Msi1基因沉默对人肝癌HepG2细胞的影响

目的:探讨siRNA沉默Msi1基因表达后,人肝癌HepG2细胞的增殖与凋亡的情况。方法:设计合成针对Msi1 mRNA序列的siRNA多条序列,分别转染人肝癌HepG2细胞。使用RT–PCR方法检测各组细胞Msi1基因表达情况;Western blot法检测survivin蛋白、caspase3蛋白表达情况;MTT法、平皿克隆实验检测HepG2细胞增殖情况;流式细胞术检测各组HepG2细胞凋亡情况。结果:(1)RT-PCR结果:转染后24 h后,序列a,b,c转染效率分别为65%,64%及62%。其抑制率分别是Msi1 siRNAa(68%)、Msi1 siRNAb(56%)、Msi1 siRNAc(35%)。(2)流式细胞术检测各组细胞凋亡:空白组、阴性组、脂质体组、Msi1 siRNAa组凋亡率分别为(4.14±0.26)%、(4.51±0.78)%、(4.19±1.21)%,(16.8±0.26)%,Msi1 siRNAa组与空白对照组比较差异有统计学意义(P<0.05)。(3)Msi1 siRNAa最能有效抑制肝癌HepG2细胞中Msi1表达,与空白对照组比较,Msi1 siRNAa组HepG2细胞生长、增殖速度明显减缓(P<0.05);survivin蛋白表达水平显著下调(P<0.05),而caspase3蛋白表达水平上调(P<0.05);Msi1 siRNAa组凋亡率增高(P<0.05)。结论:沉默Msi1可抑制人肝癌HepG2细胞的增殖,诱导其凋亡。

香蕉MaPRMT1基因的分离及表达分析(英文)

[Objective] The aim of experiment was to lay molecular foundation for studying maturity mechanism of banana after harvest. [Method] The combined method of suppressing subtractive hybridization and cDNA micro-array were used to obtain cDNA segment of one PRMT gene in banana and the whole cDNA sequence of the gene was cloned.The bioinformatics analysis was operated on it,in addition, the expression profile analysis was conducted in different organs and different mature periods of banana.[Result] The whole length of cDNA in MaPRMT1 was 1 158 bp and possessed a complete open reading frame,which could encode 385 amino acids.It had high homology with PRMT in plant,containing one Methyltransf_1 domain.The MaPRMT1 gene was expressed in root,stem,leaf and fruit of banana and the expression levels in stem and leaf were relatively high.As the increase of days after harvest,the expression level declined gradually,however it reached maximum when ethylene release was biggest,then it declined.[Conclusion] MaPRMT1 belonged to the first kind of arginine methyltransferase and it was expressed differently in different organs and fruits at different mature periods.

Expression pattern of matrix metalloproteinases-13 in a rat model of alcoholic liver fibrosis

BACKGROUND: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fi-brosis, because type Ⅰ and Ⅲ collagens increase predominantly in liver fibrosis. The present study aimed to demonstrate the gene expression of matrix metalloproteinases-13 (MMP-13) in the progressive phases of ethanol induced experimental liver fibrosis in rats. METHODS: Thirty-four Sprague-Dawley rats were randomly divided into two groups. The experimental group (24 rats) was given ethanol (44% , 7 g/kg) every day and the control group (10) was given normal saline. Liver samples were harvested from experimental rats at 4, 12 and 24 weeks respectively. The kinetics of MMP-13 mRNA expression was assayed by semi-quantity reverse transcriptase-poly-merase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR (0.24±0.41). The gene expression of MMP-13 was increased in the liver of the rats treated with ethanol for 4 weeks (0. 62 ±0. 54), but it was not considered statistically significant (P >0.05). And the livers from 12-week-treated rats showed a marked mRNA expression(1.65 ±0.47, P <0. 01). Once fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39±0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, and it was induced in a distinct time frame.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

Expression of Transforming Growth Factor-β in Cultured Normal Human Lens Epithelia Cells

Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.

Differential gene expression between asymptomatic HBV carriers and normal adults

BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.

Polypeptide Daintain as a New Biomarker for Detecting Breast Tumor

Daintain, a novel bioactive peptide produced and secreted by macrophages, was expressed in breast tumor tissues. The spatial distributions of daintain in 66 breast tumor specimens were investigated with immuno-histochemistry method. Reverse transcription polymerase chain reaction （RT-PCR） and in situ hybridization inspection system were also used to detect daintain in 45 cases of malignant breast tumors. The final results show that 93% high positive responses to daintain on breast cancer tumors. RT-PCR demonstrated that, no smear of daintain transcripted in benign tissues was found, and light smear in peri-cancer tissue was observed. Distribution of daintain was distinguishable among benign tissues, hyperplasia tissues, immature hyperplasia and invasive breast cancer, which can be used to mark the progression of the malignant lesion development. We conclude that the expression of daintain is up-regulated in breast cancers, which indicates that the peptide is closely associated with the disease progression. So daintain could be used as the biomarker for detecting breast cancer.

Quantified gene expression levels for phase Ⅰ/Ⅱ metabolizing enzyme and estrogen receptor levels in benign prostate from cohorts designated as high-risk （UK） versus low-risk （India）for adenocarcinoma at this organ site：a preliminary study

Risk of clinically significant prostate adenocarcinoma （CAP） varies worldwide,although there is a uniform prevalence of latent disease. A hormone-responsive tissue,the prostate possesses the metabolizing capacity to biotransform a variety of environmental procarcinogens or endogenous hormones. Whether such metabolizing capacity or estrogen receptor （ER） status underlies these demographic differences in susceptibility to CaP remains unclear. With appropriate ethical permission,verified-benign tissues were obtained following transurethral resection of the prostate from a high-risk region （n = 12 UK-resident Caucasians） and a typically low-risk region （n = 14 India-resident Asians）. Quantitative gene expression analysis was employed for cytochrome P450 （CYP）1B1,N-acetyltransferase （NAT）1,NAT2,catechol-O-methyl transferase （ COMT）,sulfotransferase （ SULT） 1A1,ERα,ERβ and aromatase （CYP To quantify the presence or absence of CYP1B1,ERα or ERβ,and to identify ther in situ localization,immunohistochemistry was carried out. The two cohorts had reasonably well-matched serum levels of prostate-specific antigen or hormones. Expression levels for the candidate genes investigated were similar.However,clear differences in protein levels for CYP1B1 and ERβ were noted. Staining for CYP1B1 tended to be nuclear-associated in the basal glandular epithelial cells,and in UK-resident Caucasian tissues was present at a higher （P = 0.006） level compared with that from India-resident Asians. In contrast,a higher level of positive ERβ staining was noted in prostates from India-resident Asians. These study findings point to differences in metabolizing capacity and ER status in benign prostate tissues that might modulate susceptibility to the emergence of clinically significant CaP in demographically distinct populations.

EXPRESSION AND SIGNIFICANCE OF PTEN IN ENDOMETRIAL CARCINOMA

Objective: To investigate the expression of PTEN in endometrial carcinoma and its clinical significance. Methods: Reverse transcriptase-polymerase chain reaction and Western-blot methods were used to detect PTEN expression in 28 cases of endometrial carcinoma. Results: mRNA and protein expression levels of PTEN in endometrial carcinomas were significantly lower than those in normal endometrium (P<0.01). Conclusion: PTEN may play an important role in the tumorigenesis of endometrial carcinoma.

Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.

Molecular characterization of yolk proteins in the female crab Neptunus pelagicus(A.Milne-Edwards,1861) from the Mediterranean Sea of Alexandria,Egypt

This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis.Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017.Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test.Native polyacrylamide gel electrophoresis(PAGE)Bis-Tris Gels was applied to identify vitelloginin(VN)and vitelline(VL)molecules.Protein Analyses were done by PAGE-SDS.The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins.Primary and secondary vitellogeneses consisted of 8 phases.Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis.Two protein fractions(VLI&VLII)were identified in secondary vitellogenic oocytes.The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa.In stage II and III oocyte,4 subunits were presented of 180,195,140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII.Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa.Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180,125,90 and 85 kDa in each of the two VLs.The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis.The result of the reverse transcription-polymerase chain reaction(RT-PCR)analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas.

Aberrant Expression of TNF-α and TGF-β_1 mRNA in Spontaneous Abortion

Objective To investigate the aberrant expressions of TNF-α and TGF-β1 in peripheral blood mononuclear cells （PBMCs） and placental tissues in patients with early spontaneous abortion Methods Using the technique of semi-quantitative reverse transcript-polymerase chain reaction （RT-PCR）, TNF-α mRNA and TGF-β1 mRNA in PBMCs were measured in spontaneous abortion group （30 cases）, normal pregnancy group （25 cases） and nonpregnant group （25 cases）. The expressive intension of TNF-α protein and TGF-β1 protein in placental tissues was also identified by immunohistochemistry. Results Both levels of TNF-α mRNA and TGF-β1 mRNA expressed in PBMCs were significantly different between the three groups respectively （P〈0.05）. Levels of TNF-α in syncytiotrophoblastic and cytotrophoblastic cells of the two aborted groups were substantially higher than those of the nonregnant group （P〈0.01）, but the levels of TGF-β1 in syncytiotrophoblastic cells of the two aborted groups were markedly lower than those of the non-pregnant group （P〈0.01）. Conclusion There is potential relation between TGF-β at the fetomaternal interface and spontaneous abortion. TGF-β1 may contribute to the maintenance of pregnancy, and low-level expression of TGF-β1 may be associated with pregnancy failure.

OVEREXPRESSION OF Akt-1 GENE IN HUMAN HEPATOCELLULAR CARCINOMA

Objective: To investigate the expression difference of protein kinase B/Akt (Akt-1) between hepatocellular carcinoma (HCC) and adjacent normal liver tissues through the use of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. Methods: RT-PCR of 24 pairs of specimens and Northern blot of 4 pairs of specimens were performed to investigate the expression of Akt-1. Results: Akt-1 gene was overexpressed in 15 of 24 HCC (63.3%) by RT-PCR and in all HCC (4 paired tissues) by Northern blot. Conclusion: Akt-1 activation may play a role in the pathogenesis and progression of HCC. Akt-1 gene is reported to have changed in HCC for the first time. The precise relationship between Akt-1 and HCC is a matter of further investigation.

Long-term culture of primary porcine mature hepatocytes in the medium supplemented with ascorbic acid 2-phosphate

In this study, the effect of ascorbic acid 2-phosphate (Asc2P) was tested on porcine and rat mature hepatocytes in vitro. a). Asc2P increased the porcine, but not rat, albumin secretion and mRNA expression. The enhancing effect of Asc2P on porcine C/EBP alpha mRNA was observed in porcine mature hepatocytes. These data suggested that Asc2P played an important role in the regulation of porcine albumin mRNA level. b). The enhancing effect of Asc2P on ammonium metabolic activity was also observed in porcine, but not rat, mature hepatocytes. The porcine ornithine transcarbamylase (OTC) and arginase mRNAs were augmented by Asc2P, indicating that Asc2P had a direct effect on the urea cycle. c). The porcine collagen type I and type III mRNA, but not type XII mRNA, were detected as well, sugessting that Asc2P did not have the effect on the non-parenchymal hepatocytes to induce collagen type I and III mRNA expression. d). Our RT-PCR analysis demonstrated that the porcine hepatocytes expressed the sodium-ascorbate co-transporters SVCT1 and SVCT2, however, the intensities of porcine sodium-ascorbate co-transporters SVCT1 and SVCT2 bands were not changed markedly. These findings indicated that the Asc2P had no effect on SVCT1 and SVCT2 mRNA expression. e). The enhancing effect of Asc2P on porcine albumin mRNA was inhibited by staurosporine, a portein kinase inhibitor. We conclude that the enhanced albumin mRNA by Asc2P might be due to activation of tyrosine protein kinase and/or PKC and the Asc2P enhanced porcine albumin mRNA mainly at the transcriptional step.

Effects of Mangiferin on the Expression of TNF-α,iNos,ICAM-1 and Its mRNA in the Heart,Brain and Kidneys of SHR

[Objectives] To study the effects of Mangiferin( MGF) on TNF-α,iNOS,ICAM-1 and its mRNA expression in the heart,brain and kidneys of spontaneously hypertensive rats( SHR),and reveal the mechanism of its anti-inflammatory injury in hypertension target organs.[Methods]SHRs were randomly divided into 5 groups: the model group,the high-dose,medium-dose,low-dose MGF groups and the Benazepril group,with 8 rats in each group,WKY rats were used for the normal control group. Besides,on-invasive blood pressure( BP) instruments were used to measure systolic blood pressure in the rats' tail artery,western blot was used to analyze the expression of TNF-α,iNOS,ICAM-1 and reverse transcription-polymerase chain reaction( RT-PCR) was used to analyze the expression of TNF-α,iNOS,ICAM-1 mRNA.[Results]Compared with the normal control group,the model group's BP level was significantly increased( P <0. 01)),but the MGF had no significant lowering BP effect( P > 0. 05); compared with the normal control group,the expression of TNF-α,iNOS,ICAM-1 and its' mRNA in the model group was significantly increased( P < 0. 05 or P < 0. 01),and MGF could reduce the level of expression of these inflammatory cytokines( P < 0. 05 or P < 0. 01); between the Benazepril group and high-dose,medium-dose,low-dose MGF groups,most of the indicators had no significant difference( P > 0. 05). [Conclusions]MGF had no significant lowering BP effect,SHR showed inflammatory injury in the heart,brain and kidneys,MGF showed improvement on the inflammatory injury,and the anti-inflammation mechanism may be associated with lowering TNF-α,i NOS and ICAM-1 and its mRNA expression.

Callus Cultures Of Beans Infected With Virus As A Model For Testing Antiviral Compaunds

In the work,bean callus raised from a leaves of Bean common mosaic virus infected bean plant was obtained and adapted for the testing of antiviral activity of liposomal glycan-glycolipid complexes.Ganoderma adspersum glucans and Pseudomonas spec.rhamnolipids were constituents of liposomal compaunds.It has been shown that under the long-term cultivation(up to 3 months)in the presence of a liposomal preparation containing(10-100 mg/l),the virus is eliminated from the tissue.This is evidenced by the absence of 391 bp sequence amplification product established by RT-PCR in the callus tissue,cultured on a medium containing the liposomal complex.The proposed model system is analogous to plant tumors and has obvious advantages over similar systems in vivo,since the callus growth is controlled and independent of environmental factors.

A novel cytogenetic abnormality r(7)(::p11.2->q36.3::) in a Philadelphia-positive chronic myeloid leukemia case

The so-calledPhiladelphia(Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. It results in juxtaposition of the 5' part of the BCR gene on chromosome 22 and the 3' part of the ABL1 gene on chromosome 9. An additional acquired monosomy 7 or deletion of 7q is associated with poor prognosis in a variety of myeloid disorders. Here we report a novel Ph chromosome positive CML case with a ring chromosome 7 [r(7)]. Immunophenotyping was compatible with CML, although 4.5% of total leucocytes appeared like acute myelogeneous leukemia (AML) subtype M2. The r(7) was characterized in detail by array-proven multicolor banding (aMCB), the latter being of enormous significance to characterize breakpoint regions in detail. Underlying mechanisms and prognostic are discussed, as ring chromosomes are rare cytogenetic abnormalities in hematopoietic malignancies.

Livin在原发性肝癌中的表达及与Caspase-3的关系

目的 研究凋亡抑制基因Livin在原发性肝癌中的表达,并探讨其与Caspase-3表达的相关性.方法 采用逆转录多聚酶链式反应(RT-PCR)方法检测Livin和Caspase-3在46例原发性肝癌,28例癌旁组织,12例正常肝组织中的表达,分析二者之间相关性.结果 Livin基因在肝癌组织、癌旁组织、正常肝组织表达依次降低,其表达与原发性肝癌患者的年龄、性别、肿瘤大小、是否合并乙肝肝硬化、分化程度、甲胎蛋白等差异均无统计学意义(P＞0. 05),而与肝癌是否发生转移有关(P<0. 05);Caspase-3在上述组织中依次升高,差异均有统计学意义,二者在肝癌组织中表达负相关(rs=-0. 333, P<0.05).结论 Livin在肝癌组织中高表达,可能和Caspase-3在原发性肝癌细胞凋亡中起重要作用,可能是原发性肝癌生物学行为和预后重要标记物之一.

Molecular detection of EWS-Ets fusion transcripts and their clinicopathologic significance in Ewing’s sarcoma/peripheral primitive neuroectodermal tumor

Background Ewing＇s sarcoma/peripheral primitive neuroectodermal tumor （ES/pPNET） is often difficuh to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Flil and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET. Methods Thirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features （including rhabdomyosarcoma, neuroblastoma, lymphoma, small ceil carcinoma, and desmoplastic small round cell tumor ） were used as negative controls. β-actin and β2- microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction （RT- PCR）-based assay was performed to detect the EWS-Flil and EWS-ERG fusion transcripts. Results β-aetin and β2-mieroglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS- Flil fusion transcripts were detected in 11 of 13 （85%） ES/pPNETs. Three chimeric transcripts, all EWS-Flil, were detected in ES/pPNET samples. Among 11 EWS-Flil-positive cases, 7 eases had a type Ⅰ fusion transcript involving fusion of EWS exon 7 with Flil exon 6, 2 eases had a type Ⅱ fusion transcript involving EWS exon 7 with Flil exon 5, and 2 eases expressed fusion transcripts involving EWS exon 7 and Flil exon 8. Type Ⅰ EWS- Flil fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 eases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Flil fusion transcripts and CD99 expression. Conclusions Molecular detection of EWS-Flil fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is useful for the diagnosis and differential diagnosis of ES/pPNETs.