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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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ObjectiveTo assess the effectiveness of tele-medicine and self-management goal(SMG) setting technique used in the diabetes management in the community setting.Methods It is a control-group study.415 type 2 diabetic residents were recruited from the Shanghai Wuliqiao community based on existing medical records.The subjects were divided into two groups,the study group was cared by general practitioners (GPs) specialists cooperation through the tele-medicine mechanism,the other was a control group.For the study group,a cooperation pathway between community health care centers and general hospitals were established.Standardized training and guidelines were provided to community health workers,regarding the setting of management goals of blood glucose and blood pressure,treatment plan,patient education,and SMG techniques.Fasting blood glucose ( FBG ) and 2 h postprandial blood glucose (2hBG) in the study group were monitored,followed by community health workers visiting monthly with seminars for diabetes education.At the baseline and the 12tb month,FBG,2hBG,HbA1C,blood pressure,triglyceride,total cholesterol,body mass index,waist-hip ratio were determined in each group.A survey was conducted to evaluate the costs of diabetes treatments,the knowledge base related to their disease,lifestyle,and the awareness of the new care model.The rates of achieving the goal of blood glucose,blood pressure,and HbA1Ccontrol were calculated.Internet case discussion between GPs-Specialists and referral to certain specialists were implemented when some patients did not reach the control goal.ResultsBy the 12 month follow up,FBG,2hBG,HbAIc,blood pressure of the study group were lower than the baseline,and as well as the control group with statical significance (P＜0.05).There are other improvcments:diabetes knowledge (29.1％ vs 5.5％ ),healthy diet (9.6％ vs -10.4％ ),blood glucose monitoring (30.3％ vs 10.8％ ),support for diabetes care in community (35.7％ vs 9.4％ ),and the preference of the new model (63.8％ vs 17.9％ ) with statistical significauce (P＜0.01 ).As for the medical costs,the study group's monthly costs were consistently lower than the control's.( -3.39Yuan vs 32.26 Yuan,P＜0.05).ConclusionsThe new diabetes care model based on GPs-Specialists tele-medicine and SMG in community opens the door to the community based care model formulation in regard to the health quality and costs control.The deployment of more technologies and management techniques could be explored further to improve the outcomes of community based chronic disease care model.
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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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Objective To understand patient pathway and clinical characteristics of chronic diseases in urban areas of Shanghai. Methods A total of 10 002 residents were enrolled and assigned to the chronic disease group (including hypertension, diabetes, coronary heart disease, myocardial infarction, and ischemic stroke) and the non-chronic disease group. Body mass index,fasting blood glucose, triglyceride,total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were tested.Difference of patient pathway and clinical characteristics of those chronic diseases was compared. Results Above chronic diseases were observed in 37.7% participants. About 2/3 diseases were confirmed and 80% patients were followed up in healthcare units not far away from home. Patients with coronary heart disease and myocardial infarction showed more outpatient visit to tertiary hospitals (P＜0. 05 ). However, patients with ischemic stroke had health check, rehabilitation and pharmacy done mainly in local healthcare centers (P＜0. 05 ). Diastolic blood pressure of patients visiting local doctors was significantly decreased (P＜0. 05). Conclusion Some differences in patient pathway were found in this study. Communication and cooperation between medical institutions should be intensified for effective chronic disease control.
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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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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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Humans , Carcinoma, Squamous Cell , Genetics , Pathology , Esophageal Neoplasms , Genetics , Pathology , Gene Expression Profiling , Methods , Gene Expression Regulation, Neoplastic , Oligonucleotide Array Sequence Analysis , Methods , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
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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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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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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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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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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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.