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AIM:To establish and characterize the patient-derived esophageal squamous-cell carcinoma xeno-graft (PDECX) in mice.METHODS:The samples of human esophageal cancer were grafted into severe combined immu-nodeficient ( SCID) mice.The xenografts were transferred to SCID mice when the first passage of xenografts grew up .The growth of tumors in the first, second and third passages was observed .HE staining was performed.The expression of CK5/6, p63 and p40 in the patient samples , and the first and third passages of the xenografts were detected by immunohisto-chemical analysis.The expression of mTOR, p-mTOR, p70S6K, p-p70S6K, Akt1, p-Akt (Ser473), Erk1/2 and p-Erk1/2 were determined by Western blot .RESULTS:The PDECX was successfully established .The positive expression of CK5/6, p63 and p40 in the xenografts was consistent with that in the patients ’ samples.The levels of phosphorylated and total proteins of proliferation-related signaling pathways were different in the xenografts from different patients .CONCLU-SION:The PDECX model adequately reflects the tumal heterogeneity that is observed in the patients .
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AIM:To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interfer-ence on the proliferation and apoptosis of human breast cancer cells .METHODS:The short hairpin RNA ( shRNA) targe-ting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection .The knockdown effi-ciency was assessed by Western blotting .The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays .The cell apoptosis induced by staurosporine was detected by flow cytometry . RESULTS:The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells ( P<0.01).Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01).Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION:Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemothera-peutic drug-induced cell apoptosis , suggesting that PAK2 might be a new therapeutic target in breast cancer treatment .
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Objective To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd. 3 antigen against U14 cervical cancer cell of mice. Methods Mouse brain microvascular endothelial cell bEnd. 3 was cultured and identified for preparation endothelial cell bEnd. 3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd. 3 antigen 4 times in 4 weeks (bEnd. 3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+ CD+8 surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. Results The expression of VEGF-R2 and integrin αV gene in bEnd. 3 cells were expressed highly.After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd. 3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11± 0.13) cm3 versus (3.38 ±0.34) cm3]. The CTL response of spleen iymphocytes in vitro showed that bEnd. 3-DC cells could kill bEnd. 3 cells, the special lysis rate was more than 60% . The percentage of CD+3 CD+8 spleen lymphocytes in bEnd. 3-DC group[(38.6 ± 0.7) %] was higher than those in other groups (P ＜ 0.05). The titer of serum antibody of Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd. 3 cell in bEnd. 3-DC group. Western blot analysis revealed that there were specific bands at 220 000 (VEGF-R2).Conclusions bEnd. 3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humorai immunity are induced by bEnd. 3-DC antigen which maybe have some antigens in bEnd. 3 cells that reacts with endothelial cell proliferation-related antigens.
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BACKGROUND: Further studies are needed to understand the cytobiological character, functional regulation, gene changes and expression difference of CD34~+ and CD133~+ stem cells induced by basic fibroblast growth factor (bFGF) using gene chip. OBJECTIVE: To compare the differences of gene expression and the response to bFGF of human umbilical cord CD34~+ and CD133~+ cells, and to explore gene expression changes of bFGF-induced umbilical cord CD34~+ and CD133~+ hematopotic stem cells/hemapoietic progenitor cells in vitro. METHODS: Human umbilical cord blood CD34~+ and CD133~+ cells were isolated and purified by MiniMACS immunomagnetic beads selection. The CD34~+ and CD133~+cells were cultured for 10 to 15 days in DMEM/F12 medium, supplemented with bFGF and B27. Total RNA from these cells was extracted and the genetic level of these cells was performed using Oligo GEArray(r) chip and GEArray software. Selected rate of CD34~+ and CD133~+ hematopoietic stem cells was detected using flow cytometry. CD34~+ and CD133~+ cell morphological changes were measured before and after bFGF induction. The concentration and purity of RNA were determined by agarose gel electrophoresis degeneration. Gene-chip test results were analyzed.
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AIM: To investigate whether Flk1~+ CD31~- CD34~- cells isolated from human adult adipose tissue have characteristics of hemangioblasts in vivo. METHODS: After sublethally irradiated (300cGy) with a caesium source, the female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were injected with human adipose tissue-derived Flk1~+ CD31~- CD34~- cells (10~5 cells per mouse) via tail vain with 0.4 mL Roswell Park Memorial Institute medium (RPMI-1640). The control mice received the same volume of RPMI-1640 medium. All mice were killed 2 months after transplantation for further study. The differentiation potential of Flk1~+ CD31~- CD34~- cells was assessed in bone marrow and gastrointestinal tract by the methods of flow cytometry, RT-PCR, FISH, and triple-color immunofluorescence. RESULTS: Flk1~+ CD31~- CD34~- human adipose tissue-derived adult stem cells differentiated into endothelial cells and hematopoietic cells at the single-cell level in vivo. CONCLUSION: Human adult adipose tissue-derived Flk1~+ CD31~- CD34~- cells bear characteristics of hemangioblast in vivo and may have potential application for the treatment of hematopoietic and vascular diseases.
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This research ws carried out to construct a medicine screening system targeting at human promoter of CCR5. The gene Human promoter of CCR5 was inserted into the rebuilt vector pGL3-neo. The pGL3-neo-CCR5 plasmids were transfected into Jurkat cells (the cell line of acute T lymphocyte leukemia). The lasting transfected cells were screened by G418. After seven kinds of traditional Chinese medicine had acted separately on the lasting transfected cells for 16h, the expression levels of CCR5 promoter in the cells were detected. The results showed that the level of luciferase activity of Shuanghuanglian-injectio group was remarkably lower than that of control (P < 0.05), and the levels of luciferase activity of Chuanhuning group, Baical skullcap root group, and Milkvetch root group were remarkably higher than that of control (P < 0.01). Shuanghuanglian-injectio depressed the activity of the transfected CCR5 promoter in cells cultivated in vitro; Chuanhuning, Baical skullcap root and Milkvetch root boosted the activity of the transfected CCR5 promoter in cells cultivated in vitro. Thus a medicine screening system based on Human promoter of CCR5 was initially constructed.
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Humanos , Avaliação Pré-Clínica de Medicamentos , Métodos , Medicamentos de Ervas Chinesas , Farmacologia , Vetores Genéticos , Genética , Células Jurkat , Regiões Promotoras Genéticas , Genética , Receptores CCR5 , Genética , TransfecçãoRESUMO
Objective To investigate the differentially expressed genes of primary esophageal squamous cell carci-noma and of normal esophageal mucosa. Methods LCM-GMA-cDNA microarray was used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 cases of human esophageal squamous cell carcinoma (ESCC). After high-stringent washing, the cDNA microarray was scanned for the fluores-cent signals. Results Among the 886 target genes, 34 genes had significant difference in Ⅰ / Ⅱ and Ⅲ/Ⅳ group. Cell cycle regulators possibly promoting the growth of tumor cells were highly expressed in the early stages of ESCC, whereas adhesion molecules and extracellular matrix-related molecules possibly promoting invasiveness increased in the later stages. Conclusion More than one gene contributed to esophageal cancer. The profiles of gene expression will bring us chance to understanding the molecular mechanism of tumor progression and to support clinical treat-ment.
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Objective To discuss the feasibility on building lewis lung carcinoma mouse models through different methods and improve the methods. Methods The method of culture LLC cells in vitro, trypsin digestion method, Ⅳ collagenase method and homogenate method were compared to make the different dose of cell suspension injected into C57BL/6 mice. The feasibility of the improved method was determined through observing the cell count, the tumor formation ratio, the tumor formation time, tumor volume, weight and life habit. Results The method of culture LLC cells in vitro could get needed cells and its tumor formation ratio was 100 %. Trypsin digestion method and homogenate method could get less cells and its tumor formation ratio was about 80 %～90 % and 60 %～75 %. Whereas 1V collagenase method could get most cell count and its tumor formation ratio was 100 %. Conclusion IV collagenase method is a preferred method which is simple,high efficiency and make a strong base on the cancer experimental study.
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Objective To investigate the etfect of microenvironment simulated by colon carcinoma homogenate supernatant on the differentiation and development of human dendritic ceils (DCs), and to investigate the function of vascular endothelial growth factor A (VEGF-A) during this process . Methods Fresh colon carcinoma and peri-cancer tissues were collected to prepare homogenate supernatant. The pe-ripberal blood mononuclear cells were isolated and cultured with 1640 medium including rhGM-CSF and rhIL-4. Then the colon carcinoma homogenate supernatant, peri--carcinoma homogenate supernatant and VEGF-A were added to the cultures at day 2. Antigen of colon carcinoma cell line SW620 was added at day 4 and lipopolysaccharide (LPS) was added at day 6. DCs were collected at day 8 for further study. The con-tent of VEGF-A was tested by ELISA. The morphology and the immunopbenotype of DCs were checked by microscope and flow cytometry, respectively. The expression of CDIa was tested by RT-PCR, and the prolif-eration and killing rate of T cell was measured by CCK-8. Results The content of VEGF-A in the homoge-nate supernatant of colon carcinoma was significantly higher than that of the peri-carcinoma (P ＜ 0. 05). Compared with normal DCs, the cell morphology of colon carcinoma homogenate aupernatant group was in-hibited, and the cell number was decreased. Besides, the positive expression rate of DC surface markers de-creased (P ＜ 0.01). The capacity of mixed lymphocyte reaction (MLR) and killing capacity of T cells de-creased(P ＜0.01). However, there was almost no difference between VEGF-A group and normal DCs on the cell morphology and cell number, and VEGF-A had no obvious inhibition on the expression of DCs sur-face markers (P ＞ 0.05). But VEGF-A group had significantly inhibitory effect on the MLR and T cells kill-ing. Conclusion The tumor microenvironment simulated by the colon carcinoma homogenate supernatant obviously has inhibitory effect on the differentiation and function of DCs, and VEGF-A has the inhibitory effect on DC function, but the inhibitory effect is not through the inhibition of the expression of DC costimu-lators.
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To screen the genes associated with esophageal cancer, a cDNA microarray technique was established and used for the analysis of the gene expression profile in human esophageal cancer cell line ECa109. The results showed that 107 (12.08%) genes differentially expressed among 886 target genes were identified between ECa109 cell line and normal human esophageal epithelial cells (HEEC), of which 51 (5.76%) were up-regulated and 56 (6.32%) down-regulated. Two genes were validated by quantitative RT-PCR (Q-RT-PCR) and the results were identical. The RNA amplification technique based-T7 RNA polymerase was established. The gene expression profile revealed better consistency between the amplified samples and those without amplification by T7 RNA polymerase, which provides a method for studying the profile of minute quantities of tumor cells in primary esophageal cancers. And the preliminary study on differential expression gene profile also enables us to have an understanding of the pathogenesis and pathomechanism of esophageal cancer.
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Humanos , Carcinoma de Células Escamosas , Genética , Patologia , Neoplasias Esofágicas , Genética , Patologia , Perfilação da Expressão Gênica , Métodos , Regulação Neoplásica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The differentially expressed genes between esophageal squamous cell carcinoma (ESCC)with or without lymphatic metastasis were investigated by gene chip, and the lymphatic metastasisassociated genes were screened out. Expression array was used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 cases of human ESCC. The lymphatic metastasis-associated genes were screened by bioinformatics between ESCC with or without lymphatic metastasis. The results showed that 43 (4.85%) genes significantly differed between the ESCC with and without lymphatic metastasis (P＜0.05), of which 18(2.03%)were upregulated and 25 (2.82 %) down-regulated. The up-regulated genes were involved in cell adhesion molecules and cell membrane receptors and the down-regulated genes were mostly cell cycle regulators and intracellular signaling molecules. It was suggested that lymphatic metastasis-associated genes were screened by gene chip, which was helpful to understand the molecular mechanism of ESCC lymphatic metastasis and lymphatic metastasis-associated genes might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.
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The differentially expressed genes between esophageal squamous cell carcinoma (ESCC) with or without lymphatic metastasis were investigated by gene chip, and the lymphatic metastasis-associated genes were screened out. Expression array was used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 cases of human ESCC. The lymphatic metastasis-associated genes were screened by bioinformatics between ESCC with or without lymphatic metastasis. The results showed that 43 (4.85 %) genes significantly differed between the ESCC with and without lymphatic metastasis (P<0.05), of which 18 (2.03 %)were upregulated and 25 (2.82 %) down-regulated. The up-regulated genes were involved in cell adhesion molecules and cell membrane receptors and the down-regulated genes were mostly cell cycle regulators and intracellular signaling molecules. It was suggested that lymphatic metastasis-associated genes were screened by gene chip, which was helpful to understand the molecular mechanism of ESCC lymphatic metastasis and lymphatic metastasis-associated genes might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.
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Objective To study the effect of gene engineering adenovirus H101 for treating esophagus carcinoma EC9706 induced by CEA gene specific silencing and to explore internal influential factor of H101 sensitivity. Methods To construct the siRNA expression vector of CEA and to inhibit the expression of CEA through RNA interference and gene transfection in EC9706 cell,stable CEA gene silencing system was set up,compared with empty vector group and non-transfected EC9706 cell,the model of athymic mouse subcutaneous transplantation tumor of human esophagus carcinoma EC9706 cell was established followed by injection with H101. The mRNA and protein expressions of CEA were detected by real time PCR and immunohistochemistry,the tumor size was measured. Results Silencing CEA gene by applying RNAi can inhibit CEA mRNA and protein expression in nude mice model with transplanted human esophageal cancer cells,there was no evident influence on tumor growth and mass oftumor. After using H101,the tumor size of interfering group was much smaller than that of empty vector group and normal control group(P
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Objective To study the effects of Alternariol(AOH) on DNA polymerase ?(DNA POL?)expression in NIH3T3 cells.Methods RT-PCR,Immunocytochemistry and Western blot were used to detected mRNA and the protein levels of DNA POL? in NIH3T3 cell line induced by AOH.Results The expression of DNA POL? in NIH3T3 cells contaminated by AOH was significantly higher than that in the control group(P
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Aim To evaluate the value of expressions of CD63 on basophils in diagnosing allergy to penicillins.Methods The expressions of CD63 on basophils activated with 9 kinds of antigens(PG:penicillin G,PV:phenoxomethylpenicillin,AMP:ampicillin,AX:amoxicilloyl,6-APA:6-aminopenicillanic acid,PHA:phenylacetic acid,PHOA:phenoxacetic acid,PHPG:N-(?-hydroxyphenyl)glycine,NPG:N-phenylglycine) were detected by FAST(Flow cytometric allergen stimulation test) in 43 patients with penicillins allergy.Eight types of specific IgE and IgG antibodies to penicillins were determinated by RAST and ELISA,respectively.Results The sensitivity and specificity of CD63 used as a marker in patients with penicillins allergy were 65.12% and 93.33%,respectively.Moreover,the sensitivities of CD63 and specific IgE were higher than those of specific IgG in patients with positive skin test(P
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AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA +) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro . After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA +) was observed. RESULTS: G418 test showed that about 14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA +) was prolonged 1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.
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Purpose:To detect DNA polymerase ? gene (pol?) mutations in human gastric cancer specimens and the relationship between H.pylori infection and pol? mutation. Methods:Extracting total RNA from gastric carcinoma,corresponding cancer specimens tissues and normal tissues, synthesizing cDNA and then using them as templates proceed PCR,The products of PCR were checked by SSCP. Extracting DNA from the specimen, we could detect the H.Pylori from the tissue of gastric carcinoma and the tissue adjacent to them by PCR.Results:There were 7 abnormal SSCP of 32 gastric carcinoma samples, and the mutation rate was 21.9%, but nothing abnormal was found in the tissues adjacent to the tumor. The results of H.Pylori DNA were positive in 15 samples from 32 gastric carcinoma tissues. Positive rate was 46.9(15/32).Detection result of tissues adjacent to tumor was consistent with gastric carcinoma. Comparing pol? SSCP to H.pylori-DNA in gastric carcinoma,we found the positive samples of pol? SSCP correlated with H.pylori-DNA.Conclusions:It is suggested that the pol?gene mutation may be associated with carcinogenesis and development of human gastric cancer. H.pylori infection is possibly related to pol? mutation in gastric carcinoma.
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Objective To observe the effect of RNAi that silences MTA1 gene on invasion and migration of esophageal carcinoma 9706 cells. Methods The siRNA expression vector that silences MTA1 gene was transfected into EC9706 cells by liposome. MTA1 mRNA and protein expressions were detected through quantitative RT-PCR and Western blot, respectively. The invasion and migration of EC9706 cells were evaluated by scrape wound healing assay and cell invasion assay in vitro. Results MTA1 gene expression significantly decreased. The scrape wound of EC9706 cells healed more slowly and the cell population that cut through Matrigel were less in the EC9706 cells transfected with siRNA expression vector than non-transfected EC9706 cells and the EC9706 cells transfected with blank vector (P
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AIM: To construct a mouse IFN-? expression vector and observe the antitumor effects of mouse peritoneal macrophages transfected with IFN-? in vivo and in vitro. METHODS: The IFN-? mRNA was amplified by RT-PCR. The open reading frame of mouse IFN-? gene was recombinanted with eukaryotic expression vector pcDNA3.1 through subcloning. Mouse peritoneal macrophages were transfected with recombinant vector pcDNA3.1-IFN-?. The expression of INF-? mRNA was measured by RT-PCR. Another group of peritoneal macrophages were cultured with the culture medium from pcDNA3.1-IFN-? transfecting groups, and its antitumor effect was measured by MTT. pcDNA3.1-IFN-? plasmid was peritoneally injected inte mouse with tumor. The appearance of ascites of pcDNA3.1-IFN-? plasmid injected mice and survival time were observed. RESULTS: The mouse IFN-? expression vector pcDNA3.1-IFN-? was constructed. The sequence was demonstrated to be the same as on GenBank. The recombinant vector was introduced into mouse peritoneal macrophages. IFN-? mRNA was detected by RT-PCR. The supernatant from cultured macrophages transfected with pcDNA3.1-IFN-? plasmid stimulated the antitumor effects of the macrophages without transfection. The appearance of ascites in pcDNA3.1-IFN-? plasmid injected mouse was delayed and survival time was longer than that in other groups. CONCLUSION: We have successfully constructed the mouse IFN-? expression vector pcDNA3.1-IFN-?. Mouse peritoneal macrophages transfected with pcDNA3.1-IFN-? have antitumor effects in vivo and in vitro.
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The effect of Alternariol Monomethyl Ether (AME), a suspected carcinogen in human being, on the lipid peroxidation in the epithelia of fetal esophagus and stomach was studied. The epithelia of esophagus and stomach of human fetus induced labor with water-bag were treated with AME. Lipid peroxides were measured by thiobarbituric acid test for malondialdehyde (MDA). There was a positive correlation between the values of MDA levels and AME dosaeges. It demonstrated that the lipid peroxdation could be initiated with AME. Lipid peroxidation levels were increased following treatment time elongating. The values of MDA levels in esophagus were higher than those in stomach, which were treated with the same dosaege of AME. It showed that AME had an organ selectivity. These results indicate that AME may be one of genetoxic etiological factors of esophageal carcinoma in Linxian county.