AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon(Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination.However,there are few reports on the regulation of stigma color in watermelon.In this study,a genetic analysis of the F2 population derived from ZXG1553(P1,with orange stigma)and W1-17(P2,with yellow stigma)indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma.Bulk segregant analysis sequencing(BSA-seq)revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color.Also,a major stable effective QTL Clqsc6.1(QTL stigma color)was detected in two years between cleaved amplified polymorphic sequencing(CAPS)markers Chr06_8338913 and Chr06_9344593 spanning a~1.01 Mb interval that harbors 51 annotated genes.Cla97C06G117020(annotated as zinc finger protein CONSTANS-LIKE 4)was identified as the best candidate gene for the stigma color trait through RNA-seq,quantitative real-time PCR(qRT-PCR),and gene structure alignment analysis among the natural watermelon panel.The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference.A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data.Stigma color formation is often related to carotenoids,and we also found that the expression trend of ClCHYB(annotated asβ-carotene hydroxylase)in the carotenoid metabolic pathway was consistent with Cla97C06G117020,and it was expressed in low amounts in the orange stigma accession.These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color.This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Thymosin <i>β</i>4 Improves Neurological Outcome and Enhances Induced Oligodendrogenesis in the Rat after ICH

Thymosin β4 (Tβ4), a G-actin binding protein, has diverse biological functions. This study tested the effects of Tβ4 on oligodendrogenesis in a rat model of intracerebral hemorrhage (ICH). ICH was induced by stereotactic injection of 100 μm of autologous blood into the striatum in 32 male Wistar rats. The rats were randomly divided into four groups: 1) saline control group (n = 8);2) 3 mg/kg Tβ4-treated group (n = 8);3) 6 mg/kg Tβ4-treated group (n = 8);and 4) 12 mg/kg Tβ4treated group (n = 8). Tβ4 or saline was administered intraperitoneally starting at 24 h post ICH and then every 3 days for 4 additional doses. The neurological functional outcome was evaluated by behavioral tests (i.e., modified Neurological Severity Score and corner turn test) at multiple time points after ICH. Animals were sacrificed at 28 days post ICH, and histological studies were completed. Tβ4 treatment improved neurological functional recovery significantly and increased actively proliferating oligodendrocytic progenitor cells and myelinating oligodendrocytes in the ICH-affected brain tissue, compared with the saline-treated group. The high-dose treatment of Tβ4 showed better restorative effects compared with the low-dose treatment. Tβ4 treatment enhanced ICH-induced oligodendrogenesis that may contribute to the enhanced functional recovery after ICH. Further investigation is warranted to determine the associated underlying mechanisms of Tβ4 treatment for ICH.

Cellular Senescence and SENEX Gene on the Peripheral CD4+CD25+ Treg Cells Enhancement in Elderly

Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. However, this process is also associated with apoptosis, upregulated secretion of inflammatory cytokine, and promoted surrounding tissue damage. When cellular senescence accumulates to a certain extent, it triggers geriatric diseases, such as chronic inflammation, immune senescence-associated tumors and incontrollable infections. Cellular senescence gene SENEX, which was cloned in 2004, has been demonstrated to play a unique gatekeeper function in human endothelial cells when stress-induced pre-mature senescence and apoptosis occurr. The phenomenon that CD4+CD25+ Treg cells accumulated in the aged population has been well studied in recent years. Now Treg accumulation related to immune-pathology has attracted more interest. CD4+CD25+ Treg did not decline and age, but accumulated and suppressed immunoreaction. The enhanced Treg number and function may be associated with stress-induced premature senescence-mediated unique cellular senescence protection mechanisms, and SENEX may play a critical role in this process. In this article, we summarize the cellular senescence and SENEX gene in the accumulation and functional activity of CD4+CD25+ Treg in the elderly.

Thymosin β4 promotes angiogenesis and increases muscle progenitor cell density in ischemic skeletal muscle in a mouse model of hind limb ischemia

Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arteries and internal iliac artery. Tβ4 was dissolved in sterile saline and intramuscularly injected into the centre and periphery of ligation area in the treatment group (n = 10) starting from the surgery day until 4 weeks after surgery, while control animals received saline injection only (n = 9). All animals were sacrificed at 6 weeks after surgery and used for immunohistochemistry studies. Results: Tβ4 stimulated angiogenesis was evidenced by increased vascular density based on CD31 immunostaining, which was sig- nifycantly increased in Tβ4 group (562.5 ± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2;p 0.05) groups. Tβ4 increased Pax3/7+ skeletal muscle progenitor cell density. Pax3/7+ cell density of Tβ4 group (13.7% ± 2%) was significantly higher than that of the control group (4.3% ± 1.6%, p < 0.05). However, the numbers of central nuclei fiber and central nuclei per fiber were insignificantly increased in Tβ4 group as compared to control group. The numbers of central nuclei fiber were 8.9 ± 2.1 and 9.5 ± 1.6, and the central nuclei per fiber were 0.25 ± 0.07 and 0.48 ± 0.09 for control and Tβ4 groups, respectively. Conclusions: This preliminary study suggests that localized delivery of Tβ4 increased angiogenesis and skeletal muscle progenitor cell density in ischemic skeletal muscle, but failed to promote skeletal muscle regeneration.

Genetic Polymorphism of TLR4 Gene and Correlation with Mastitis in Cattle

Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRBR2, of Chinese Holstein, Sanhe cattle, and Chinese Simmental was amplified by polymerase chain reaction （PCR）, respectively. The genetic polymorphisms in the three populations were detected by Single-Strand Conformational Polymorphism （SSCP） in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results showed that both alleles （A and B） of two loci were found in all the three populations and the value of polymorphism information content （PIC） indicated that these were a moderate polymorphism. Statistical results of X^2 test indicated that two polymorphism sites in the three populations fitted with Hardy-Weinberg equilibrium （P 〉 0.05）. After sequencing, A-G single nucleotide polymorphism （SNP） was identified at nucleotide 4,525 in intron 1 of TLR4 gene and C-T SNP was identified at nucleotide 1,397 in exon 3 of TLR4 gene. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score （SCS） was analyzed, the results indicated that the cattle with allele A in T4CRBR1 showed lower somatic cell score than that of allele B （P 〈 0.05）. In short, the allele A might play an important role in mastiffs resistance in bovine.

胸腺素β_4在心脏保护和修复中的作用

胸腺素β4在多种组织和细胞中都有表达,在心脏的发育过程中起着重要的作用,促进冠状血管的发生,诱导新生血管形成。胸腺素β4在损伤组织的再生、重构和愈合中发挥着重要的作用,促进了血管生成、内皮细胞的分化和生长,促进心肌细胞和内皮细胞的迁移,促进冠心病患者侧支循环的形成,增加心肌细胞的存活和修复,改善心脏功能。这些均显示了胸腺素β4在心肌保护和修复作用中的重要地位。

胸腺素β_4-脯氨酸寡肽酶-AcSDKP轴在肝内生物学功能的研究

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-Ser-Asp-Lys-Pro AcSDKP)由脯氨酸寡肽酶水解其前体胸腺素β4生成,三者均存在于肝内并发挥重要生物学功能。本文就胸腺素β4-脯氨酸寡肽酶-AcSDKP轴在肝内病理生理功能,特别是其与肝损伤修复和肝脏细胞外基质沉积关系的研究做一概述。

重组人胸腺素β_4基因的克隆、表达、纯化、鉴定及活性检测

目的:利用基因工程的方法原核表达重组人胸腺素β4(Tβ4)并对其进行纯化和鉴定.方法:使用大肠杆菌偏爱密码子合成人Tβ4全长基因,构建了Tβ4的二串联体基因(Tβ4②),将其克隆入表达载体pET-22b(+)中,重组质粒转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达后,经盐析、疏水层析和离子交换层析纯化重组蛋白,采用免疫印迹和基质辅助激光解吸电离飞行时间质谱对重组蛋白进行鉴定.结果:获得了纯化的重组Tβ4②蛋白,并具有生物学活性.结论:成功构建、表达和纯化了Tβ4②,为其功能研究奠定基础.

血清4型禽腺病毒的分离鉴定及致病性分析

旨在从送检的病鸡中分离鉴定出禽腺病毒(fowl adenovirus,FAdV),为临床诊断和疫病防控提供参考依据。本研究将阳性样品接种SPF鸡胚并传代,利用PCR检测、基因序列分析、动物回归试验等方法鉴定病毒。结果:经过鸡胚传代及病毒特异性检测获得了纯净的FAdV,命名为HB2306;分析Hexon基因序列可知,HB2306株属于血清4型分支,与国内外分离的FAdV-4毒株核苷酸序列同源性为98.2%~99.9%,HB2306株与国内外的4型高致病性毒株氨基酸序列相似性在97.9%~99.8%;受试动物临床病理变化显示,HB2306株以104.5EID50/只的剂量经胸部肌肉注射后,引起宿主心包积液,肝脏变黄肿胀、淤血出血,肾出血、肿胀等典型的病理变化,与流行的高致病性毒株所致临床症状相似,且死亡率为100%。综上,本研究成功分离并鉴定出一株FAdV-4高致病力毒株,丰富了毒种库,为该病毒感染的流行情况调查和综合防控奠定了基础。

香蕉枯萎病菌内源报告基因Foc4carS的鉴定及其应用

香蕉枯萎病是由尖孢镰刀菌古巴转化型(Fusarium oxysporum f. sp. cubense, Foc)引起的香蕉毁灭性土传病害,其中4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。carS基因通过调控下游car结构基因参与调控镰刀菌类胡萝卜素的生物合成,本研究克隆鉴定了Foc4carS基因(FOIG_05085),Foc4carS蛋白具有典型的RING-finger蛋白结构域。利用分割标记法(Split-marker PCR)获得Foc4carS基因的融合片段,同时构建含有Foc4carS基因sgRNA591序列的pUC-fFuCas9-HTBNLS-hph-Foc4carS基因编辑载体,通过PEG介导的原生质体转化获得该基因的敲除突变体、回补突变体以及基因编辑敲除体,并对敲除和回补突变体的生物学特性和致病力进行分析。结果显示:ΔFoc4carS突变体的菌落直径、产孢量和致病力等生物学表型与野生菌株Foc4无显著差异,而ΔFoc4carS突变体菌落颜色呈深橙色,Foc4carS基因的缺失影响了次生代谢产物类胡萝卜素的生物合成;基因编辑的ΔFoc4carS(HDR)突变体不论是再生筛选板还是继代后的PDA平板,其菌落均出现典型的深橙色,表明Foc4carS可作为内源报告基因,在香蕉枯萎菌Foc4中进行基因质粒型CRISPR/Cas9编辑可行。

冠脉支架内再狭窄患者TLR4基因rs4986790、rs4986791位点多态性及其与临床关系的研究

目的 探讨冠脉支架内再狭窄(ISR)患者Toll样受体4(TLR4)外显子基因突变情况及其与临床的关系。方法 选取2019年1月至2022年12月在右江民族医学院附属医院治疗的ISR患者137例,检测体重指数(BMI)、血压、血脂、血糖、血尿酸、C反应蛋白(CRP)、β2微球蛋白(β2MG)、白细胞介素6(IL-6)和TLR4基因rs4986790、rs4986791位点碱基。并与131例同期行冠脉支架植入术支架内无再狭窄(NISR)患者比较。结果 ISR组患者BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于NISR组(P <0.05);ISR组TLR4基因rs4986790位点碱基突变率高于NISR组(P <0.05),TLR4基因rs4986791位点碱基突变率高于NISR组(P <0.05);TLR4基因突变表型组BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于TLR4野生表型组(P <0.05)。结论 冠脉支架内再狭窄患者TLR4基因外显子突变率高,TLR4基因外显子突变的患者其代谢和炎症因子异常情况有叠加作用。

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema Correlation with variation in apparent diffusion coefficient

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 pL shRNA- aquaporin-4 （control group） or siRNA- aquaporin-4 solution （1：800） （RNA interference group） into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25- 6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5 4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson＇s correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient （r = -0.806, P 〈 0.01）. These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracelfular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema.

Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma

INTRODUCTION Hepatocellular carcinoma(HCC)is the mostcommon cause of death from cancer in China.Themechanisms of hepatocarcinogenesis are not yetknown clearly,p16INK4a gene,the multiple tumorsuppressor gene 1(MTS1),encodes P16 protein,which acts as an inhibitor by binding directly toCDK4 and CDK6 and preventing its association

Recombinant adeno-associated virus carrying thymosin β4 suppresses experimental colitis in mice

AIMTo investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4 (AAV-Tβ4) on murine colitis via intracolonic administration.METHODSAAV-Tβ4 was prepared and intracolonically used to mediate the secretory expression of Tβ4 in mouse colons. Dextran sulfate sodium (DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to establish a mouse colitis model resembling Crohn’s disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tβ4 on colitis. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis and proliferation were detected by TUNEL assay and immunochemistry, respectively.RESULTSRecombinant AAVs efficiently delivered LacZ and Tβ4 into the colonic tissues of the mice, and AAV-Tβ4 led to a strong expression of Tβ4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ4 significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tβ4 also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice.CONCLUSIONTβ4 exerts a protective effect on murine colitis, indicating that AAV-Tβ4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.

Serum thymosin β4 levels in patients with hepatitis B virus-related liver failure

AIM:To investigate whether serum thymosinβ4 can provide diagnostic or prognostic information in liver failure patients caused by chronic hepatitis B virus(HBV) infection. METHODS:Serum thymosinβ4 levels were measured in 30 patients with acute-on-chronic liver failure(ACLF), 31 patients with chronic liver failure(CLF),30 patients with compensated liver cirrhosis(CR)and 32 patients with chronic hepatitis B and 30 healthy controls.Serum thymosinβ4 levels were measured by enzyme-linked immunosorbent assay and Child-Pugh and model for end-stage liver disease(MELD)scores were calculated for each patient on admission.RESULTS:Compared with healthy controls,serum thymosinβ4 levels in ACLF,CLF,CR and chronic hepatitis B patients were significantly lower,6.5047 (4.7879-10.5314)μg/mL vs 0.4632(0.2759-0.8768) μg/mL,0.6981(0.5209-1.2008)μg/mL,1.8053 (0.8110-2.3397)μg/mL,3.7803(1.8570-6.4722)μg/mL, respectively(P<0.001).The levels of thymosinβ4 in liver failure(ACLF or CLF)patients were markedly lower than that in CR(P<0.001),and a difference was also found between CLF and ACLF patients(P=0.038).In patients with chronic liver disease,there was a positive relationship between thymosinβ4 levels and albumin, choline esterase,and platelet(P<0.001),and negative relationship with alanine aminotransferase(P=0.020), aspartate aminotransferase,total bilirubin,international normalized ratio of prothrombin time,and Child-Pugh and MELD scores(P<0.001).Of the 61 liver failure patients,the thymosinβ4 levels of non-survivors were significantly lower than that of survivors(P=0.007). Receiver operating characteristics analysis identified a thymosinβ4 cutoff level of 0.5708μg/mL for predicting poor prognosis in all liver failure patients.The serial thymosinβ4 values were observed in 13 liver failure inpatients.Lower initial values were observed in the death.While greater improvement in thymosinβ4 value was found in those who recovered from the disease. CONCLUSION:Serum thymosinβ4 can be used as an important potential predictor for liver failure caused by chronic HBV infection.

Association of Graves’ disease and Graves’ ophthalmopathy with the polymorphisms in promoter and exon 1 of cytotoxic T lymphocyte associated antigen-4 gene

Objective: To investigate the association of Graves’ disease and Graves’ ophthalmopathy with the C/T transition polymorphism at position –318 of promoter and the A/G transition polymorphism at position 49 of exon 1 within cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene. Methods: Thirty-three patients with ophthalmopathy of Graves’ disease, fifty-six Graves’ patients without ophthalmopathy and sixty normal subjects as control were involved in the present case-control study. The polymorphisms were evaluated by polymerase chain reaction fragment length polymorphism (PCR-RFLP). Com-parisons were made of gene frequencies and allele frequencies between the groups. Results: The gene frequencies of CT and allele frequencies of T were much higher in Graves’ patients with ophthalmopathy than that in the group without ophthalmopathy (P=0.020, P=0.019). The gene frequencies of GG and allele frequencies of G in patients with Graves’ disease were significantly increased as compared with control group (P=0.008, P=0.007). The data suggest that smokers with Graves’ disease seemed to be more predisposed to ophthalmopathy than non-smokers (P=0.018). Conclusion: Our results suggest that an allele of T at position –318 of promoter is associated with genetic susceptibility to Graves’ ophthalmopathy while an allele of G at position 49 of exon 1 is associated with genetic susceptibility to Graves’ disease instead. Smoking is believed to be a major risk factor for ophthalmo-pathy.

人胸腺素β_4 RNA干扰载体的构建及其对293T细胞目的基因表达的影响

目的探讨人胸腺素β4(thymosin β4,TMSB4)基因RNA干扰慢病毒载体的构建,观察病毒感染293T细胞后TMSB4 mRNA和蛋白表达,为进一步研究TMSB4基因的功能提供基础。方法设计特异性干扰人TMSB4基因的小发卡RNA序列,合成含有干扰序列的双链DNAoligo,与经AgeI和EcoRI双酶切后的pGCSIL-GFP载体连接产生慢病毒载体。转化感受态大肠杆菌DH5a并对阳性克隆进行PCR扩增测序鉴定。慢病毒载体、包装系统质粒共转染包装细胞293T细胞,产生慢病毒颗粒并测定病毒滴度。将获得的慢病毒感染293T细胞,分别采用实时定量PCR和免疫细胞化学染色方法观察TMSB4 mRNA和蛋白的表达情况,利用生成的TMSB4 shRNA慢病毒感染原代成纤维细胞。结果感染重组慢病毒的293T细胞与空病毒转染阴性对照细胞的TMSB4 mRNA表达分别为0.56±0.11和1.00±0.06(P=0.0006),实验组细胞的敲减率为44%。实验组TMSB4蛋白表达水平0.042±0.007比阴性对照组细胞0.093±0.009的表达降低55%(H=461.342,P<0.001)。感染重组慢病毒的原代培养的成纤维细胞与空病毒转染的对照细胞相比,TMSB4蛋白表达水平亦明显降低。结论TMSB4基因干扰慢病毒构建成功并能显著降低TMSB4基因和蛋白在293T细胞中的表达,成功感染原代成纤维细胞使其目的基因蛋白减少,为进一步研究TMSB4基因的功能奠定研究基础。

Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.