OVER-EXPRESSION OF A TRUNCATED TYPE I TGF-P RECEPTOR IN NORMAL DERMAL FIBROBLASTS DECREASES TGF-β1 AUTOPRODUCTION

Objective To determine over-expression of a truncated type ⅡTGF-β receptor in down-regulating TGF-β1 auto production in normal dermal fibroblasts. Methods In vitro cultured dermal fibroblasts were treated with rhTGF-β1 (5ng/ml) or recombinant adenovirus containing α truncated type Ⅱ TGF-β receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-β1 were observed with Northern Blot. Results rh TGF-β1 up-regulated the gene expression of TGF-β1, (34 %-150%) and type Ⅰ pro-collagen( 13 %- 190%). Overexpression of a truncated receptor Ⅱ decreased the gene expression of TGF-β1 (53%-66%). Conclusion Over-expression of the truncated TGF-β receptor Ⅱdown-regulated TGF-β1 autoproduction via blocking signal transduction of TGF-β. This study may provide a new strategy for scar gene therapy.     

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.     

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.

     

Mutation of P53 tumor suppressor gene in colorectal cancer and its relationship with histologic grade and Dukes＇ stage

目的:探讨大肠癌组织类型、临床分期与P53基因突变的相关关系.方法:采用PCR-SSCP方法对30例大肠癌P53基因突变状况进行了检测.结果:发现6例(20%)P53基因突变,其中,1例在第4外显子,5例在第5外显子,第2,3外显子末检出突变.在不同组织分化程度中,P53基因突变率分别为高分化腺癌和中分化腺癌均为1/8(12.5%),低分化腺癌为3/10(30.0%),未分化腺癌为1/4(25.0%).在临床分期中,Dukes＇B期为1/10(10.0%),C期为2/12(16.7%),D组为3/8(37.5%).结论:P53基因突变与Dukes＇分期有关,并且P53基因突变很少发生第2,3外显子.     

Association of GYS1 and β3-AR gene with postprandial hyperglycemia and ser um uric acid in type 2 diabetes mellitus

Objective To determine the relationships of Met416Val and XbaI polymorphism of muscle glycogen synthase (GYS1) gene and Trg64Arg variant of the β3-adrenergic-receptor (β3-AR) gene with type 2 diabetes mellitus (DM) and its intermediate phenotypes in the Chinese population. Methods Polymerase chain reaction-oligonucleotide ligation assay and restriction fragment length polymorphism assay were used to evaluate the GYS1 and β3-AR gene polymorphisms in 102 pairs of case-control Chinese spouses.Results Subjects with Met416Val variant had a significantly higher 2-hour post-glucose level than subjects without this variant had in diabetic group (P=0.032).The Met416Val polymorphism of GYS1 gene was not significantly associated with the risk of type 2 DM (adjusted OR=1.67; 95% CI: 0.73-3.81, P=0.223). Subjects with Trp64Arg variant had a significantly higher serum uric acid level than subjects without this variant had in diabetic group (P=0.034). The combination of BMI and Arg64 allele carrier of the β3-AR gene increased the diabetic risk over four-fold (adjusted OR=4.00; 95%CI: 1.53-10.45, P=0.005).Conclusions In the Chinese population, Met416Val polymorphism is identified in a subgroup of diabetic subjects with high 2-hour post-glucose.It will explain why some diabetic patients appear to be genetically predisposed to developing high postpradial glucose level.The presence of the Arg64 allele in the β3-AR gene may predispose patients to higher serum uric acid level.     

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…     

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.     

Osteogenic Potential of Cultured Bone Marrow Stromal Cells Transfected with Transforming Growth Factor β_1 Gene in vitro

Bone tissue engineering has a broad prospect intreating bone defects. It is a crucial link to chooseand optimize seed cells.In recent years,bone mar-row stromal cells(BMSCs) have been regarded to beone kind of preferred seed cells[1,2 ] . We have trans-fected BMSCs with TGF- β1gene and then examinedtheirosteogenic potential in orderto improve theirbi-ological properties.1　 MATERIALSAND METHODS1 .1　 Experimental MaterialsThe materials used in the experiment includedpc DNA3 - T…     

巨幼细胞贫血骨髓幼红细胞百分比

目的 分析巨幼细胞贫血(MA)患者的骨髓幼红细胞百分比.方法 本研究包括53例未治疗的初诊MA患者.骨髓涂片经瑞氏染色后,显微镜下计数每例患者200个骨髓有核细胞.结果 53例MA患者中,36例骨髓幼红细胞百分比≥40%,占67.9%;40例骨髓幼红细胞百分比在30%～60%之间,占75.5%;全部患者骨髓幼红细胞百分比均数为45.32%,约为参考值(19.65%)的2倍多,其中位数为45.00%;幼红细胞百分比最小值为24.0%,最大值为89.0%.早幼红细胞百分比均数增加最显著,高达11.19%,为参考值(0.92%)的12.16倍,其中位数为9.50%.结论 MA患者骨髓幼红细胞百分比范围广泛,需与其他贫血相鉴别. Abstract： Objective To analyze the percentage of bone marrow erythroblast from patients with megaloblastic anemia(MA). Methods Fifty-three cases of newly diagnosed MA patients untreated were included in this study. Bone marrow smears were handled with wright's stain. For each case, 200 bone marrow nucleated cells were counted with microscope. Results The percentage of bone marrow erythroblast from 36(67.9%) of them was ≥40%. The percentage from 40(75.5%) of them was between 30% and 60%. The mean percentage of all the cases was 45.32%, more than twice the reference values(19.65%). The median percentage of all the cases was 45.00%. The minimum percentage of erythroblast was 24.0%, the maximum percentage was 89.0%. The mean percentage of basophilic erythroblasts was increased most obviously, that was as high as 11.19%, about 12.16 times of the reference values(0.92%). The percentage median of basophilic erythroblasts was 9.50％. Conclusions The percentage of bone marrow erythroblast from MA is wide in range. MA should be differentiated from other anemias.     

Stable clonal expansion of the T-cell receptor Vβ6， Vβ17 and Vβ19 T cells in a cGVHD case using genescan analysis

Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variab le regions was amplified in peripheral blood mononuclear cells drawn from one cG VHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 o r 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ re pertoire T cells.The PCR products were further analyzed by genescan to evaluat e clonality of T cells.Results Fourteen or 16 TCR Vβ subfamily T cells were detected in each sample of cGVHD c ase. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamili es.The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies. Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cG VHD cases and it may be related to the initiation of cGVHD.     

Bone marrow biopsy findings in brucellosis patients with hematologic abnormalities

Background  Brucellosis can mimic various multisytem diseases, showing wide clinical polymorphism that frequently leads to misdiagnosis and treatment delay, further increasing the complication rates. In this study, we aimed to examine bone marrow biopsy findings in brucellosis cases presenting with hematologic abnormalities.

Methods  Forty-eight brucellosis cases were prospectively investigated. Complaints and physical examination findings of patients were recorded. Patients’ complete blood count, routine biochemical tests, erythrocyte sedimentation rate, C-reactive protein and serological screenings were performed. Bone marrow biopsy and aspiration was performed in patients with cytopenia, for bone marrow examination and brucella culture, in accordance with the standard procedures from spina iliaca posterior superior region of pelvic bone.

Results  Of the 48 patients, 35 (73%) were female and 13 (27%) were male. Mean age was (34.8±15.4) years (age range: 15–70 years). Anemia, leukopenia, thrombocytopenia and pancytopenia were found in 39 (81%), 28 (58%), 22 (46%) and 10 patients (21%), respectively. In the examination of bone marrow, hypercellularity was found in 35 (73%) patients. Increased megacariocytic, erythroid and granulocytic series were found in 28 (58%), 15 (31%) and 5 (10%) patients, respectively. In addition, hemophagocytosis was observed in 15 (31%) patients, granuloma observed in 12 (25%) and increased eosinophil and plasma cells observed in 9 (19%) patients.

Conclusion  According to the results of our series, hemophagocytosis, microgranuloma formation and hypersplenism may be responsible for hematologic complications of brucellosis.

     

β-FIBRINOGEN PROMOTER -455 G/A(HaeIII)POLYMORPHISM PREDICTION OF PLASMA FIBRINOGEN BUT NOT OF ISCHEMIC CEREBROVASCULAR DISEASE

ANelevatedplasmafibrinogenlevelisariskfactorforcardiovasculardiseaseincidenceandmortality.12β-fi-brinogengenepolymorphismsinfluenceplasmafibr-Ainogenlevels.3SurveysgenerallybutnotuniformlyindicatedadultswiththeAalleleforthe-455G/ApolymorphismHaeⅢhaveincreasedfibrinogenlevels.4-7Somestudiesobservedassociationof-455Aallelewithelevatedplasmafibrinogenwaspredomin-antinsmokers89butotherstudiesalsofoundthisas-sociationtoonlybeobservedincurrentnonsmokers.10TheassociationbetweenpresenceoftheAalle…     

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.     

Expression of USP15, TβR- Ⅰ and Smad7 in Psoriasis

Summary： The deubiquitinating enzyme ubiquitin specific peptidase 15 （USP15） is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type Ⅰ TGFβ receptor （TβR- Ⅰ ）, one of the receptors of TGFβ. The expression level of USP 15 seems to play vital roles in the pathogenesis of many neoplasms, but so far there has been no report about USP15 in psoriasis. In this study, immunohistochemical staining of USP15, TβR- Ⅰ and Smad7 was performed in 30 paraffin-embedded psoriasis specimens and 10 normal specimens to investigate the expression of USP15, TβR- Ⅰ and Smad7 in psoriasis and to explore the relevance among them. And USP 15 small interfering RNA （USP 15 siRNA） was used to transfect Hacat cells to detect the mRNA expression of TβR- Ⅰ and Smad7. Of 30 cases of psoriasis in active stage, 28, 24 and 26 cases were positive for USP15, TβR- Ⅰ and Smad7 staining, respectively. The positive rates of USP15 and Smad7 were significantly higher in psoriasis specimens than in normal skin specimens （44.1%±26.0% vs. 6.1%±6.6%, 47.2%±27.1% vs. 6.6%±7.1%）, and positive rate of TβR- I （20.3%±22.2%） in psoriasis was lower than that in normal skin specimens （46.7%±18.2%）. There was a significant positive correlation between USP15 and Smad7 expression, and significant negative correlations between USP15 and TβR- Ⅰ expression, and between TβR- Ⅰ and Smad7 expression in psoriasis. After transfection of USP15 siRNA in Hacat ceils, the expression ofTβR- Ⅰ mRNA was up-regulated and that of Smad7 was down-regulated. It is concluded that USP15 may play a role in the pathogenesis of psoriasis through regulating the TβR- Ⅰ/Smad7 pathway and there may be other cell signaling pathways interacting with USP 15 to take part in the development of psoriasis.     

The Expression of Integrinβ1 and FAK in Pituitary Adenomas and Their Correlation with Invasiveness

The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 (INTβ1) and focal adhesion kinase (FAK) by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was ana- lyzed. The results showed that INTβ1 was expressed in 46 cases (93.9%) and FAK in 36 cases (73.5%), respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.     

Construction of Eukaryotic Expression Vector pBlacz and Its Expression Both in Vitro and In Vivo

（屈伸）（杨仕林）（尤颖健）（刘绍春）（何善述）ＣｏｎｓｔｒｕｃｔｉｏｎｏｆＥｕｋａｒｙｏｔｉｃＥｘｐｒｅｓｓｉｏｎＶｅｃｔｏｒｐＢｌａｃｚａｎｄＩｔｓＥｘｐｒｅｓｓｉｏｎＢｏｔｈｉｎＶｉｔｒｏａｎｄＩｎＶｉｖｏＱＵＳｈｅｎ；ＹＡＮＧＳｈｉｌｉｎ；Ｙ...     

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…