KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

Objective: To establish two stably KGF-transfected, immortalized cell lines. Methods: HaCaT-keratinocytes and KMST-6-fibroblasts were transfected by liposome mediated gene transfer. Transfection effectivity, gene integration and configuration of the transgenic protein were investigated by ELISA, DANN-PCR and β-Gal-staining. Results: Most effective GF producing clones were tested by a colorimetric XTT-test. Conclusion: This is a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system.     

Is the human dystrophin gene’s intron structure related to its intron instability?

Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene’s intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5’ and 3’ breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.     

Adenovirus-mediated transfer of β-galactosidase and prourokinase genes into vein grafts

Objective To study the feasibility of adenovirus mediated gene transfer into vein grafts and the role of the prourokinase gene in protecting vein grafts from thrombosis.Methods Fifty-two Wistar rats underwent implantation of reversed autologous jugular vein interposition grafts in the common carotid arteries. Jugular veins were excised and distended with solution containing three different adenovirus vectors (Adv(5)-CMV, group Ⅰ; Adv(5)-CMV/LacZ, group Ⅱ; Adv(5)-CMV/Pro-UK, group Ⅲ) for 30 min, then the jugular veins were reversed and interposed into the divided carotid arteries, and end-to-end anastomoses were performed. The amount of (51)Cr-labeled platelets in vein grafts of group Ⅰ and group Ⅲ was counted 24 hours postoperatively. On the 14th day, the vein grafts were harvested to examine β-galactosidase activity and prourokinase (Pro-UK) activity and observe thrombosis in vein grafts. Results Extensive blue coloration in the area of intima and media of each vein graft in group Ⅱ was observed. No blue coloration was seen in group Ⅰ. Pro-UK activity was not detected in the vein grafts of group Ⅰ. In group Ⅲ, the amount of Pro-UK gene expression was 308 IU/g tissue. The amount of (51)Cr labeled platelets in group Ⅰ and group Ⅱ was (123.7±19.4) ×10(6)/g dry wt, (34.4±5.3) ×10(6)/g dry wt, respectively. The thrombosis rate and occlusion rate of the vein grafts in group Ⅰ were 30% and 10%, respectively. In group Ⅲ, all vein grafts were patent and free of thrombosis. Conclusions Ex vivo gene transfer before vein grafting is feasible using replication deficient recombinant adenovirus and results in a high level of gene expression in vivo. Direct transfer of the Pro-UK gene into vein grafts may prevent thrombosis.     

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

Hepatocellularcarcinoma (HCC)isamajorcauseofcancerdeathwithmorethan 1.2millionglobalan nualincidences[1] .Surgeryandlivertransplantationaretheonlyeffectivetreatments ,butmostHCCpa tientsarenoteligiblebecauseofdiagnosisatalaterstageorunderliverinsufficiencyinthesettingofcir rhosis[2 5] .Thedevelopmentofnoveltreatmentstrategiesisneeded .TheAFPisanHCC associatedoncofetalantigen .ThemajorityofhumanHCCsoverexpresstheoncofetalantigenAFP .ThisMr 700 0 0 glycoprotein ,producedathighlevelsbyth…     

Identification and analysis of mutations in WTX and WT1 genes in peripheral blood and tumor tissue of children with Wilms’ tumor

Background  Wilms’ tumor (nephroblastoma) is the most common pediatric kidney cancer. Only one Wilms’ tumor gene is known, WT1 at 11p13, which is mutated in 5%–10% of Wilms’ tumors. Recently, mutations were reported in WTX at Xq11.1 in Wilms’ tumors. This study investigated the mutation proportion, type, and distribution in WTX and WT1 in children with Wilms’ tumor. The role of WTX/WT1 in the development of Wilms’ tumor, and the relationship between clinical phenotype and genotype, were also studied.

Methods  Wilms’ tumor specimens (blood samples from 70 patients and tumor tissue samples from 52 patients) were used. A long fragment of WTX and 10 exons and intron sequences of WT1 were amplified by polymerase chain reaction (PCR) from extracted genomic DNA and sequenced. A chi-square test compared the difference between the WTX mutation group and the no mutation group. The relationship between the mutations and clinical phenotype was analyzed.

Results  WTX mutations were found in 5/52 tumor tissues and in 2/70 peripheral blood samples (five cases in total, all point mutations). Two patients had a WTX mutation in both samples. WT1 mutations were found in 2/52 tumor tissues and in 4/70 peripheral blood samples (five cases in total, all point mutations). One patient had a WT1 mutation in both samples. Ten cases had WTX or WT1 mutation (19.2% of Wilms’ tumors). No overlapping WTX and WT1 mutations were found. No significant differences in clinical parameters were found between patients with and without a WTX mutation.

Conclusions  WTX mutations occur early in Wilms’ tumor development, but at a low proportion. There was no evidence that WTX is the main cause of Wilms’ tumor. Clinical parameters of patients with WTX mutations are not related to the mutation, indicating a limited impact of WTX on tumor progression. WTX and WT1 mutations occur independently, suggesting a relationship between their gene products.

     

Multichannel piezoelectric genesensor for the detection of human papilloma virus

Ｗｉｔｈｉｎｔｈｅｐａｓｔｔｅｎｙｅａｒｓ,ｔｈｅｒｅｈａｓｂｅｅｎｉｎｃｒｅａｓｉｎｇｉｎｔｅｒｅｓｔｉｎｔｈｅｄｅｔｅｃｔｉｏｎｏｆｓｐｅｃｉｆｉｃＤＮＡｓｅｑｕｅｎｃｅｓｕｓｉｎｇｍｅｔｈｏｄｓｗｈｉｃｈｄｏｎｏｔｒｅｑｕｉｒｅｔｈｅｕｓｅｏｆｒａｄｉｏｉｓｏｔｏｐｅｓ ,ｅｎｚｙｍｅｓｏｒｆｌｕｏｒｏｐｈｏｒｅｓ Ｂｉｏｓｅｎｓｏｒｓｙｓｔｅｍｓｂａｓｅｄｏｎｎｕｃｌｅｉｃａｃｉｄｎｏｔｏｎｌｙｅｌｉｍｉｎａｔｅｔｈｅｎｅｅｄｆｏｒｓｕｃｈｌａｂｅｌｓｂｕｔａｌｓｏｏｆｆｅｒｔｈｅｐｏｔｅｎ…     

The Effectand Mechanism of Forsinopril on Ventricular Hypertrophy of SHR and Left Ventricular Pressure overloading Rat

Ventricular remodeling,the structural andmorphological changes of ventricle occurring dur-ing heart function transfer from compensation todecompensation in the processof heartdisease,is akind of self- evolved process which will lead toheartfunction failure[1] .Renin- Angiotensin system(RAS) plays a very importantrole in this process,especially the RAS in local heart tissue (localRAS) .After local RAS was activated,the activa-tion will last outeven when the nosogenesis disap-pears.As the m…     

The gene diagnosis and mutation survey of autosomal dominant polycystic kidney disease Ⅰ

Theprevalenceofautosomaldominantpoly-cystickidneydiseaseI(ADPKDI)inChineseisaboutl.l/1ooo['],itisthethirdcauseofadultre-nalfailure.Theetiologyandpathogenesisarestillnotclearandnoprofitabletherapycanbeofchoice'Inrecenttenyears,althoughalotofad-vancementofADPKDIgeneresearchhadbeenmade,andthisisthemostpossiblewayforcuringthedisease,ithassti1lnotreachedtheidealaim[2j-ThispaperobjectiveistoexploregenediagnosisofADPKDIandtolookfortypicalmutationinordertoimprovethegenediagnosis.MATERIALSA…     

Rearranged Patterns of IgH and TcRγ Genes in Patients with Acute Lymphoblastic Leukemia

TherearrangementofIgHandToRYgenesinpatientswithacutelymphoblasticleukemia(ALL)generatedspecific--tumormarke..[11,andtherearrangedpatternsreflectedstatusofcloneformationofleukemiccells.Byusingpolymerasechainreaction(PCR)technique.weexaminedtheregrrangementofIgHandToRYgenesin30patientswithALLinanattempttoanalyzerearrangedpatternsofIgHandToRYgenesatthemolecularlevel.IMATERIALSANDMETHODS1.1PatientsThirtypatients,(20maleand10female)withanaverageageof25.6years,werediagnosedashaving…     

Cloning of human brevican cDNA and expression of its mRNA in human glioma

Objective: To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma. Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human anaplastic astrocytoma by RT-PCR, and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization. Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained. In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas (100%), whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined. Conclusion: The results suggest that brevican mRNA is highly and specifically expressed in human glioma.     

Analysis of the T cell receptor Vδ region gene repertoire in bronchoalveolar lavage fluid (BALF) and peripheral blood of asthmatics

Objective To explore the role of γδT cells in the airway of asthmatics and to identify t he forces which induce and maintain the inflammatory process.Methods Peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) were obtained from 7 asthmatic subjects and 7 nonsmoker control subjects. The percentage of γδT cells in the PB and BALF was measured by immunofluorescent staining and flow cy tometry. The frequency of usage and the clonality of Vδ subfamilies (Vδ(1)-V δ(3)) were assessed by RT- PCR and gene scanning. Results A higher proportion of γδT cell was detected in the BALF of asthmatic subjects (7.8%±4.7%) than that from control subjects (3.3%±3.0%, P=0.04). No selective usage for a particular Vδ subfamily was found, but the relative ex pression level of Vδ(1) was significantly higher in the asthmatic airway (44%± 13%) than in the control (19%±5%, P=0.0002). In asthmatic subjects, the m onoclonal or oligoclonal expansion of γδT lymphocytes was predominant in the B ALF, especially Vδ(1)(+) T lymphocytes.Conclusions Antigenic specific γδT cells might play an important role in the inducement an d maintenance of airway inflammation. Persistent antigenic stimulation may be t he key factor that maintains chronic airway inflammation in asthma.     

Association of coagulation factor Ⅶ with the risk of myocardial infarction in the Chinese

Objective To elucidate the association of plasma factor Ⅶ coagulant activity (FⅦc) w it h the risk of myocardial infarction (MI) and to assess the influence of factor Ⅶ gene MspI polymorphism and lipid metabolism on FⅦc in the Chinese.Methods A total of 137 patients with angiographically confirmed MI and 125 healthy indiv iduals were evaluated retrospectively. Plasma FⅦc was measured by one-sta ge prothrombin time, and FⅦ genotype was determined after MspI digestion of polym erase chain reaction-amplified genomic DNA. Serum lipid levels were assessed b y routine methods.Results MI patients had significantly higher levels of FⅦc (119.5%±22.7% vs 99. 9% ±21.8%, P<0.01) and total serum cholesterol (5.80±1.06 mmol/L vs 5.5 3±1.08 mmol/L, P<0.05) than controls, but only FⅦc independently co rrelated with the risk of MI (OR=1.04, P<0.01). There were no significant di fferences in FⅦ genotype or allele frequency between patients and controls ( P>0.05). Subjects with the Gln353 allele were associated with significantly lower FⅦc levels than Arg353 homozygotes (99.7%±19.3% vs 111.4%±24.6% , P<0.05). Serum triglyceride was positively correlated with plasma FⅦc in both control (r=0.25, P<0.01) and case (r=0.87, P<0.01) gro ups, but this correlation was restricted to Arg/Arg genotype (r=0.68, P <0.01). A significant correlation of total serum cholesterol with FⅦc onl y appeared in Arg/Arg homozygotes (r=0.17, P<0.01).Conclusions Our findings support the role of plasma FⅦc as a risk factor for MI in Chin es e. Plasma triglyceride and FⅦ gene MspI polymorphism are two independent d eter minants of FⅦc. Assay of this polymorphism will be helpful in determining who will benefit most from lipid-lowing therapy.     

Antitumor Effects of the Fibroblasts Transfected TNF-α Gene and its Mutants

Summary: To compare the anti-tumor effects of transmembrane TNF-α (TM-TNF) and secreted TNF-α (S-TNF) in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mu tant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-α gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-α or its mutants and effectively kill H22 in vitro. The trans fected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation ac cording to a ratio of 5: 1 or 1: 1 (effector/target cells, E/T) after the third day of H22 challenge,respectively. At the E/T= 5 : 1, the NIH3T3/TM-TNFm induced the highest tumor regression,while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T= 1 .: 1 in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.     

Effects of transfection of ICAP-1α and its mutants on adhesion and migration of 2H-11 cells

This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed.After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting.Adhesion ability was evaluated by using MTT.Cell migration was determined by using Boyden chamber method.Tube formation test was conducted on Matrigel.The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased.In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group.ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group.Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively.In ICAP-1α group, the ratio was decreased to 0.1005±0.0073.In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group.In ICAP-1α group, the number was decreased to 12.06±1.72.The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71.In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24.It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect.These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.     

Improvement of Chemically—activated Luciferase Gene Expression Bioassay for Detection of Dioxin—Iike Chemicals

To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs. Method A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-Odeethylase (EROD) activity induction assay. Result The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.1 lpmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%,Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.     

Study of Rat Osteoblasts Transfected by Transforming Growth Factor β1 Gene

Summary: In order to investigate the effect of TGFβ1 gene transfer on the biological characteristics,the effects of gene transfer and supernatant of transfected osteoblasts on the proliferation and ALP activity of osteoblasts were detected by 3H-TdR and MTT. Our results showed that TGFβ1 gene transfer had no effect on the biological characteristics and the activated supernatant of transfected os-teoblasts stimulated proliferation and inhibited ALP activity of osteoblasts. TGFβ1 gene transfer could promote the expression of TGFβ1 and the biological characteristics of transfected osteoblasts were sta-ble, which might be helpful for gene therapy of bone defects in vivo.     

The Binding Ability Analysis of the Normal VLDL Receptor and Its Mutant

VLDL receptor,which belongs to the LDL re-ceptor family[1] ,is made up of 846amino acids. Itsstructure issimilar to thatof the LDL receptor.Bothof them contain five functional domains:ligand bind-ing domain,pre- EGF homologous domain,O- linksugar domain,transmembrane and cytoplasmic re-gion[2 ] . The key structuraldifference between VLDLreceptor and LDL receptor is in the ligand- bindingdomain.LDL receptor's ligand binding domain hasseven cysteine- rich repeats and that of VLDL rece…