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.     

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.     

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…     

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.     

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…     

Clinical efficacy and molecular mechanism of nourishing shen and supplementing marrow principle in treating β-thalassemia

Objective: To explore the possibility of using traditional Chinese medicine (TCM) in treating β-thalassemia, its clinical effect and molecular mechanism of the action.Methods: According to the TCM theory of “Shen producing marrow”, the composite recipe, Yisui Shenxueling Granule (YSSXL), consisting of Chinese drugs for nourishing Shen and supplementing marrow (NS & SM) was given orally to 78 patients with β-thalassemia (49 of the severe type and 29 of moderate type), 3 times a day, 10 g each time (for children, the dose would be reduced properly), with 3 months as one therapeutic course, and no blood transfusion used in the course. The clinical therapeutic efficacy and hematologic parameters in patients were observed, and systemic gene analysis was conducted with PAGE, PCR, PCR-SSCP, RT-PCR and DNA sequences analysis and mRNA detection, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation.Results: YSSXL showed obvious therapeutic effect in treating β-thalassemia. Gene analysis revealed that it did not change the genetic mutation type, but could obviously increase hemoglobin, fetal hemoglobin (HbF), γ/(β+γ) globin ratio, γ-globin mRNA expression and GM-CSF mRNA expression in patients, as well as the GM-CSFmRMA in marrow of mice after60Co radiation.Conclusion: YSSXL has a remarkable therapeutic effect on β-thalassemia, and its possible mechanism is its action in unlocking γ-gene, increasing the γ-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has opened a new path for the treatment of β-thalassemia with TCM. This item was supported by National Funds of Natural Sciences (No. 30171199) and Natural Science Foundation of Guangxi Autonomous Region (No. 014402C)     

米托蒽醌配合生脉注射液治疗老年性急性髓性白血病

目的：观察生脉注射液防止米托蒽醌治疗老年性急性髓性白血病所致骨髓抑制的临床疗效。方法：35例患者随机分为治疗组17例和对照组18例。两组均采用米托蒽醌联合阿糖胞苷治疗。治疗组加用生脉注射液治疗。结果：治疗组与对照组总有效率分别为70．6％、66．7％（P＞0．05）。治疗组和对照组完全缓解率分别为52．9％,44．4％（P＞0．05）,治疗组完全缓解时间和白细胞降低最低持续时间均短于对照组（P＜0．01）。化疗后感染率及出血发生率治疗组明显低于对照组（P＜0．01）。结论：生脉注射液能明显减轻和缩短米托蒽醌对骨髓的抑制作用。     

小鼠骨髓来源树突细胞体外扩增和鉴定

目的　探讨从小鼠骨髓分离纯化及大量扩增树突细胞 (DC)的方法 ,并对其行免疫表型和形态鉴定。　方法　制备 Balb/ c小鼠骨髓细胞 ,利用 0 .83% NH4 Cl- Tris溶液、抗鼠 CD4 / CD8/ CD4 5R单克隆抗体和补体溶液 ,依次去除红细胞、淋巴细胞、单核 -巨噬细胞、粒细胞等混杂细胞以获得纯化的 DC,再将其培养于含有小鼠重组的粒细胞 -巨噬细胞集落刺激因子 (m GM- CSF)和白细胞介素 4 (m IL - 4 )的 RPMI 1 6 4 0营养液中 ,培养第 6～ 8天收集悬浮的细胞 ,电子显微镜观察细胞形态并用流式细胞术检测小鼠 DC细胞表面特有的 CD1 1 b抗原和 MHC- 类分子的表达量。　结果　每只小鼠可扩增到 (2～ 4 )× 1 0 6个 DC细胞 ,细胞纯度 >85 % ;细胞表面高水平表达小鼠 DC细胞特异性抗原 CD1 1 b和 MHC- 类分子 ,电镜下细胞形态符合典型的树突细胞形态。　结论　联合应用 m GM- CSF和m IL- 4刺激小鼠骨髓细胞可扩增到大量高纯度的树突细胞     

先天性角化不良症患儿的临床特征和基因分析：附1例报告

目的：探讨先天性角化不良症（dyskeratosis congenita, DC）患儿的临床特征和基因特点。方法回顾我院收治的1例DC患儿的临床资料，提取患儿外周血DNA，PCR扩增DC的7个热点基因，包括DKC1、TERT、TERC、TINF2、NOP10、NHP2、WRAP53，进行DNA测序和基因分析。结果患儿外周血标本检测出DKC1基因中一个半合子变异：c.85-15T>C。其母亲为相应变异杂合携带者，并出现部分先天性角化不良的临床表现如指（趾）甲变形等。结论当低龄患儿出现典型皮肤黏膜改变、骨髓衰竭、肿瘤易感性、有肿瘤家族史时，应考虑到DC可能。早期行相关基因检测可提高临床诊断的诊断率，减少误诊、漏诊。     

人骨形成蛋白7基因的克隆及其在兔骨髓基质干细胞中的瞬时表达

目的克隆人骨形成蛋白7(BMP-7基因,并构建其真核表达载体,观察其在兔骨髓基质干细胞(rBMSC)中的表达并探讨其应用于骨科局部基因治疗的可能性.方法用RT-PCR法从人胎肾组织中克隆人BMP-7基因全长cDNA并测序.将BMP-7基因cDNA克隆到穿梭载体pShuttle中构建表达载体pShuttle-BMP-7,经鉴定后利用LipofectAMINE2000瞬时转染rBMSC,用RT-PCR方法及免疫组化检测BMP-7基因的表达.结果用RT-PCR法从胎肾组织中克隆出1296 bp的cDNA,测序证实为人BMP-7基因.RT-PCR方法及免疫组化检测证实其能在rBMSC中表达.结论成功克隆人BMP-7基因并证实其在rBMSC的表达,为下一步腺病毒介导的局部基因治疗打下基础.     

深师、梅师及其著作考

据现存资料记载,僧深（深师）为南北朝时期宋齐间道人;梅师,号文梅,为隋广陵僧人。两人看似风马牛不相及,好像没有任何关系,但李时珍在《本草纲目》的＂引据古今医家书目＂称：＂深师脚气论即梅师＂,一句话将事情变得复杂起来。历史上深师和梅师到底有没有联系,是否是一个人,笔者通过对《隋书.经籍志》、《备急千金要方》、《外台秘要》和《医心方》等书中有关僧深和《僧深方》的记载进行考证,结合其他历史事实综合分析,发现李时珍的结论是正确的,即僧深就是梅师。     

MDR1基因修饰的自体骨髓移植后体内表达及时限的研究

目的 研究多药耐药基因1(multidrug resistanle gene 1, MDR1)转染的骨髓单个核细胞自体移植后,外源性MDR1基因在骨髓中的功能性表达及时限.方法 体外浓缩病毒上清转染法将MDR1基因导入兔骨髓单个核细胞;经大剂量环磷酰胺化疗预处理后,将转染的骨髓单个核细胞行自体骨髓移植;采用PCR法、免疫组织化学法和柔红霉素(daunorubicin, DNR)排出试验检测MDR1基因在受体骨髓中的整合及功能性表达.结果 自体骨髓移植后1～4个月,PCR法检测到外源性MDR1基因在骨髓细胞基因组中的整合;免疫组化法测得骨髓单个核细胞中P-gp阳性率分别为9.5%、8.5%、6.0%、3.5%;柔红霉素排除试验检测到定植的MDR1基因能功能性的表达.结论 转染MDR1基因的骨髓单个核细胞自体移植后,MDR1基因能定植于骨髓中并功能性的表达4个月.     

HLX基因在AML中的表达及预后分析

目的 分析H2.0样同源盒基因（H2.0-like homeobox gene,HLX）在急性髓系白血病（acute myelogenous leukemia,AML）患者中的临床表达情况,探究HLX基因与AML疾病预后的关系。方法 收集56例AML患者的骨髓单个核细胞,实时荧光定量PCR （quantitative real-time polymerase chain reaction,qRT-PCR）检测HLX的表达水平,并结合预后基因进行分析。结果 在临床初发的AML患者中,HLX的表达水平高于正常人（P<0.01）,HLX表达水平与白血病组患者年龄、骨髓原始细胞数显著相关（P<0.05）,HLX表达水平与染色体核型代表的预后分层无明显关联（P>0.05）。结论 HLX基因在AML患者中的表达水平是上调的,可能拥有独立的预后信息,是AML中潜在的干预靶点。     

VEGF基因在兔骨髓基质细胞中的表达及活性测定

目的 探讨人血管内皮细胞生长因子(VECF)基因修饰的免骨髓基质细胞对VEGF基因的表达及表达产物的生物学活性。方法 制备真核表达质粒pAdCMV-VEGF165,以Superfect转染试剂介导VFGF基因转染兔骨髓基质细胞,收获转染后24h至6d的转染上清和细胞,采用RT-PCR技术、免疫组化SABC法检测转染细胞中外源性VECF165基因的转录及表达,用血管内皮细胞增殖试验测定转染细胞培养上清中VEGF的生物活性。结果 VEGF基因转染的兔骨髓基质细胞可有效转录VEGF165,其分泌于培养上清中的表达产物可促进血管内皮细胞增殖,具有很强的生物活性。结论 VEGF基因转染的兔骨髓基质细胞可有效表达具有生物活性的VEGF。     

人糖基化磷脂酰肌醇特异性磷脂酶D的基因结构初探

探讨人糖基化磷脂酰肌醇特异性磷脂酶D (GPI PLD)的cDNA及其基因组基因的结构。从人骨髓基质细胞克隆出的GPI PLDcDNA中发现 ,它能编码信号肽 ,其编码的酶蛋白成熟肽为 817个氨基酸残基。该GPI PLDcDNA与从人肝组织和胰腺组织克隆到的GPI PLDcDNA的碱基序列的同源性分别为 95 %和 99%。人GPI PLD基因组基因定位于 6p2 2 .1～ 2 2 .3区域 ,包含 2 5个外显子 ,其上游调控区具有启动子 (TATA盒与CAAT盒 )、增强子核心序列 (TGGAAG)及同源转换盒Pit 1/GHF 1结合序列 (TATGCATAA)等顺式作用元件。