正常皮肤和瘢痕组织表皮干细胞定位与增殖分化特征的比较性研究

目的采用特殊染色法研究少儿与成年人大面积重度烧伤后瘢痕组织表皮干细胞分布与表达β1整合素和角蛋白19、14、10(K19,K14,K10)的特征与规律,在此基础上确定表皮干细胞及短暂扩充细胞分布、数量的差异以及这些改变与瘢痕愈合关系.方法分别取4～12岁少儿及35～53岁成年人2组健康皮肤及大面积深度烧伤后瘢痕组织.采用免疫组织化学SP法检测表皮干细胞、短暂扩充细胞特异表达的β1整合素和K19以及分化表皮细胞表达的K14和K10.结果瘢痕组织表皮基底层表达β1整合素与K19的阳性细胞数较健康皮肤明显减少,阳性强度降低.瘢痕组织表皮中表达K14的阳性细胞仅位于表皮底部2～3层,明显少于健康皮肤,而K10表达阳性细胞则较健康皮肤分布广泛.结论瘢痕组织表皮基底层具有增殖能力的表皮干细胞和短暂扩充细胞明显少于健康皮肤,且瘢痕组织表皮干细胞的分化过程与健康皮肤不同,处于有丝分裂后分化阶段的细胞比例降低,而终末分化细胞的比例明显增高.提示瘢痕组织表皮的增殖能力下降,细胞的分化行为紊乱,这可能是瘢痕组织表皮结构与功能改变、愈合能力下降的原因之一.     

成人正常皮肤和瘢痕组织表皮干细胞定位与表达特征的比较研究

目的采用免疫组织化学染色方法研究成人正常皮肤和瘢痕组织表皮干细胞定位与表达β1整合素和角蛋白19,14,10(K19,K14,K10)的特征与规律,探讨两种组织表皮干细胞表达特征的差异与烧伤后瘢痕愈合的关系.方法分别取成年人健康皮肤6例和大面积深度烧伤后瘢痕组织6例.采用免疫组织化学Elivision两步法检测表皮干细胞、短暂扩充细胞特异表达的β1整合素和K19以及分化表皮细胞表达的K14和K10.结果瘢痕组织表皮基底层表达β1整合素与K19的阳性细胞数较健康皮肤明显减少,阳性强度降低.瘢痕组织表皮中表达K14的阳性细胞仅位于表皮底部2～3层,明显少于健康皮肤,而K10表达阳性细胞则较健康皮肤分布广泛.结论瘢痕组织表皮基底层干细胞和短暂扩充细胞明显少于健康皮肤,且瘢痕组织表皮干细胞的分化过程与健康皮肤不同,处于有丝分裂后分化阶段的细胞比例降低,而终末分化细胞的比例明显增高.提示瘢痕组织表皮的修复能力下降,细胞的分化行为紊乱,这可能是瘢痕组织表皮结构与功能改变、愈合能力下降的原因之一.     

成人正常皮肤和瘢痕组织表皮干细胞定位与表达特征的研究

目的：观察成人正常皮肤和瘢痕组织中表皮干细胞定位与β1整合素和角蛋白19、14、10（K19、K14、K10）的表达，探讨两种组织表皮干细胞表达特征的差异。方法：取6例大面积深度烧伤患伤后1年的增生性瘢痕组织，另取6例健康志愿对应部位的全层皮肤。采用免疫组织化学Eivision两步法，检测表皮干细胞、短暂扩充细胞特异表达的β1整合素和K19以及分化表皮细胞表达的K14和K10。结果：瘢痕组织表皮基底层表达β1整合素与K19的阳性细胞数较正常皮肤明显减少，阳性强度降低，其表皮中表达K14的阳性细胞仅位于表皮底部2－3层，而K10表达阳性细胞则较正常皮肤分布广泛；瘢痕组织皮肤的分化过程亦不相同，处于有丝分裂后分化阶段的细胞比例降低，而终末分化细胞的比例明显增高。结论：瘢痕组织表皮的修复能力下降。细胞的分化行为紊乱，可能是导致瘢痕组织表皮结构与功能改变、愈合能力下降的原因之一。     

不同发育阶段人皮肤表皮干细胞增殖分化特征及其与创面修复结局关系的研究

目的从细胞分化角度研究人胎儿期、少儿期、成年人期皮肤中表皮干细胞增殖分化特征,以及这些特征与创面修复结局的关系.方法分别取因创伤等原因致流产的22～24周龄胎儿和4～12周岁少儿、35～53岁成年人3组全层皮肤.采用免疫组化方法检测表皮干细胞特异表达的β1整合素和细胞角蛋白19(K19).结果胎儿期皮肤表皮基底层细胞β1整合素和K19染色均为强阳性.少儿期表皮基底层细胞中60%～80%的细胞表达β1整合素和K19.成年人表皮基底层中表达β1整合素和K19的细胞较少儿组进一步减少,且染色强度较弱.结论本实验结果提示,胎儿期表皮基底层增殖细胞均为表皮干细胞和短暂扩充细胞,而少儿期表皮基底层中部分细胞为于细胞和短暂扩充细胞,成年人期干细胞与短暂扩充细胞所占的比例则进一步降低.这种干细胞增殖分化的差异可能与三种不同发育阶段皮肤损伤的完全与不完全修复有关.     

皮肤扩张过程中p63和细胞角蛋白的表达特征及意义

目的:初步观察皮肤扩张术对表皮细胞增殖分化的影响并探讨其机制。方法:用iWstar大鼠制作扩张动物模型,将72个扩张器埋于36只wistar鼠背部浅筋膜下,利用表皮干细胞特异表达p63和角蛋白19及短暂扩充细胞表达角蛋白14的特点,采用免疫组织化学Powervision M二步法检测扩张过程中表皮干细胞和短暂扩充细胞的分布与数量差异。结果:扩张皮肤表皮层增厚,表皮干细胞和短暂扩充细胞的数量明显增多,在棘层和颗粒层可见表皮干细胞和短暂扩充细胞的阳性表达。结论:皮肤扩张术能诱导表皮干细胞增殖分化,表皮干细胞在扩张过程中对机械应力和创伤修复的响应机制可能是其主要的生物学基础。     

黑皮素-1受体在增生性瘢痕组织中的表达与分布

目的:研究黑皮素-1受体(mel anocort in-1 recept or,MC-1R)在增生性瘢痕组织中的表达和分布特点,探讨它与增生性瘢痕形成的关系。方法:利用兔抗人MC-1R抗体进行免疫组织化学染色,并测定8例增生性瘢痕,8例表浅性瘢痕和16例同体的正常皮肤标本切片染色后的平均灰度值,分别检测MC-1R在上述不同组织中表达的差异。结果:在正常皮肤、表浅瘢痕和增生性瘢痕中,MC-1R免疫阳性产物呈棕黄色颗粒状,主要分布于表皮基底层细胞胞浆中。在增生性瘢痕组织中,MC-1R灰度值(159.2±5.5)明显高于正常皮肤(134.7±5.9)和表浅性瘢痕(135.9±9.3),差异显著(P<0.01)。而后两者无明显差异(P>0.05)。结论:增生性瘢痕的形成可能与MC-1R在增生性瘢痕组织中表达明显减少,由其介导的α-黑色素细胞刺激素(α-melanocyte stimul ating hormone,α-MSH)对胶原蛋白的代谢和细胞外基质沉积的调节作用减弱有关。     

人体正常皮肤及瘢痕组织中CD3~ 表皮内T细胞的形态学研究

目的通过对 CD3~ 表皮内 T 细胞在人体正常皮肤及瘢痕组织中的分布密度的研究,阐明表皮内 T 细咆对表皮细胞发育分化、分裂再生的影响。方法利用组织学和免疫组织化学法,观察表皮内 T 细胞在表皮组织中的分布。结果正常表皮组织中存在有少量 CD3~ 表皮内 T 细胞,增生性瘢痕组织中可见较正常组织数量多的 CD3~ 表皮内 T 细胞,表皮细胞增生活跃,萎缩性瘢痕组织中未发现有 CD3~ 表皮内 T 细胞存在,表皮细胞层平坦。结论正常皮肤组织和瘢痕组织中CD3~ 表皮内 T 细胞的分布情况不同,提示 CD3~ 表皮内 T 细胞可能与表皮细胞的发育分化、分裂再生有关,CD3~ 表皮内 T 细胞在表皮组织中的正常分布,对维持表皮组织的正常形态可能具有特殊意义。     

人体正常皮肤及瘢痕组织中CD3~ 表皮内T细胞的形态学研究

目的　通过对ＣＤ３ ＋ 表皮内Ｔ细胞在人体正常皮肤及瘢痕组织中的分布密度的研究,阐明表皮内Ｔ 细胞对表皮细胞发育分化、分裂再生的影响。方法　利用组织学和免疫组织化学法,观察表皮内Ｔ细胞在表皮组织中的分布。结果　正常表皮组织中存在有少量ＣＤ３＋ 表皮内Ｔ 细胞,增生性瘢痕组织中可见较正常组织数量多的ＣＤ３ ＋ 表皮内Ｔ细胞,表皮细胞增生活跃,萎缩性瘢痕组织中未发现有ＣＤ３ ＋ 表皮内Ｔ细胞存在,表皮细胞层平坦。结论　正常皮肤组织和瘢痕组织中ＣＤ３＋ 表皮内Ｔ细胞的分布情况不同,提示ＣＤ３＋ 表皮内Ｔ细胞可能与表皮细胞的发育分化、分裂再生有关,ＣＤ３＋ 表皮内Ｔ细胞在表皮组织中的正常分布,对维持表皮组织的正常形态可能具有特殊意义     

表皮干细胞表型的成纤维样细胞在瘢痕中的表达

目的：探寻正常皮肤与增生性瘢痕中表皮干细胞标记物的表达,从组织学角度探讨表皮干细胞参与瘢痕增生的证据。方法：术中切取8例伤后6月增生性瘢痕患者的瘢痕组织及同例患者的正常皮肤,将其制备切片并分组,用E1iviSion二步法免疫组织化学检测表皮干细胞表面标志物β1整合素和CK19的表达,计数阳性细胞并进行统计学处理。结果：瘢痕组织真皮层中,表达阳性细胞数β1整合素和CK19较正常皮肤β1整合素和CK19明显增多（P〈0．05）,成纤维细胞数显著增多。结论：在病理性瘢痕的发生中,表皮干细胞可能在细胞因子作用下分化为成纤维细胞,从而参与瘢痕形成。     

肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P＜0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在肝癌SP细胞中的含量分别为(92.65±3.92)%、(12.75±1.62)%、(17.35±2.31)%、(9.57±1.71)%和(28.39±5.28)%,在NSP细胞中的含量分别为(0.26±0.06)%、(2.51±0.17)%、(1.74±0.38)%、(1.52±0.41)%和(3.37±1.02)%,差异有统计学意义(P＜0.01).结论 肝癌SMMC-7721细胞中的SP细胞可能富集了肝癌干细胞,联合应用多种干细胞标记筛选肝癌SP细胞可能会获得纯化的肝癌干细胞.

Abstract：

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P ＜0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P ＜ 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.     

Expression of stem cell markers on fetal and tumoral human liver cells in primary culture

Hepatic stem cells can be identified by the expression of putative markers such as CD117 (c-kit), CD90 (Thy-1), CD34, and HLA-DR. We have identified populations expressing these markers in both fetal and tumoral human liver by flow cytometry, using monoclonal antibodies against CD90, CD117, CD34, and HLA-DR. In tumoral liver CD117+/CD90+ cells were found in decreasing number from the neoplastic (2.48 +/- 0.67) and peritumoral region (0.88 +/- 0.12) to the area of para-tumoral (normal) parenchyma (0.13 +/- 0.04). The CD117+/CD34+ cells showed the following distribution: 0.35 +/- 0.05% in the tumoral region, 1.01 +/- 0.23% in the peritumoral region and 0.35 +/- 0.01 in the para-tumoral region. Using the same markers on fetal liver cells we have also identified small populations of CD117+/CD90+ cells (0.28 +/- 0.07%) and CD117+/CD34+ cells (1.13 +/- 0.24%), presumably resident stem cells or hematopoietic stem cells. Immunomagnetic negative separation was then performed on fetal liver cells using monoclonal antibodies against specific markers of hematopoietic lineages such as CD3, 14, 16, 19, 22, and CD56 to eliminate this population. The remaining cells were then incubated with fluorescently labeled monoclonal antibodies against CD90 and CD117 and analyzed using fluorescence microscopy. As expected these markers were expressed on the majority of the selected cells (89.28 +/- 9.56%). Isolation using appropriate markers and initiation of primary cultures is a first step to the therapeutic use of fetal stem cells and for the study of adult liver stem cells involvement in carcinogenesis.     

Expression of embryonic stem cell markers in cultured JKT-1, a cell line derived from a human seminoma

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (αFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2γ and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.     

肝癌干细胞标志物研究的进展

肝癌是常见的恶性肿瘤,其目前的治疗手段是以手术为主的综合治疗,但是术后易复发、转移,预后较差。肿瘤干细胞学说认为只有杀灭肝癌干细胞才能从根本上治愈肝癌,因此分离和鉴定肝癌干细胞成为研究的热点。笔者就目前的肝癌干细胞表面标志物研究进展进行综述。     

Acquired renal scars in children

To determine the important factors involved in the etiology of renal scarring we studied 37 children with renal scars seen at our hospital since 1965. This is the second largest series reported to date. Children who had neurogenic bladders or any structural abnormalities of the urinary tract other than vesicoureteral reflex were excluded. The study group included 36 girls and 1 boy. The average age at first detection of renal scars was 5.7 years. Acute pyelonephritic episodes, which were treated early and aggressively, infrequently led to renal scarring. However, the initial prolonged or poorly treated episode of acute pyelonephritis was followed invariably by the development of renal scarring. The severity of renal scarring was related to the grade of vesicoureteral reflux (p less than 0.05), although some scars did develop in the absence of reflux. Neither the shape and position of the ureteral orifice nor the ureteral tunnel length correlated with the severity of renal scarring. Treatment with prophylactic antibiotics may have lessened the severity of renal scarring (0.1 less than p less than 0.2) but treatment with reimplantation surgery did not appear to alter the course of renal scarring. This study suggests that the key to the prevention of renal scarring is the early and aggressive treatment of acute pyelonephritis.     

大鼠的不同纯度胰岛中干细胞标志物CK-19及PDX-1 mRNA的表达

目的探讨大鼠的不同纯度胰岛中干细胞标志物细胞角质蛋白19(CK-19)和胰十二指肠同源盒基因1(PDX-1)mRNA的表达。方法将30只雄性SD大鼠随机平均分成3组,均采用胶原酶Ⅴ型通过胰管对胰腺进行灌注,切取胰腺,剪碎、吹打、消化、离心获得胰岛细胞沉淀物。A组胰岛沉淀物不进行纯化;B组胰岛沉淀物中加入体积分数为25%的Ficoll-400液纯化;C组胰岛沉淀物依次加入25%及11%的Ficoll-400纯化。分别将上述3组纯化后的胰岛抽提RNA行逆转录聚合酶链反应(RT-PCR),分析胰岛干细胞标志物CK-19及PDX-1mRNA的表达。结果采用不同的纯化方法后,A、B、C三组获得胰岛的纯度分别为(43.6±6.29)%、(65.3±4.40)%和(77.6±6.36)%,三组间比较,差异有统计学意义(P<0.05)。A组胰岛中CK-19和PDX-1mRNA的表达明显高于B组和C组,而C组表达最弱。结论不同纯度的胰岛中均有CK-19及PDX-1mRNA的表达,表明在纯化胰岛中均有干细胞存在。     

人胰腺癌细胞系表达干细胞标志物研究

目的 观察胚胎干细胞和胰腺相关干细胞标志物在胰腺癌细胞系中基因和蛋白水平的表达,探讨胰腺癌起源.方法 应用逆转录聚合酶链反应(RT-PCR)检测胚胎干细胞标志物Oct4、abc2、c-kit以及c-kit配体干细胞因子(SCF)和胰腺相关干细胞标志物角蛋白19(ck19)、巢蛋白(nestin)、波形蛋白(vimentin)、胰十二指肠同源盒基因-1(PDX-1)等在胰腺痛细胞系sw1990、BxPC3、pc3和jt305中基因水平(mRNA)的表达;利用免疫细胞化学(ICC)方法检测Oct4、abcs2、c-kit和ck19、nestin在上述细胞系中蛋白水平的表达.同时结合异硫氰荧光素(FITC)标记抗体进行流式分选检测上述指标在胰腺癌细胞中的表达率.结果 除BxPC3、pc3和j1305不表达c-kit以外,上述4种胰腺癌细胞系在mRNA水平表达其余所有的标志物;在蛋白水平,上述4种细胞均表达Oct4、abcg2、ck19、nestin,而除sw1990以外的3株胰腺癌细胞株小表达c-kit.结论 人胰腺癌细胞系表达胚胎干细胞标志物Oct4、abcg2和胰腺干细胞标志物ck19、nestin、vimentin、PDX-1,部分表达c-kit.胰腺癌可能来源于成体胰腺干细胞或胰腺前体细胞.