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Objective To obtain the sequence of recombinant human basic fibroblast growth factor (rhbFGF) with endonuclease sites of BamHI and Pst Ⅰ by PCR based gene assembly and construct the recombining vector of rhbFGF gene by TA cloning technique.Methods The rhbFGF gene sequence which was designed for lactococcus with endonuclease sites of BamHI and Pst Ⅰ was divided into 22 oligonucleotides by DNASTAR 6.0 (bFGFl-bFGF22).The 22 oligonucleotides were spliced by PCR based gene assembly to get the rhbFGF eDNA with endonuclease sites of BamHI and Pst Ⅰ.The PCR product was inserted into the PMD18-T VECTOR.The recombining vector were converted to the competent E.coli TOP10.The clones generated from LAB were analyzed by miniprep isolation from LAB host.They were identified by the restriction enzyme cuuing and sequencing.Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst Ⅰ was verified by 2％ agarose electrophoresis.The recombining vector of rhbFGF gene by TA cloning technique was identified by enzyme digestion and gene sequencing.Conclusions The TA cloning vector of recombining hbFGF with endonuclease sites of BamHI and Pst Ⅰ was constructed successfully.
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ObjectiveTo investigate the protection effect of bifidobacterial adhesin for intestine ischemia/reperfusion (I/R) injury on gut barrier function in rat.MethodsSeventy-two male SD rats were randomly divided into sham operation group (n =24), I/R model group (n =24) and pretreatment group of bifidobacterial adhesin (pretreatment group, n = 24).Six rats were anatomized at 6 h, 1 d, 4 d and 7d after inducing I/R model in each group, respectively.The pathological changes of the terminal ilea and the blood levels of TNFα, IL-6, IL-10, diamine oxidase (DAO), and the activity and content of D-lactic acid were observed.ResultsThe blood levels of TNFα, IL-6, DAO and D-lactic acid in I/R model group were significantly higher than sham operation group at all time points (P ＜0.05) , while the blood level of IL-10 was no significantly change.The activity of IL-6 and DAO in pretreatment group was significantly lower than I/R model group at all time points (P ＜ 0.05), the blood level of TNFαt in pretreatment group was significantly lower than I/R model group at 1 d, the blood level of D-lactic was significantly lower than I/R model group at 4 d and 7 d (P ＜ 0.05). Intestinal pathological damages were obviously milder in pretreatment group than I/R model group at all time points (Chiu's pathological scores: 6 h, 3.22 ±0.22 vs 3.57 ±0.20;1 d,3.77 ±0.13 vs 3.90 ±0.12;4 d,2.93 ±0.23 vs 3.07 ±0.21;7 d,2.10 ±0.30 vs 2.22 ±0.17,all P ＜ 0.05).ConclusionThe pretreatment of bifidobacterial adhesin could protect the intestinal mucosa from I/R injury, and alleviate intestinal ischemic reperfusion injury.
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ObjectiveTo observe the effects of bifidobacterial sectory adhesin on NF-κB DNA binding activity,cytoplasm IκB,nuclei NF-κBp65,TNF-α,IL-1β,and IL-8 expression in intestinal epithelial cell post stress response.MethodsThe cultured intestinal epithelial cell line Lovo cells were divided into 5 groups,which included directly stimulated by lypopolysaccharide (LPS) (100 ng/ml) or H2O2 (200 μmol/L) for 3 hours groups,bifidobacterial sectory adhesin (30μg/ml) pre-incubation for 30 minutes and then treated by LPS (100 ng/ml) or H2O2 (200 μmol/L) for 3 hours groups and control group with no treatment.NF-κB DNA binding activity was evaluated by electrophoretic mobility shift assay (EMSA).The cytoplasm IκB and nuclei NF-κBp65 expression were determined by Western blot.The expressions of TNF-α,IL-1β and IL-8 at mRNA level were detected by semi-quantatitive assay of RT-PCR.ResultsThe expressions of NF-κB DNA binding activity [(6.20±0.35) times and (4.16 ± 0.52) times of that of non treated control group,respectively] and nuclei NF-κBp65 [ (0.64±0.05) and (0.67±0.06)] were higher in cells after LPS or H2()2 stimulated,however the cytoplasm IκB expression [ (0.28 ± 0.10) and (0.39 ± 0.12) respectively] was weaker.After bifidobacterial sectory adhesin pre-incubation,the expressions of NF-κB DNA binding activity and nuclei NF-κBp65decreased obviously,but cytoplasm IκB expression increased.After treated with LPS or H2O2,the mRNA expressions of TNF-α,IL-1β and IL-8 were significantly increased [LPS treated group2 (0.92±0.10)、(0.38±0.03)、(1.44±0.25),H2O2 treated group:(0.89±0.13)、(0.36±0.06)、(1.42±0.18)].After bifidobacterial sectory adhesin pre-incubation,their expressions decreased.The mRNA expressions of TNF-α,IL-1β and IL-8 have significantly positive correlation with NF-κB DNA binding activity.ConclusionsThere is a significant inhibition role of bifidobacterial sectory adhesin in LPS and H2O2 induced NF-κB DNA binding activity in intestinal epithelia cells.The activation of NFκB may associate with the regulation of TNF-α,IL-1β and IL-8 proinflammatory cytokines expression.
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Objective To investigate the effects of angiotensin Ⅱ (AngⅡ) on the expression of albumin and the synthesis of type Ⅰ collagen in human normal hepatic cells. Methods HL-7702 cells (human normal hepatocyte) were cultured and divided into control group, Ang Ⅱ treated group, an AngⅡ+irbesartan (co-stimulated) group. The expressions of albumin and type Ⅰ collagen were detected by immunofluorescence and Western blotting, respectively. The mRNA level of type Ⅰ collagen was measured by real time-PCR(qRT-PCR). Results After stimulated with 10-7 mol/L Ang Ⅱ for 72 hours, the expression of albumin significantly decreased in Ang Ⅱ treated group compared with control group (0.85±0.11 vs 1. 41±0.23,P=0.000), while the mRNA expression increased in AngⅡ treated group compared with control group (1.00±0.08 vs 3.72±0.19,P=0.000). In costimulated group, however, the expression of albumin significantly increased (0.85 ± 0.11 vs 1.38 ±0.32,P=0.000),and mRNA expression (3. 72±0.19 vs 2.86±0.13,P=0.000) and synthesis of type Ⅰ collagen were reduced when compared with Ang Ⅱ treated group. Conclusions The reduction of albumin and elevated systhesis of type Ⅰ collagen in HL-7702 cells are induced via Ang Ⅱ AT1 receptor.
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Objective To investigate the effects of silenced Racl on invasion and migration in LOVo cells.Methods The expression of Racl mRNA and protein in colorectal cancer cells(including LoVo SW480.SW620.SW1116,HT29)were detected by RT-PCR and Western blot,respectively.The changes of cytoskeleton were observed in LoVO cells after transfected with Racl-shRNA.then invasion and migration were recorded respectively in LoVo cells after transfected with Racl-N17 and Racl-L61.Results Racl mRNA and protein were overexpressed in all selected colorectal cancer cells.Deletion of Racl decreased the cross-linked actin network and pseudopodia,and inhibited the invasion and migration in LoVo cells.The migration experiment showed that the migrated cells were higher in Racl-shRNA[(75±5)cells].Racl-N17 [(93±5)cells]and Racl-L61[(267±7)cells]groups compared with control group[(214±8)cells,P＜0.01,＜O.01 and＜0.05,resprectively].The invasion experimental study revealed that the migrated cells were higher in Racl-shRNA[(35±5)cells],Racl-N17[(42±5)cells]and Racl-L61[(86±7)cells] groups compared with control group[(73±6)cells,P＜0.01,＜0.01 and＜0.05,resprectively].Condusion Deletoin of Racl can inhibit the invasion and migration in LDVo cells.
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BACKGROUND: Hepatic oval cells (HOCs) possess the potential of self-renewal, replication, and clone, proliferation and differentiation into mature hepatocytes under a certain condition. HOCs can be used as biomaterial for constructing biological artificial liver in vitro, employed for in vivo transplantation, as well as for tissue engineering as seed cells. HOCs can be widely used for improving clinical treatment of liver diseases. OBJECTIVE: To establish adult Wistar rat models of HOC proliferation, to perform/n vitro isolation and culture of HOCs, and to study the possibility of induction and differentiation of HOCs into hepatucytes. DESIGN: Observational study. SETTING: Institute of Gastroenterology, Nanfang Hospital, Southern Medical University. MATERIALS: Experiments were performed at the Laboratory of Institute of Gastroenterology of Nanfang Hospital from December 2003 to February 2006. Thirty-six healthy male Wistar rats aged 3-4 months (150-200 g) were provided by Experimental Animal Center of Southern Medical University. METHODS: Male Wistar rats were orally fed with ethionine received two-thirds partial hepatectomy (2/3 PH). HOCs were harvested and purified by two-steps perfusion and Percoll density gradient centrifugation, and then cultured in vitro and induced with hepatocyte growth factor (HGF), oncostatin M (OSM) and fibroblast growth factor-4 (FGF4). MAIN OUTCOME MEASURES: Identification and differentiation of HOCs. RESULTS: The concentration of HOCs was about 1.34×108 L-1 in each rat model after in vitro isolation. These cells were round, oval or polygon, about 1/6 1/3 the size of normal hepatocytes. The nucleus-cytoplasm ratio was relatively large. After 2 weeks, clone-like proliferation of HOCs could be observed. Laser scanning confocal microscopy indicated positive expression of stem cells markers Thy-1 and C-kit in cytoplasm and membrane of HOCs. Immunocytochemistry demonstrated positive stem cells marker alpha fetoprotein (AFP) in cytoplasm of HOCs. HOCs can stably passage and its shape gradually changed after inducing with HGF, OSM and FGF4. HOC volume became larger and HOCs lost their ability of sticking to the wall of culture flask. Apparent positive stain of cytoplasm albumin (Alb) was detected 14 days after induction, and the positive ratio increased along with the extension of inducing duration. Results of cytochemistry indicated a brown or black deposit after glucose-6-phosphotase (G-6-P) staining and red particles after periodic acid-Schiff (PAS) staining. CONCLUSION: Adult Wistar rat models of HOC proliferation are replicated by ethionine feeding combined with 2/3 PH. HOCs can be obtained through collagenase perfusion and Percoll density gradient centrifugation. Rat HOCs can be passaged and cultured in vitro. Under a certain condition, HOCs can be induced and differentiated into hepatocytes.
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To study adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells in vitro mediated by purified adhesin. Results showed that the binding of human bifidobacterial strains to human intestinal epithelial cells appeared to be specific. The adhesion was time and dose-dependent.These data suggested adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells in vitro can be mediated specifically by purified adhesin.
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The aim of this experiment was to construct a human colorectal carcinoma cDNA phage expression library. Total RNA was extracted from the cancer tissue of human colorectal carcinoma, and the mRNA was purified. The single-strand and double-strand of cDNA were synthesized through reverse transcription-PCR and LD-PCR. cDNA fragments, after removal of those smaller than 500bp, were combined with ?TriplEx2 phage vector. The recombinant cDNA were packaged in vitro with MaxPlax TM Packaging Extract, then a small portion of packaged phage was used to infect E.coli XL1-blue for titration and determination of the percentage of recombinant clones. PCR method was used to identify the size of inserted cDNA. A human colorectal carcinoma cDNA phage library consisting of 2.07?10 6 pfu/ml recombinant bacteriophages was successfully constructed, the recombinant percentage was 94.5%, and the range of the fragment length of exogenous inserted cDNA was between 600bp～4kb, with an average of about 1.4kb. It met the universally accepted standards, and it could be useful in screening cDNA clones to find out the human colorectal carcinoma associated antigen genes.
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To investigate the correlation between the expression of vascular growth factor (VEGF) and its receptor KDR and angiogenesis in human colorectal cancer. VEGF and KDR were detected in 68 cases colorectal cancer by immunohistochemical SABC technique. Microvessel density was determined by immunostaining for factor Ⅷ related antigen. Results showed that the positive rate of VEGF was 55 9% (38/68) in 68 cases of colorectal carcer, and VEGF was mainly located at the cytoplasm or the membrane of the colorectal cancerous cell; KDR positive rate was 45 6% (31/68), and KDR was located in the vascular endothelial cell of colorectal cancer tissues and their related Peri Cancerous tissues but also in the cytoplasm or the membrane of the colorectal cancerous cell. VEGF was closely related to Dukes stage. Microvessel density (MVD) was significantly higher in VEGF positive tumor than in VEGF negative tumor ( P
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Objective To establish human colon carcinoma LoVo/Adr cell line with multidrug resistance (MDR) and to study its MDR mechanism. Methods MDR cell line (LoVo/Adr) was induced by stepwise selection on exposure to increasing doses of adriamycin (ADR). The MDR of LoVo/Adr was detected by MTT assay and the distribution of its cell cycle was detected by flow cytometry. The expression of MDR related genes, including mdr1, MRP, GST ? and TopoⅡ was measured by RT PCR and the level of P gp was detected by immunohistochemistry. Results Compared with parental cells, the resistance line had a slower growth rate and longer doubling time. It was larger and mixed with giant cells in different sizes and the number of cells in S phase decreased while that in G1, G2 phase increased. The LoVo/Adr cell line showed 61 fold, 14 fold, 3 fold, 9 fold and 1 fold higher resistance to ADR, VCR, MMC, CTX and 5 FU respectively than its parental cell line. It was also cross resistant to VCR, MMC and CTX, but not to 5 FU. The parental LoVo cells showed no mdr1 expression and the level of mdr1 mRNA expression increased gradually according to the concentration of ADR in resistant cell lines, and the level of GST ? mRNA was only increased significantly in the induced initial stage, although the parental LoVo cells expressed a low level of GST ?. MRP mRNA expression was not detected in both parental cell line and resistant cell lines. The level of Topo ⅡmRNA remained stable. Conclusions LoVo/Adr cell line offers a model with a typical MDR phenotype for the study of MDR in human colon cancer. Its drug resistance was mediated by mdr1 and GST ?, not MRP and TopoⅡ.
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Objective To investigate the expression of vascular endothelial growth factor (VEGF) in colon cancer and its relation with the microvessel density (MVD) and p53 gene. Methods The expression of VEGF, p53 gene and MVD of different region of colon cancer were studied by immunohistochemical method from 68 by surgical resected specimens. Results The positive rates of VEGF, p53 and MVD in colon cancer were significantly higher than those in peritumor and normal tissue. The expression of VEGF and p53 positively correlated with depth of invasion, lymph node metastasis, distant metastasis, vessel invasion and Dukes' stage, but their relation to the histologic types was not significant. The MVD in p53(+) ( 34.6 ?12.2 ) and VEGF(+) (31.2?12.6) tumors was significantly higher than that in p53(-) and VEGF(-) tumors (15.0?7.9; 12.7?6.3, respectively, P
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Objective To investigate the effects of percutaneous radio frequency ablation of liver cancer, and evaluate the indications, contraindications, and side effects. Methods One hundred and fifty four patients with hepatocellular carcinoma or metastases liver cancer, with the average diameter of the tumours 7.07 cm were treated with by radio frequency RF 2000TM instrument, the results were evaluated. Results Average theraputic time was 1.18 times, the theraputic point was 5.74. The symptoms were improved after therapy, and the average concentration of AFP was droped from 1 553.68 ng/ml to 883.70 ng/ml. The percentage of tumour reduction was 56.8%. There were no serious complications observed. Conclusion Percutaneous radio frequency ablation of liver cancer is a simple, safe and effective method. For larger liver cancer several times action will be needed, or other intervantion therapy is added to enhance effectiveness.
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Objective To establish a proliferating model of hepatic oval cells (HOCs) with adult Wistar rats, isolate and culture in vitro the HOCs, and to approach the possibility of inducing the HOCs differentiated into hepatocytes. Methods Rats were fed with 0.07% wt/wt ethionine. On day 8, 2/3 partial hepatectomy (2/3 PH) was performed. Isolate, harvest and purify HOCs by type Ⅳ collagenase perfusion with semi in situ 2-step method and Percoll density gradient method. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to complete Williams' medium E (WE) to culture the HOCs. HOCs was induced differentiation with HGF, oncostatin m (OSM) and fibroblast growth factor-4 (FGF4) into hepatocytes. Results The concentration of cell after purification was about 1.34?105/ml. Most cells were small, about 1/6~1/3 the size of normal hepatocyte. Ovoid, elliptical or polygonal in shap, the nucleus-cytoplasm ratio was relatively large, and clone-like proliferation appeared 2 weeks later. LSCM revealed positive expression of Thy-1 and C-kit in cytoplasm and membrane of HOCs. ICC showed AFP in cytoplasm of HOCs. Stimulated by inducing system, the shape of HOCs changed gradually. The volume enlarged and cells lost their adherence ability. ICC indicated apparent positive stain of cytoplasm Alb 14 days after differentiated induction, and the positive ratio increased along with the extension of induction duration. Cytochemical tests indicated brown or black sediment with G-6-P staining and red particles with PAS staining, respectively. Conclusion The proliferation model of rats' HOCs was established after ethionine feeding and 2/3 PH. HOCs can be obtained with type IV collagenase perfusion and Percoll density gradient isolation and purification. Clone proliferation can be achieved through culturing HOCs in vitro. Under certain conditions, HOCs can be induced and differentiated into certain typical hepatocyte phenotype.
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Objective To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells by purified adhesin derived from bifidobacterium adolescentis 1027. Methods The number of bacteria adherent to intestinal epithelial cell line Lovo was counted and the results were analyzed by comparison. Results The purified adhesin in the concentration of 10?g/ml, 20?g/ml and 30?g/ml could significantly inhibit the adhesion of ETEC and EPEC to intestinal epithelial cell line Lovo. The higher the concentration of adhesin the higher inhibition rate was observed. Concentrations lower than 10?g/ml had no such effect. Conclusion The purified adhesin of bifidobacterium adolescentis 1027 could inhibit the adhesion of ETEC and EPEC to intestinal epithelial cell line Lovo, and the effect was dose-dependent.
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Objective To assemble fusion gene of c-myc tagged human trefoil factor family 2(hTFF2)(in vitro) and construct a cloning vector of this fusion gene.Methods Based on amino acid sequence of hTFF2 and codon of lactococcus lactis,cDNA of htff2 sequence was designed and extended at their 5' ends with a sequence encoding the c-myc tag;and the sequence of fusion gene c-myc-htff2 was designed.According to restriction enzyme sites of pBluescript Ⅱ sk(+),the SalⅠ and BamHⅠ were arranged at 5′ and 3′ ends of the fusion gene respectively.Instructed by DNAWORKS program,the fusion gene sequence of c-myc-htff2 was designed as 14 oligonucleotides that overlapped each other.The target gene fragment of cmyc-htff2 fusion gene was obtained by the means of polymerase chain reaction(PCR) based gene assembly.Then,the c-myc-htff2 was subcloned into vector of pBluescript II sk(+) for identification of the fusion gene by restricting enzyme excision and DNA sequencing.Results Assembly of c-myc-TFF2 fusion gene(in vitro) and construction of pBS-TFF2 had been completed successfully;and DNA sequencing showed that the sequence of synthetic gene accorded with the expectation.Conclusion With the help of DNAWORKS program,the design and assembly of oligonucleotides (in vitro) is effective to synthesize a target gene,and the cloning vector of c-myc-htff2 fusion gene has been successfully constructed.