高表达GRIM-19诱导结肠癌SW480细胞凋亡

目的：研究干扰素/维甲酸诱导死亡基因（retinoidinterferoninduced mortality,GRIM19）对结肠癌SW480细胞凋亡的影响。方法：构建GRIM19真核表达载体（pCMVFlagGRIM19）,转染入SW480细胞中,Western blotting检测GRIM19及凋亡相关蛋白Balxl的表达,采用AnnexinV/PI和线粒体膜电位JC-1染色检测SW480细胞的凋亡。结果：成功构建pCMVFlagGRIM19真核表达载体。pCMV-Flag-GRIM-19质粒转染SW480细胞后,GRIM19的表达上调,凋亡抑制蛋白Bclxl的表达则下调。转染空质粒pCMVFlag组SW480细胞凋亡率为（7.7±1.39）%,转染pCMV-Flag-GRIM-19质粒后SW480细胞凋亡率为（15.0±2.52）%（P<0.05）。线粒体膜电位检测显示转染空质粒pCMVFlag组SW480细胞膜电位降低细胞为（7.5±2.09）%,而转染pCMVFlagGRIM19后细胞线粒体膜电位降低细胞为（17.5±3.07）%（P<0.05）。结论：GRIM-19体外转染可有效诱导结肠癌SW480细胞凋亡。     

基因对胃癌MGC 803细胞的抑制作用及其可能的机制

目的:研究沉默乳腺癌相关抗原1（breast cancerassociated antigen 1,BRCAA1)基因对胃癌细胞株MGC803的抑制作用及其可能的机制。方法：构建BRCAA1基因shRNA载体，将构建的shRNABRCAA1质粒与阴性对照质粒shRNAN转染胃癌MGC803细胞，24 h后用荧光显微镜观察转染效率，实时定量PCR检测 BRCAA1和GAPDH基因mRNA表达水平。MTT法检测转染后24、48与72 h的细胞增殖水平，AnnxinV PE/7AAD检测转染24 h后的细胞凋亡水平，Western blotting检测转染48 h后细胞的凋亡相关蛋白表达水平。结果：BRCAA1 siRNA表达质粒转染MGC803细胞24 h 的转染效率为（81.2±2.6）％ 。转染后48 h MGC803细胞的BRCAA1 mRNA水平下降了61.4％，MGC803细胞增殖的抑制率达45.0%，转染siRNA细胞的凋亡率明显高于未转染细胞和对照质粒转染细胞\[（14.4±１.6）% vs（5.4±2.0）％，（4.4±2.5）％，P<0.05\]。转染siRNA细胞的凋亡相关蛋白Rb与Bax的表达量显著增加（P<0.05），Bcl2的表达量显著减少（P<0.05）。结论：BRCAA1基因的沉默可有效抑制人胃癌MGC803细胞的增殖和诱导细胞凋亡，其机制与其促进Rb和Bax蛋白表达、抑制Bcl2蛋白表达有关。     

含AFP基因调控序列的pAFP-P53-EGFP质粒诱导AFP阳性肝癌细胞凋亡

目的：观察含AFP基因调控序列的载体对AFP阳性肝癌细胞的靶向致凋亡作用。方法：将AFP启动子、沉默子和远端增强子Ⅲ组合为1.2 kb的AFP基因调控序列,构建pAFPEGFP载体,分别转染人肝癌HepG2（AFP阳性）、人肝癌SMMC7721（AFP阴性）和人宫颈癌HeLa（AFP阴性）细胞,荧光显微镜下观察EGFP荧光蛋白表达强度。引入P〖STBX〗53〖STBZ〗基因片段,构建pAFPP53EGFP重组质粒,转染HepG2、SMMC7721和HeLa细胞,Western blotting检测各组细胞P53蛋白的表达,流式细胞术分析各组细胞凋亡率及细胞周期。结果：成功构建了pAFPEGFP和pAFPP53EGFP重组质粒。pAFPEGFP转染后,AFP阳性的HepG2细胞中EGFP荧光蛋白表达显著高于AFP阴性的SMMC7721和HeLa细胞。pAFPP53EGFP转染后,HepG2细胞中P53蛋白的表达量明显高于SMMC7721和HeLa细胞;HepG2细胞的G1期细胞及细胞凋亡率明显高于SMMC7721和HeLa细胞\[(66.7±0.25)% vs（50.5±0.18）%,（51.0±0.20）%,P<0.05;（2.65±008）% vs（0.42±003）%,（0.39±0.02）%,P<0.05\], 但S期细胞明显低于转染后SMMC7721和HeLa细胞\[(20.1±022)% vs（29.8±018）%,（37.8±0.21）%,P<0.05\]。结论：含AFP基因调控序列的pAFPP53EGFP载体可专一性地作用于AFP阳性肝癌细胞,引起肝癌细胞周期阻滞和凋亡。     

pGL3-hTERT-tk/GCV对胃癌细胞的促凋亡作用

目的:探讨重组质粒pGL3hTERTtk/GCV对胃癌细胞的促调亡作用。方法：以基因工程方法构建重组质粒pGL3hTERTtk和相应的荧光报告质粒pGL3hTERTtkLuc+;脂质体LipofectamineTM2000瞬时转染胃癌细胞系SGC7901并用GCV干预,荧光显微镜观察细胞形态变化和转染效率,TUNEL标记和流式细胞术观察转染后胃癌细胞的凋亡;以上实验均以正常肝细胞L02为对照。结果：经鉴定,重组质粒pGL3hTERTtk中tk片段的长度为1 100 bp。荧光素酶标记的阳性、阴性对照及治疗报告质粒pGL3hTERTtkLuc+均能有效转染高表达端粒酶活性的胃癌细胞SGC7901,转染效率为(8.2±114)%。重组质粒转染胃癌细胞后与GCV共育4 d,细胞的凋亡率为(60.0±1.56)%;被pGL3hTERTtk转染的肿瘤细胞细胞周期发生了变化,处于细胞周期早期的细胞大量凋亡,早期凋亡率为（47.1±1.35）%。〖HT5W〗结论：〖HT5"SS〗pGL3hTERTtk/GCV对胃癌细胞有强烈的杀伤作用,但不影响正常细胞的生长,有潜在临床应用前景。     

沉默HuR的表达增加人乳腺癌耐药MCF-7/Adr细胞对多柔比星的敏感性

目的：研究RNA干扰人抗原R（human antigen R,HuR）基因的表达对人乳腺癌耐药细胞株MCF-7/Adr对多柔比星（doxorubicin）敏感性的影响。 方法： 构建靶向 HuR基因 的shRNA表达质粒（pGenesil-siHuR）,稳定转染至MCF-7/Adr细胞,real-time PCR检测细胞中 MDR1 mRNA的表达,Western blotting检测MCF-7/Adr细胞中由 MDR1 基因编码的P糖蛋白（P-glycoprotein,P-gp）的表达,MTT法检测pGenesil-siHuR 转染后MCF-7/Adr细胞在多柔比星作用后的存活率和IC50,流式细胞术检测MCF-7/Adr细胞的凋亡率。 结果： 与未转染的MCF-7/Adr细胞比较,pGenesil-siHuR质粒转染MCF-7/Adr细胞中 MDR1 mRNA的表达水平明显减低\[（0184±0.029） vs （1.203±0.026）,P<0.01\],P-gp表达水平明显降低。pGenesil-siHuR质粒转染MCF-7/Adr细胞后,MCF-7/Adr细胞对多柔比星的IC50从未转染的(148.2±2.3）nmol/L降至(42.9±0.4）nmol/L;经多柔比星处理后,pGenesil-siHuR质粒转染组MCF-7/Adr细胞的凋亡率明显上升\[（34.6±1.1）% vs （1.1±0.2）%,P<001\]。 结论： RNA干扰HuR的表达能抑制 MDR1基因 的表达,增加耐药乳腺癌MCF-7/Adr细胞对多柔比星的敏感性。     

PUMA基因转染胰腺癌AsPC-1细胞增强对5-FU致凋亡的敏感性

目的: 探讨PUMA基因转染是否增强胰腺癌AsPC1细胞对5FU致凋亡的敏感性。方法: 采用脂质体转染法将PUMApCEP4和空载体pCEP4质粒转染入胰腺癌AsPC1细胞中,G418筛选阳性细胞。将系列浓度（0.01～100 μmol/L）的5FU 分别作用于AsPC1、AsPC1/PUMA和AsPC1/pCEP4细胞72 h,MTT法测定各组细胞的存活率并计算IC50, FCM、断裂DNA琼脂糖凝胶电泳和TUNEL法检测细胞凋亡情况,Western blotting检测各组细胞PUMA蛋白表达的变化。〖HT5W〗结果: 〖HT5"SS〗AsPC1、AsPC1/PUMA和AsPC1/pCEP4细胞的5FU IC50分别为(12±1.9)、（1.6±04）和(10.4±1.6)μmol/L,AsPC1/PUMA细胞对5FU作用的敏感性增加了7.5倍。5FU以剂量依赖方式诱导AsPC1细胞凋亡,但对AsPC1/PUMA细胞所诱导的凋亡比AsPC1和AsPC1/pCEP4 更明显。低浓度5FU(0.1 μmol/L)轻微引起AsPC1/pCEP4\[(1.14±0.28)%\]和AsPC1细胞凋亡\[（0.9±0.23)%\],但能诱导AsPC1/PUMA细胞明显凋亡\[（6.47±1.42)%\];高浓度5FU (1 μmol/L)诱导各组细胞凋亡,但AsPC1/PUMA细胞凋亡率\[（34.54±9.36)%\]明显高于AsPC1\[（12.8±3.74)%\]和AsPC1/pCEP4细胞\[（15.8±5.15)%\],差异均有统计学意义（P<0.01）;FCM、断裂DNA琼脂糖凝胶电泳和TUNEL方法检测显示相同的结果。PUMA蛋白在AsPC1/PUMA细胞中的表达明显高于AsPC1和AsPC1/pCEP4细胞。结论:PUMA基因转染明显地增强了胰腺癌AsPC1细胞对5FU致凋亡作用的敏感性。     

Raf激酶抑制蛋白增强卵巢癌细胞的化疗敏感性

摘 要　目的：探讨Raf激酶抑制蛋白（Raf kinase inhibitor protein,RKIP）对卵巢癌SKOV-3细胞化疗敏感性的影响。方法：以脂质体法将含有人全长RKIP基因的真核表达质粒pcDNA3.1-ssRKIP转染入SKOV-3细胞中,Western blotting检测SKOV-3细胞中RKIP蛋白的表达。不同浓度顺铂作用转染后的SKOV-3细胞,MTS法观察RKIP基因转染对顺铂处理后SKOV-3细胞增殖的影响,流式细胞仪检测RKIP基因转染对顺铂诱导SKOV-3细胞凋亡及细胞周期的影响。结果：pcDNA3.1-ssRKIP转染的SKOV-3细胞RKIP表达明显升高。不同浓度顺铂处理细胞24、48、72 h后,RKIP基因转染细胞增殖抑制率显著高于对照细胞（P<0.05）。用2.5 μg/ml顺铂作用SKOV-3细胞24 h后,RKIP转染细胞的凋亡率为（10.86±0.73）%,明显高于未转染细胞的（4.27±0.67）%和空质粒转染细胞的（4.02±0.43）%（P<0.01);在无顺铂作用情况下,RKIP转染细胞的凋亡率为（3.11±0.78）%,仍然高于未转染细胞的（1.51±0.13）%和转染空质粒细胞的（1.97±0.46）%（P<0.01)。细胞周期检测结果显示,RKIP转染细胞G0/G1期的比例下降,S期的比例增加,转染的SKOV-3细胞发生S期阻滞。结论：RKIP基因的转染可以增加卵巢癌SKOV-3细胞对化疗药物顺铂的敏感性。     

人血管生成素-1对血管内皮细胞增殖及凋亡影响的研究

目的:探讨人血管生成素1（angiopoietin1, Ang1）对人脐静脉内皮细胞(human umbilican vein endothelia cell, HUVEC)增殖和凋亡的影响，以进一步研究其生物学作用及其在肿瘤发生中的作用机制。方法构建pcDNA3.1 V5HisCAng1真核表达载体，并瞬时转染293细胞;取新生儿脐带经胶原酶消化等方法分离培养HUVEC;分别通过MTT比色和细胞计数法，分析Ang1瞬时转染上清对HUVEC增殖的影响;通过流式细胞仪分析在血浆饥饿实验条件下，Ang1对HUVEC凋亡的影响。结果： 成功克隆了Ang1基因，构建了其正义真核表达载体，并在293细胞中瞬时表达;成功进行了HUVEC的原代分离及传代培养；MTT法检测HUVEC增殖结果：只加培养液组、加空载体转染上清组、加Ang1转染上清组HUVEC OD490值分别为0.36±0.11， 0.40±0.03，0.68±0.10(P<0.05)；细胞计数法检测结果依次为：（10.13±2.06）×104，（8.7±1.73）×104，（15.03±1.98）×104(P<0.05)。流式细胞仪检测HUVEC凋亡率依次为： 21%，19%和6%。结论： Ang1能显著促进血管内皮细胞体外增殖，抑制血浆饥饿时HUVEC的凋亡。     

KLK7 siRNA对胃癌AGS细胞的抑制作用

目的： 体外合成4条靶向人组织激肽释放酶7（kallikrein-related peptidase 7, KLK7 ）基因的片段,并筛选最有效siRNA片段,观察沉默 KLK7 表达对胃癌AGS细胞增殖和凋亡的影响。 方法： 设计4条靶向 KLK7 的siRNA片段（KLK7-siRNA-416、KLK7-siRNA-596、KLK7-siRNA-474、KLK7-siRNA-795）,瞬时转染AGS细胞,qRT-PCR检测各干扰组 KLK7 mRNA表达的变化,Western blotting检测AGS细胞中HK7蛋白（由 KLK7 基因编码）的表达,MTT法检测转染后AGS细胞的增殖,流式细胞术检测AGS细胞的细胞周期及凋亡。 结果： 4条KLK7-siRNA片段中以KLK7-siRNA-416的干扰效率最高,KLK7-siRNA-416组 KLK7 mRNA表达率显著低于NC组\[(0.32±0.049）% vs (0.93±0.071)%, P<0.01\],KLK7-siRNA-416组转染48 h后AGS细胞HK7蛋白的表达水平显著降低\[（1.18±0.198） vs（0.52±0.096）,P<0.01\]。KLK7-siRNA-416转染72 h后对AGS细胞增殖的抑制率达（37.70±0.12）%（P<0.05）,该转染阻滞AGS细胞于G0/G1期但不影响AGS细胞的凋亡。 结论： KLK7-siRNA沉默 KLK7 的表达可抑制AGS细胞的增殖,可阻滞细胞于G0/G1期,对细胞凋亡的作用不明显。     

In-vivo transfection of pcDNA3.1-IGFBP7 inhibits melanoma growth in mice through apoptosis induction and VEGF downexpression

Background

Genome-wide RNA interference screening study revealed that loss of expression of insulin-like growth factor binding protein 7 (IGFBP7) is a critical step in development of a malignant melanoma (MM), and this secreted protein plays a central role in apoptosis of MM. In this study we constructed pcDNA3.1-IGFBP7 to obtain high expression of IGBPF7 and to inhibit the growth of MM in C57BL/6J mice.

Methods

pcDNA3.1-IGFBP7 was transfected into B16-F10 cell, the expression of IGFBP7 was detected by RT-PCR and western blot. The proliferations and apoptosis rates of transfected and control cells were measured by CCK8 and FCM, respectively. The tumorigenicity and tumor growth in both pcDNA3.1-IGFBP7 group and control groups were studied in C57BL/6J mice model. IGFBP7, caspase-3, and VEGF expressions in tumor tissue were measured by immunohistochemistry. Apoptosis of tumors were detected by TUNEL.

Results

We demonstrated this plasmid inhibited proliferation of B16-F10 melanoma cells efficiently in vivo, exploiting the high expression of IGFBP7. More importantly, in-vivo transfection of pcDNA3.1-IGFBP7 inhibited MM growth in C57BL/6J mice. The inhibition of MM growth was proved owing to apoptosis and reduced expression of VEGF induced by pcDNA3.1-IGFBP7.

Conclusions

These results suggest a potential new clinical strategy for MM gene treatment.     

IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma

Insulin-like growth factor-binding protein 7 (IGFBP7) functions in several cellular processes including proliferation, senescence and apoptosis. This study analyzed IGFBP7 function in hepatocellular carcinoma (HCC) cells by gene manipulation and investigated the prognostic significance of IGFBP7 expression in clinical HCC samples. In this study, we investigated changes in malignant potential such as cell growth and invasiveness in an HCC cell line, PLC/PRF/5, after transfection with shRNA against IGFBP7. The extent of apoptosis and cell cycle progression were examined after the transfection. The correlation between immunohistochemically determined IGFBP7 expression and long-term postoperative prognosis after curative resection was also investigated in clinical HCC specimens obtained from 104 patients. PLC/PRF/5 cells transfected with shRNA against IGFBP7 showed significantly more rapid growth and stronger invasiveness than control cells. Annexin V assays showed that the IGFBP7-depleted cells were significantly more resistant to apoptosis than the control cells, and showed decreased expression of cleaved caspase-3 and PARP. Cell cycle progression was more rapid in the IGFBP7-suppressed cells. In clinical HCC specimens, IGFBP7 expression was judged as positive in 67 patients (64.4%) and negative in the remaining 37 patients (35.6%). The IGFBP7 downregulation correlated significantly with poor postoperative prognosis, and IGFBP7 status was identified as an independent significant prognostic factor. Our results indicated that IGFBP7 expression correlated significantly with the malignant potential in HCC cells, suggesting that the expression could be a useful prognostic marker for HCC.     

Purification of a colon cancer cell growth inhibitor and its identification as an insulin-like growth factor binding protein.

We have purified a protein from serum-free conditioned medium of the HT29 human colon adenocarcinoma cell line based on its ability to inhibit the proliferation of the same cell line. The purification procedure consisted of acid gel permeation, semipreparative, and analytical reversed-phase chromatographies. The high-pressure liquid chromatography-purified colon cancer cell growth inhibitor migrates as a single band of 27 and 34 kDa on sodium dodecyl sulfate/polyacrylamide gels under nonreducing and reducing conditions, respectively. NH2-terminal amino acid sequence analysis of the first 32 residues has demonstrated that this protein belongs to the insulin-like growth factor-binding protein (IGFBP) family. More precisely, this growth inhibitor appeared to be identical to the recently cloned human IGFBP-4. This IGFBP (HT29-IGFBP) has been characterized by performing ligand blotting and competitive binding experiments. The affinity of HT29-IGFBP for insulin-like growth factor (IGF) II (approximately 3.4 x 10(10) M-1) is slightly greater than its affinity for IGF-I (approximately 1.4 x 10(10) M-1). HT29 cells also produce two other isoforms (28 and 31 kDa, nonreduced) of the HT29-IGFBP having the same partial NH2-terminal amino acid sequence as the 27-kDa protein. The monoclonal antibody alpha IR-3 is known to block the mitogenic actions of IGFs. alpha IR-3 inhibited the growth of HT29 cells, thus suggesting that IGFs are required for the growth of these colon cancer cells.     

IGFBP7 is a p53 target gene inactivated in human lung cancer by DNA hypermethylation

Insulin-like growth factor binding protein 7 (IGFBP7) was considered a tumor suppressor gene in lung cancer. However, the mechanism responsible for the downregulation of this gene has not yet been fully understood. In this study, we analyzed the epigenetic inactivation of IGFBP7 expression in human lung cancer. We found that 14 out of 16 lung cancer cell lines showed decreased expression of IGFBP7 compared to control cells by real-time RT-PCR, and 42 out of 90 patients (46.7%) with primary lung tumor exhibited negative staining of IGFBP7 by immunohistochemistry analysis. The IGFBP7 expression could be restored by demethylation agent 5-aza-2′-deoxycytidine (DAC) in 7 cancer cell lines. Methylation status of IGFBP7 was further evaluated by bisulfite sequencing (BS) and methylation-specific-PCR (MSP). It turned out that low expression of IGFBP7 was associated with DNA methylation in lung cancer cell lines and in primary lung tumors (P = 0.019). To explore the regulatory role of p53 on IGFBP7, we transfected a wild type p53 expression vector into lung cancer cell lines H1299, H2228, and H82. Forced expression of p53 increased IGFBP7 expression only in H82 harboring no IGFBP7 methylation, while transfection in combination with DAC induced the expression of IGFBP7 in H1299 and H2228, in which IGFBP7 was methylated. Additionally, treatment with p53 inducer adriamycin (ADR) alone or in combination with DAC increased the expression of IGFBP7 in the 3 cell lines. Our data suggest that IGFBP7 is inactivated in lung cancer by DNA hypermethylation in both lung cancer cell lines and primary lung tumors, and IGFBP7 might be regulated by p53 in lung cancer cells.     

CCL21/CCR7轴促进人肺癌A549细胞的趋化与侵袭

目的：研究CCL21/CCR7轴对肺癌A549细胞定向趋化与侵袭活性的影响.方法：RT-PCR法从临床肺腺癌标本中扩增出CCR7编码区序列,定向克隆至载体pEGFP-N1中,稳定转染A549细胞,Boyden小室法检测转染前后A549细胞对CCL21的趋化和侵袭活性的改变.结果：CCL21作用下多聚碳酸酯膜背面的转染后A549细胞数明显多于转染前的A549细胞数.结论：CCL21/CCR7轴能够促进A549细胞的趋化与侵袭,其可能参与了肺癌淋巴结转移的过程.进一步研究CCL21/CCR7在肺癌中的作用将有助于阐明肺癌淋巴结转移的机制.     

Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) has been found to be a tumor suppressorin several human cancers, but the role of IGFBP7 in gastric cancer has not yet been fully investigated. Herein, weexamined the epigenetic downregulation of IGFBP7 expression in gastric cancer. Methods: Expression and methylationof IGFBP7 in gastric cancer cells and primary gastric cancer patients were determined using qRT-PCR, western blot,immunohistochemistry, and methylation specific-PCR, respectively. The effects of IGFBP7 on gastric cancer cellswere investigated by various experimental conditions, such as proliferation, colony formation, apoptosis, invasion,and migration assay. Results: IGFBP7 methylation was inversely correlated with IGFBP7 expression in gastriccancer. Univariate and multivariate analysis showed that IGFBP7 expression and tumor stage were independentprognostic factors. IGFBP7 knockdown increased gastric cancer cell growth, invasion, and migration, whereas IGFBP7overexpression in gastric cancer cells induced cell growth inhibition and apoptosis. Conclusion: Our data suggest thatIGFBP7 functions as a tumor suppressor in gastric cancer via an epigenetic pathway.     

Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.     

B7-1/GFP基因真核表达载体的构建及其在骨肉瘤细胞中的表达

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.     

胰岛素样生长因子结合蛋白5对人骨肉瘤细胞增殖与侵袭的影响

  目的  探讨胰岛素样生长因子结合蛋白5(insulin-like growth factor binding protein 5, IGFBP5)对人骨肉瘤细胞株143B增殖及侵袭力的影响。  方法  用IGFBP5过表达重组腺病毒(Ad-IGFBP5)、IGFBP5 RNA干扰重组腺病毒(Ad-siIGFBP5)和空载红色荧光蛋白重组腺病毒(Ad-RFP)感染人骨肉瘤细胞株143B。通过细胞增殖、划痕愈合、细胞迁移室(Transwell)侵袭等多种实验方法, 研究IGFBP5对人骨肉瘤细胞株生长、迁移与侵袭的影响。  结果  Ad-IGFBP5组感染后143B细胞生长速度低于Ad-RFP组(P < 0.05);Ad-IGFBP5组细胞在划痕后16h即完全愈合, 划痕愈合速度显著低于Ad-RFP组(P < 0.05);24 h后Ad-IGFBP5组侵袭至Matrigel胶上的平均细胞数量较Ad-RFP组减少(P < 0.05)。而Ad-siIGFBP5感染后, 143B细胞表现为生长率升高, 划痕愈合速度加快及侵袭力增强。  结论  IGFBP5能抑制骨肉瘤细胞增殖、迁移、侵袭。提示IGFBP5在抑制骨肉瘤发生及转移中可能起到重要作用。      

Biochemical mechanism of acetylsalicylic acid (Aspirin) selective toxicity toward melanoma cell lines

In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a molecular cancer therapeutic target. At 2 h, ASA was oxidized 88% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The 50% inhibitory concentration (48 h) of ASA and salicylic acid toward SK-MEL-28 cells were 100 micromol/l and 5.2 mmol/l, respectively. ASA at 100 micromol/l was selectively toxic toward human melanocytic SK-MEL-28, MeWo, and SK-MEL-5 and murine melanocytic B16-F0 and B16-F10 melanoma cell lines. However, ASA was not significantly toxic to human amelanotic C32 melanoma cell line, which does not express tyrosinase enzyme, and human nonmelanoma BJ, SW-620, Saos, and PC-3 cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased ASA toxicity toward SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA-induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine, inhibitor of permeability transition pore in mitochondria, prevented ASA toxicity. ASA led to significant intracellular GSH depletion in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ASA also led to significant reactive oxygen species (ROS) formation in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ROS formation was exacerbated by dicoumarol and 1-bromoheptane in SK-MEL-28. Our investigation suggests that quinone species, intracellular GSH depletion, ROS formation, and mitochondrial toxicity significantly contributed toward ASA selective toxicity in melanocytic SK-MEL-28 melanoma cells.