Preliminary study of endocytosis-related gene mRSD-9

AIM: To study the function of the gene mRSD-9 . METHODS: The techniques of immunoprecipitation and immunofluorescence were applied to verify the interaction between mRSD-9 and endophlin 3. The ATP/GTP combined experiment and the clathrin releasing experiment were conducted to investigate the effect of mRSD-9 on endocytosis. RESULTS: Expressed Myc-mRSD-9 was precipitated by Flag-endophilin 3. Conversely, the expressed Flag-endophilin 3 was also precipitated by Myc-mRSD-9. Under laser scanning confocal microscope, mRSD-9-GFP fusion protein and endophilin 3-RFP fusion protein were observed to co-localize in CHO cells. The combination of mRSD-9 protein with ATP/GTP was found and was specific because no combination was detected using mutant ΔmRSD-9. In empty vector transfected group, the quantity of transferrin in red fluorescent expressed cells was roughly the same as the untransfected cells. In pDsRed1-N1-mRSD-9 transfected group, the quantity of transferrin in mRSD-9 protein expressed cells was obviously reduced. In pDsRed1-N1-ΔmRSD-9 transfected group, the quantity of transferrin in ΔmRSD-9 protein expressed cells was significantly increased. CONCLUSION: The protein coded by mRSD-9 gene can suppress endocytosis of transferrin.     

Progress on DAB2IP gene

Human DAB2 interaction protein (DAB2IP) is a novel member of Ras GTPase-activating protein family. It interacts directly with disabled-2 protein (DAB2/DOC2) which suppresses growth of cancers derived from different tissues, including mammary, prostate and ovarian cancers. DAB2IP was identified as an immediate downstream effector mediated by DAB2/DOC2. DAB2IP and DAB2/DOC2 form a unique protein complex that has a negative regulatory effect on the Ras-mediated signal pathway. It is demonstrated that DAB2IP is a tumor suppressor gene inactivated by methylation in several cancers. This article reviews the structure and biological functions of DAB2IP gene as well as its potential roles in carcinogenesis and evolution.     

Experimental study of a new hypertension-related gene Slc7a8

AIM: To investigate the disease related genes in SHR.METHODS: The total RNA samples were obtained from second-order mesenteric arteries and kidney of SHR and WKY.Microarray containing over 10 000 genes was used to determine the level of mRNA expression in two groups.The genes were identified using real time quantitative RT- PCR.RESULTS: 19 down-regulated genes were determined by microarray,which were classified as chaperones,transport,growth factors,signal transduction,nuclear factor and lipoprotein.The result was confirmed by the method of real time quantitative RT- PCR.It was found that the Slc7a8 gene was up-regulated 9.3 fold in SHR.CONCLUSION: Slc7a8 gene may relate to hypertension.Further study on the Slc7a8 gene and its function would help us wholly understand the mechanism of hypertension and provide new clue to hypertension causes.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs

AIM: To construct lentiviral vector carrying the angiopoietin-1 (Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells (rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.     

Study on genetic instability of nm23-H1 gene and expression of hMLH1,hMSH2 in sporadic gallbladder carcinoma

AIM: To examine the MSI and LOH of locus D17S396 and their influence on the expression of nm23-H1 in gallbladder tumors,and to examine the protein expression of hMLH1/hMSH2,which may provide experimental evidence for the tumor occurrence and metastasis.METHODS: Techniques such as DNA extraction,CR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision IHC was used to assess the expression of nm23-H1 and hMLH1/hMSH2.RESULTS: ① The frequency of heredity instability of gallbladder carcinoma was 42.55%.The frequency of LOH in liver and lymph node metastasis cases and in stage Nevin IV and V was significantly higher than that without metastasis and stage I,II and III.However,the frequency of MSI showed contrary correlation with some clinicopathologic characteristics.② The expression of nm23-H1 was 46.81%.The case with lymph node metastasis and Nevin stage IV and V showed significantly lower expression than that without lymph node metastasis and stage I,II and III.③ The expressions of hMLH1 and hMSH2 were 51.06% and 42.55% respectively.hMLH1 in lymph node and liver metastasis cases and in stage Nevin IV and V were significantly lower than that without metastasis and in stage I,II and III.④ Positive frequency of hMLH1 in MSI positive group was higher than that in MSI negative group.The positive frequency of nm23-H1 and hMSH2 protein in LOH positive group was lower than that in negative group.CONCLUSION: The heredity instability of nm23-H1 gene may be implicated pathogenesis and progression of gallbladder carcinoma.Both MSI and LOH of nm23-H1 control the development of gallbladder carcinoma independently in different paths.Abnormal expression of hMLH1/hMSH2 may be a molecule marker in early stage of gallbladder carcinoma.     

Effects of a pentanucleotide repeat polymorphism of the apolipoprotein (a)gene on serum levels of lipoproteins in healthy Han population

AIM:To understand the relationship between pentanucleotide repeat(PNR) polymorphism of the apolipoprotein(a) gene and lipoprotein natures of normal Han population. METHODS:The serum levels of TG, TC, LDL-C,HDL-C, apo AI, apo B and Lp(a) were measured and the polymorphism of the apo(a) PNR was studied by using PCR-SSCP in 153 random normal individuals in Hubei Han population respectively. RESULTS:The relative frequencies of apo(a) PNR allele were significantly different from western population. The apo(a) gene which copy number of PNR is 5 was associated with high Lp(a) levels. No marked differences in the levels of TG, TC,LDL-C, HDL-C, apo AI and apo B were found among the various genotype groups of apo(a) PNR in Hubei Han. CONCLUSION:The data of lipids and PNR polymorphism of the apo(a) gene from healthy Hubei Han were obtained. The apo(a) allele with 5(TTTTA)-repeats may be related to high serum Lp(a) levels in the Hubei Han population.     

Significance and alterations of p14ARF gene of CDKN2A locus in gastric cancer

AIM:To investigate the significance and changes of p14^ARF gene in gastric cancer.METHODS:The tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. The homozygous deletions, mutations, methylation of the CpG islands, and mRNA expression of p14^ARF gene were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.RESULTS:①The homozygous deletion rate of p14^ARF was 31.3% (15/48), and no homozygous deletions were examined in all the gastric tissues neighboring tumor. ②There were no point mutations of p14^ARF in 33 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. ③Methylation rate of the CpG islands of p14^ARF was significantly higher in gastric cancers(47.9%, 23/48) than that in gastric tissues neighboring cancer (4.2%, 2/48)(P＜0.01).④ No expression of p14^ARF mRNA was detected in 45.8%(22/48) of gastric cancers. Moreover, the negative rate (100%, 3/3) of p14^ARF mRNA of gastric cancers with the combined methylation of exons 1β and 2 was significantly higher than that (15%, 3/20) of the sole methylation of exon2(P＜0.05). CONCLUSION:p14^ARF gene is frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which may play an important role in the development of gastric cancer.     

Function of Shh gene in the development of embryos

Mammals have three genes with homology to the hh gene. These comprise Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh has been shown to play a crucial role in embryogenesis and the development of ectoderm. Shh has the great relationship with the forming all the system. Shh-Patched-Smoothened signal pathway will lead the embryogenesis in the normal way. Otherwise, the abnormal embryogenesis, malformation, and tumor will be happened.     

龙眼授粉生物学研究

龙眼花粉萌发的适温为２０￣３０℃，添加２，４－Ｄ１０×１０＾－６￣２０×１０＾－６、ＧＡ３１５×１０＾－６￣３０×１０＾－６、ＮＡＡ２０×１０＾－６￣４０×１０＾－６，三十烷醇２×１０＾－６￣３×１０＾－６以及硼砂５０×１０＾－６￣２００×１０＾－６、钼酸铵１×１０＾－４￣５×１０＾－６，均可显著提高其花粉育性。龙眼授粉的有效时间为开花后１￣４天，以开花后２天内授粉着果率最高；授粉后５小时已有少量     

Recent advances in nm-23 gene and hepatocellular carcinoma

nm-23 gene is the first antimetastatic gene which was found to have close relationship with metastasis and prognosis of many malignant carcinoma. This review briefly summarizes the development of nm-23 gene and hepatocellular carcinoma in recent years.     

Effect of weaver gene on biology function of CAD cells

AIM:To assess the impact of weaver gene on neuronal development, protein expression and vitality. METHODS: DNA encoding the wild-type and mutant ion channel was introduced into immortalized tyrosine hydroxylase-positive CNS-derived neurons named CAD(Cath.a-differentiated, a variant of Cath.a. Cath.a was established by targeted oncogenesis in transgenic mice) cells. DNA clone, immunostaining and Western blotting were used. Three different concentrations (0.25 mg/L, 0.5 mg/L, or 1.0 mg/L) of Girk2 and wvGirk2 expression plasmids were transfected into the CAD cells. The number of transfected cycling cells, protein synthesis and neurites growth were observed between two groups. RESULTS:The number of transfected cycling CAD cells with high concentration of wvGirk2 reduced to about 60%, compared to Girk2-transfected cells. Low concentration of wvGirk2 did not cause cell death but reduced the protein production of transfected genes. Neurite growth was also affected by wvGirk2. MK-801 and Kir2.3 altered the effect of wvGirk2. CONCLUSION:The results indicate that wvGirk2 functions as a blocker in weaver animals, which blockes the wvGirk2 channel to rescue the cells from death. Our data also suggest that the presence of channels and the level of wvGirk2 may have a significant impact on the fate of cells containing wvGirk2.     

Protective effect of the bone marrow cells transfected with multidrug resistance gene on the reconstruction of murine hematopoietic function

AIM: To investigate the protective effect of the bone marrow cells transfected with human multidrug resistance gene (MDR1) on the reconstruction of murine hematopoietic function.METHODS: The mononuclear cells of the bone marrow from donors, BALB/C mice, treated with 5-Fu previously, were isolated and transfected with human multidrug resistance gene in vitro , then transplanted to the tertiary recipients. After lethal irradiation(8.5 Gy) and bone marrow transplantation, the recipients were selected with Taxol 7 mg/kg intraperitoneal injection, VCR 5 mg/kg or DNA 5 mg/kg intravenous injection. The survival rate and blood pictures of mice as well as the integration and expression of target gene MDR1 were studied. RESULTS: The lethal irradiated murine hematopoietic function could be reconstructed and protected from toxicity of high doses Taxol, VCR and DNR selection after reinfusing the hematopoietic progenitor cells containing human multidrug resistance gene (MDR1). The survival rate and survival time of experimental mice were higher than that in the control group. The integration and expression of MDR1 gene in recipients were confirmed by PCR, RT-PCR and FCM. CONCLUSION: The integration and expression of human multidrug resistance gene in recipients may play an important role in the reconstruction and protection of murine hematopoietic function.