Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Hepatoprotective Effect of Guava (<i>Psidium guajava</i>L.) Leaf Extracts on Ethanol-Induced Injury on Clone 9 Rat Liver Cells

Guava (Psidium guajava L.), a tropical fruit, belongs to Myrtaceae family. Leaves and fruits of guava have been reported to have an anti-diarrheal, hypoglycemic, lipid lowering, anti-bacterial in addition to antioxidant activities. The aim of this study was to investigate several guava leaf extract cytotoxic effects on healthy clone 9 liver cells and its hepatoprotective effects on ethanol-induced heap-toxicity. It was discovered that when the clone 9 liver cells were treated with guava (Psidium guajava Linn.) extracts for 24 hours, there was no retardation of growth as well as when ethanol and acetone extracts at low concentrations 100 μg/mL and 50 μg/mL were administered however cytototoxic effects were detected at higher concentrations. Water and hot water extracts in concentrations lower than or equal to 500 μg/mL revealed no cytotoxic effects. Injury induction to healthy clone 9 liver cells using 5% alcohol concentration for 30 minutes revealed the hepatoprotective properties of guava (Psidium guajava Linn.) extracts. This was significant in concentrations of 100 μg/mL or lower for ethanol and all concentrations for hot water extracts. Hot water extracts showed higher hepatoprotective and lower cytotoxic properties than other extracts.

重组人BAFF_(134～285)的克隆及在大肠杆菌中的高效表达

目的克隆人TNF家族的B细胞激活因子(BcellactivatingfactortotheTNFfamily,BAFF)胞外区cDNA,并进行高效表达和纯化。方法提取人新鲜扁桃体组织总RNA,经RTPCR扩增编码人BAFF胞外区134～285氨基酸残基cDNA,经序列测定后,克隆至pQE80L载体中,并转化大肠杆菌DH5α,经IPTG诱导表达及Ni2+NTA柱层析纯化目的蛋白,最后经SDSPAGE和Westernblot检测。结果RTPCR扩增得到了459bp的cDNA片段,序列分析与GenBank中报道的编码人BAFF134～285的cDNA序列一致,SDSPAGE及Westernblot证实表达蛋白确实为6×HisBAFF134～285融合蛋白,并存在于包涵体中。结论利用大肠杆菌可高效表达rhBAFF134285,为进一步研究其生物学活性奠定了基础。

Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T】C, 1146C】T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9. METHODS: cDNA of UGT1 A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E.Coli DH5(alpha). The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III /Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9-UGT1A9, and selected by G418 (400 mg x L(-1)) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples forUGT1A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9-UGT1A9, contains the entire coding region, along with 18 bp of the 5' and 55 bp of the 3' untranslated region of theUGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was found to be 101+/- 24 pmol x min(-1) x mg(-1) protein (n=3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL cells. The CHL-UGT1 A9 cell lines established efficiently expressed the protein ofUGT1A9 for the further enzyme study of drug glucuronidation.

高低不同分化特性人成骨肉瘤MG-63克隆细胞株的建立

目的:建立分化程度高低不同的骨肉瘤细胞株.方法:用有限稀释法将人成骨肉瘤MG-63细胞系单克隆化,并作如下鉴定:(1)细胞增殖;(2)细胞形态学;(3)软琼脂克隆形成率;(4)染色体分析;(5)细胞周期时相分析;(6)碱性磷酸酶的活性;(7)细胞运动能力;(8)裸鼠成瘤实验.结果:共得到23个克隆,命名为MG-63-1～MG-63-23.其中的5、18号细胞株具有明显差异,18号为大核长梭形细胞,无接触抑制,克隆形成率高,体内成瘤时间短,代表低分化的骨肉瘤细胞株.5号以短梭形为主,核质比例变小,有一定的接触抑制,致瘤能力较低,为高分化的骨肉瘤细胞株.结论:通过对骨肉瘤细胞系MG-63的单克隆化,建立了具有高、低不同分化程度的两个人骨肉瘤细胞株.

人谷氧还蛋白基因的分子克隆及表达

目的:克隆人脐静脉内皮细胞谷氧还蛋白(glutaredoxin, Grx)编码区的cDNA序列并进行序列测定,构建原核表达载体并在大肠杆菌BL21(DE3)中表达. 方法:从人脐静脉内皮细胞中提取总RNA,采用RT- PCR技术,获得该基因编码区的cDNA,并重组入原核克隆表达载体pRSETA,构建重组质粒pRSET- Grx,通过茵落PCR筛选及限制性内切酶鉴定,选择阳性克隆并测序.将测序正确的重组质粒pRSET-Grx 转化大肠杆菌BL21(DE3),用IPTG诱导表达. 结果:将所得序列与GenBank提供的序列(NM002064)比较,测出的序列在核苷酸序列上有一处碱基不同,但在氨基酸序列上与已知序列一致.IPTG诱导4-5 h后, 150 g/L SDS-PAGE分析,表达出Mr16000的蛋白. 结论:从人脐静脉内皮细胞中成功地获得Grx编码区的cDNA,成功构建了原核融合表达载体pRSET-Grx并获得表达.

信号通路对胃癌干细胞及胃癌发生的影响

背景:Hedgehog与Wnt/β-catenin信号通路对胃癌干细胞的影响及在胃癌发生发展中的作用机制少见报道。目的:探讨Hedgehog与Wnt/β-catenin信号通路在胃癌发生发展中的作用机制。方法:采用肿瘤球悬浮分选法从胃癌组织标本中分选胃癌干细胞。采用免疫组化SP法检测Hedgehog及Wnt/β-catenin信号通路主要分子SHH、GLI1、Wnt2及β-catenin在胃癌干细胞中的表达。Spearman相关分析各细胞因子间的相关性分析。结果与结论:SHH、GLI1、Wnt2及β-catenin在胃癌干细胞中的阳性表达率分别为74.7%,78.3%,85.5%和83.3%,均显著高于癌旁组织的阳性表达率(P<0.05)。各细胞因子间在胃癌干细胞中的表达均呈正相关(P<0.05)。说明在胃癌干细胞中Hedgehog及Wnt/β-catenin信号通路均被激活,二者互相作用可能参与了胃癌的发生发展,为胃癌的干细胞治疗提供了新的研究方向。

Induction of Chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

lncRNA NEAT1降表达人喉鳞状细胞癌细胞增殖凋亡变化及与miR-214靶向关系

目的观察长链非编码RNA(lncRNA)核富集转录体1(NEAT1)降表达的人喉鳞状细胞癌(LSCC)细胞增殖凋亡变化,探讨其与微小RNA-214(miR-214)的靶向关系。方法采用实时荧光定量PCR(RT-qPCR)法检测人永生化表皮细胞Hacat和人LSCC细胞系(FD-LSC-1、Hep-2、TU177)中lncRNA NEAT1、miR-214,选择TU177细胞为受试细胞。取对数生长期TU177细胞,分为甲、乙组,分别转染si-lncRNA NEAT1(敲低lncRNA NEAT1表达)、si-NC(乱序无意义序列),培养至24 h时采用CCK8法观察两组细胞增殖情况、采用平板克隆形成实验观察两组细胞克隆形成情况、采用流式细胞术观察两组细胞周期和细胞凋亡情况、采用RT-qPCR法检测两组细胞lncRNA NEAT1及miR-214。另取对数生长期TU177细胞,分为一二三四组,一组顺序转染WT-lncRNA NEAT1、miR-214 mimic,二组顺序转染WT-lncRNA NEAT1、miR-NC,三组顺序转染MUT-lncRNA NEAT1、miR-214 mimics,四组顺序转染MUT-lncRNA NEAT1、miR-NC。采用双荧光素酶报告基因实验验证lncRNA NEAT1及miR-214的靶向关系。结果与乙组相比,培养24、48、72 h时甲组TU177细胞OD值低,培养14 d时甲组TU177细胞克隆形成数少,甲组TU177细胞G0/G1期细胞比例高、S期细胞比例低,细胞凋亡率高(P均<0.05);与乙组相比,转染24 h时甲组TU177细胞lncRNA NEAT1相对表达量低、miR-214相对表达量高(P均<0.05)。培养48 h时一、二、三、四组TU177细胞细胞荧光素酶活性分别为0.63±0.08、0.99±0.01、1.02±0.02、0.98±0.03,与二组相比,一组细胞荧光素酶活性低(P<0.05)。结论敲低lncRNA NEAT1表达可抑制TU177细胞的增殖和克隆,阻滞细胞周期于G0/G1期,并促进细胞的凋亡。TU177细胞中lncRNA NEAT1与miR-214靶向相关。lncRNA NEAT1可能通过与miR-214靶向结合,抑制TU177细胞的增殖克隆,阻滞细胞周期于G0/G1期,促进细胞的凋亡。

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O^6-METHYLGUANINE-DNA-METHYLTRANS cDNA INTO HUMAN UMBILICAL CORD BLOOD CD34^+ CELLS

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

培养方式对自原始生殖细胞分离克隆小鼠胚胎干细胞的影响

目的 比较与同源胎鼠生殖嵴共培养及其传统饲养层培养的方式 ,从小鼠原始生殖细胞中分离克隆胚胎干细胞的差异。方法 获取 8～ 14d龄的胎鼠 ,制备同源胎鼠成纤维细胞饲养层 ,采用常规饲养层培养的方式和与生殖嵴共同培养的方式 ,分离克隆小鼠胚胎干细胞。结果 在初期分离克隆的过程中 ,采用共同培养的方式优于传统饲养层培养模式。

ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC CELL CLONE AFTER CFU-MIX CULTURE

By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leukemlc celb still showed defects In differentiation and maturation. These were characterized by abnormal colony which consisted of smooth cells, bizarre shape, nuclear-cytoplasmic asynchrony In development, and appearance of nuclear bleb. However, chronic myelogenous leukemlc celb were more nature than the acute ones, manifesting in normal colony with finger- like projections and ruffled membrane. Macrophages and eosinophils could be observed. It b suggested that there b a difference In differentiation between acute and chronic leukemia cells.

HPV18 E6 and E7 Intratumour Heterogeneity in Esophageal Cancer

The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and migrations, we examined esophageal cancer clonality in the context of intra-tumor heterogeneity. In this research, we have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the HPV positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control. Quantitative real-time PCR (QRT-PCR) was run to detect the expression levels of HPV E6 and E7, Cell Counting Kit-8 (CCK-8) assay was used to examine cell proliferation, invasion assays performed using Costar chambers and wounding assay to study cell migrations in vitro. We investigated the intra-tumor heterogeneity of HPV E6 and E7 in esophageal cancer and the evaluation of the growth and migrations at the clonal level, using 10 single cell clones. In particular clones, C7 & C10 displayed a highly variable expression in both HPV E6 and E7 and weak in four clones (C1, C3, C4, and C9) consequently, the cell invasion, proliferation, and migration increase with increasing the level of HPV expression and inverse. In conclusion, the resulting based on single cell cloning showed the relationship between HPV and cell growth and migration in esophageal cancer. Future study in HPV DNA integration needed to explore the mains specific integration site of HPV DNA in esophageal cancer and molecular monitoring of the HPV for future prevention researches and also effective therapeutic strategies.

戊地昔布抑制clone 26肿瘤细胞增生及机制

目的探讨戊地昔布对小鼠肠癌clone26细胞的生长抑制作用及机制。方法用噻唑蓝（MTT）法检测戊地昔布对clone26细胞生长的作用。用流氏细胞仪检测clone26细胞凋亡率和细胞周期分布。用Western blot检测clone26细胞caspase-3，PCNA和COX-2的表达．结果①戊地昔布可抑制clone26细胞生长并呈时间和浓度依赖性。②50-400μmol·L^-1戊地昔布可明显提高clone26细胞的凋亡率，从对照的3．1％提高到4．4％～11．0％。给予戊地昔布后，细胞的增殖指数，S期的细胞比例和G2／M期细胞有下降趋势，但只有在400μmol·L^-1戊地昔布组时才有统计学意义。③给予戊地昔布后，细胞PCNA的表达降低，caspase-3的表达升高，COX-2的表达没有变化。结论戊地昔布通过诱导凋亡和细胞周期停滞而抑制clone26细胞生长，小剂量时主要与凋亡有关，大剂量时与诱导凋亡和细胞周期停滞有关、Caspase-3参与戊地昔布诱导的凋亡。