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.

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.

ESTABLISHMENT OF CELL CLONE OF LYMPHOMA AND A CELL LINE INFECTED WITH LEUKEMIA VIRUS AND STUDY ON ITS INDUCTED DIFFERENTIATION

Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.

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.

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.

Bcl-1 Rearrangement and Cyclin D1 Protein Expression in Multiple Myeloma Precursor Cells

The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.

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。

家蚕glial cell missing(BmGcm)基因鉴定、表达、亚细胞定位和功能

【目的】鉴定、克隆家蚕(Bombyx mori)glial cell missing(Bm Gcm)基因,分析其m RNA表达及亚细胞定位特征。制备多克隆抗体,同时在细胞水平进行过表达,检测Gcm对细胞增殖和周期的影响,为探究Bm Gcm功能打下基础。【方法】利用RACE方法克隆获得Bm Gcm全长c DNA序列,利用ORF Finder和SMART等在线工具对Bm Gcm基本序列特征和结构信息进行分析,运用Clustalx和MEGA 6.0等软件对多物种Gcm蛋白进行同源序列比对和进化分析。采用RT-PCR和q RT-PCR方法检测Bm Gcm的表达情况。利用原核表达系统获得重组蛋白,通过蛋白纯化和免疫小鼠制备多克隆抗体,运用Western blot对抗体进行检测。构建Bm Gcm表达载体,转染家蚕胚胎细胞系,分析其亚细胞定位情况,同时利用EDU细胞增殖标记和流式细胞仪对其功能进行探索。【结果】Bm Gcm(BGIBMGA006182)定位于4号染色体的nscaf2847上,其基因全长4 046 bp,包含4个外显子和3个内含子。其c DNA全长1 734 bp,包含166 bp的5′UTR、227 bp的3′UTR和1 341 bp的完整开放阅读框(ORF)。该基因编码446个氨基酸残基,预测蛋白分子量为50.61 k D,等电点5.557,含有典型的GCM结构域。多重比对结果显示GCM结构域在不同物种间具有高度的保守性,进化分析显示昆虫Gcm蛋白单独聚为一支,其中Bm Gcm蛋白与帝王蝶同源蛋白亲缘关系最为接近。表达分析结果显示Bm Gcm在胚胎发育第4天表达达到峰值,随后表达水平逐渐下调,而在幼虫阶段,Bm Gcm主要表达于中肠、精巢和卵巢。将Bm Gcm完整的开放阅读框序列构建至原核表达系统,经IPTG诱导和亲和层析纯化获得高纯度重组蛋白,通过免疫小鼠获得了多克隆抗体,Western blot检测该抗体可以特异性识别重组蛋白。在家蚕细胞系中过表达Bm Gcm蛋白,结果显示其定位于细胞核。在细胞水平,过表达Bm Gcm会明显抑制细胞增殖,将细胞周期阻滞于G1/S期。【结论】克隆鉴定得到Bmgcm全长序列,获得其表达和亚细胞定位信息。通过原核表达、蛋白纯化和免疫小鼠制备了可用的多克隆抗体。细胞实验发现Bm Gcm可以显著抑制增殖和影响正常的细胞周期进程。

Moving toward an understanding of the metastatic process in hepatocellular carcinoma

INTRODUCTIONClinical factors contributing to the therapeutic challenge of hepatocellular carcinoma (HCC) are manifold :tumors arise often in patients with compromised liver function ,therefore limiting therapeutic options ;symptoms develop only at later stages of tumor progression ,and tumors tend to invade normal stuctures or occur in multiple locations simultaneously.

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.

Activating interactions of sulfanilamides with T cell receptors

Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell receptors (TCR) itself, the pharmacological interaction [p-i] concept. To analyze whether the drug sulfamethoxazole (SMX) interacts directly with the TCR and thereby contributing to signaling and T cell activation, we analyze two SMX specific T cell clones (TCC “1.3”and “H13”). Proliferation to SMX and 11 related sulfanilamides, Ca++ influx in drug stimulated T-cells and the inhibitory effect of non-reactive sulfanilamides on SMX stimulation were analyzed. In silico docking of SMX and related sulfanilamide to the TCR were used to identify possible drug binding sites, and correlated to in vitro data to find the correct docking. In Ca++ influx assays, reactions occurred as early as 14 sec after adding SMX to TCC and APC. The broadly reactive clone (“H13”) was stimulated by 5 additional sulfanilamide, while one TCC (“1.3”) was reactive exclusively with SMX but not other sulfanilamides. Competition experiments with sulfanilamide inhibited SMX induced Ca++ influx and proliferation of the TCC1.3 ina dose dependent way. Docking experiments with SMX and related sulfanilamides confirmed and explained the in vitro data as docking localized binding sites for SMX and the 5 stimulating sulfanilamides on the CDR2β domain of the clone H13, while the 6 non-stimulatory SA failed to bind. In TCC 1.3, SMX could be docked on the CDR3α of the TCR. The other, non-stimulatory but inhibitory SA could also be docked to the same site. The combined analysis of in vitro and in silico data show that sulfanilamide can bind directly to TCRs. It shows that TCR, like other receptors, appear to be reamenable to manipulations by small molecules.

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.

China Succeeded in Somatic Cell Cloning

Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.……

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.

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。

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

To explore a novel strategy for antisense gene therapy of cancer,the coding sequence of hum an proliferating cell nuclear antigen(PCNA) c DNA was reversely inserted into the eukaryotic vector p L XSN by molecular cloning techniques and transferred into bladder cancer EJcells with li- posome. The PCNA expression in transferred cells was dynamically detected by immunofluo- rescence and RT- PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorim etric and cloning formation m ethods.In the experiment,the antisense eukaryotic vector was successfully constructed and nam ed as p L APSN.After transfection with it for1- 7 days,PCNA protein and m RNA levels in cancer cells were blocked by16 .74 % - 84 .2 1% (P< 0 .0 5 ) and2 3.2 7% - 86 .15 % (P<0 .0 5 ) respectively.The proliferation activities of transferred cells were inhibited by 2 7.91% - 6 2 .0 7% (P<0 .0 1) ,with cloning formation abilities being de- creased by 5 0 .81% (P<0 .0 1) . Itwas concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique,which could serve as an ideal strategy for gene therapy of bladder cancer.

STUDY OF ECK GENE EXON-3 FROM HUMAN NORMAL TISSUE AND BREAST CANCER CELL LINE

Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR 75 1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR 75 1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR 75 1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR 75 1 cell line respectively. ④ ZR 75 1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.

七彩神仙鱼催乳素基因的克隆、定位及表达分析

【目的】七彩神仙鱼(Symphysodon aequifasciatus)具有特殊的亲代抚育行为,克隆并定位七彩神仙鱼催乳素基因,分析其在亲代抚育中的表达模式,为理解催乳素在七彩神仙鱼亲代抚育中的作用提供依据。【方法】通过BLASTP对七彩神仙鱼全基因组进行分析,鉴定出1个催乳素基因,命名为dfprl。基于dfprl基因的CDS区,设计克隆和PCR引物,通过RACE技术克隆获得dfprl基因全长,并利用生物信息学对dfprl基因进行结构分析,对其氨基酸序列进行理化性质和进化分析。对不同抚育阶段七彩神仙鱼的脑、性腺和皮肤进行转录组分析,探究dfprl基因在亲代抚育中的表达特征,并利用原位杂交技术对dfprl基因在七彩神仙鱼皮肤中的表达进行定位。【结果】dfprl全长为1282 bp,cDNA序列开放阅读框长度为639 bp,共编码212个氨基酸,5'-UTR为309 bp,3'-UTR为334 bp。dfprl蛋白存在PRL家族的典型结构域Hormone_1,dfprl蛋白与慈鲷科其他鱼类PRL蛋白相似性较高,与尼加拉瓜湖始丽鱼(Archocentrus centrarchus)对应的氨基酸序列同源性最高、为96.70%。亲鱼进入抚育阶段后,dfprl在性腺和皮肤中的表达水平逐渐上升,抚育结束时表达水平下降。原位杂交结果显示,dfprl在皮肤粘液细胞中表达,且与七彩神仙鱼催乳素受体(dfprlr)表达位点重叠,在抚育早期阶段高表达。【结论】七彩神仙鱼dfprl基因高度保守,存在稳定的Hormone_1结构域。七彩神仙鱼dfprl基因在亲代抚育阶段的皮肤中高表达,且在亲代抚育阶段的皮肤粘液细胞中与dfprlr表达位点重合,表明dfprl可能通过作用于dfprlr进而促进七彩神仙鱼独特抚育行为的发生。