采用罗丹明123对肝癌细胞系MHCC97(Rholow)干细胞亚群的分离及鉴定

目的 建立分离肝癌细胞系MHCC97细胞中对罗丹明123染料具有不同排斥能力细胞亚群的方法,并探讨其在肝癌干细胞和异质性研究中的意义.方法 利用流式细胞分选术(FCM)并结合不同浓度的罗丹明123(Rho123)染料,分析和分选肝癌细胞系MHCC97细胞中具有不同染料排斥能力的肝癌细胞亚群,再通过细胞生长的测定、软琼脂克隆形成以及免疫组织化学检测和裸鼠成瘤实验等方法,以比较不同肝癌细胞亚群的差异.结果 根据不同染料排斥能力可以将肝癌细胞系MHCC97细胞分为Rholow和Rhohigh2个亚群,2个亚群在增殖能力、克隆形成率以及甲胎蛋白(AFP)和角蛋白-19(CK-19)表达以及裸鼠成瘤实验上差异有统计学意义,Rholow亚群具有干细胞特性.结论 罗丹明123结合FCM可以富集具有干细胞特性的Rholow亚群,同时为进一步揭示肝癌异质性机制奠定基础.     

氟尿嘧啶致肠黏膜损伤修复过程中肠上皮干细胞的动态观察

目的观察氟尿嘧啶（5-FU）致肠黏膜损伤修复过程中肠上皮干细胞的动态变化。方法成年C57BL／6J小鼠50只，其中40只给予连续5d腹腔注射5-Fu（30mg·kg^-1·d^-1），另10只腹腔注射等量PBS作为空白对照。于处理后1、3、5和7d剖杀取出中段小肠，光镜下观察肠黏膜病理改变：流式细胞术检测肠黏膜细胞中Rhdamine123（Rho）染色阴性细胞比例变化情况：免疫组化分析Musashi-1fmsi-1）表达阳性细胞数量及其百分比。结果与对照组比较，5-Fu作用后小鼠肠黏膜萎缩，间质有少量炎性细胞浸润：Rho染色阴性细胞比例显著增高，以处理后1d最高，随后逐渐下降（P〈0．011；msi-1表达阳性细胞数量略有降低，但差异无统计学意义（p〉0．05）；msi．1表达阳性细胞百分比显著增高（P〈0．01）R与Rho染色阴性细胞比例呈显著正相关（P〈0．01，r=0．7551。结论Rho染色阴性细胞中含有丰富的肠上皮干细胞．msi-1表达阳性的肠上皮干细胞可能起着修复5-FU致肠黏膜损伤的作用。     

流式细胞仪富集大鼠A1～A4型精原干细胞

目的:探讨A型精原干细胞的A1~A4亚型细胞的分离纯化方法。方法:先使用不连续密度梯度法分离A型精原细胞,再经流式细胞分选技术分选有干细胞因子受体(c-kit)表达的亚型细胞。并采用免疫组化检测c-kit在睾丸中的表达,电镜下观察分选后细胞超微结构。结果:经密度梯度分离后的精原细胞内含c-kit阳性细胞比例为(18.65±1.69)%,未经密度梯度分离的睾丸细胞内含c-kit阳性细胞比例为(3.16±0.84)%,两者比较差异有显著性(P<0.01);c-kit阳性细胞经流式细胞术分选,阳性细胞回收率(65.90±1.24)%,活性(85.60±1.14)%。结论:以c-kit为标记物,经不连续密度梯度分离初步纯化后,使用流式细胞分选技术能有效分离A型精原干细胞亚型。     

大剂量5-FU致肠黏膜严重损伤时肠上皮细胞msi-1的表达及其意义

目的 观察大剂量5-Fu致肠黏膜严重损伤时肠上皮细胞musashi-1(msi-1)的表达及其意义.方法 成年C57BL/6J小鼠40只,分为对照组(n=8,D组)和处理组(n=32).D组给予腹腔注射PBS,处理组给予腹腔注射大剂量5-Fu(150 mg·kg-1·d-1×5 d),分别于处理后1 d(A组)、3 d(B组)、5 d(C组)剖杀濒死小鼠取出小肠,用来常规HE染色,免疫组化检测肠上皮干细胞标记物msi-1的表达;制备A组小鼠肠黏膜细胞悬液,流式细胞术检测msi-1阳性细胞比例. 结果 大剂量5-FU作用后,小鼠肠黏膜被严重破坏,黏膜萎缩,绒毛、隐窝结构消失;免疫组化结果显示msi-1表达上升,msi-1阳性细胞比例显著增高(P＜0.01);流式检测提示A组小鼠肠黏膜细胞悬液中前向角(FSC)数值较大的细胞群msi-1阳性细胞比例较高,约为67.75%. 结论 小鼠大剂量5-Fu腹腔注射后,肠黏膜细胞中表达msi-1的肠上皮干细胞比例大幅增加,可为肠上皮的进一步分离纯化及其生物学特征的研究奠定基础.     

显微分离培养与免疫磁珠法分离纯化人毛囊干细胞

目的 探讨从人毛囊隆突区细胞(bulge cells,BCs)获取高纯度有活性的人毛囊干细胞(hair folliclestem cells,HFscs)的方法及条件. 方法 取自愿捐献的成人头皮标本,显微分离培养获得BCs,应用免疫磁珠纯化BCs中CD200阳性的HFSCs.苔盼蓝染色比较纯化前后细胞活性;流式细胞仪检测细胞纯化效率;免疫荧光检测纯化前后细胞CD200的表达情况. 结果 显微分离培养获得的人毛囊BCs,培养6 d后细胞生长融合,呈铺路石样.所得细胞角蛋白19组织化学染色阳性.CD200免疫磁珠分选法纯化细胞后,苔盼蓝染色显示纯化前细胞活力为95.0%±0.6%,纯化后为94.2%±1.0%,差异无统计学意义(P＞0.05).流式细胞仪及免疫荧光检测显示纯化前CD200阳性细胞的纯度为8.31%,纯化后CD200阳性细胞纯度为82.31%,纯化后CD200阳性细胞回收率为65.39%. 结论 联用显微分离培养与免疫磁珠法,可获得高纯度HFSCs,且细胞活性不受影响.     

人表皮细胞中侧群细胞表型的初步分析

目的:检测人表皮细胞中是否存在侧群(side population,SP)细胞,并研究该表型细胞是否表达通用干细胞标志物ABCG2和表皮干细胞的标志物整合素α6和β1.方法:运用中性蛋白酶和胰蛋白酶两步消化法从手术切除的人包皮组织中分离获得表皮细胞.经Hoechst33342荧光染料和PI染色,流式细胞仪分析并分选SP细胞.采用流式细胞仪分析SP细胞ABCG2、整合素α6和β1的表达情况.结果:人表皮细胞中存在SP细胞,其比例为0.2%～0.3%.用流式细胞仪可检测到在SP细胞以及总表皮细胞中均有少量细胞表达通用干细胞标志物ABCG2以及表皮干细胞的标志物整合素α6和β1,但二者阳性细胞百分比差异无统计学意义(P>0.05).结论:表皮细胞中的SP细胞是否是富集的表皮干细胞仍存在疑问,还需要进一步的动物体内移植实验、克隆形成能力和增殖能力的研究证实.     

CD133作为肝癌干细胞表面标记的初步研究

目的 研究人肝癌SMMC7721和bcl-7402细胞株中CD133的表达,探讨CD133作为肝癌干细胞表面标记的可能性.方法 采用流式细胞仪检测SMMC7721和bcl-7402细胞株中CD133的表达;免疫磁珠细胞技术分离SMMC7721和bcl-7402细胞株中CD133阳性和CD 133阴性细胞,检测其细胞周期,观察并采用单因素方差分析和u检验分析体外培养过程中CD133阳性和CD133阴性细胞的细胞形态、增殖和分化能力的差异.结果 CD133阳性细胞在SMMC7721和bcl-7402细胞株中所占比例分别为0.7%～1.0%、1.7%～8.9%;流式细胞仪检测显示两种细胞株中处于G0/G1期的CD133阳性细胞分别为85.3%、89.4%,明显高于CD133阴性细胞和未分选细胞(F=14.49,38.84,P<0.05);CD133阳性细胞体外增殖能力强于相同条件下CD133阴性细胞和未分选细胞,在1～3 d和5～7 d曲线变化更明显(F=49.32,784.04,89.91,152.83,P<0.05);体外培养过程中,CD133阳性细胞的比例逐日下降,至扩增的第15天,与未分选细胞含量相似(u=0.271,P<0.05).结论 人肝癌SMMC7721和bcl-7402细胞株中,CD133阳性细胞体外增殖和分化能力较强,CD133是肝癌干细胞的表面标记之一.     

胃癌CD133阳性细胞亚群肿瘤起始细胞样特性的检测

目的 探讨人胃癌细胞CD133＋亚群的分选及其特性的鉴定.方法 对50例胃癌原发灶和癌旁胃黏膜组织标本行免疫组织化学染色及Western blot检测CD133蛋白的表达.采用流式细胞仪检测不同分化胃癌细胞系CD133所占百分比,免疫磁珠法分选CD133＋细胞亚群,悬浮培养并观察其生长特性,并检测CD133阳性细胞裸鼠皮下接种致瘤能力.单细胞克隆观察单个CD133＋细胞生长特性,半定量反转录聚合酶链反应法鉴定相关干细胞标记的表达.结果 CD133蛋白多定位于胃癌原发灶黏膜及黏膜下层的肿瘤细胞膜表面,其相对表达量高于癌旁胃黏膜组织（P＜0.05）.不同分化程度的人胃癌细胞系KATO-Ⅲ、SGC-7901、AGS及MKN-45中CD 133＋亚群所占相对百分比为（28±2）％、（17±2）％、（6±2）％及（4±2）％.人胃癌细胞系KATO-Ⅲ分选后阳性组中CD133＋亚群比例分别为（91±3）％;培养1周后,达到（95±2）％,并不断增殖形成细胞球.而且其增殖能力强于阴性细胞[群体倍增时间分别为（21±3）h和（40±8）h,P＜0.05].CD133阳性组和未分选组细胞裸鼠皮下接种时成瘤率分别为100％和80％;而CD133阴性组不成瘤,CD133阳性组移植瘤平均体积及重量均大于未分选组（P＜0.05,P＜0.05）.单克隆形成实验示单个CD133＋细胞可形成新的细胞克隆.半定量RT-PCR检测示其表达干细胞标记物Oct-4、Nanog、Sox-2、Musashi-1及EGFR.结论 体外可成功分离、纯化和扩增人胃癌细胞CD133＋亚群,其具有自我更新、增殖能力及较强的致瘤能力,并表达部分干细胞相关基因.     

ABCG2在人肝癌细胞株中的表达及其相关功能

目的探讨干细胞标志物三磷酸腺苷结合盒超家族G家族第2个成员(ATP-binding cassette super-family G member 2,ABCG2)在人肝癌细胞株中的表达及其与肝癌耐药的关系,观察人肝癌细胞株中ABCG2阳性表达和阴性表达细胞的功能差异。方法流式细胞仪检测ABCG2在人肝癌细胞株PLC/PRF/5、7402、7701及7721中的阳性表达,计算5-氟尿嘧啶(5-FU)、阿霉素的半数抑制浓度(IC50)值。采用免疫荧光染色观察ABCG2在肝癌7721细胞株中的表达,并进行无菌流式分选,观察ABCG2阳性表达与ABCG2阴性表达的肝癌7721细胞的功能差异。结果在4种肝癌细胞株中7721细胞株ABCG2表达阳性率最高(P<0.05),且流式细胞图中阳性表达和阴性表达细胞呈明显双峰。5-FU、阿霉素对7721细胞株的IC50值明显高于其他3种细胞株,除与5-FU对7701细胞的IC50值差异无统计学意义(P>0.05)外,其余比较差异均有统计学意义(P<0.05)。分选后的ABCG2阳性表达细胞与阴性表达细胞的增殖差异无统计学意义(P>0.05);ABCG2阳性表达细胞较阴性表达细胞更多处于相对静止期(P<0.05)。结论 ABCG2在人多种肝癌细胞株中均表达,ABCG2表达阳性率越高的细胞较表达阳性率越低的细胞更具有耐药性,ABCG2阳性表达细胞处于相对静止期。     

非小细胞肺癌循环肿瘤细胞的定量检测

目的 探讨非小细胞肺癌循环肿瘤细胞(CTCs)定量检测方法 .方法 CD45免疫磁珠阴性分选组20例,CD326免疫磁珠阳性分选组25例,均为明确诊断的非小细胞肺癌患者.磁性分离富集后肺静脉与外周静脉血标本应用多参数流式细胞仪对CTCs进行定量检测.结果 阴性分选由于只能去除CD45阳性细胞,回收目的 细胞纯度低.阳性分选组中25例术中肺静脉血CTCs定量检测阳性率为64%(16/25),外周静脉血CTCs阳性率40%(10/25,P＜0.05).结论 免疫磁珠富集联合流式细胞分析检测CTCs的敏感性和特异性较高.     

Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells

BACKGROUND: The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. METHODS: Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). RESULTS: Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. CONCLUSIONS: Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake.     

溴隐亭联合肿瘤坏死因子-α对人肝癌耐药细胞株化疗敏感性的影响

目的 研究溴隐亭联合肿瘤坏死因子-α（TNF—α）基因处理人肝癌耐药细胞株HepG2／ADM后对其化疗敏感性的影响。方法 脂质体转染TNF-α基因至肝癌耐药细胞HepG2／ADM;实验分4组：空白对照组HepG2（A组）、耐药组HepG2／ADM（B组）、转染TNF—α基因组（C组）及联合溴隐亭和TNF—α组（D组）;流式细胞仪检测各组罗丹明123细胞内潴留率变化、四唑盐比色法（MTT）检测C、D两组细胞耐药指数及用免疫组织化学染色、Western blot和RT—PCR方法测定各组PKC-α、P-gP蛋白、MDR1 mRNA表达的变化,流式细胞仪检测Bcl-2蛋白的表达,AnnexinV-FITC,PI双标检测各组肿瘤细胞化疗后凋亡的变化。结果 MTT测定C、D两组之间耐药逆转率有显著差异（P〈0.01）,罗丹明123潴留率实验发现C、D两组均能明显增强其潴留作用且两组差异有统计学意义（P〈0．01）;P-gP蛋白和MDR1基因表达显示C、D两组之间MDR1基因和P—gp蛋白的表达无统计学意义（P〉0．05）,但C、B两组之间比较差异有统计学意义（P〈0．01）;PKC—α蛋白表达在D组明显下调（P〈0．01）;B组细胞Bcl-2蛋白表达明显增强,C、D两组细胞Bcl-2蛋白表达明显下调,与A组比较均有统计学意义（P〈0．01）,但C、D两组细胞Bcl-2蛋白表达无统计学意义（P〉0．05）。细胞凋亡率C、D两组比较有统计学意义（P〈0．01）,D、A两组比较无统计学意义（P〉0．05）。结论 溴隐亭和TNF—α通过不同的机制逆转肿瘤的多药耐药性,两者联合明显加强抗肿瘤药物对细胞的毒力。     

胰腺导管上皮细胞来源的胰岛样细胞移植治疗1型糖尿病

目的 观察小鼠胰腺导管上皮细胞向胰岛样细胞转分化及其在治疗糖尿病小鼠中的作用.方法 分离培养昆明小鼠胰腺导管上皮细胞,经体外扩增及诱导培养后,进行体外和体内功能评价.结果 小鼠胰腺导管上皮细胞经体外扩增和诱导分化后,逆转录-聚合酶链反应(RT-PCR)显示培养1周和2周后胰岛素mRNA的IA值为(1.892±0.119比3.135±0.092,P＜0.05),胰高血糖素为(1.564±0.087比2.271±0.042,P＜0.05),表达明显上调;胰岛样细胞对高糖刺激(15.0 mmol/L)的胰岛素释放较低糖(5.6 mmol/L)时增加了1.7倍[(52.3±10.5)mmol/L比(30.2±9.7)mmol/L,P＜0.05];DTZ染色阳性;将其移植入糖尿病小鼠体内后,移植组小鼠较对照组血糖下降,差异有统计学意义(P＜0.05).结论 昆明小鼠胰腺导管上皮细胞在体外培养条件可转分化胰岛素分泌细胞,该细胞团可在体内环境下发挥生物学功能.

Abstract：

Objective To investigate the differentiation of stem cells deived from mouse pancreatic ductal epithelial cells toward insulin secreting cells and the potential application for diabetes therapy.Methods Pancreatic ductal epithelial cells were separated and differentiated into islet-like cells. Study was performed to determine whether these islet-like clusters could secrete insulin in response to glucose both in vivo and in vitro. Results Pancreatic ductal epithelial cells were separated and cultured in vitro.The expression of insulin mRNA in the stem cells was significantly up-regulated ( 1. 892 ±0. 119 vs 3. 135± 0. 092,P ＜ 0. 05 ), and also in glucagon ( 1. 564 ± 0. 087 vs 2. 271 ± 0. 042, P ＜ 0. 05 ). Immunofluoresence staining indicated that there were a lot of insulin positive cells. Insulin released from cells in response to glucose stimulation in vitro was increased [ ( 52. 3 ± 10. 5 ) mmol/L vs ( 30. 2± 9. 7 ) mmol/L, P ＜0. 05 ]. Hyperglycemia in diabetic animals was alleviated after cell transplantation. Conclusion Islet-like cell clusters generated in vitro from Kunming mice pancreatic ductal epithelial cells could secrete insulin and have some effects on reversing the diabetes in diabetic mice.     

Evidence for Rho protein regulation of renal tubular epithelial cell function

BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton. The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known. METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells. RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis. A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis. A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells. Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis. The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor. CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis. Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.     

Signaling through tumor necrosis receptor 2 induces stem cell marker in CD133+ regenerating tubular epithelial cells in acute cell‐mediated rejection of human renal allografts

Tumor necrosis factor receptor 2 (TNFR2) is strongly upregulated on renal tubular epithelial cells by acute cell‐mediated rejection (ACR. In human kidney organ culture, TNFR2 signaling both upregulates TNFR2 expression and promotes cell cycle entry of tubular epithelial cells. We find significantly more cells express CD133 mRNA and protein, a putative stem cell marker, in allograft biopsy samples with ACR compared to acute tubular injury without rejection or pretransplant “normal kidney” biopsy samples. Of CD133⁺ cells, ~85% are within injured tubules and ~15% are interstitial. Both populations express stem cell marker TRA‐1‐60 and TNFR2, but only tubular CD133⁺ cells express proximal tubular markers megalin and aquaporin‐1. TNFR2⁺CD133⁺ cells in tubules express proliferation marker phospho‐histone H3^S10 (pH3^S10). Tubular epithelial cells in normal kidney organ cultures respond to TNFR2 signaling by expressing CD133 mRNA and protein, stem cell marker TRA‐1‐60, and pH3^S10 within 3 hours of treatment. This rapid response time suggests that CD133⁺ cells in regenerating tubules of kidneys undergoing ACR represent proliferating tubular epithelial cells with TNFR2‐induced stem cell markers rather than expansion of resident stem cells. Infiltrating host mononuclear cells are a likely source of TNF as these changes are absent in acute tubular injury .