人胰腺癌细胞系SW1990中肿瘤干细胞样侧群细胞的分离及生物学特性研究

目的 从人胰腺癌细胞系SW1990中分离肿瘤干细胞样侧群(side population,SP)细胞,并进一步研究其生物学特性.方法 细胞常规培养后应用Hoechst 33342和PI染色、荧光激发流式细胞仪检测并分选SP细胞,MTT法检测细胞活性,流式细胞仪分析干细胞标志物CD133的表达,克隆平板测定细胞克隆形成能力,Western blot检测细胞ABC超家族成员G2(ABCG2)蛋白表达.结果 人胰腺癌细胞系SW1990中SP细胞比例为2.70%,可完全被维拉帕米阻断.培养9 d后,SP细胞的A492值为2.1,克隆形成率为(38.7±6.8)%,CD133阳性率为69.63%,均显著高于非SP细胞的0.5,(15.5±2.8)%,16.71%;SP细胞ABCG2表达也较非SP细胞强.结论 胰腺癌细胞系SW1990存在SP细胞.     

Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma

AIM： To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1. METHODS： The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 （1：1 volume） （DMEM-F12） supplemented with 20% fetal bovine serum （FBS）. Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl- 2H-tetrazolium bromide （MTT） method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex （ABC） immunohistochemical staining. RESULTS： The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44^＋ CD24^＋. Compared with CD44-CD24- cells, CD44^＋CD24^＋ cells had a lower growth rate in vitro. Implantation of 104 CD44 CD24 cells in nude mice showed no evidenttumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 103 CD44^＋CD24^＋ cells at wk 4 （2/8）, a 20-fold increase in tumorigenic potential （P 〈 0.05 or P 〈 0.01）. There was no obvious histological difference between the cells of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells. CONCLUSION： CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44^＋CD24^＋ have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new dr     

胃癌MKN-28肿瘤细胞系SP细胞亚群初步分析

目的:分析胃癌细胞株MKN-28中是否包含肿瘤干细胞相关的SP(side population)细胞亚群.方法:采用免疫荧光方法检测干细胞标记ABcG2在胃癌细胞株MKN-28的表达;制备MKN-28细胞悬液,经Hoechst33342染色,流式细胞仪分析SP亚群.结果:ABCG2在胃癌细胞系MKN-28中有少量表达,ABCG2阳性细胞约占1.7%.SP亚群占MKN-28细胞的0.25%,维拉帕米阻断后减少为0.05%.结论:人胃癌细胞株MKN-28中存在SP细胞亚群,提示胃癌干细胞的存在.     

阻断PI3K/Akt通路增强缺氧环境胰腺癌细胞化疗敏感性的实验研究

目的研究LY294002联合吉西他滨是否能增强缺氧环境下胰腺癌细胞株PANC-1的化疗敏感性。方法缺氧条件下培养胰腺癌细胞株PANC-1,运用MTT检测LY294002联合吉西他滨对胰腺癌细胞株细胞PANC-1生长的影响,运用Western blot检测AKT及磷酸化AKT蛋白表达的水平。结果 MTT检测在缺氧环境下LY294002联合吉西他滨作用的胰腺癌细胞PANC-1与单纯吉西他滨作用组相比细胞生存率显著下降(P=0.003),且具有时间依赖性。Western blot检测显示在缺氧环境下,LY294002联合吉西他滨作用的胰腺癌细胞PANC-1磷酸化AKT蛋白水平与单纯吉西他滨作用组相比显著降低(P=0.002)。结论阻断PI3K/Akt通路能增强缺氧环境下胰腺癌细胞化疗敏感性,为胰腺癌的治疗方法及逆转耐药提供了新的实验依据。     

侧群细胞在胃癌细胞株中的分布及其致瘤特性

目的 研究侧群细胞的致瘤特性及其在胃癌细胞株和胃癌组织中的分布.方法 用荧光激活细胞分选法分析SGC-7901、MKN28和BGC-823三种胃癌细胞株中的侧群细胞.取36只裸鼠,分为6组,将SOC-7901分离出的侧群细胞和非侧群细胞分别以每只500、5000、50 000的数量接种到裸鼠皮下,8周后观察成瘤情况.实时定量PCR检测胃癌组织和胃癌细胞株中三磷酸腺苷结合转运蛋白超家族成员G2(ABCG2)mRNA的表达,免疫组化法检测胃癌组织中ABCG2蛋白的表达.结果 SGC-7901细胞株中侧群细胞比例为1.0%,BGC-823为1.3%,MKN28则为阴性.从SGC-7901中分离的侧群细胞最少可成瘤细胞数是500/只,非侧群细胞为50000/只.胃癌细胞株SGC-7901和BGC-823的ABCG2 mRNA相对量高于MKN28(分别为0.162、0.096和0.005).ABCG2 mRNA和蛋白在胃癌和胃炎组织中有不同程度的表达.结论 胃癌细胞株侧群细胞的致瘤能力明显强于非侧群细胞.在胃癌组织和部分胃炎组织中检测到ABCG2的表达,在胃癌细胞株中侧群细胞比例高的细胞株ABCG2表达高.     

Gastric mucosal injury due to hemorrhagic reperfusion and efficacy of Salvia miltiorrhizae extract F and cimetidine

AIM: To observe the gastric mucosal injury caused by hemorrhagic shock and reperfusion and to compare the effect between Salvia miltiorrhizae extract F (SEF) and cimetidine (CI) on it. METHODS: A model of hemorrhage/reperfusion injury was produced by Itoh method. Wistar rats were randomly divided into three groups: 0.9% sodium chloride treatment group (NS group), SEF treatment group (SEF group), and CI treatment group (CI group). Saline, SEF and CI were injected respectively. The index of gastric mucosal lesions (IGML) was expressed as the percentage of lesion area in the gastric mucosa. The degree of gastric mucosal lesions was categorized into grades 0, 1, 2, 3. Atom absorption method was used to measure the intracellular calcium content. Radioimmunoassay was used to measure the concentrations of prostaglandins. RESULTS: IGML (%) and grade 3 (%) were 23.18±6.82, 58.44±9.07 in NS group, 4.42±1.39, 20.32±6.95 in SEF group and 3.74±1.56, 23.12±5.09 in CI group, and the above parameters in SEF group and CI group decreased significantly (IGML: SEF vs NS, t=6.712, P=0.000<0.01; CI vs NS, t=6.943, P=0.000<0.01; grade 3: SEF vs HS, t=8.386, P=0.000; CI vs HS, t=8.411, P= 0.000), but the grade 0 and grade 1 damage in SEF group (22.05±5.96, 34.12±8.12) and CI group (18.54±4.82, 30.15±7.12) were markedly higher than those in NS group (3.01±1.01, 8.35±1.95; grade 0: SEF vs HS, t=8.434, P=0.000<0.01; CI vs NS, t=7.950, P=0.000<0.01; grade 1: SEF vs NS, t =8.422, P=0.000<0.01; CI vs NS, t=8.448, P=0.000<0.01). The intracellular calcium content (μg/mg) in SEF group (0.104±0.015) and CI group (0.102±0.010) was markedly lower than that in NS group (0.131±0.019, SEF vs NS, t=2.463, P=0.038<0.05; CI vs HS, t=3.056, P=0.017<0.05). The levels (pg/mg) of PGE_2, 6-keto-PGF_(1α) and 6-keto-PGF_(1α)/TXB_2 were 540±183, 714±124,17.38±5.93 in NS group and 581±168, 737±102, 19.04±8.03 in CI group, 760±192,1 248±158, 33.42±9.24 in SEF group, and the above parameters in SEF group markedly raised (PGE_2: SEF vs NS, t=2.282, P=0.046<0.05; SEF vs CI, t=2.265, P=0.047<0.05; 6-keto-PGF_(1α): SEF vs NS, t=6.583, P=0.000<0.000; SEF vs CI, t=6.708, P=0.000<0.01; 6-keto-PGF_(1α)/TXB_2: SEF vs NS, t=3.963, P=0.003<0.001; SEF vs Cl, t=3.243, P=0.009<0.01), whereas TXB_2 level in SEF group (45.37±7.54) was obviously lower than that in NS group (58.28±6.74, t=3.086, P=0.014<0.05) and CI group (54.32±6.89, t=2.265, P=0.047<0.05). No significant difference was shown between NS group and CI group (PGE_2: t=0.414, P=0.688>0.05; 6-keto-PGF_(1α): t=0.310, P=0.763>0.05; TXB_2: t=1.099, P=0.298>0.05; 6-keto-PGF_(1α)/TXB_2: t=0.372, P=0.718>0.05). CONCLUSION: Both SEF and CI could inhibit reperfusioninduced injury in gastric mucosa, but with different mechanisms. SEF could not only enhance the protective effect of gastric mucosa, but also abate the injury factors, while CI can only abate the injury factors.     

Isolation of Side Population Cells in B-Cell Non-Hodgkin's Lymphomas

Background: Side population (SP) cells are characterized by the ability to exclude Hoechst 33342 dye due to high expression of the ATP-binding cassette transporter. This ability is associated with drug-resistant characteristics of cancer stem cells. Methods: We analyzed SP cells from human B-cell non-Hodgkin's lymphoma cell lines and primary cells derived from patients and compared them with non-SP (NSP) cells. Results: SP cells comprised a minor fraction of all cells ranging from 1.5 ± 1.8 to 8.3 ± 5.7% in cell lines and had higher ABCG2 expression than NSP cells. SP cells had better cell viability, colony-forming ability and drug resistance than NSP cells. The SP cells also showed stem cell-like characteristics, including elevated telomerase activity and higher expression of OCT4 and NANOG. A cDNA microarray demonstrated that SP cells had decreased expression of genes associated with apoptosis and cell death compared to NSP cells. Conclusions: The presence of SP cells might imply the possibility of lymphoma stem cells and be associated with a malignant potential of B-cell lymphoma.     

Antitumor effects of all-trans-retinoic acid on cultured human pancreatic cancer cells

BACKGROUND AND AIM: Although it is uncommon, pancreatic cancer is known to have a poor prognosis. The aim of the present study was to determine the inhibitory effects of all-trans-retinoic acid (ATRA) on cell growth, cell cycle and alkaline phosphatase (ALP) activity in the human pancreatic cancer cell line PANC-1 in vitro. METHODS: Human pancreatic cancer PANC-1 cells were treated by various concentrations of ATRA, and then the cell growth was determined by MTT viability assay. Cell cycle distribution and ALP activity were analyzed by flow cytometry and chemical analyzer, respectively. RESULTS: ATRA inhibited the growth of PANC-1 cells grown in culture; a dose-dependent inhibitory influence was found. ATRA arrested PANC-1 cells at G2/M phase. The ALP activity of PANC-1 cells was significantly increased by 1-50 micromol/L ATRA. CONCLUSIONS: The antitumor effects of ATRA on human pancreatic cancer cells are associated with G2/M phase arrest and increased ALP activity.     

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases

AIM：To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro.
METHODS： Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 μg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase （LDH） assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction （PCR） and Western blotting. Cell cycle stages were evaluated by flow cytometry.
RESULTS： The growth inhibitory rates of MK615 at 150, 300, and 600 μg/mL were 2.3% ± 0.9%, 8.9% ± 3.2% and 67.1% ± 8.1% on PANC1 cells, 1.3% ± 0.3%, 8.7% ± 4.1% and 45.7 ± 7.6% on PK1 cells, and 1.2 ± 0.8%, 9.1% ± 2.1% and 52.1% ± 5.5% on PK45H cells, respectively （P 〈0.05）. The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 μg/mL were 19.6% ± 1.3%, 26.7% ± 1.8%, 25.5% ± 0.9% and 26.4% ± 0.9% in PANC1 cells, 19.7% ± 1.3%, 24.7% ± 0.8%, 25.9% ± 0.9% and 29.9% ± 1.1% in PK1 cells, and 28.0% ± 0.9%, 31.2% ± 0.9%, 30.4% ± 1.1% and 35.3 ± 1.0% in PK45H cells, respectively （P 〈 0.05）. Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase.
CONCLUSION： MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.     

A distinct "side population" of cells with high drug efflux capacity in human tumor cells

A subset of stem cells, termed the "side population" (SP), has been identified in several tissues in mammalian species. These cells maintain a high efflux capability for antimitotic drugs. We have investigated whether functionally equivalent stem cells also may be detected in human cancers. We initially examined primary tumor cells from 23 patients with neuroblastoma and cell lines derived from a range of other tumors. A distinct SP was found in neuroblastoma cells from 15 of 23 patients (65%). The SP was capable of sustained expansion ex vivo and showed evidence for asymmetric division, generating both SP and non-SP progeny. These cells also expressed high levels of ABCG2 and ABCA3 transporter genes and had a greater capacity to expel cytotoxic drugs, such as mitoxantrone, resulting in better survival. A SP also was detected in breast cancer, lung cancer, and glioblastoma cell lines, suggesting that this phenotype defines a class of cancer stem cells with inherently high resistance to chemotherapeutic agents that should be targeted during the treatment of malignant disease.     

ABCB1、ABCG2与食管癌患者多药耐药的关系

目的探讨多药耐药蛋白三磷酸腺苷结合转运蛋白G2(ABCG2)蛋白和三磷酸腺苷结合转运蛋白B1(ABCB1)在食管癌组织中的表达及其意义。方法选用80例食管癌患者手术切除标本,采用流式细胞术(FCM)方法检测80例原发性食管癌患者的癌组织、癌旁组织(距离癌2 cm)及食管切缘正常黏膜(距离癌5 cm以上)ABCG2及ABCB1蛋白的表达量。结果 ABCG2及ABCB1蛋白在食管癌组织中有较高程度的表达,表达量分别为(467.30±37.85)、(444.79±22.83),而正常食管黏膜中表达量分别为(418.41±21.70)、(401.35±16.09);其在食管癌中的表达显著高于正常黏膜(P<0.01)。ABCG2蛋白及ABCB1蛋白在低分化鳞癌中的表达量显著高于中高分化鳞癌(P<0.05),在侵及纤维膜的癌组织中表达显著高于未侵及纤维膜的癌组织(P<0.05),在有淋巴结转移的癌组织中表达显著高于无淋巴结转移癌组织(P<0.01),在年龄和性别间表达无显著性差异(P>0.05)。结论 ABCG2及ABCB1参与食管癌多药耐药的形成,对于临床上食管癌的化疗具有一定的指导意义。     

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells

AIM： To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS： KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane （Matrigel） assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student＇s two tailed t test.RESULTS： By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h （MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P 〈 0.05）. Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells （MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P 〈 0.05）. In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively （P 〈 0.05）.CONCLUSION： High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting     

Side population does not define stem cell-like cancer cells in the adrenocortical carcinoma cell line NCI h295R

Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 +/- 18.1 vs. 100.0 +/- 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.     

Oncogene-specific formation of chemoresistant murine hepatic cancer stem cells

At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of activation of v-akt murine thymoma viral oncogene homolog (AKT) and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This "side population" (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers, and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ATP binding cassette drug transporter proteins that also contribute to chemoresistance in cancer. We found that the multidrug resistance gene 1 (MDR1) transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1. Conclusion: The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer. (HEPATOLOGY 2012).     

Characterization of CD133^＋ parenchymal cells in the liver： Histology and culture

AIM: To reveal the characteristics of CD133~+ cells in the liver.METHODS: This study examined the histological characteristics of CD133~+ cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133~+ cells derived from human hepatocellular carcinoma (HCC) or cholangiocarcinoma cell lines.RESULTS: Immunostaining revealed constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133~+/CK19+/HepPar-1~-. A small number of CD133~+/CK19~-/HepPar-1~+ cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC (HuH7, PLC5 and HepG2) and two cholangiocarcinoma cell and HepG2) and two cholangiocarcinoma cell lines (HuCCT1 and CCKS1). Fluorescence-activated cell sorting (FACS) revealed that CD133~+ and CD133~-cells derived from HuH7 and HuCCT1 cells similarly produced CD133~+ and CD133~-cells during subculture. To examine the relationship between CD133~+ cells and the side population (SP) phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133~+/CD133~-cells were almost identical in the SP and non-SP in HuH7. In addition, four different cellular populations (SP/CD133~+, SP/CD133~-, non-SP/CD133~+, and non-SP/CD133~-) could similarly produce CD133~+ and CD133~-cells during subculture. CONCLUSION: This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines.     

Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively

There is increasing evidence that cancers contain their own stem-like cells called cancer stem cells (CSCs). A small subset of cells, termed side population (SP), has been identified using flow cytometric analysis. The SP cells have the ability to exclude the DNA binding dye, Hoechst33342, and are highly enriched for stem cells in many kinds of normal tissues. Because CSCs are thought to be drug resistant, SP cells in cancers might contain CSCs. We initially examined the presence of SP cells in several human thyroid cancer cell lines. A small percentage of SP cells were found in ARO (0.25%), FRO (0.1%), NPA (0.06%), and WRO (0.02%) cells but not TPC1 cells. After sorting, the SP cells generated both SP and non-SP cells in culture. The clonogenic ability of SP cells was significantly higher than that of non-SP cells. Moreover, the SP prevalence was dependent on cell density in culture, suggesting that SP cells preferentially survived at lower cell density. Microarray experiment revealed differential gene expression profile between SP and non-SP cells, and several genes related to stemness were up-regulated. However, non-SP population also contained cells that were tumorigenic in nude mice, and non-SP cells generated a small number of SP cells. These results suggest that cancer stem-like cells are partly, but not exclusively, enriched in SP population. Clarifying the key tumorigenic population might contribute to the establishment of a novel therapy for thyroid cancer.     

从胎儿胰腺组织建立单克隆胰腺前体细胞的研究

目的 探索一种新的人胰腺干细胞分离纯化体系。方法 从胎儿胰腺中分离获得巢蛋白阳性细胞群体，荧光激活细胞分选(FACS)技术分选单个side population(SP)细胞后进行单克隆细胞培养。应用逆转录-聚合酶链反应(RT-PCR)及放射免疫分析(RIA)方法，研究单克隆SP细胞在体外增殖和向胰岛内分泌细胞分化的能力。结果 胎儿胰腺组织经过分离和体外培养后，可获得巢蛋白阳性细胞，对其进行Htoechst 33342染色，分析显示SP细胞约占0．1％；RT-PCR证实SP细胞有ATP结合盒转运子G2(ABCG2)表达，而非SP细胞则未见表达；SP细胞的克隆形成率约为2．7％，而非SP细胞没有克隆形成。在多种细胞因子和无血清的条件下，单克隆SP细胞经诱导后出现胰岛素、胰升糖素和胰十二指肠同源盒基因-1(PDX-1)mRNA的表达，而巢蛋白、neurogenin3 (Ngn3)和ABCG2 mRNA表达消失；RIA分析可检测到诱导后的细胞内有胰岛素产生。结论 首次报道从胎儿胰腺组织中分离并建立了单克隆胰腺前体细胞。该细胞在体外具有很强的增殖能力，并可分化为胰岛内分泌细胞。