P-糖蛋白(P-gp)表达水平,Real-time PCR检测多药耐药相关基因MDR1表达水平。结果:MACS免疫磁珠法分选后,CD44+CD24-/low细胞比例为93.85%,其成球能力明显强于non-CD44+CD24-/low细胞亚群。MCF-7/ADR细胞株和CD44+CD24-/low乳腺癌干细胞P-gp表达强度分别为101 177.10±2 171.86和114 906.70±2 560.19,P<0.05。CD44+CD24-/low乳腺癌干细胞MDR1基因表达水平为MCF-7/ADR细胞株的(1.07±0.02)倍,P<0.05。结论:经MACS免疫磁珠法分选所得CD44+CD24-/low细胞亚群有更强的自我更新能力,高表达P-gp蛋白和MDR1基因可能是引起乳腺癌多药耐药的重要原因。     

乙醛脱氢酶阳性乳腺癌干细胞的分离培养及其生物学特性分析

 目的　证实人类乳腺癌MCF-7细胞系中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性。方法　应用流式细胞术从MCF-7细胞中分离并培养ALDH+ 乳腺癌干／祖细胞,通过划痕试验、MTT法生长曲线测定、以及Transwell法等检测ALDH+乳腺癌干细胞的生物学特性。结果　MCF-7细胞系中,CD-／low24 CD+44 细胞比例约为1.4 %,ALDH+ CD-／low24 CD+44 细胞比例约为1.2 %; ALDH+乳腺癌干细胞与CD-／low24 CD+44 细胞两者的生长曲线基本一致;CD-／low24 CD+44 ,ALDH+ CD-／low24 CD+44 组细胞划痕区细胞间距离明显缩短,其迁移能力明显强于对照组,且两群干细胞之间存在差异;Transwell实验结果,与对照组相比,CD-／low24 CD+44 、ALDH+ CD-／low24 CD+44 两组细胞有大量细胞过膜,三组MTT检测吸光度值分别为1.05±0.098、1.56±0.075、1.67±0.032。结论　MCF-7细胞系中存在CD-／low24 CD+44 和ALDH+ CD-／low24 CD+44 乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一。     

基于CD19/IgM亚群分析套细胞淋巴瘤骨髓中T细胞的构比异常

目的:研究T细胞在初诊IV期套细胞淋巴瘤(mantle cell lymphoma, MCL)骨髓单个核细胞中的差异表达。方法:首先流式检测健康供者及MCL组中CD19^-/IgM^-,CD19^-/IgM⁺,CD19⁺/IgM^-,CD19⁺/IgM⁺亚群的比例;其次检测CD45⁺CD3⁺T细胞在两组中的总比例;最后检测T细胞在两组四个亚群中的占比。结果:四个亚群在组内有极其显著的差异(P<0.000 1),两组间无统计学差异。T细胞在MCL组占(0.46±0.13)%较健康供者组(1.10±0.06)%显著减少(P=0.025 1)。四个亚群中T细胞占比在健康供者组内有极其显著的差异(P<0.000 1),而MCL组内差异消失(P=0.329 4)。T细胞在MCL组CD19^-/IgM^-亚组中占(0.02±0.01)%较...     

小鼠衰老所致“正虚”过程中免疫功能衰退表征的特点

目的：基于小鼠渐进衰老模型探讨衰老所致“正虚”的免疫功能衰退表征的特点。方法：使用不同月龄(2、6、15月龄)C57BL/6小鼠,通过流式细胞术检测并比较小鼠外周血和脾组织中T细胞、髓源性抑制细胞(MDSC)及其亚群的丰度变化。结果：外周血中T细胞亚群表型为CD3⁺CD4⁺CD44-CD62L⁺的幼稚CD4⁺T细胞(2 vs 6月龄,P=0.137;2 vs 15月龄,P=0.004;6 vs15月龄,P=0.105)和表型为CD3⁺CD8⁺CD44-CD62L⁺的幼稚CD8⁺T细胞(2 vs 6月龄,P=0.179;2 vs 15月龄,P=0.001;6 vs15月龄,P=0.015)出现与衰老有关的细胞比例降低,差异具有统计学意义。表型为CD3⁺CD4⁺CD44⁺CD62L⁺的中央记忆CD8⁺T细胞出...     

结直肠癌及腺瘤患者CD4+、CD8+和CD28+T淋巴细胞的亚群变化及临床意义

背景与目的：结直肠癌（colorectal cancer,CRC）严重影响患者生存。探讨肿瘤微环境（tumor microenvironment,TME）T细胞亚群在CRC和腺瘤中的表达及意义。方法：用免疫组织化学法和流式细胞术对51例健康人（对照组）、46例结直肠腺瘤（腺瘤组）、100例CRC（癌症组）和15例CRC术后（癌术后组）患者进行T细胞亚群检测。结果：① 对照组、腺瘤组及癌症组3组中CD4⁺T细胞的阳性率分别是90.00%、43.75%及32.65%,CD8⁺T淋巴细胞的阳性率分别是30.00%、56.25%及75.51%,CD28⁺T淋巴细胞的阳性率分别是42.86%、30.00%及20.00%。② 对照组、腺瘤组及癌症组3组中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺逐渐降低,CD8⁺、CD8⁺CD28^－逐渐增加（P＜0.05）;癌术前术后T细胞亚群差异有统计学意义（P＜0.05）。结论：① CRC微环境T细胞亚群中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺呈递减趋势,CD8⁺、CD8⁺CD28^－呈递增趋势,且在癌前病变腺瘤中已逐步出现上述趋势变化。② CRC患者行肿瘤切除术后,其T细胞亚群有所恢复,故在一定程度上,CRC中T细胞亚群的变化可以早期预测结直肠疾病的发展。     

结直肠癌肿瘤微环境中调节性T细胞CD39、CD73亚群及二者双阳性亚群的临床意义

目的 观察结直肠癌肿瘤组织微环境中浸润的调节性T细胞的临床意义,并探究其CD39、CD73及CD39,CD73双阳性亚群与肿瘤微环境中其他淋巴细胞及临床病理参数之间的关系。方法 收集24例结直肠癌患者根治术后组织标本,剪碎并分离培养组织内肿瘤浸润淋巴细胞(Tumor infiltrating lymphocyte,TIL),以流式细胞术观察调节性T细胞CD39⁺、CD73⁺及CD39⁺CD73⁺亚群与患者临床病理参数之间的关联。结果 结直肠癌肿瘤组织微环境中浸润的调节性T细胞CD73⁺亚群,CD39⁺CD73⁺亚群与淋巴结转移及分化程度不良具有相关性,并且其机制与肿瘤相关性炎症密切相关。结论 相比Treg中的其他亚群,CD39⁺CD73⁺阳性的结直肠肿瘤浸润Treg具有更为独特的生物学活性,本研究为预测结直肠癌预后提供了新思路与理论依据。     

ALDH+乳腺癌干细胞的原代分离培养及生物学特性分析

目的 证实乳腺癌组织中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性.方法 应用流式细胞法从人乳腺癌组织细胞中分离并培养ALDH+乳腺癌干/祖细胞,通过克隆形成实验,生长曲线测定,以及transwell法等实验方法检测ALDH+乳腺癌干细胞的生物学特性.结果 在乳腺癌组织内,我们检测到ALDH+ CD24-/lowCD44+约占总细胞量的16％,无血清培养72 h后逐渐出现细胞球,2～3周时细胞球数量达到高峰.Transwell法检测ALDH+乳腺癌干细胞侵袭性较ALDH-乳腺癌干细胞侵袭性强.结论 乳腺癌组织中存在ALDH+ CD24-/low CD44+乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一.     

乳腺浸润性导管癌组织中ALDH1+/CD133+干细胞样细胞与血管生成的关系

目的 探讨乳腺浸润性导管癌组织中肿瘤干细胞标志物ALDH1、CD133的表达及其与肿瘤血管生成的关系。方法 应用免疫组织化学双染法检测120例乳腺浸润性导管癌组织中ALDH1⁺/CD133⁺ 干细胞样细胞,单染法检测血管性标记CD34、CD105及VEGF的表达情况。统计ALPH1⁺/CD133⁺干细胞样细胞与临床病理因素;CD34、CD105与VEGF的关系。结果 25.83％（31/120）的病例存在ALDH1⁺/CD133⁺干细胞样细胞,ALDH1⁺/CD133⁺干细胞样细胞与ER、VEGF的表达及MVD均相关（P＜0.05）,但与年龄、肿瘤直径、PR、Her-2、组织学分级及淋巴结转移均无关（P＞0.05）。结论 乳腺浸润性导管癌组织中ALDH1⁺/CD133⁺干细胞样细胞可能通过调节VEGF的表达促进肿瘤新生血管的生成。     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: I, Diverse Patterns of Expression in Mature (Post-Thymic) T-Cell Proliferations

By simultaneous two- and three-colour flow cytometry, this study analysed the expression of membrane CD45RA (2H4) and CD45RO (UCHL1) determinants by normal thymocytes (n = 5) and peripheral blood lymphocyte subpopulations (CD4⁺, n = 21; CD8⁺, n = 12; CD8^dim+, n = 12) and compared these patterns with those of T-cells from representative CD4⁺CD8^- (n = 8), CD4⁺CD8⁺ (n = 2), CD4^-CD8⁺ (n = 10) and CD4^-CD8^- (n = 1) proliferations. These comprised cases of prolymphocytic leukaemia (T-PLL, n = 5), adult T-cell leukaemia-lymphoma (ATLL, n = 2), Sezary Syndrome (SS, n = 4), chronic lymphocytic leukaemia (T-CLL, n = 4), and lymphoproliferative disease of granular lymphocytes (LDGL, n = 5). Normal thymocyte fractions, of which a mean of 85% cells co-expressed membrane CD4 and CD8, were predominantly (mean 89%) 2H4^-UCHL1⁺ with the remaining cells consisting of 2H4^intUCHL1⁺ and 2H4⁺UCHL1^- components. Further analysis showed that virtually all CDla⁺ thymocytes were UCHL1⁺ whereas the CD1a^- fraction comprised similar proportions of both UCHL1^- and UCHL1⁺ subpopulations. Similarly, normal blood CD4⁺, CD8⁺ and CD8^dim+ lymphocytes showed reciprocal CD45RA/CD45RO expression and could be phenotypically grouped into 2H4⁺UCHL1^- 2H4^intUCHL1⁺ and 2H4^-UCHL1⁺ subpopulations. Mean proportions of 48% and 68%, for CD4⁺ and CD8⁺ lymphocytes respectively, showed a composite 2H4⁺UCHL1^- phenotype, whereas the percentage of NK-associated CD8^dim+ cells with this phenotypic pattern was considerably higher (mean, 85%). Normal lymphocyte subpopulations lacking both determinants (2H4^-UCHL1^-) were only rarely noted. Comparing normal patterns of CD45RA/CD45RO expression with those of the T-cell proliferations revealed diverse and abnormal patterns of staining for 3/6 of the CD4⁺CD8^- SS and ATLL, and for 5/5 of the T-PLL (CD4⁺CD8^-, n = 2; CD4⁺CD8⁺, n = 2; and CD4^-CD8⁺, n = 1) cases studied. In contrast, the nine cases of CD4^-CD8⁺ T-CLL and LDGL all showed CD45RA/CD45RO staining patterns similar to that of normal CD8⁺/CD8^dim+ blood lymphocytes (i.e. a predominance of 2H4⁺UCHL1^- cells). Although the variant CD45RA/CD45RO pattern types of the CD4⁺ proliferations did not appear to be related to either the diagnostic category or other phenotypic characteristics, the high proportion of abnormal patterns within this case group suggests that recognition of these abnormalities may be potentially relevant to the differentiation of benign and malignant CD4⁺ proliferations and, in addition, may be of aetiological importance with respect to the diverse acquired defects in immunity commonly seen in patients with such disorders.     

CD44+CD24-/low在基底样乳腺癌中过表达与其恶性预后的相关性研究

目的探讨乳腺癌干细胞样标志物CD44⁺CD24^-/low在基底样乳腺癌(basal-like breast carcinoma, BLBC)中过表达与BLBC恶性预后的相关性。方法 在乳腺癌基因表达分型的基础上, 根据雌激素受体(ER)、孕激素受体(PR)、人类表皮生长因子受体2(Her-2)免疫表型的表达选取乳腺癌组织四组:管腔A组、管腔B/C组、Her-2高表达组及三阴性组;对三阴性组检测CK5/6、EGFR, 分为正常乳腺样型和BLBC型两组;对上述5组进行免疫组化Envision法染色, 选用抗体为CD44、CD24, 观察CD44⁺CD24^-/low表型表达并比较BLBC组与其它各组的差异。结果 (1)三阴性组共60例, CK5/6和或EGFR阳性者41例(68.3%), 确定为BLBC;CK5/6、EGFR阴性者19例(31.7%), 确定为正常乳腺样组;(2)CD44⁺CD24^-/low表型在BLBC组中占78.0%(32/41), 相对于管腔A组37.9%(11/29)、管腔B组25.9%(7/27)、Her-2高表达组17.2%(5/29)、正常乳腺样组26.3%(5/21), 表达增高并具有显著性(P＜0.05);(3)所有150例乳腺癌中(可评价145例)具有CD44⁺CD24^-/low免疫表型者其Ki-67指数增高相对于非CD44⁺CD24^-/low表型具有统计学差异(P＜0.001)。结论 BLBC型乳腺癌表达乳腺癌干细胞样标志物CD44⁺CD24^-/low显著高于其它各型乳腺癌, CD44⁺CD24^-/low与BLBC独特的恶性生物学行为相关。     

Homing-Associated Cell Adhesion Molecule (H-CAM/CD44) on Human CD34+ Hematopoietic Progenitor Cells

Human CD34⁺ hematopoietic progenitor cells (HPCs) express CD44 and can directly adhere to hyaluronate (HA) via CD44. Furthermore, CD44 may also be involved in the regulation of CD34⁺ HPC proliferation and development. The expression of CD44 molecules on CD34⁺ hematopoietic progenitor cells is significantly lower on bone marrow (BM) CD34⁺ cells compared with circulating CD34⁺ cells in cord blood and peripheral blood. Myeloid and erythroid progenitor cells are found predominantly in CD34⁺CD44^- cell fractions. More interestingly, CD34⁺CD44⁺ cells expressing B-lymphocyte-associated CD10 and CD19 would represent unique B-lymphocyte committed precursors in the BM, which might undergo apoptotic cell death in the early steps of B-cell differentiation.     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: II, Aberrant HLA-ABC Expression by CD45RA and CD45RO Cell Subpopulations of Mature CD4+ T-Cell Leukaemias

Six thymocyte suspensions, 10 normal blood CD4⁺ CD8^- lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4⁺ mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4⁺ CD8^- blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a ⁺ 2H4^- UCHL1⁺ (25 HLA-ABC fluorescent units), (b)CD1a^-2H4^intUCHL1⁺ (134 units), and (c) CD 1a^- 2H4 ⁺ UCHL1 ^- (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4⁺ 2H4⁺ (318 units) and CD4 ⁺ 2H4^- (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4⁺ and 2H4^- components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a^- thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4⁺ 2H4⁺ (710 units) and CD4⁺ 2H4^- (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 ⁺ components, with only 14% of 2H4^- fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4^-UCHL1⁺ subpopulation in CD4⁺ malignancies had a lower HLA-ABC level than the 2H4⁺UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.     

Multi-drug resistance mediated by P-glycoprotein overexpression is not correlated with ZAP-70/CD38 expression in B-cell chronic lymphocytic leukemia

ZAP-70 and CD38 expression can identify B-cell chronic lymphocytic leukemia with an inferior clinical outcome. Many groups have investigated the meaning of the expression of these two proteins and the correlation with the bad prognosis in B-CLL. But nobody has investigated the relation between the multidrug resistance mediated by Pgp overexpression (MDR1) and ZAP-70/CD38 coexpression. Forty-one untreated and stage A patients, either ZAP-70⁺CD38⁺ or ZAP-70^-CD38^-, were tested to determine the MDR1 status. MDR1 was observed in 41% of CLL ZAP-70⁺CD38⁺ and in 37% of CLL ZAP-70^-CD38^-. The difference was not significant (p = 0.745). Patients with ZAP-70 and CD38 positive CLL can not be candidates for MDR1 antagonists.     

CD34+ CD90+ cells and late hematopoietic reconstitution after autologous peripheral blood stem cell transplantation

Autologous peripheral blood stem cell transplantation (PBSCT) has been demonstrated to result in rapid, stable long-term engraftment. However, there has been considerable debate concerning the cells responsible for early and late hematopoietic reconstitution after PBSCT. Recently, CD34⁺ hematopoietic stem and progenitor cells have been clearly divided into two subpopulations by flow cytometry; namely undifferentiated pluripotent stem cells and differentiated committed progenitor cells. However, only a few studies have defined which subset contained in graft products might be the most predictive for late hematopoietic reconstitution after PBSCT. In this review, we present updated information regarding the relationships between the number of infused CD34⁺ cells or their immature subsets such as CD34⁺CD90⁺ cells and the late hematopoietic reconstitution after PBSCT, and discuss the threshold dose of CD34⁺CD90⁺ cells required for sustained long-term engraftment.     

Enhanced antitumor effects by the coculture of allotumor RNA-pulsed dendritic cells with autologous cytokine-induced killer cells on hormone-refractory prostate cancer

In this study, we evaluated antitumor effects of allotumour RNA-transfected dendritic cells (DCs) cocultured with autologous cytokine-induced killer cells (CIKs) on hormone-refractory prostate cancer. The cocultured cells enhanced prostate cancer cytolysis from 26% (CIKs-induced cytolysis) to 80.8%. They also increased the productions of CD4⁺ Th1 (IFN-γ⁺IL-4^-, 55.52%) and CD8⁺ T (IFN-γ⁺, 69.59%) cells determined by intracellular cytokines IFN-γ /IL-4 staining and reduced the rate of CD4⁺ CD25⁺ cells from 18.72% (in CIKs) to 9.72%. The cocultured cells significantly inhibited tumor growth in SCID mouse and induced cancer cells necrosis and apoptosis. Our study indicates that tumor RNA-pulsed DCs cocultured with autologous CIKs significantly enhance antitumor immunity, which can be induced by increased CD4⁺ Th1 and CD8⁺ T cells and decreased CD4⁺CD25⁺ regulatory T (T_reg) cells. This provides a potential immunotherapy strategy for HRPC.