Side population cells from HXO-Rb44 retinoblastoma cell line have cancer-initiating property

AIM: To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. METHODS: We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell line by Hoechst 33342 staining on flow cytometry. SP and NSP cells were determined their ability of proliferation and self-renewal by SP reanalysis, soft agar assay and tumor sphere assay in vitro. Clone formation was detected by seeding HXO-Rb44 and HXO-Rb44 -RFP cells into soft agar. The expression of ABCGZ MDRI Bmi-1 and Oct-4 was determined by RT-PCR between SP and non-SP (NSP) cells. Moreover, they were injected into nude mice to determine their tumorigency in vivo. RESULTS: SP from HXO-Rb44 retinoblastoma cell line could grow clonally in soft agar assays and form tumor spheres from single cells in conditioned media. The expressions of ABCG2, MDRI Bmi-1 and Oct-4 were significantly higher in SP than NSP cells. As few as SP cells resulted in tumor formation in 6 of 12 injected sites, however, the injection of NSP cells failed to form new tumor. CONCLUSION: SP cells isolated by Hoechst 33342 from the HXO-Rb44 retinoblastoma cell line had property of high tumorigency in vivo and in vitro Therefore, SP might be a target while developing retinoblastoma therapies.

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

AIM To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell （HUVEC） monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocldng studies using anti CD44 monodonal antibodies or by hyaluronan digestion. RESULTS： Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice vadants CD44v4, CD44v5, and CD44v7 were all upregulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.

Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

PEI-PEG as a siRNA Genetic Vector Demonstrating Interference in the Expression of CD44v6 Protein in Gastric Cancer Cells

OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.

Characteristics of an Established Retinoblastoma Cell Line HXO-Rb_(44)

Purpose:To study the retinoblastoma cell culture and to establish a new retinoblastoma cellline.Methods:22 retinoblastomes were cultured by using the method of single cellsus-pension.Characteristics of the cultured cells were studied in the following pro-grams:tumor cell morphology in vitro,electron microscopic,growth curve,cloning in soft agar,immunohistochemistry,karyotype and tumorigenicity.Results.22 retinoblastoas were cultured successfully in ivtro,only a cotinued cell line HXO-Rb44was established(more than3years).The characteristics of this cell line are that it grew as a suspension of round cell in graps like clusters in vitro,its population doubling time was 44hours,and it could be cloned in softa-gar.Histopathologic and ulatastructured pictures showed the characteristics of Rb.HXO-Rb44cell was positive to NSE and negative to GFAP in immunohis-tochemical staining.A subcutaneous injection of HXO-Rb44cells produced a retinoblastoma in BALB/C athumic nude mice.Conclusions:HXO-Rb44 has the characteristice of retinoblastoma and is a new retinoblastoma cell line.It is a useflu material for study this tumor both in basic and clinical fields.

The expression and clinical significance of β-catenin and colorectal cancer stem cells marker EpCAM^(high)/CD44^+ in colorectal cancer

Objective： The aim of the study was to explore the role of Wnt βcatenin signalling pathway in the maintenance, invasion and metastasis of colorectal cancer stem cells. Methods： Double immunohistochemical staining was used to detect the expression of EpCAMhigh/CD44~ which is regarded as the marker of colorectal cancer stem cells in 80 cases of colorectal cancer and their corresponding liver metastases. The SP method of immunohistochemistry was used to detect the expression of the key protein βcatenin in the Wnt pathway in these tissue. The expression and correlation of ^-catenin and EpCAMh^gh/ CD44＋ in colorectal cancer were analyzed and their role on the biological behavior of colorectal cancer was explored. Results： The abnormal expression of βcatenin was significantly higher in colorectal cancer than in the paraneoplastic normal intes- tinal mucosa [55% （44/80） vs 10% （2/20）, P 〈 0.05]. The positive expression of EpCAMhigh/CD44＋ was significantly higher in colorectal cancer than in the paraneoplastic normal intestinal mucosa [66.25% （53/80） vs 0% （0/20）, P 〈 0.05]. In the 80 cases of colorectal cancer, the abnormal expression of ^-catenin has no correlation with gender （P = 0.079）, age （P = 0.416） and the magnitude （P = 0.816） of the tumor （P 〉 0.05）, but it was significantly correlated with degree of differentiation （P = 0.001）, depth of invasion （P = 0.001）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the colorectal cancer, the expression of EpCAMhi^h/CD44~ cells has no correlation with gender （P = 0.934） and the magnitude （P = 0.160） of the tumor （P 〉 0.05）, but was significantly correlated with age （P = 0.021）, degree of differentiation （P = 0.013）, depth of invasion （P = 0.000）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the corresponding liver metastases, we could also detecte EpCAMhih/CD44＋ cells. In cases with abnormal expression of βcatenin, the positive expression rate of EpCAMhigh/CD44＋ was significantly higher than those with normal expression of β-catenin （84.1% vs 44.4%）, and the difference was statistically significant （P 〈 0.05）. Conclusion： The abnormal activation of Wnt β-catenin signalling pathway may prompt the abnormal proliferation of the colorectal cancer stem cells, which leads to the recurrence and metastasis of the cancer.

视网膜母细胞瘤HXO-RB44细胞裸鼠皮下移植瘤的建立

目的建立视网膜母细胞瘤HXO-RB44细胞裸鼠皮下移植瘤模型,观察不同浓度HXO-RB44细胞的致瘤效果。方法将90只试验动物随机分成A、B、C3个组,每组裸鼠分别皮下注射浓度为1.0×108/mL、2.0×108/mL和3.0×108/mL的HXO-RB44细胞悬液各0.2mL。观察HXO-RB44细胞的成瘤时间、肿瘤生长情况、肿瘤大小及裸鼠摄食和活动状况。结果观察4周后,A组裸鼠无一成瘤。B组裸鼠仅5只成瘤。C组裸鼠有27只造模成功,成瘤率达90.00%。B组裸鼠的平均成瘤时间为21、34、5d,C组裸鼠的平均成瘤时间为18、32、3d,B、C两组时间之间进行比较,P<0.05,差异有显著性。小鼠成瘤后,出现消瘦、体重减轻、食欲精神不佳等症状。结论HXO-RB44细胞裸鼠皮下移植瘤的致瘤效果与细胞浓度密切相关。

褪黑素对裸鼠HXO-RB_(44)细胞皮下移植瘤的抑制作用

目的研究褪黑素(melatonin,MLT)对裸鼠HXO-RB44细胞皮下移植瘤的生长抑制作用及其相关机制。方法50只裸鼠视网膜母细胞瘤(retinoblastoma,RB)皮下移植瘤模型,随机分成5组,每组10只:A组为阴性对照组;B组、C组、D组分别为低、中、高浓度MLT治疗组;E组为阳性对照组。低、中、高浓度MLT治疗组分别按每只每次腹腔注射MLT20 mg.kg-1、40mg.kg-1、60 mg.kg-1各2 mL进行给药,阴性对照组注射等量生理盐水,阳性对照组每只每次腹腔注射等量足叶乙甙(4 mg.kg-1);每天给药1次,共7 d。观察各组裸鼠皮下移植瘤生长情况、裸鼠体质量的变化。采用组织病理学检查,观察裸鼠移植瘤的形态学特征;采用免疫组织化学、Western blotting检测肿瘤细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)、细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)的表达情况。结果E组白细胞、血红蛋白、血小板计数值及谷丙转氨酶、谷草转氨酶、次黄嘌呤核苷值与A组相比,差异均有统计学意义(P均<0.05),MLT各治疗组裸鼠血常规结果及肝、肾功能与A组相比,差异无统计学意义(P>0.05);不同时间点B组、C组、D组裸鼠体质量与A组比较,差异无统计学意义(P>0.05);B组裸鼠皮下移植瘤肿瘤质量与A组相比,差异无统计学意义(P>0.05),表明低剂量的MLT对裸鼠移植瘤的生长无抑制作用,C组、D组与A组进行比较,差异均有显著统计学意义(P均<0.01),表明大剂量的MLT对裸鼠移植瘤的生长有抑制作用;低、中、高浓度MLT治疗组及阳性对照组裸鼠移植瘤抑瘤率分别为12.50%、27.89%、40.72%、44.52%;组织病理学检查发现MLT治疗组及E组肿瘤内出血、坏死的程度明显高于阴性对照组,免疫组织化学及Western blotting检测发现MLT治疗组VEGF、ICAM-1及其蛋白的表达明显低于阴性对照组;MLT的治疗剂量与VEGF、ICAM-1蛋白的表达呈高度负相关。结论MLT对HXO-RB44细胞皮下移植瘤生长有抑制作用;MLT能抑制裸鼠移植瘤内VEGF、ICAM-1蛋白的表达,其浓度与VEGF、ICAM-1蛋白的表达呈高度负相关。

8-Br-cAMP对人视网膜母细胞瘤HXO-Rb44细胞癌基因的表达效应

目的研究８ＢｒｃＡＭＰ对培养的人视网膜母细胞瘤ＨＸＯＲｂ４４细胞癌基因表达的效应及其与该细胞生长的关系。方法ｃｆｏｓｍＲＮＡ、ＮｍｙｃｍＲＮＡ及ｐ２１ｒａｓｍＲＮＡ均以原位杂交和ＲＮＡ斑点印迹技术检测，对繁殖细胞核抗原（ＰＣＮＡ）、ｃＦｏｓ、ＮＭｙｃ和Ｐ２１ｒａｓ蛋白表达的免疫反应性（ＩＲ）则采用免疫组化及蛋白质斑点印迹技术检测。结果在人ＨＸＯＲｂ４４细胞，ｃｆｏｓｍＲＮＡ、ＮｍｙｃｍＲＮＡ及ｐ２１ｒａｓｍＲＮＡ定位于胞质，ｃＦｏｓＩＲ及ＮＭｙｃＩＲ定位于胞核，而Ｐ２１ｒａｓＩＲ则位于胞质。斑点印迹的相对扫描数值显示实验组（８ＢｒｃＡＭＰ处理）均低于相应各对照组（未经８ＢｒｃＡＭＰ处理），Ｐ＜０．０５～０．０１。结论（１）８ＢｒｃＡＭＰ可抑制人ＨＸＯＲｂ４４细胞的生长增殖；（２）８ＢｒｃＡＭＰ可下调ｃｆｏｓ、Ｎｍｙｃ及ｐ２１ｒａｓ癌基因的表达；（３）８ＢｒｃＡＭＰ也可下调ｃＦｏｓ、ＮＭｙｃ及Ｐ２１ｒａｓ蛋白的表达，而且其变动各与其ｍＲＮＡ基因表达的变动相一致；（４）结果提示８ＢｒｃＡＭＰ可能从不同的途径而协同抑制ＨＸＯＲｂ４４细胞的生长增殖。

Inhibition of all-trans retinoic acid on MDM2 gene expression in astrocytoma cell line SHG-44

Objective To investigate the impact of all-trans retinoic acid （ATRA） on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma. Methods The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade Ⅱ and grade Ⅳ astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis. Results The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to β-actin in ATRA-treated and untreated SHG-44 were 14.02±0.35 and 21.40±0.58 （t = 24.728, P = 0.000）, respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% （6/25） and 56.52% （13/23） （x^2 = 5.298, P = 0.021） in WHO grade Ⅱ and grade Ⅳ astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy. Conclusion ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

8-Br-cAMP可上调人视网膜母细胞瘤HXO-Rb44细胞抑癌基因的表达(英文)

目的 探讨8BrcAMP对人视网膜母细胞瘤HXORb44细胞抑癌基因表达的效应。方法 应用RNA斑点印迹技术检测细胞p16、p21wafl、wp53、mp53和Rb的mRNA,应用免疫组化斑点印迹技术检测细胞P16、P21wafl、PRb、cdk2、cdk4和PCNA蛋白质表达的免疫反应性(IR)。结果 在斑点印迹标本上,人HXORb44细胞p16、p21wafl、wp53及Rb的mRNA信号和P16IR、P21waflIR及PRbIR均是实验组高于对照组,P<005～001,而mp53mRNA信号、PCNAIR、cdk2IR和cdk4IR均是实验组低于对照组,P<005～001。结论 8BrcAMP上调人HXORb44细胞p16、p21wafl、wp53、Rb抑癌基因的表达及P16、P21wafl、PRb蛋白的表达,并下调mp53mRNA、PCNAIR、cdk2IR和cdk4IR的表达,提示8BrcAMP可通过阻抑细胞周期进展相关基因的表达而抑制人HXORb44细胞的生长增殖。

HXO-RB_(44)细胞上清液对ARPE-19细胞内VEGF表达的影响

目的探讨HXO-RB44细胞上清液对人视网膜色素上皮(RPE)细胞系ARPE-19细胞内血管内皮生长因子(VEGF)表达的影响及其相关机制。方法分别利用含有10%胎牛血清的RPMI-1640培养基及MEM培养基对HXO-RB44细胞及ARPE-19细胞进行培养,在ARPE-19细胞内加入100μL HXO-RB44细胞上清液,分别干预0、4、8、12、24 h,利用RT-PCR、免疫荧光细胞化学、Western blot等方法,检测不同浓度的MLT干预后24 h ARPE-19细胞内VEGF mRNA及蛋白的表达情况。结果HXO-RB44细胞上清液加入后的不同时间点,VEGF mRNA的表达差异有统计学意义(F=195.072,P=0.000)。随着HXO-RB44细胞上清液作用时间的延长,ARPE-19细胞内VEGF mRNA表达逐渐增高(P<0.01)。HXO-RB44细胞上清液加入后4、8、12、24 h,ARPE-19细胞内VEGF蛋白表达显著增加(F=160.687,P=0.000),其表达与ARPE-19细胞内VEGF mRNA表达方式相同(P<0.05)。随着HXO-RB44细胞上清液作用时间的延长,ARPE-19细胞内绿荧光蛋白强度逐渐增加。结论HXO-RB44细胞上清液能促进ARPE-19细胞内VEGF的表达。

SMAD3和CD44在甲状腺乳头状癌组织中的表达水平与临床病理特征及预后的关系

目的探讨信号转导分子3(SMAD3)和白细胞分化抗原44(CD44)在甲状腺乳头状癌(PTC)组织中的表达水平与临床病理特征及预后的关系。方法选取2019年6月-2020年6月收治的PTC患者88例为研究对象,同期取距离癌组织3 cm以上癌旁组织作为对照,实时荧光定量PCR(qRT-PCR)检测SMAD3和CD44的mRNA表达水平;免疫组化法检测SMAD3和CD44的阳性表达情况。Spearman相关性分析PTC患者癌组织中SMAD3和CD44的mRNA表达水平的关系。SMAD3和CD44与PTC患者预后的关系采用Kaplan-Meier法进行分析。COX回归分析影响PTC患者预后的危险因素。结果与癌旁组织相比,PTC组SMAD3的表达水平显著降低(t=8.484,P<0.05),CD44表达水平显著升高(t=11.232,P<0.05)。相关性分析显示,PTC患者癌组织中SMAD3和CD44的mRNA表达水平呈负相关(r s=-0.519,P<0.05)。与癌旁组织相比,PTC组SMAD3阳性表达率显著降低(χ^(2)=41.905,P<0.05),CD44阳性表达率显著升高(χ^(2)=62.888,P<0.05)。SMAD3表达与TNM分期(χ^(2)=7.678,P=0.006)、淋巴结转移(χ^(2)=20.398,P<0.05)、侵犯包膜(χ^(2)=9.881,P=0.002)有关。CD44表达与TNM分期(χ^(2)=3.959,P=0.047)、淋巴结转移(χ^(2)=20.702,P<0.05)、侵犯包膜(χ^(2)=10.363,P=0.001)有关。术后随访3年发现,SMAD3阳性表达PTC患者生存率高于阴性表达患者生存率(χ^(2)=4.644,P=0.031)。CD44阳性表达PTC患者生存率显著低于阴性表达患者生存率(χ^(2)=5.331,P=0.021)。多因素COX回归分析显示,淋巴结转移、SMAD3和CD44表达是影响PTC患者预后的危险因素(P<0.05)。结论PTC组织中SMAD3呈异常低表达,CD44呈高表达,其水平与PTC患者临床病理特征及预后有关。

重度子痫前期患者胎盘组织中CD44 mRNA和CD24 mRNA及蛋白表达水平的临床价值研究

目的探讨重度子痫前期(severe preeclampsia,SPE)患者胎盘中细胞表面跨膜糖蛋白分子(cell surface transmembrane glycoprotein molecules,CD)44 mRNA,细胞表面跨膜糖蛋白分子24(CD24)mRNA及蛋白表达水平表达的临床价值研究。方法选取2019年6月~2022年6月在聊城市第二人民医院接受剖宫产分娩的SPE患者,根据发病孕龄的不同,进一步将其分为早发型SPE组(孕龄≤34周,n=45)和晚发型SPE组(孕龄>34周,n=55)。选取同期产检正常者100例为对照组。采用荧光定量PCR和免疫组织化学检测SPE患者胎盘中CD44和CD24表达,Pearson法分析其表达水平差异以及与SPE疾病临床特征的相关性,多因素Logistic回归分析发生SPE的影响因素。结果相较于对照组,SPE胎盘组织中CD44 mRNA(0.55±0.12 vs 1.02±0.33),CD24 mRNA的表达水平(0.68±0.19vs 1.05±0.11)均降低,差异具有统计学意义(t=13.385,16.853,P<0.05)。免疫组织化学染色结果显示,CD44,CD24在SPE组胎盘组织中多呈阴性表达或弱阳性表达,而在对照组中多呈阳性表达,且SPE胎盘组织中CD44,CD24阳性率低于对照组,差异具有统计学意义(χ^(2)=9.696,14.346,P<0.05)。相较于早发型SPE组,晚发型SPE胎盘组织中CD44(0.65±0.17 vs 0.42±0.11),CD24(0.77±0.23 vs 0.58±0.13)mRNA的表达水平均较高,差异具有统计学意义(t=7.830,4.932,P<0.05)。相较于对照组,SPE组BMI,收缩压、舒张压、尿蛋白、Cr,LDH和BUN均显著升高,差异有统计学意义(t=5.360~30.241,均P<0.05);SPE组分娩孕周较早、MPV,ALB较低、新生儿出生身长较短和体质量较轻均低于对照组,差异具有统计学意义(t=3.232~11.109,均P<0.05)。且SPE胎盘组织中CD44与CD24表达呈正相关(r=0.698,P<0.05),SPE胎盘组织中CD44的表达分别与CD24,分娩孕周、MPV和新生儿出生身长呈正相关(r=0.611,0.639,0.612,0.465,均P<0.05);与收缩压、尿蛋白和LDH呈负相关(r=-0.604,-0.569,-0.593,均P<0.05)。CD24的表达分别与分娩孕周、MPV和新生儿出生身长呈正相关(r=0.605,0.584,0.640,均P<0.05);与收缩压、尿蛋白和LDH呈负相关(r=-0.637,-0.593,-0.561,均P<0.05)。经Logistic回归分析结果显示,MPV(95%CI:1.429~4.350),尿蛋白(95%CI:1.529~2.709),LDH(95%CI:1.425~3.932)均是发生SPE的独立危险因素(均P<0.05)。高水平CD44(95%CI:0.561~0.940),CD24(95%CI:0.495~0.814)是发生SPE的独立保护因素(均P<0.05)。结论SPE患者胎盘中CD44,CD24表达水平较低,高水平CD44,CD24均是SPE发生的独立保护因素,可为后续SPE的治疗提供方向。

Effects of salvianolic acid B on in vitro growth inhibition and apoptosis induction of retinoblastoma cells

AIM: To observe the effects of salvianolic add B (SalB) on in vitro growth inhibition and apoptosis induction of retinoblastoma HXO-RB44 cells. METHODS: The effects of SalB on the HXO-RB44 cells proliferation in vitro were observed by MTT colorimetric method. The morphological changes of apoptosis before and after the treatment of SalB were observed by Hoechst 33258 fluorescent staining method. Apoptosis rate and cell cycle changes of HXO-RB44 cells were detected by flow cytometer at 48 hours after treated by SalB. The expression changes of Caspase-3 protein in HXO-RB44 cells were detected by Western Blot. RESULTS: SalB significantly inhibited the growth of HXO-RB44 cells, while the inhibition was in a concentration-and time-dependent manner. The results of fluorescent staining method indicated that HXO-RB44 cells showed significant phenomenon of apoptosis including karyorrhexis, fragmentation and the formation of apoptotic bodies, etc. after 24, 48 and 72 hours co-culturing of SalB and HXO-RB44 cells. The results of flow cytometer showed that the apoptosis rate and the proportion of cells in S phase were gradually increased at 48 hours and 72 hours after treated by different concentrations of SalB. Western Blot strip showed that the expression of Caspase-3 protein in HXO-RB44 cells was gradually increased with the increase of the concentration of SalB. CONCLUSION: SalB can significantly affect on HXO-RB44 cells growth inhibition and apoptosis induction which may be achieved through the up-regulation of Caspase-3 expression and the induction of cell cycle arrest.

Expression of cell adhesion molecule CD44 in gastric adenocarcinoma and its prognostic importance

AIM: To evaluate the relation of cluster of differentiation 44 (CD44) expression with clinicopathological features of gastric adenocarcinoma, and also its effect on prognosis with an emphasis on the differences between intestinal and diffuse types. METHODS: From 2000 to 2006, 100 patients with gastric adenocarcinoma, who had undergone total or subtotal gastrectomy without any prior treatment, were studied. Haematoxylin & eosin (HE) staining was used for histological evaluation, including the type (Lauren's classifi cation) and grading of the tumor. The expression of CD44 in the gastric adenocarcinoma mucosa and the adjacent mucosa were determined by immunohistochemistry. The survival analysis was obtained using the Kaplan-Meier test. RESULTS: Of 100 patients, 74 (74%) patients were male. The tumors were categorized as intestinal type (78%) or diffuse type (22%). Sixty-five percent of patients were CD44-positive. CD44 expression was not detected in normal gastric mucosa. Rather, CD44 was more commonly expressed in the intestinal subtype (P = 0.002). A signifi cant relation was seen between the grade of tumor and the expression of CD44 (P = 0.014). The survival analysis showed a poor prognosis of patients with CD44-positive tumors (P = 0.008); and this was more prominent in the intestinal (P = 0.001) rather than diffuse type. CONCLUSION: Cell adhesion molecule CD44 is highly expressed in gastric adenocarcinoma. CD44 expression is correlated with a poor prognosis in patients with the intestinal type of gastric adenocarcinoma. CD44 can, therefore, be utilized as a prognostic marker for this group of patients.

Expression of human chorionic gonadotropin, CD44v6 and CD44v4/5 in esophageal squamous cell carcinoma

AIM： To study the relationship between the expression of human chorionic gonadotropin （HCG）, CD44v6, CD44v4/5 and the infiltration, metastasis of esophageal squamous cell carcinoma. METHODS： By labeled streptavidin-biotin technique, the expressions of HCG, CD44v6, and CD44v4/5 in 42 patients with esophageal squamous cell carcinoma were examined. RESULTS： The positive rate of HCG expression in patients with lymph node metastasis was 85.71% （18/21）, higher than that （57.14%, 12/21） in those without lymph node metastasis （P〈0.05）. The positive rate of CD44v6 expression was 71.43% （15/21） in lymph node metastasis group, and 38.09% （8/21） in nonmetastasis group; there was a significant difference between the two groups （P〈0.05）. The positive rate of CD44v4/5 expression was 76.19% （16/21） in lymph node metastasis group, and 42.86% （9/21） in non-metastasis group; there was also a significant difference between them （P〈0.05）. From grade Ⅰ to grade Ⅲ in differentiation, the positive rate of HCG expression was 84.62% （11/13）, 70.59% （12/17） and 58.33% （7/12）, respectively, there was no significant difference among them （P〉0.05）. The positive rate of CD44v6 expression in grades Ⅰ-Ⅲ of cancer tissues was 76.92% （10/13）, 52.94% （9/17）, and 33.33% （4/12） respectively; there was no significant difference among them. The positive rate of CD44v4/5 expression in grades Ⅰ-Ⅲ of cancer tissues was 69.23% （9/13）, 64.71% （11/17）, and 41.67% （5/12） respectively; there was no significant difference among the three groups. There was no correlation between the positive rates of HCG and CD44v6, CD44v4/5 expression. Cancer cells in carcinomatous emboli and those infiltrating into vascular wall strongly expressed HCG, CD44v6, and CD44v4/5. CONCLUSION： Expression of HCG, CD44v6, and CD44v4/5 in esophageal squamous cell carcinoma is related to its infiltration and metastasis. HCG, CD44v6, and CD44v4/5 have different effects on the infiltration and metastasis of esophageal squamous cell carcinoma.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.

HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling

BACKGROUND Breast cancer is a common malignant tumor that seriously threatens women’s health.Breast cancer stem cell(CSC)-like cell population may be the main factor for breast cancer metastasis.Therefore,targeted therapy for CSCs has great potential significance.Hypoxia-inducible factor is a transcription factor widely expressed in tumors.Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxiainducible factor-2α(HIF-2α).However,the specific mechanism remains unclear and further research is necessary.AIM To investigate the effect of HIF-2αdown-regulation on stem cell markers,microsphere formation,and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.METHODS Immunohistochemistry was used to detect the expression of HIF-2αand CD44 in triple-negative breast cancer(TNBC)and non-TNBC tissues.Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2αand CD44 in MDA-MB-231 cells and MCF-7 cells.HIF-2αwas silenced by RNA interference,and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR.Further,flow cytometry,TdT-mediated X-dUTP nick end labeling,and mammosphere formation assays were used to evaluate the effect of HIF-2αon CSCs and apoptosis.The possible mechanisms were analyzed by Western blot.RESULTS The results of immunohistochemistry showed that HIF-2αwas highly expressed in both TNBC and non-TNBC,while the expression of CD44 in different molecular types of breast cancer cells was different.In in vitro experiments,it was found that HIF-2αand CD44 were expressed almost in the same cell.Compared with hypoxia+negative-sequence control,HIF-2αsmall interfering ribonucleic acid transfection can lower the expression of HIF-2αand CD44 mRNA(P<0.05),increase the percentage of apoptotic cells(P<0.05),and resulted in a reduction of CD44+/CD24−population(P<0.05)and mammosphere formation(P<0.05)in hypoxic MDA-MB-231 cells.Western blot analysis revealed that phosphorylated protein-serine-threonine kinase(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)levels in MDA-MB-231 decreased significantly after HIF-2αsilencing(P<0.05).CONCLUSION Down-regulation of HIF-2αexpression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis,and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.