Objective To explore the pathogenesis of the level of gene and therapeutic target genes associated with intestinal obstruction by analyzing the differential expression gene. Methods The gene expression data that came from public database gene expression omnibus （GEO） which provided adhesion formation’ gene expression data on 1， 3， 7，and 14 days after operation （n=8） and normal intestinal tissues’ gene expression data （n=2） of mouse were collected. The gene function and differential expression of genes were analyzed by using gene ontology （GO） and significance analysis of microarray （SAM）. Results There were a lot of response stimulated up-regulation of gene expression when occurrence of adhesion， and the products of these genes were distributed on cell membrane. The analysis results of gene expression at different time point after operation showed that expression up-regulated of Hmgcs 2 gene occurred on 3-14 days ofter operation and expression up-regulated of Stxbp 5 gene occurred on 14 days ofter operation. Conclusions The adhesion formation may be closely associated with the genes of response to stimulus and the gene product in membrane. The Hmgcs 2 and Stxbp 5 genes may be closely associated with the occurrence of other diseases which induced by adhesion formation.This provides a basis for the discovery of potential therapeutic targets.
Objective To summarize the current advancement of preoperative radiotherapy for rectal cancer. Methods Relevant literatures about current advancement of preoperative radiotherapy for rectal cancer published domesticly and abroad recently were collected and reviewed. Results The lower local recurrence rate and longer disease-free survival time were observed in preoperative radiotherapy， compared with postoperative radiotherapy for rectal cancer. The recurrence rate was higher in short-course radiotherapy， compared with conventionally radiotherapy for stageⅢrectal cancer， but there was no significant difference for stageⅡrectal cancer. The biology molecular such as p53， CEA， Cox-2， EGFR， and VEGF had shown to be radiosensitive. Conclusions The proposal of preoperative radiotherapy for rectal cancer， could be prone to conventionally radiotherapy. There are more screening targets for preoperative radiotherapy in extensive exploration of diverse radiosensitivity. Biology molecular， developed gene expression profiling， and gene chips for rectal cancer may contribute to the individualization treatment.
Objective To establish hepatocellular carcinoma (HCC) cell lines which olig-expressed IGF1R gene stably． Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents． After transferred, cells were selected with G418 to obtain positive clones. The expressions of IGF1R, cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot． Cell growth curve were painted. Results Two cell lines clones were screened olig-expressing IGF1R gene stably． The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased (P＜0.05). Conclusion The HCC cell lines for olig-expressing IGF1R gene stably are established successfully．The plasmid pSUPER-IGF1R-siRNA can inhibit the growth of SMMC7721 and Hep3B cell lines, and the expression of cyclin D1.
Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12 (mIL-12) which was regulated with mifepristone (RU486) and explore its expression in vitro. Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction (PCR) and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR. The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which contains RU486 regulator cassette. The positive clone named pRS-RUmIL-12 was identified by restriction endonuclease digestion and PCR. Lipofectamine 2000 was used to transfect the pRS-RUmIL-12 to HEK293 cells followed by manufacturer’s recommendations. The protein concentration of mIL-12 induced with RU486 in supernatant of the transfected HEK293 cells was measured by ELISA. Results The sequence of single chain mIL-12 what we obtained was the same as the expected result. The results of restriction endonuclease digestion and PCR showed that the RU486-inducible regulatory vector (pRS-RUmIL-12) was successfully constructed. No significant mIL-12 protein concentration in supernatant of HEK293 cells activation was measured without the inducer RU486, whereas higher concentration of the mIL-12 protein was observed in the presence of RU486. The relationship of concentration of the mIL-12 protein and RU486 was positive correlated under definite range. Conclusion A regulatable eukaryotic expression plasmid of mIL-12 single chain fusion gene was constructed, which could be used in the further research of gene regulation and gene therapy.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
【Abstract】ObjectiveTo detect p27 expression in rectal carcinoma and serum transforming growth factor-β1 (TGF-β1) level in these patients, and to elucidate the modulatory effect of serum TGF-β1 on p27 expression in rectal carcinoma. MethodsExpression of p27 was measured in 37 cases of rectal carcinoma, 22 of rectal adenoma and 19 of normal control specimens by immunohistochemical staining using antibodies to p27. Serum level of TGFβ1 was measured in these patients by enzymelinked immunosorbent assay (ELISA) method. Resultsp27 protein was expressed in normal rectal tissue, rectal adenoma and rectal carcinoma, and the positive rate was 89.47%, 90.91% and 64.87%, respectively. The positive rate of p27 in rectal carcinoma was significantly lower than that of normal rectal tissue and rectal adenoma (P=0.025). p27 was mainly located in nucleolus of normal rectal tissue and rectal adenoma, and the positive rate of p27 in cytoplasm of rectal carcinoma was higher than that of normal and rectal adenoma. The positives rates of serum TGF-β1 in normal group, rectal adenoma group and rectal carcinoma group were 21.05%, 27.27% and 51.35%(P=0.045),respectively. The expression of p27 related to histological differentiation, lymph node metastasis and infiltration depth. Serum level of TGF-β1 related to lymph node metastasis, infiltrated depth and CEA level. The positive rate of p27 in TGF-β1 negative group and positive group was 88.89% and 42.11%(MantelHaenszel χ2=6.755,P=0.009), respectively. ConclusionTGF-β1 may be useful in assessment of malignance and prognosis of rectal carcinoma. TGF-β1 can downregulate p27 expression in rectal carcinoma.
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
【Abstract】Objective To establish and assess the rat model of postoperative fatigue syndrome (POFS). Methods The rat model of POFS was developed by the partial resection of the liver. The behavioral changes prior and post to operation, the disorder of nutritive intake after operation, stress reaction (pathological changes of mucous membrane in small intestine) and the hepatic albumin gene expression were observed. Results Low body temperature, lower sensitivity and reactivity were found. The serum levels of the iron, total protein, albumin, globulin and so on as the indexes of nutrition obviously dropped. The injury of the mucous membrane resulted from the stress reaction after the resection of the liver. The gene expression of the albumin decreased in the model group.Conclusion The experimental rat model of POFS by partial resection of the liver can be used for the investigation of POFS.
【Abstract】Objective To design the hammerhead ribozyme gene according to the hTR sequence in the gallbladder cancer cell, and build it into the eukaryon expression vector pTriEx-4. Methods According to the hTR cDNA sequence, the authors designed the primers and take the hTR template area gene from the gallbladder cancer cells by RT-PCR.The hammerhead ribozyme gene was synthesize according to the result of sequencing, and combine them with eukaryon expressing vector. Identified the exactitude of recombine vector by digestion.Results The 68 bp sequence extracted from the cell through the RT-PCR had the same template sequence comparing with the hTR cDNA. The recombinant plasmid with the hammerhead ribozyme gene was correct by digestion identification. Conclusion The RT-PCR method can extract the gallbladder cancer cell’s hTR gene. We construct the eukaryon expression vector containing the hammerhead ribozyme gene successfully which is the foundation for gene therapy of gallbladder cancer.
ObjectiveTo investigate the effects of somatostatin8 (SS8) on the apoptosis and the expression of cmyc protein of hepatocellular carcinoma cell SMMC7721. MethodsCultured in vitro, hepatocellular carcinoma cells SMMC7721 were incubated with SS8 (10 μg/ml). The apoptosis rate and expression of cmyc protein were detected by flow cytometry (FCM). ResultsSS8 can cause the spanonumber in S and G2/M phase and the auxonumber in G0/G1 phase of SMMC7721 cells . The apoptosis rate was 14.2% in the study group and 6.1% in the control group, and there was significant difference (P＜0.05); The level of expressions of cmyc protein was 0.833±0.035 after action by SS8 for 24 h. Compared with control group, there was no significant difference in the study group（P＞0.10）.But after the cells were incubated with SS8 for 48，72，96，120，144 h, the level of expressions of cmyc protein was 0.818±0.04，0.721±0.029，0.669±0.026，0.648±0.045，0.642±0.028 respectively in the study group, and there was significant difference as compared with the control group (P＜0.05). Conclusion The SS8 can induce the apoptosis and lower expression of cmyc protein of hepatocellular carcinoma cell SMMC7721.