BACKGROUND Dystrophic epidermolysis bullosa is characterized by fragile ulcerations of the skin caused by mutations in specific genes.However,genetic typing of this con-dition is rare.CASE SUMMARY An 11-year-old femal...BACKGROUND Dystrophic epidermolysis bullosa is characterized by fragile ulcerations of the skin caused by mutations in specific genes.However,genetic typing of this con-dition is rare.CASE SUMMARY An 11-year-old female suffered from recurrent fever,visible ulcerations of the entire skin,and severe malnutrition.Genetic testing revealed a frameshift mu-tation in the coding region 4047 of the 35th intron region of COL7A1,and she was diagnosed as malnutrition-type epidermolysis bullosa.Drug therapy(immu-noglobulin,fresh frozen plasma),topical therapy(silver ion dressing),fever redu-ction,cough relief,and promotion of gastrointestinal peristalsis are mainly used for respiratory and gastrointestinal complications.The patient’s condition impro-ved after treatment.CONCLUSION Dystrophic epidermolysis bullosa caused by a new framework shift mutation in COL7A1 should be taken seriously.展开更多
BACKGROUND Hypertrophic cardiomyopathy(HCM)is one of the most prevalent inherited myocardial disorders and is charac-terized by considerable genetic and phenotypic heterogeneity.A subset of patients with HCM progress ...BACKGROUND Hypertrophic cardiomyopathy(HCM)is one of the most prevalent inherited myocardial disorders and is charac-terized by considerable genetic and phenotypic heterogeneity.A subset of patients with HCM progress to a dilated phase of HCM(DPHCM),which is associated with a poor prognosis;however,the underlying pathogenesis remains inadequately understood.CASE SUMMARY In this study,we present a case involving a pedigree with familial DPHCM and conduct a retrospective review of patients with DPHCM with identified gene mutations.Through panel sequencing targeting the coding regions of 312 genes associated with inherited cardiomyopathy,a heterozygous missense mutation(c.746G>A,p.Arg249Glu)in the MYH7 gene was identified in the proband(III-5).Sanger sequencing subsequently confirmed this pathogenic mutation in three additional family members(II-4,III-4,and IV-3).A total of 26 well-documented patients with DPHCM were identified in the literature.Patients with DPHCM are commonly middle-aged and male.The mean age of patients with DPHCM was 53.43±12.79 years.Heart failure,dyspnoea,and atrial fibrillation were the most prevalent symptoms observed,accompanied by an average left ventricular end-diastolic size of 58.62 mm.CONCLUSION Our findings corroborate the pathogenicity of the MYH7(c.746G>A,p.Arg249Glu)mutation for DPHCM and suggest that the Arg249Gln mutation may be responsible for high mortality.展开更多
[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activi...[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.展开更多
[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify t...[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.展开更多
AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene....AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using <sup>32</sup>p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.展开更多
The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the...The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.展开更多
AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tum...AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.展开更多
In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene...In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.展开更多
The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion i...The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.展开更多
BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no ef...BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.AIM To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.METHODS We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.RESULTS We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice(small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63;large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).CONCLUSION The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice.Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.展开更多
AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L0...AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L02. METHODS： We constructed the recombinant replication-incornpetent Ad.rnda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the rnRNA expressing in cells, by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.rnda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS： Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RTPCR showed that the Ad.rnda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.rnda-7-infected L02 and HepG2 ceils, respectively. Ad.mda-7 infection obviously induced apoptosis （from 2.604±0.72% to 33.64±13.2%, P=0.00012） and growth suppression in HepG2 （inhibition ratio IR=68%） and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle （from 6.44% to 32.29%, P〈 0.01）, but not in L02 cells.CONCLUSION： These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.展开更多
Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 wa...Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.展开更多
文摘BACKGROUND Dystrophic epidermolysis bullosa is characterized by fragile ulcerations of the skin caused by mutations in specific genes.However,genetic typing of this con-dition is rare.CASE SUMMARY An 11-year-old female suffered from recurrent fever,visible ulcerations of the entire skin,and severe malnutrition.Genetic testing revealed a frameshift mu-tation in the coding region 4047 of the 35th intron region of COL7A1,and she was diagnosed as malnutrition-type epidermolysis bullosa.Drug therapy(immu-noglobulin,fresh frozen plasma),topical therapy(silver ion dressing),fever redu-ction,cough relief,and promotion of gastrointestinal peristalsis are mainly used for respiratory and gastrointestinal complications.The patient’s condition impro-ved after treatment.CONCLUSION Dystrophic epidermolysis bullosa caused by a new framework shift mutation in COL7A1 should be taken seriously.
基金Supported by National Natural Science Foundation of China,No.81770379.
文摘BACKGROUND Hypertrophic cardiomyopathy(HCM)is one of the most prevalent inherited myocardial disorders and is charac-terized by considerable genetic and phenotypic heterogeneity.A subset of patients with HCM progress to a dilated phase of HCM(DPHCM),which is associated with a poor prognosis;however,the underlying pathogenesis remains inadequately understood.CASE SUMMARY In this study,we present a case involving a pedigree with familial DPHCM and conduct a retrospective review of patients with DPHCM with identified gene mutations.Through panel sequencing targeting the coding regions of 312 genes associated with inherited cardiomyopathy,a heterozygous missense mutation(c.746G>A,p.Arg249Glu)in the MYH7 gene was identified in the proband(III-5).Sanger sequencing subsequently confirmed this pathogenic mutation in three additional family members(II-4,III-4,and IV-3).A total of 26 well-documented patients with DPHCM were identified in the literature.Patients with DPHCM are commonly middle-aged and male.The mean age of patients with DPHCM was 53.43±12.79 years.Heart failure,dyspnoea,and atrial fibrillation were the most prevalent symptoms observed,accompanied by an average left ventricular end-diastolic size of 58.62 mm.CONCLUSION Our findings corroborate the pathogenicity of the MYH7(c.746G>A,p.Arg249Glu)mutation for DPHCM and suggest that the Arg249Gln mutation may be responsible for high mortality.
文摘[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
基金Supported by the National Key Technology Research and Development Program of China (2012BAK08B07)the National Natural Science Foundation of China (31201919)~~
文摘[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.
基金the National Natural Science Foundation of China,No.39770379the National Basic Research Project("973")SUGEN,USA.
文摘AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using <sup>32</sup>p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.
基金Supported by Grants-in-Aid for Young Scientists(B)(No.15K18454 to Tsujimura T)Scientific Research(B)(No.15H03001 to Hishikawa K)Scientific Research(C)(Nos.25461208 to Takase O,15K09244 to Yoshikawa M and 26462400 to Idei M)from the Japan Society for the Promotion of Science
文摘The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.
基金Supported by the Medical Scientific Foundation of Jiangsu Province, No. H200147
文摘AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.
基金a grant of the Clinical Key Subject Foundation from Ministry of Health of China (No. 2004CB518705)
文摘In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.
基金supported by the National Natural Science Foundation of China,No.81460193
文摘The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.
基金the National Natural Science Foundation of China,No.81372585 and No.81772557Beijing Health System High Level Training Plan of Health Technical Personnel,No.2014-3-048
文摘BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.AIM To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.METHODS We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.RESULTS We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice(small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63;large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).CONCLUSION The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice.Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.
文摘AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L02. METHODS： We constructed the recombinant replication-incornpetent Ad.rnda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the rnRNA expressing in cells, by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.rnda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS： Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RTPCR showed that the Ad.rnda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.rnda-7-infected L02 and HepG2 ceils, respectively. Ad.mda-7 infection obviously induced apoptosis （from 2.604±0.72% to 33.64±13.2%, P=0.00012） and growth suppression in HepG2 （inhibition ratio IR=68%） and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle （from 6.44% to 32.29%, P〈 0.01）, but not in L02 cells.CONCLUSION： These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.
基金This study was supported by a grant from the youth dawn program of Wuhan (Grant No. 20025001028).
文摘Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.
